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Sample records for high-affinity phosphate transporter

  1. Na+-Dependent High-Affinity Nitrate, Phosphate and Amino Acids Transport in Leaf Cells of the Seagrass Posidonia oceanica (L. Delile

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    Lourdes Rubio

    2018-05-01

    Full Text Available Posidonia oceanica (L. Delile is a seagrass, the only group of vascular plants to colonize the marine environment. Seawater is an extreme yet stable environment characterized by high salinity, alkaline pH and low availability of essential nutrients, such as nitrate and phosphate. Classical depletion experiments, membrane potential and cytosolic sodium measurements were used to characterize the high-affinity NO3−, Pi and amino acids uptake mechanisms in this species. Net uptake rates of both NO3− and Pi were reduced by more than 70% in the absence of Na+. Micromolar concentrations of NO3− depolarized mesophyll leaf cells plasma membrane. Depolarizations showed saturation kinetics (Km = 8.7 ± 1 μM NO3−, which were not observed in the absence of Na+. NO3− induced depolarizations at increasing Na+ also showed saturation kinetics (Km = 7.2 ± 2 mM Na+. Cytosolic Na+ measured in P. oceanica leaf cells (17 ± 2 mM Na+ increased by 0.4 ± 0.2 mM Na+ upon the addition of 100 μM NO3−. Na+-dependence was also observed for high-affinity l-ala and l-cys uptake and high-affinity Pi transport. All together, these results strongly suggest that NO3−, amino acids and Pi uptake in P. oceanica leaf cells are mediated by high-affinity Na+-dependent transport systems. This mechanism seems to be a key step in the process of adaptation of seagrasses to the marine environment.

  2. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

    OpenAIRE

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-01-01

    Zr(IV) can form phosphate and Zr(IV) (?PO3 2??Zr4+?) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). Aft...

  3. Novel and high affinity fluorescent ligands for the serotonin transporter based on (s)-citalopram

    DEFF Research Database (Denmark)

    Kumar, Vivek; Rahbek-Clemmensen, Troels; Billesbølle, Christian B

    2014-01-01

    Novel rhodamine-labeled ligands, based on (S)-citalopram, were synthesized and evaluated for uptake inhibition at the human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) and for binding at SERT, in transiently transfected COS7 cells. Compound 14 demons...... demonstrated high affinity binding and selectivity for SERT (K i = 3 nM). Visualization of SERT, using confocal laser scanning microscopy, validated compound 14 as a novel tool for studying SERT expression and distribution in living cells....

  4. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

    Science.gov (United States)

    Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

    2016-10-12

    Zr(IV) can form phosphate and Zr(IV) (-PO₃ 2- -Zr 4+ -) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A 520nm / A 650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  5. Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV

    Directory of Open Access Journals (Sweden)

    Wenjing Qi

    2016-10-01

    Full Text Available Zr(IV can form phosphate and Zr(IV (–PO32−–Zr4+– complex owing to the high affinity between Zr(IV with phosphate. Zr(IV can induce the aggregation of gold nanoparticles (AuNPs, while adenosine triphosphate(ATP can prevent Zr(IV-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRAsensor for ATP have been developed using AuNPs based on the high affinity between Zr(IVwith ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV. After the addition of ATP, ATP reacts with Zr(IV and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV, ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945 with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

  6. Humic Acid Confers HIGH-AFFINITY K+ TRANSPORTER 1-Mediated Salinity Stress Tolerance in Arabidopsis.

    Science.gov (United States)

    Khaleda, Laila; Park, Hee Jin; Yun, Dae-Jin; Jeon, Jong-Rok; Kim, Min Gab; Cha, Joon-Yung; Kim, Woe-Yeon

    2017-12-31

    Excessive salt disrupts intracellular ion homeostasis and inhibits plant growth, which poses a serious threat to global food security. Plants have adapted various strategies to survive in unfavorable saline soil conditions. Here, we show that humic acid (HA) is a good soil amendment that can be used to help overcome salinity stress because it markedly reduces the adverse effects of salinity on Arabidopsis thaliana seedlings. To identify the molecular mechanisms of HA-induced salt stress tolerance in Arabidopsis, we examined possible roles of a sodium influx transporter HIGH-AFFINITY K+ TRANSPORTER 1 (HKT1). Salt-induced root growth inhibition in HKT1 overexpressor transgenic plants (HKT1-OX) was rescued by application of HA, but not in wild-type and other plants. Moreover, salt-induced degradation of HKT1 protein was blocked by HA treatment. In addition, the application of HA to HKT1-OX seedlings led to increased distribution of Na+ in roots up to the elongation zone and caused the reabsorption of Na+ by xylem and parenchyma cells. Both the influx of the secondary messenger calcium and its cytosolic release appear to function in the destabilization of HKT1 protein under salt stress. Taken together, these results suggest that HA could be applied to the field to enhance plant growth and salt stress tolerance via post-transcriptional control of the HKT1 transporter gene under saline conditions.

  7. Blockage of High-Affinity Choline Transporter Increases Visceral Hypersensitivity in Rats with Chronic Stress

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    Chen Zhao

    2018-01-01

    Full Text Available Background. Visceral hypersensitivity is a common feature of irritable bowel syndrome. Cholinergic system involves in the development of visceral hypersensitivity, and high-affinity choline transporter (CHT1 is of crucial importance in choline uptake system. However, involvement of CHT1 in visceral hypersensitivity remains unknown. The research aimed to study the CHT1 expression in dorsal root ganglions (DRGs and the role of CHT1 in visceral hypersensitivity. Methods. Repetitive water avoidance stress (WAS was used to induce visceral hypersensitivity in rats. Colorectal distension (CRD was determined, and the abdominal withdrawal reflex (AWR and threshold intensity data were recorded to measure the visceral sensitivity. After intraperitoneal injection of hemicholinium-3 (HC-3, the specific inhibitor of CHT1, CRD data were also recorded. The CHT1 expression of DRGs was investigated by Western blotting, immunohistochemistry, and quantitative RT-PCR. Acetylcholine levels in the DRGs were detected by the assay kit. Results. Repetitive WAS increased the AWR score of CRD at high distension pressure and decreased the mean threshold of rats. The CHT1 expression and acetylcholine concentration of DRG were significantly increased in WAS rats. After the administration of HC-3, the AWR score in WAS group was significantly increased at higher distension pressure while the threshold intensity was significantly reduced compared to the normal saline group. Acetylcholine concentration was significantly lower than the normal saline rats. Conclusion. Our research firstly reports that CHT1 is overexpressed in noninflammatory visceral hypersensitivity, and blockage of CHT1 can enhance the visceral hypersensitivity. CHT1 may play an inhibitory role in visceral hypersensitivity.

  8. Blockage of High-Affinity Choline Transporter Increases Visceral Hypersensitivity in Rats with Chronic Stress

    Science.gov (United States)

    2018-01-01

    Background Visceral hypersensitivity is a common feature of irritable bowel syndrome. Cholinergic system involves in the development of visceral hypersensitivity, and high-affinity choline transporter (CHT1) is of crucial importance in choline uptake system. However, involvement of CHT1 in visceral hypersensitivity remains unknown. The research aimed to study the CHT1 expression in dorsal root ganglions (DRGs) and the role of CHT1 in visceral hypersensitivity. Methods Repetitive water avoidance stress (WAS) was used to induce visceral hypersensitivity in rats. Colorectal distension (CRD) was determined, and the abdominal withdrawal reflex (AWR) and threshold intensity data were recorded to measure the visceral sensitivity. After intraperitoneal injection of hemicholinium-3 (HC-3), the specific inhibitor of CHT1, CRD data were also recorded. The CHT1 expression of DRGs was investigated by Western blotting, immunohistochemistry, and quantitative RT-PCR. Acetylcholine levels in the DRGs were detected by the assay kit. Results Repetitive WAS increased the AWR score of CRD at high distension pressure and decreased the mean threshold of rats. The CHT1 expression and acetylcholine concentration of DRG were significantly increased in WAS rats. After the administration of HC-3, the AWR score in WAS group was significantly increased at higher distension pressure while the threshold intensity was significantly reduced compared to the normal saline group. Acetylcholine concentration was significantly lower than the normal saline rats. Conclusion Our research firstly reports that CHT1 is overexpressed in noninflammatory visceral hypersensitivity, and blockage of CHT1 can enhance the visceral hypersensitivity. CHT1 may play an inhibitory role in visceral hypersensitivity. PMID:29849603

  9. Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

    Science.gov (United States)

    Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.

    2013-01-01

    Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing. PMID:23382943

  10. Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose.

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    Shahin S Ali

    Full Text Available Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km((glucose was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

  11. A rhodamine-labeled citalopram analogue as a high-affinity fluorescent probe for the serotonin transporter

    DEFF Research Database (Denmark)

    Zhang, Peng; Jørgensen, Trine Nygaard; Løland, Claus Juul

    2013-01-01

    A novel fluorescent ligand was synthesized as a high-affinity, high specificity probe for visualizing the serotonin transporter (SERT). The rhodamine fluorophore was extended from an aniline substitution on the 5-position of the dihydroisobenzofuran ring of citalopram (2, 1-(3-(dimethylamino......)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile), using an ethylamino linker. The resulting rhodamine-labeled ligand 8 inhibited [3H]5-HT uptake in COS-7 cells (Ki = 225 nM) with similar potency to the tropane-based JHC 1-064 (1), but with higher specificity towards the SERT relative...

  12. Flavin binding to the high affinity riboflavin transporter RibU

    NARCIS (Netherlands)

    Duurkens, Hinderika; Tol, Menno B.; Geertsma, Eric R.; Permentier, Hjalmar P.; Slotboom, Dirk Jan

    2007-01-01

    The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin ( vitamin B-2) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when

  13. Novel and High Affinity 2-[(Diphenylmethyl)sulfinyl]acetamide (Modafinil) Analogues as Atypical Dopamine Transporter Inhibitors

    DEFF Research Database (Denmark)

    Cao, Jianjing; Slack, Rachel D.; Bakare, Oluyomi M.

    2016-01-01

    The development of pharmacotherapeutic treatments of psychostimulant abuse has remained a challenge, despite significant efforts made toward relevant mechanistic targets, such as the dopamine transporter (DAT). The atypical DAT inhibitors have received attention due to their promising pharmacolog...

  14. Localization of high affinity [3H]glycine transport sites in the cerebellar cortex

    International Nuclear Information System (INIS)

    Wilkin, G.P.; Csillag, A.; Balazs, R.; Kingsbury, A.E.; Wilson, J.E.; Johnson, A.L.

    1981-01-01

    A study was made of [ 3 H ]glycine uptake sites in a preparation greatly enriched in large pieces of the cerebellar glomeruli (glomerulus particles) and in morphologically well preserved slices of rat cerebellum. Electron microscopic autoradiography revealed that of the neurones in the cerebellar cortex only Golgi cells transported [ 3 H]glycine at the low concentration used. Glial cells also took up [ 3 H]glycine but to a lesser extent than the Golgi neurons. It was also confirmed that under comparable conditions Golgi cells transport [ 3 H]GABA. Kinetic studies utilizing the Golgi axon terminal-containing glomerulus particles showed that glycine is a weak non-competitive inhibitor of [ 3 H]GABA uptake (Ksub(i) over 600 μM vs the Ksub(t) of about 20 μM) and that GABA is an even weaker inhibitor of [ 3 H]glycine uptake. (Auth.)

  15. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

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    Thomas Stockner

    Full Text Available The high-resolution crystal structure of the leucine transporter (LeuT is frequently used as a template for homology models of the dopamine transporter (DAT. Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii LeuT and DAT share a rather low overall sequence identity (22% and (iii the extracellular loop 2 (EL2 of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  16. A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

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    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

  17. Glucose uptake and growth of glucose-limited chemostat cultures of Aspergillus niger and a disruptant lacking MstA, a high-affinity glucose transporter

    DEFF Research Database (Denmark)

    Jørgensen, Thomas R; vanKuyk, Patricia A; Poulsen, Bjarne R

    2007-01-01

    This is a study of high-affinity glucose uptake in Aspergillus niger and the effect of disruption of a high-affinity monosaccharide-transporter gene, mstA. The substrate saturation constant (K(s)) of a reference strain was about 15 microM in glucose-limited chemostat culture. Disruption of mst......-affinity uptake system of A. niger. The mstA disruptant and a reference strain were cultivated in glucose-limited chemostat cultures at low, intermediate and high dilution rate (D=0.07 h(-1), 0.14 h(-1) and 0.20 h(-1)). Mycelium harvested from steady-state cultures was subjected to glucose uptake assays...

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  19. Blockade of the high-affinity noradrenaline transporter (NET) by the selective 5-HT reuptake inhibitor escitalopram: an in vivo microdialysis study in mice

    Science.gov (United States)

    Nguyen, Hai T; Guiard, Bruno P; Bacq, Alexandre; David, Denis J; David, Indira; Quesseveur, Gaël; Gautron, Sophie; Sanchez, Connie; Gardier, Alain M

    2013-01-01

    BACKGROUND AND PURPOSE Escitalopram, the S(+)-enantiomer of citalopram is the most selective 5-HT reuptake inhibitor approved. Although all 5-HT selective reuptake inhibitors (SSRIs) increase extracellular levels of 5-HT ([5-HT]ext). some also enhance, to a lesser extent, extracellular levels of noradrenaline ([NA]ext). However, the mechanisms by which SSRIs activate noradrenergic transmission in the brain remain to be determined. EXPERIMENTAL APPROACH This study examined the effects of escitalopram, on both [5-HT]ext and [NA]ext in the frontal cortex (FCx) of freely moving wild-type (WT) and mutant mice lacking the 5-HT transporter (SERT−/−) by using intracerebral microdialysis. We explored the possibilities that escitalopram enhances [NA]ext, either by a direct mechanism involving the inhibition of the low- or high-affinity noradrenaline transporters, or by an indirect mechanism promoted by [5-HT]ext elevation. The forced swim test (FST) was used to investigate whether enhancing cortical [5-HT]ext and/or [NA]ext affected the antidepressant-like activity of escitalopram. KEY RESULTS In WT mice, a single systemic administration of escitalopram produced a significant increase in cortical [5-HT]ext and [NA]ext. As expected, escitalopram failed to increase cortical [5-HT]ext in SERT−/− mice, whereas its neurochemical effects on [NA]ext persisted in these mutants. In WT mice subjected to the FST, escitalopram increased swimming parameters without affecting climbing behaviour. Finally, escitalopram, at relevant concentrations, failed to inhibit cortical noradrenaline and 5-HT uptake mediated by low-affinity monoamine transporters. CONCLUSIONS AND IMPLICATIONS These experiments suggest that escitalopram enhances, although moderately, cortical [NA]extin vivo by a direct mechanism involving the inhibition of the high-affinity noradrenaline transporter (NET). PMID:22233336

  20. Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source.

    Science.gov (United States)

    Fraisier, V; Gojon, A; Tillard, P; Daniel-Vedele, F

    2000-08-01

    The NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate (NO3-) uptake system in Nicotiana plumbaginifolia. Here we report functional and physiological analyses of transgenic plants expressing the NpNRT2.1 coding sequence fused to the CaMV 35S or rolD promoters. Irrespective of the level of NO3- supplied, NO3- contents were found to be remarkably similar in wild-type and transgenic plants. Under specific conditions (growth on 10 mM NO3-), the steady-state NpNRT2. 1 mRNA level resulting from the deregulated transgene expression was accompanied by an increase in 15NO3- influx measured in the low concentration range. This demonstrates for the first time that the NRT2.1 sequence codes a limiting element of the inducible high-affinity transport system. Both 15NO3- influx and mRNA levels decreased in the wild type after exposure to ammonium, in agreement with previous results from many species. Surprisingly, however, influx was also markedly decreased in transgenic plants, despite stable levels of transgene expression in independent transformants after ammonium addition. We conclude that the conditions associated with the supply of a reduced nitrogen source such as ammonium, or with the generation of a further downstream metabolite, probably exert a repressive effect on NO3- influx at both transcriptional and post-transcriptional levels.

  1. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin

    DEFF Research Database (Denmark)

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across...... the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes...

  2. The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings.

    Science.gov (United States)

    Suchak, Sachin K; Baloyianni, Nicoletta V; Perkinton, Michael S; Williams, Robert J; Meldrum, Brian S; Rattray, Marcus

    2003-02-01

    The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

  3. Fluorinated phenmetrazine "legal highs" act as substrates for high-affinity monoamine transporters of the SLC6 family.

    Science.gov (United States)

    Mayer, Felix P; Burchardt, Nadine V; Decker, Ann M; Partilla, John S; Li, Yang; McLaughlin, Gavin; Kavanagh, Pierce V; Sandtner, Walter; Blough, Bruce E; Brandt, Simon D; Baumann, Michael H; Sitte, Harald H

    2018-05-15

    A variety of new psychoactive substances (NPS) are appearing in recreational drug markets worldwide. NPS are compounds that target various receptors and transporters in the central nervous system to achieve their psychoactive effects. Chemical modifications of existing drugs can generate NPS that are not controlled by current legislation, thereby providing legal alternatives to controlled substances such as cocaine or amphetamine. Recently, 3-fluorophenmetrazine (3-FPM), a derivative of the anorectic compound phenmetrazine, appeared on the recreational drug market and adverse clinical effects have been reported. Phenmetrazine is known to elevate extracellular monoamine concentrations by an amphetamine-like mechanism. Here we tested 3-FPM and its positional isomers, 2-FPM and 4-FPM, for their abilities to interact with plasma membrane monoamine transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). We found that 2-, 3- and 4-FPM inhibit uptake mediated by DAT and NET in HEK293 cells with potencies comparable to cocaine (IC 50 values 80 μM). Experiments directed at identifying transporter-mediated reverse transport revealed that FPM isomers induce efflux via DAT, NET and SERT in HEK293 cells, and this effect is augmented by the Na + /H + ionophore monensin. Each FPM evoked concentration-dependent release of monoamines from rat brain synaptosomes. Hence, this study reports for the first time the mode of action for 2-, 3- and 4-FPM and identifies these NPS as monoamine releasers with marked potency at catecholamine transporters implicated in abuse and addiction. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.' Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  4. pH, Lactate, and Hypoxia: Reciprocity in Regulating High-Affinity Monocarboxylate Transporter Expression in Glioblastoma

    Directory of Open Access Journals (Sweden)

    James P. Caruso

    2017-02-01

    Full Text Available Highly malignant brain tumors harbor the aberrant propensity for aerobic glycolysis, the excessive conversion of glucose to lactic acid even in the presence of ample tissue oxygen. Lactic acid is rapidly effluxed to the tumor microenvironment via a group of plasma-membrane transporters denoted monocarboxylate transporters (MCTs to prevent “self-poisoning.” One isoform, MCT2, has the highest affinity for lactate and thus should have the ability to respond to microenvironment conditions such as hypoxia, lactate, and pH to help maintain high glycolytic flux in the tumor. Yet, MCT2 is considered to not respond to hypoxia, which is counterintuitive. Its response to tumor lactate has not been reported. In this report, we experimentally identify the transcription initiation site/s for MCT2 in astrocytes (normal and glioma (tumor. We then use a BACmid library to isolate a 4.2-kbp MCT2 promoter-exon I region and examine promoter response to glycolysis-mediated stimuli in glioma cells. Reporter analysis of nested-promoter constructs indicated response of MCT2 to hypoxia, pH, lactate, and glucose, the major physiological “players” that facilitate a tumor's growth and proliferation. Immunoblot analysis of native MCT2 expression under altered pH and hypoxia reflected the reporter data. The pH-mediated gene-regulation studies we describe are the first to record H+-based reporter studies for any mammalian system and demonstrate the exquisite response of the MCT2 gene to minute changes in tumor pH. Identical promoter usage also provides the first evidence of astrocytes harnessing the same gene regulatory regions to facilitate astrocyte-neuron lactate shuttling, a metabolic feature of normal brain.

  5. A novel high-affinity sucrose transporter is required for virulence of the plant pathogen Ustilago maydis.

    Directory of Open Access Journals (Sweden)

    Ramon Wahl

    2010-02-01

    Full Text Available Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1 from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host.

  6. [Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

    Science.gov (United States)

    Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

    2012-01-01

    It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

  7. The high affinity K+ transporter AtHAK5 plays a physiological role in planta at very low K+ concentrations and provides a caesium uptake pathway in Arabidopsis.

    Science.gov (United States)

    Qi, Zhi; Hampton, Corrina R; Shin, Ryoung; Barkla, Bronwyn J; White, Philip J; Schachtman, Daniel P

    2008-01-01

    Caesium (Cs(+)) is a potentially toxic mineral element that is released into the environment and taken up by plants. Although Cs(+) is chemically similar to potassium (K(+)), and much is known about K(+) transport mechanisms, it is not clear through which K(+) transport mechanisms Cs(+) is taken up by plant roots. In this study, the role of AtHAK5 in high affinity K(+) and Cs(+) uptake was characterized. It is demonstrated that AtHAK5 is localized to the plasma membrane under conditions of K(+) deprivation, when it is expressed. Growth analysis showed that AtHAK5 plays a role during severe K(+) deprivation. Under K(+)-deficient conditions in the presence of Cs(+), Arabidopsis seedlings lacking AtHAK5 had increased inhibition of root growth and lower Cs(+) accumulation, and significantly higher leaf chlorophyll concentrations than wild type. These data indicate that, in addition to transporting K(+) in planta, AtHAK5 also transports Cs(+). Further experiments showed that AtHAK5 mediated Cs(+) uptake into yeast cells and that, although the K(+) deficiency-induced expression of AtHAK5 was inhibited by low concentrations of NH(4)(+) in planta, Cs(+) uptake by yeast was stimulated by low concentrations of NH(4)(+). Interestingly, the growth of the Arabidopsis atakt1-1 mutant was more sensitive to Cs(+) than the wild type. This may be explained, in part, by increased expression of AtHAK5 in the atakt1-1 mutant. It is concluded that AtHAK5 is a root plasma membrane uptake mechanism for K(+) and Cs(+) under conditions of low K(+) availability.

  8. 2,2'-Dithiobis(N-ethyl-spermine-5-carboxamide) is a high affinity, membrane-impermeant antagonist of the mammalian polyamine transport system.

    Science.gov (United States)

    Huber, M; Pelletier, J G; Torossian, K; Dionne, P; Gamache, I; Charest-Gaudreault, R; Audette, M; Poulin, R

    1996-11-01

    We have synthesized 2,2'-dithiobis(N-ethyl-spermine-5-carboxamide) (DESC), its thiol monomer (MESC), and the mixed MESC-cysteamine disulfide (DEASC) as potential inhibitors of polyamine transport in mammalian cells. DESC was the most potent antagonist of spermine transport in ZR-75-1 human breast cancer cells, with Ki values of 5. 0 +/- 0.7, 80 +/- 31, and 16 +/- 3 microM for DESC, MESC, and DEASC, respectively. DESC also strongly blocked putrescine and spermidine uptake in ZR-75-1 cells (Ki = 1.6 +/- 0.5 and 2.7 +/- 1.1 microM, respectively). While DESC and MESC were purely competitive inhibitors of putrescine transport, DEASC was a mixed competitive/noncompetitive antagonist. Remarkably, DESC was virtually impermeant in ZR-75-1 cells despite its low Ki toward polyamine transport. The marked difference in affinity between DESC and MESC was essentially due to the tail-to-tail juxtaposition of two spermine-like structures, suggesting that dimeric ligands of the polyamine transporter might simultaneously interact with more than one binding site. While DESC strongly decreased the initial rate of [3H]spermidine transport, even a 40-fold molar excess of antagonist could not completely abolish intracellular spermidine accumulation. Moreover, as little as 0.3 microM spermidine fully restored growth in ZR-75-1 cells treated with an inhibitor of polyamine biosynthesis in the presence of 50 microM DESC, thus emphasizing the importance of uptake of trace amounts of exogenous polyamines. Thus, reducing the exogenous supply of polyamines with a potent competitive inhibitor may be kinetically inadequate to block replenishment of the polyamine pool in polyamine-depleted tumor cells that display high transport capacity. These results demonstrate that polyamine analogues cross-linked into a dimeric structure such as DESC interact with high affinity with the mammalian polyamine carrier without being used as substrates. These novel properties provide a framework for the design of

  9. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1), Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo.

    Science.gov (United States)

    Sankova, Tatiana P; Orlov, Iurii A; Saveliev, Andrey N; Kirilenko, Demid A; Babich, Polina S; Brunkov, Pavel N; Puchkova, Ludmila V

    2017-11-03

    There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST) and the N-terminal domain (ectodomain) of human high affinity copper transporter CTR1 (hNdCTR1), which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell's copper metabolism and its chelating properties are discussed.

  10. The Extracellular Domain of Human High Affinity Copper Transporter (hNdCTR1, Synthesized by E. coli Cells, Chelates Silver and Copper Ions In Vivo

    Directory of Open Access Journals (Sweden)

    Tatiana P. Sankova

    2017-11-01

    Full Text Available There is much interest in effective copper chelators to correct copper dyshomeostasis in neurodegenerative and oncological diseases. In this study, a recombinant fusion protein for expression in Escherichia coli cells was constructed from glutathione-S-transferase (GST and the N-terminal domain (ectodomain of human high affinity copper transporter CTR1 (hNdCTR1, which has three metal-bound motifs. Several biological properties of the GST-hNdCTR1 fusion protein were assessed. It was demonstrated that in cells, the protein was prone to oligomerization, formed inclusion bodies and displayed no toxicity. Treatment of E. coli cells with copper and silver ions reduced cell viability in a dose- and time-dependent manner. Cells expressing GST-hNdCTR1 protein demonstrated resistance to the metal treatments. These cells accumulated silver ions and formed nanoparticles that contained AgCl and metallic silver. In this bacterial population, filamentous bacteria with a length of about 10 µm were often observed. The possibility for the fusion protein carrying extracellular metal binding motifs to integrate into the cell’s copper metabolism and its chelating properties are discussed.

  11. A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

    Science.gov (United States)

    Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

    2012-10-12

    The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

  12. Involvement of the VDE homing endonuclease and rapamycin in regulation of the Saccharomyces cerevisiae GSH11 gene encoding the high affinity glutathione transporter.

    Science.gov (United States)

    Miyake, Tsuyoshi; Hiraishi, Hiroyuki; Sammoto, Hiroyuki; Ono, Bun-Ichiro

    2003-10-10

    The Saccharomyces cerevisiae gene HGT1/GSH11 encodes the high affinity glutathione transporter and is repressed by cysteine added to the culture medium. It has been found previously that a 5'-upstream cis-element, CCGCCACAC, is responsible for regulating GSH11 expression and that several proteins bind to this element (Miyake, T., Kanayama, M., Sammoto, H., and Ono, B. (2002) Mol. Genet. Genomics 266, 1004-1011). In this report we present evidence that the most prominent of these proteins is VDE, known previously as the homing endonuclease encoded by VMA1. We show also that GSH11 is not expressed in a VDE-deleted strain and that inability to express the GSH11 of this strain is overcome by introduction of the coding region of VDE or the entire VMA1 gene. It is also found that VDE does not cut DNA in the vicinity of the GSH11 cis-element. Rapamycin, an inhibitor of the target of rapamycin (TOR) signal-transduction system, is found to enhance expression of GSH11 in a VDE-dependent manner under conditions of sulfur starvation. These results indicate that GSH11 is regulated by a system sensitive to sulfur starvation (presumably via cysteine depletion) and a more general system involving the nutritional starvation signal mediated by the TOR system. Both systems need to be operational (inhibition of TOR and sulfur starvation) for full expression of GSH11.

  13. PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family.

    Science.gov (United States)

    Quesada, A; Krapp, A; Trueman, L J; Daniel-Vedele, F; Fernández, E; Forde, B G; Caboche, M

    1997-05-01

    A family of high-affinity nitrate transporters has been identified in Aspergillus nidulans and Chlamydomonas reinhardtii, and recently homologues of this family have been cloned from a higher plant (barley). Based on six of the peptide sequences most strongly conserved between the barley and C. reinhardtii polypeptides, a set of degenerate primers was designed to permit amplification of the corresponding genes from other plant species. The utility of these primers was demonstrated by RT-PCR with cDNA made from poly(A)+ RNA from barley, C. reinhardtii and Nicotiana plumbaginifolia. A PCR fragment amplified from N. plumbaginifolia was used as probe to isolate a full-length cDNA clone which encodes a protein, NRT2;1Np, that is closely related to the previously isolated crnA homologue from barley. Genomic Southern blots indicated that there are only 1 or 2 members of the Nrt2 gene family in N. plumbaginifolia. Northern blotting showed that the Nrt2 transcripts are most strongly expressed in roots. The effects of external treatments with different N sources showed that the regulation of the Nrt2 gene(s) is very similar to that reported for nitrate reductase and nitrite reductase genes: their expression was strongly induced by nitrate but was repressed when reduced forms of N were supplied to the roots.

  14. The Mitochondrial Metallochaperone SCO1 Is Required to Sustain Expression of the High-Affinity Copper Transporter CTR1 and Preserve Copper Homeostasis

    Directory of Open Access Journals (Sweden)

    Christopher J. Hlynialuk

    2015-02-01

    Full Text Available Human SCO1 fulfills essential roles in cytochrome c oxidase (COX assembly and the regulation of copper (Cu homeostasis, yet it remains unclear why pathogenic mutations in this gene cause such clinically heterogeneous forms of disease. Here, we establish a Sco1 mouse model of human disease and show that ablation of Sco1 expression in the liver is lethal owing to severe COX and Cu deficiencies. We further demonstrate that the Cu deficiency is explained by a functional connection between SCO1 and CTR1, the high-affinity transporter that imports Cu into the cell. CTR1 is rapidly degraded in the absence of SCO1 protein, and we show that its levels are restored in Sco1−/− mouse embryonic fibroblasts upon inhibition of the proteasome. These data suggest that mitochondrial signaling through SCO1 provides a post-translational mechanism to regulate CTR1-dependent Cu import into the cell, and they further underpin the importance of mitochondria in cellular Cu homeostasis.

  15. Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1.

    Science.gov (United States)

    Li, Guowei; Tillard, Pascal; Gojon, Alain; Maurel, Christophe

    2016-04-01

    The water status and mineral nutrition of plants critically determine their growth and development. Nitrate (NO3(-)), the primary nitrogen source of higher plants, is known to impact the water transport capacity of roots (root hydraulic conductivity, Lpr). To explore the effects and mode of action of NO3(-) on Lpr, we used an extended set of NO3(-) transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1 and nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3(-) conditions. First, a strong positive relationship between Lpr and NO3(-) accumulation, in shoots rather than in roots, was revealed. Secondly, a specific 30% reduction of Lpr in nrt2.1 plants unraveled a major role for the high-affinity NO3(-) transporter NRT2.1 in increasing Lpr These results indicate that NO3(-)signaling rather than nitrogen assimilation products governs Lpr in Arabidopsis. Quantitative real-time reverse transcription-PCR and enzyme-linked immunosorbent assays (ELISAs) were used to investigate the effects of NO3(-) availability on plasma membrane aquaporin (plasma membrane intrinsic protein; PIP) expression. Whereas PIP regulation mostly occurs at the post-translational level in wild-type plants, a regulation of PIPs at both the transcriptional and translational levels was uncovered in nrt2.1 plants. In conclusion, this work reveals that control of Arabidopsis Lpr and PIP functions by NO3(-) involves novel shoot to root signaling and NRT2.1-dependent functions. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

    Science.gov (United States)

    Chen, Jiandong

    2016-01-01

    ABSTRACT The l-arabinose-inducible araBAD promoter (PBAD) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where PBAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA regulates PBAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of PBAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system and has implications for improving its usage for tunable gene expression. PMID:27849174

  17. Two zinc-binding domains in the transporter AdcA from Streptococcus pyogenes facilitate high-affinity binding and fast transport of zinc.

    Science.gov (United States)

    Cao, Kun; Li, Nan; Wang, Hongcui; Cao, Xin; He, Jiaojiao; Zhang, Bing; He, Qing-Yu; Zhang, Gong; Sun, Xuesong

    2018-04-20

    Zinc is an essential metal in bacteria. One important bacterial zinc transporter is AdcA, and most bacteria possess AdcA homologs that are single-domain small proteins due to better efficiency of protein biogenesis. However, a double-domain AdcA with two zinc-binding sites is significantly overrepresented in Streptococcus species, many of which are major human pathogens. Using molecular simulation and experimental validations of AdcA from Streptococcus pyogenes , we found here that the two AdcA domains sequentially stabilize the structure upon zinc binding, indicating an organization required for both increased zinc affinity and transfer speed. This structural organization appears to endow Streptococcus species with distinct advantages in zinc-depleted environments, which would not be achieved by each single AdcA domain alone. This enhanced zinc transport mechanism sheds light on the significance of the evolution of the AdcA domain fusion, provides new insights into double-domain transporter proteins with two binding sites for the same ion, and indicates a potential target of antimicrobial drugs against pathogenic Streptococcus species. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Slc5a8, a Na+-coupled high-affinity transporter for short-chain fatty acids, is a conditional tumour suppressor in colon that protects against colitis and colon cancer under low-fibre dietary conditions.

    Science.gov (United States)

    Gurav, Ashish; Sivaprakasam, Sathish; Bhutia, Yangzom D; Boettger, Thomas; Singh, Nagendra; Ganapathy, Vadivel

    2015-07-15

    Mammalian colon harbours trillions of bacteria under physiological conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. In the present study we show that Slc5a8 (solute carrier gene family 5a, member 8), a Na(+)-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T-cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naive T-cells into immunosuppressive forkhead box P3(+) (FoxP3(+)) Tregs (regulatory T-cells) and suppress conversion of naive T-cells into pro-inflammatory interferon (IFN)-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T-cells in response to butyrate. We also provide in vivo evidence for an obligatory role for Slc5a8 in suppression of IFN-γ-producing T-cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fibre intake but not when dietary fibre intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fibre and mucosal immune system via the bacterial metabolite butyrate and that this transporter is a conditional tumour suppressor in colon linked to dietary fibre content. © 2015 Authors; published by Portland Press Limited.

  19. In vivo high-affinity uptake and axonal transport of D-(2,3-/sup 3/H)aspartate in excitatory neurons

    Energy Technology Data Exchange (ETDEWEB)

    Storm-Mathisen, J.; Wold, J.E. (Oslo Univ. (Norway))

    1981-12-28

    D-(2,3-/sup 3/H)aspartate ((/sup 3/H)D-Asp) at ..mu..M concentrations in Krebs' solution was infused intracerebrally in rats, mice and hamsters. Neuropil sites in the hippocampal formation, septum and neostriatum, known to receive excitatory nerve inputs with glutamate and aspartate as putative transmitters, showed strong autoradiographic labeling after intraventricular infusions. There was evidence for retrograde axonal transport to pyramidal cell bodies in hippocampus CA3 and neocortex. Infusions into the hilus fasciae dentatae led to anterograde axonal transport of (/sup 3/H)D-Asp in the mossy fibers.

  20. Expression and distributeion of 'high affinity' glutamate transporters GLT1, GLAST, EAAC and of GCPII in the rat peripheral nervous system

    Czech Academy of Sciences Publication Activity Database

    Carozzi, V. A.; Canta, A.; Oggioni, N.; Ceresa, C.; Marmiroli, P.; Konvalinka, Jan; Zoia, Ch.; Bossi, M.; Ferrarese, C.; Tredici, G.; Cavaletti, G.

    2008-01-01

    Roč. 213, č. 4 (2008), s. 539-546 ISSN 0021-8782 Institutional research plan: CEZ:AV0Z40550506 Keywords : glutamate * glutamate transporters * immunoblotting * immunohistochemistry * peripheral nervous system Subject RIV: CE - Biochemistry Impact factor: 2.063, year: 2008

  1. Chronic treatment with amyloid beta(1-42) inhibits non-cholinergic high-affinity choline transport in NG108-15 cells through protein kinase C signaling

    Czech Academy of Sciences Publication Activity Database

    Nováková, Jana; Mikasová, Lenka; Machová, Eva; Lisá, Věra; Doležal, Vladimír

    2005-01-01

    Roč. 1062, č. 1-2 (2005), s. 101-110 ISSN 0006-8993 R&D Projects: GA AV ČR(CZ) IAA5011206; GA MŠk(CZ) LC554 Grant - others:Lipidiet(XE) QLK1-CT-2002-00172 Institutional research plan: CEZ:AV0Z50110509 Keywords : choline transporter * beta-amyloid * protein kinase C Subject RIV: ED - Physiology Impact factor: 2.296, year: 2005

  2. Translocation of metal phosphate via the phosphate inorganic transport system of Escherichia coli

    NARCIS (Netherlands)

    van Veen, H.W; Abee, T.; Kortstee, G.J J; Konings, W.N; Zehnder, A.J B

    1994-01-01

    P-i transport via the phosphate inorganic transport system (Pit) of Escherichia coil was studied in natural and artificial membranes. P-i uptake via Pit is dependent on the presence of divalent cations, like Mg2+, Ca2+, Co2+, or Mn2+, which form a soluble, neutral metal phosphate (MeHPO(4)) complex.

  3. Interrogating the Molecular Basis for Substrate Recognition in Serotonin and Dopamine Transporters with High-Affinity Substrate-Based Bivalent Ligands

    DEFF Research Database (Denmark)

    Andersen, Jacob; Ladefoged, Lucy Kate; Kristensen, Trine N. Bjerre

    2016-01-01

    insight into substrate recognition in SERT and DAT. An optimized bivalent ligand comprising two serotonin moieties binds SERT with 3,800-fold increased affinity compared to that of serotonin, suggesting that the human transporters have two distinct substrate binding sites. We show that the bivalent...... ligands are inhibitors of SERT and an experimentally validated docking model suggests that the bivalent compounds bind with one substrate moiety in the central binding site (the S1 site), whereas the other substrate moiety binds in a distinct binding site (the S2 site). A systematic study of nonconserved...

  4. Characterisation of [11C]PR04.MZ in Papio anubis baboon: A selective high-affinity radioligand for quantitative imaging of the dopamine transporter

    Energy Technology Data Exchange (ETDEWEB)

    Riss P. J.; Fowler J.; Riss, P.J.; Hooker, J.M.; Shea, C.; Xu, Y.; Carter, P.; Warner, D.; Ferrari V.; Kim, S.W.; Aigbirhio, F.I.; Fowler, J.S.; Roesch, F.

    2011-10-25

    N-(4-fluorobut-2-yn-1-yl)-2{beta}-carbomethoxy-3{beta}-(4{prime}-tolyl)nortropane (PR04.MZ, 1) is a PET radioligand for the non-invasive exploration of the function of the cerebral dopamine transporter (DAT). A reliable automated process for routine production of the carbon-11 labelled analogue [{sup 11}C]PR04.MZ ([{sup 11}C]-1) has been developed using GMP compliant equipment. An adult female Papioanubis baboon was studied using a test-retest protocol with [{sup 11}C]-1 in order to assess test-retest reliability, metabolism and CNS distribution profile of the tracer in non-human primates. Blood sampling was performed throughout the studies for determination of the free fraction in plasma (fP), plasma input functions and metabolic degradation of the radiotracer [{sup 11}C]-1. Time-activity curves were derived for the putamen, the caudate nucleus, the ventral striatum, the midbrain and the cerebellum. Distribution volumes (VT) and non-displaceable binding potentials (BPND) for various brain regions and the blood were obtained from kinetic modelling. [{sup 11}C]-1 shows promising results as aselective marker of the presynaptic dopamine transporter. With the reliable visualisation of the extra-striatal dopaminergic neurons and no indication on labelled metabolites, the tracer provides excellent potential for translation into man.

  5. MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity.

    Science.gov (United States)

    Lu, Shuo; Zgurskaya, Helen I

    2013-11-01

    The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic membrane fusion protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC.

  6. Phosphate transporter mediated lipid accumulation in Saccharomyces cerevisiae under phosphate starvation conditions.

    Science.gov (United States)

    James, Antoni W; Nachiappan, Vasanthi

    2014-01-01

    In the current study, when phosphate transporters pho88 and pho86 were knocked out they resulted in significant accumulation (84% and 43%) of triacylglycerol (TAG) during phosphate starvation. However in the presence of phosphate, TAG accumulation was only around 45% in both pho88 and pho86 mutant cells. These observations were confirmed by radio-labeling, fluorescent microscope and RT-PCR studies. The TAG synthesizing genes encoding for acyltransferases namely LRO1 and DGA1 were up regulated. This is the first report for accumulation of TAG in pho88Δ and pho86Δ cells under phosphate starvation conditions. Copyright © 2013. Published by Elsevier Ltd.

  7. Role of Phosphate Transport System Component PstB1 in Phosphate Internalization by Nostoc punctiforme.

    Science.gov (United States)

    Hudek, L; Premachandra, D; Webster, W A J; Bräu, L

    2016-11-01

    In bacteria, limited phosphate availability promotes the synthesis of active uptake systems, such as the Pst phosphate transport system. To understand the mechanisms that facilitate phosphate accumulation in the cyanobacterium Nostoc punctiforme, phosphate transport systems were identified, revealing a redundancy of Pst phosphate uptake systems that exists across three distinct operons. Four separate PstB system components were identified. pstB1 was determined to be a suitable target for creating phenotypic mutations that could result in the accumulation of excessive levels of phosphate through its overexpression or in a reduction of the capacity to accumulate phosphate through its deletion. Using quantitative real-time PCR (qPCR), it was determined that pstB1 mRNA levels increased significantly over 64 h in cells cultured in 0 mM added phosphate and decreased significantly in cells exposed to high (12.8 mM) phosphate concentrations compared to the level in cells cultured under normal (0.8 mM) conditions. Possible compensation for the loss of PstB1 was observed when pstB2, pstB3, and pstB4 mRNA levels increased, particularly in cells starved of phosphate. The overexpression of pstB1 increased phosphate uptake by N. punctiforme and was shown to functionally complement the loss of PstB in E. coli PstB knockout (PstB - ) mutants. The knockout of pstB1 in N. punctiforme did not have a significant effect on cellular phosphate accumulation or growth for the most part, which is attributed to the compensation for the loss of PstB1 by alterations in the pstB2, pstB3, and pstB4 mRNA levels. This study provides novel in vivo evidence that PstB1 plays a functional role in phosphate uptake in N. punctiforme IMPORTANCE: Cyanobacteria have been evolving over 3.5 billion years and have become highly adept at growing under limiting nutrient levels. Phosphate is crucial for the survival and prosperity of all organisms. In bacteria, limited phosphate availability promotes the

  8. sphingosine-1-phosphate transport and its role in immunology

    NARCIS (Netherlands)

    Reitsema, V.; Bouma, Hjalmar; Kok, Jan

    2014-01-01

    Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite with many important functions in cellular and systemic physiology, including the immune system. As it cannot traverse the membrane, it is exported from cells by transporters. Several members of the ATP-binding cassette (ABC) transporter

  9. A phosphate transporter from the mycorrhizal fungus Glomus versiforme.

    Science.gov (United States)

    Harrison, M J; van Buuren, M L

    1995-12-07

    Vesicular-arbuscular (VA) mycorrhizal fungi form symbiotic associations with the roots of most terrestrial plants, including many agriculturally important crop species. The fungi colonize the cortex of the root to obtain carbon from their plant host, while assisting the plant with the uptake of phosphate and other mineral nutrients from the soil. This association is beneficial to the plant, because phosphate is essential for plant growth and development, especially during growth under nutrient-limiting conditions. Molecular genetic studies of these fungi and their interaction with plants have been limited owing to the obligate symbiotic nature of the VA fungi, so the molecular mechanisms underlying fungal-mediated uptake and translocation of phosphate from the soil to the plant remain unknown. Here we begin to investigate this process by identifying a complementary DNA that encodes a transmembrane phosphate transporter (GvPT) from Glomus versiforme, a VA mycorrhizal fungus. The function of the protein encoded by GvPT was confirmed by complementation of a yeast phosphate transport mutant. Expression of GvPT was localized to the external hyphae of G. versiforme during mycorrhizal associations, these being the initial site of phosphate uptake from the soil.

  10. High affinity hemoglobin and Parkinson's disease.

    Science.gov (United States)

    Graham, Jeffrey; Hobson, Douglas; Ponnampalam, Arjuna

    2014-12-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra (SN) region of the midbrain. Oxidative damage in this region has been shown to play an important role in the pathogenesis of this disease. Human neurons have been discovered to contain hemoglobin, with an increased concentration seen in the neurons of the SN. High affinity hemoglobin is a clinical entity resulting from mutations that create a functional increase in the binding of hemoglobin to oxygen and an inability to efficiently unload it to tissues. This can result in a number of metabolic compensatory changes, including an elevation in circulating hemoglobin and an increase in the molecule 2,3-diphosphoglycerate (2,3-DPG). Population based studies have revealed that patients with PD have elevated hemoglobin as well as 2,3-DPG levels. Based on these observations, we hypothesize that the oxidative damage seen in PD is related to an underlying high affinity hemoglobin subtype. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Dual and Direction-Selective Mechanisms of Phosphate Transport by the Vesicular Glutamate Transporter

    Directory of Open Access Journals (Sweden)

    Julia Preobraschenski

    2018-04-01

    Full Text Available Summary: Vesicular glutamate transporters (VGLUTs fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (Pi. It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔμH+ with inversely directed Pi transport coupled to the Na+ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and Pi using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔμH+-dependent fashion, with glutamate preferred over Pi. When facing the extracellular space, Pi is transported in a Na+-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and Pi homeostasis within glutamatergic neurons. : Preobraschenski et al. show that the vesicular glutamate transporter functions as a bi-directional phosphate transporter that is coupled with different cations in each direction and hence may play a key role in neuronal phosphate homeostasis. Keywords: VGLUT, SLC17 family, type I Na+-dependent inorganic phosphate transporter, ATPase, proteoliposomes, hybrid vesicles, anti-VGLUT1 nanobody

  12. Differential expression of genes encoding phosphate transporters contributes to arsenic accumulation in shrub willow (Salix spp.)

    Science.gov (United States)

    Emily E. Puckett; Michelle J. Serpiglia; Alyssa M. DeLeon; Stephanie Long; Rakesh Minocha; Lawrence B. Smart

    2012-01-01

    Studies of arsenate and phosphate uptake by plants in hydroponic and soil systems indicate a common transport mechanism via the phosphate transporters (PHTs) due to structural similarity of the anions. Typically, the presence of phosphate decreases plant uptake and translocation of arsenate in hydroponic solution. This study quantified arsenic (As) uptake related to...

  13. Ethylene regulates phosphorus remobilization and expression of a phosphate transporter (PhPT1) during petunia corolla senescence

    Science.gov (United States)

    Chapin, Laura J.; Jones, Michelle L.

    2009-01-01

    The programmed degradation of macromolecules during petal senescence allows the plant to remobilize nutrients from dying to developing tissues. Ethylene is involved in regulating the timing of nucleic acid degradation in petunia, but it is not clear if ethylene has a role in the remobilization of phosphorus during petal senescence. To investigate ethylene's role in nutrient remobilization, the P content of petals (collectively called the corolla) during early development and senescence was compared in ethylene-sensitive wild type Petunia×hybrida ‘Mitchell Diploid’ (MD) and transgenic petunias with reduced sensitivity to ethylene (35S::etr1-1). When compared to the total P content of corollas on the day of flower opening (the early non-senescing stage), P in MD corollas had decreased 74% by the late stage of senescence (advanced wilting). By contrast, P levels were only reduced by an average of 32% during etr1-1 corolla (lines 44568 and Z00-35-10) senescence. A high-affinity phosphate transporter, PhPT1 (PhPht1;1), was cloned from senescing petunia corollas by RT-PCR. PhPT1 expression was up-regulated during MD corolla senescence and a much smaller increase was detected during the senescence of etr1-1 petunia corollas. PhPT1 mRNA levels showed a rapid increase in detached corollas (treated at 1 d after flower opening) following treatment with low levels of ethylene (0.1 μl l-1). Transcripts accumulated in the presence of the protein synthesis inhibitor, cycloheximide, indicating that PhPT1 is a primary ethylene response gene. PhPT1 is a putative phosphate transporter that may function in Pi translocation during senescence. PMID:19380421

  14. Arabidopsis copper transport protein COPT2 participates in the cross talk between iron deficiency responses and low-phosphate signaling.

    Science.gov (United States)

    Perea-García, Ana; Garcia-Molina, Antoni; Andrés-Colás, Nuria; Vera-Sirera, Francisco; Pérez-Amador, Miguel A; Puig, Sergi; Peñarrubia, Lola

    2013-05-01

    Copper and iron are essential micronutrients for most living organisms because they participate as cofactors in biological processes, including respiration, photosynthesis, and oxidative stress protection. In many eukaryotic organisms, including yeast (Saccharomyces cerevisiae) and mammals, copper and iron homeostases are highly interconnected; yet, such interdependence is not well established in higher plants. Here, we propose that COPT2, a high-affinity copper transport protein, functions under copper and iron deficiencies in Arabidopsis (Arabidopsis thaliana). COPT2 is a plasma membrane protein that functions in copper acquisition and distribution. Characterization of the COPT2 expression pattern indicates a synergic response to copper and iron limitation in roots. We characterized a knockout of COPT2, copt2-1, that leads to increased resistance to simultaneous copper and iron deficiencies, measured as reduced leaf chlorosis and improved maintenance of the photosynthetic apparatus. We propose that COPT2 could play a dual role under iron deficiency. First, COPT2 participates in the attenuation of copper deficiency responses driven by iron limitation, possibly to minimize further iron consumption. Second, global expression analyses of copt2-1 versus wild-type Arabidopsis plants indicate that low-phosphate responses increase in the mutant. These results open up new biotechnological approaches to fight iron deficiency in crops.

  15. Exploration of the dopamine transporter: in vitro and in vivo characterization of a high-affinity and high-specificity iodinated tropane derivative (E)-N-(3-iodoprop-2-enyl)-2β-carbomethoxy- 3β-(4'-methylphenyl)nortropane (PE2I)

    International Nuclear Information System (INIS)

    Guilloteau, Denis; Emond, Patrick; Baulieu, Jean-Louis; Garreau, Lucette; Frangin, Yves; Pourcelot, Leandre; Mauclaire, Laurent; Besnard, Jean-Claude; Chalon, Sylvie

    1998-01-01

    For the diagnosis and follow-up of neurodegenerative diseases, many cocaine derivatives have been proposed as radioligands to explore the dopamine transporter. As none of them have all the criteria of specificity and kinetics for human use, we have developed a new derivative, (E)-N-(3-iodoprop-2-enyl)-2β-carbomethoxy-3β-(4'-methylphenyl)nortropane (PE2I), which displays promising properties. We report the characterization of PE2I in vitro on rat striatal membranes and in vivo in rats and in monkeys. PE2I had a high affinity (Kd=0.09±0.01 nM) and high specificity for the dopamine transporter. In rats we observed a high accumulation in the striatum; by contrast, a very low fixation was measured in the cortex. Moreover, a preinjection of a saturating dose of GBR 12909 prevented the striatal accumulation of PE2I by 74%. These results confirmed the specificity of PE2I for the dopamine transporter. In vivo in monkeys, SPECT studies showed a high accumulation in striatum. Moreover, an equilibrium state was obtained 1 h after injection. PE2I seemed to be the most promising ligand for the dopamine transporter exploration by SPECT using a single-day protocol.

  16. Proton transport properties of tin phosphate, chromotropic acid ...

    Indian Academy of Sciences (India)

    The functionalized materials of tin (IV) phosphate (SnP) like chromotropic acid anchored tin ... elemental analysis (ICP–AES), thermal analysis, X-ray analysis and FTIR spectroscopy. .... nal level below 1 V, interfaced to a minicomputer for data.

  17. The transport of phosphate between the plasma and dialysate compartments in peritoneal dialysis is influenced by an electric potential difference

    DEFF Research Database (Denmark)

    Graff, J; Fugleberg, S; Brahm, J

    1996-01-01

    was not identifiable. Furthermore, it was demonstrated that the electrochemical gradient between plasma water and dialysate favours the diffusive phosphate transport, and both electric and chemical potentials must be taken into account in calculations of the transperitoneal phosphate transport.......Six kinetic models of transperitoneal phosphate transport were formulated and validated on the basis of experimental results obtained from 22 non-diabetic patients undergoing peritoneal dialysis. The models were designed to elucidate the presence or absence of diffusive, non-lymphatic convective......, and lymphatic convective phosphate transport. Calculations allowed for a 20% protein binding of phosphate. The validation procedure demonstrated that only diffusive and non-lymphatic convective phosphate transport mechanisms were identifiable. A lymphatic convective phosphate transport mechanism...

  18. An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function

    Science.gov (United States)

    Reynafarje, Baltazar; Lehninger, Albert L.

    1978-01-01

    This paper describes the properties and a possible biological role of a transport process across the inner membrane of rat liver mitochondria resulting in the exchange of ATP4- (out) for ADP3- (in) + 0.5 phosphate2- (in). This transmembrane exchange reaction, designated as the ATP-ADP-phosphate exchange, is specific for the ligands shown, electroneutral, insensitive to N-ethylmaleimide or mersalyl, inhibited by atractyloside, and appears to occur only in the direction as written. It is thus distinct from the well-known phosphate-hydroxide and phosphate-dicarboxylate exchange systems, which are inhibited by mersalyl, and from the ATP-ADP exchanger, which does not transport phosphate. During ATP hydrolysis by mitochondria, half of the phosphate formed from ATP passes from the matrix to the medium by the mersalyl-insensitive ATP-ADP-phosphate exchange and the other half by the well-known mersalyl-sensitive phosphate-hydroxide exchange. These and other considerations have led to a hypothesis for the pathway and stoichiometry of ATP-dependent reverse electron transport, characterized by a requirement of 1.33 molecules of ATP per pair of electrons reversed and by the utilization of a different membrane transport pathway for phosphate and adenine nucleotides than is taken in forward electron flow and oxidative phosphorylation. The possible occurrence of independent pathways for ATP-forming forward electron flow and ATP-consuming reverse electron flow is consonant with the fact that the opposing degradative and synthetic pathways in the central routes of cell metabolism generally have different pathways that are independently regulated. PMID:283393

  19. Oral peptide specific egg antibody to intestinal sodium-dependent phosphate co-transporter-2b is effective at altering phosphate transport in vitro and in vivo.

    Science.gov (United States)

    Bobeck, Elizabeth A; Hellestad, Erica M; Sand, Jordan M; Piccione, Michelle L; Bishop, Jeff W; Helvig, Christian; Petkovich, Martin; Cook, Mark E

    2015-06-01

    Hyperimmunized hens are an effective means of generating large quantities of antigen specific egg antibodies that have use as oral supplements. In this study, we attempted to create a peptide specific antibody that produced outcomes similar to those of the human pharmaceutical, sevelamer HCl, used in the treatment of hyperphosphatemia (a sequela of chronic renal disease). Egg antibodies were generated against 8 different human intestinal sodium-dependent phosphate cotransporter 2b (NaPi2b) peptides, and hNaPi2b peptide egg antibodies were screened for their ability to inhibit phosphate transport in human intestinal Caco-2 cell line. Antibody produced against human peptide sequence TSPSLCWT (anti-h16) was specific for its peptide sequence, and significantly reduced phosphate transport in human Caco-2 cells to 25.3±11.5% of control nonspecific antibody, when compared to nicotinamide, a known inhibitor of phosphate transport (P≤0.05). Antibody was then produced against the mouse-specific peptide h16 counterpart (mouse sequence TSPSYCWT, anti-m16) for further analysis in a murine model. When anti-m16 was fed to mice (1% of diet as dried egg yolk powder), egg yolk immunoglobulin (IgY) was detected using immunohistochemical staining in mouse ileum, and egg anti-m16 IgY colocalized with a commercial goat anti-NaPi2b antibody. The effectiveness of anti-m16 egg antibody in reducing serum phosphate, when compared to sevelamer HCl, was determined in a mouse feeding study. Serum phosphate was reduced 18% (Pegg yolk powder) and 30% (Pegg immunoglobulin. The methods described and the findings reported show that oral egg antibodies are useful and easy to prepare reagents for the study and possible treatment of select diseases. © 2015 Poultry Science Association Inc.

  20. Relative quantitative RT-PCR to study the expression of plant nutrient transporters in arbuscular mycorrhizas

    DEFF Research Database (Denmark)

    Burleigh, S.H.

    2001-01-01

    had high reproducibility and reflected trends in gene expression as observed by Northern blotting. Using this technique, it was demonstrated that both the high-affinity phosphate transporter MtPt2 and a putative nitrate transporter from Medicago truncatula were down-regulated in roots when colonized...

  1. Natural and azido fatty acids inhibit phosphate transport and activate fatty acid anion uniport mediated by the mitochondrial phosphate carrier

    Czech Academy of Sciences Publication Activity Database

    Engstová, Hana; Žáčková, Markéta; Růžička, Michal; Meinhardt, A.; Hanuš, Jan; Krämer, R.; Ježek, Petr

    2001-01-01

    Roč. 276, č. 7 (2001), s. 4683-4691 ISSN 0021-9258 R&D Projects: GA ČR GA301/95/0620; GA ČR GA301/98/0568; GA MŠk ME 085; GA MŠk ME 389 Grant - others:US(US) Czechoslovak Science and Technology Program 94043 Institutional research plan: CEZ:AV0Z5011922 Keywords : phosphate transport * fatty acids Subject RIV: CE - Biochemistry Impact factor: 7.258, year: 2001

  2. The competitive advantage of a dual-transporter system.

    Science.gov (United States)

    Levy, Sagi; Kafri, Moshe; Carmi, Miri; Barkai, Naama

    2011-12-09

    Cells use transporters of different affinities to regulate nutrient influx. When nutrients are depleted, low-affinity transporters are replaced by high-affinity ones. High-affinity transporters are helpful when concentrations of nutrients are low, but the advantage of reducing their abundance when nutrients are abundant is less clear. When we eliminated such reduced production of the Saccharomyces cerevisiae high-affinity transporters for phosphate and zinc, the elapsed time from the initiation of the starvation program until the lack of nutrients limited growth was shortened, and recovery from starvation was delayed. The latter phenotype was rescued by constitutive activation of the starvation program. Dual-transporter systems appear to prolong preparation for starvation and to facilitate subsequent recovery, which may optimize sensing of nutrient depletion by integrating internal and external information about nutrient availability.

  3. Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

    International Nuclear Information System (INIS)

    Ogura, Kenji; Shiga, Takanori; Yokochi, Masashi; Yuzawa, Satoru; Burke, Terrence R.; Inagaki, Fuyuhiko

    2008-01-01

    The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity

  4. Molecular basis of a high affinity murine interleukin-5 receptor.

    OpenAIRE

    Devos, R; Plaetinck, G; Van der Heyden, J; Cornelis, S; Vandekerckhove, J; Fiers, W; Tavernier, J

    1991-01-01

    The mouse interleukin-5 receptor (mIL-5R) consists of two components one of which, the mIL-5R alpha-chain, binds mIL-5 with low affinity. Recently we demonstrated that monoclonal antibodies (Mabs) recognizing the second mIL-5R beta-chain, immunoprecipitate a p130-140 protein doublet which corresponds to the mIL-3R and the mIL-3R-like protein, the latter chain for which so far no ligand has been identified. In this study we show that a high affinity mIL-5R can be reconstituted on COS1 cells by...

  5. Deregulated Renal Calcium and Phosphate Transport during Experimental Kidney Failure.

    Directory of Open Access Journals (Sweden)

    Wilco P Pulskens

    Full Text Available Impaired mineral homeostasis and inflammation are hallmarks of chronic kidney disease (CKD, yet the underlying mechanisms of electrolyte regulation during CKD are still unclear. Here, we applied two different murine models, partial nephrectomy and adenine-enriched dietary intervention, to induce kidney failure and to investigate the subsequent impact on systemic and local renal factors involved in Ca(2+ and Pi regulation. Our results demonstrated that both experimental models induce features of CKD, as reflected by uremia, and elevated renal neutrophil gelatinase-associated lipocalin (NGAL expression. In our model kidney failure was associated with polyuria, hypercalcemia and elevated urinary Ca(2+ excretion. In accordance, CKD augmented systemic PTH and affected the FGF23-αklotho-vitamin-D axis by elevating circulatory FGF23 levels and reducing renal αklotho expression. Interestingly, renal FGF23 expression was also induced by inflammatory stimuli directly. Renal expression of Cyp27b1, but not Cyp24a1, and blood levels of 1,25-dihydroxy vitamin D3 were significantly elevated in both models. Furthermore, kidney failure was characterized by enhanced renal expression of the transient receptor potential cation channel subfamily V member 5 (TRPV5, calbindin-D28k, and sodium-dependent Pi transporter type 2b (NaPi2b, whereas the renal expression of sodium-dependent Pi transporter type 2a (NaPi2a and type 3 (PIT2 were reduced. Together, our data indicates two different models of experimental kidney failure comparably associate with disturbed FGF23-αklotho-vitamin-D signalling and a deregulated electrolyte homeostasis. Moreover, this study identifies local tubular, possibly inflammation- or PTH- and/or FGF23-associated, adaptive mechanisms, impacting on Ca(2+/Pi homeostasis, hence enabling new opportunities to target electrolyte disturbances that emerge as a consequence of CKD development.

  6. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. PMID:23747010

  7. NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

    Energy Technology Data Exchange (ETDEWEB)

    Cowan, Catherine M. [Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza,7523 Boelter Hall, Los Angeles, CA 90095 (United States); Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Zhang, Xinli; James, Aaron W.; Mari Kim, T.; Sun, Nichole [Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Wu, Benjamin [Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza,7523 Boelter Hall, Los Angeles, CA 90095 (United States); Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Ting, Kang [Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, University of California, Los Angeles, 40833 Le Conte Ave, Los Angeles, CA 90095 (United States); Soo, Chia, E-mail: bsoo@ucla.edu [UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic, Hospital Research Center, University of California, Los Angeles, 2641 Charles E. Young Dr. South, Los Angeles, CA 90095 (United States)

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer NELL-1 accelerates extracellular matrix mineralization in MC3T3-E1 pre-osteoblasts. Black-Right-Pointing-Pointer NELL-1 significantly increases intracellular inorganic phosphate levels. Black-Right-Pointing-Pointer NELL-1 positively regulates osteogenesis but not proliferation in MC3T3-E1 cells. Black-Right-Pointing-Pointer NELL-1 regulates inorganic phosphate transporter activity. -- Abstract: NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 or rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.

  8. NELL-1 increases pre-osteoblast mineralization using both phosphate transporter Pit1 and Pit2

    International Nuclear Information System (INIS)

    Cowan, Catherine M.; Zhang, Xinli; James, Aaron W.; Mari Kim, T.; Sun, Nichole; Wu, Benjamin; Ting, Kang; Soo, Chia

    2012-01-01

    Highlights: ► NELL-1 accelerates extracellular matrix mineralization in MC3T3-E1 pre-osteoblasts. ► NELL-1 significantly increases intracellular inorganic phosphate levels. ► NELL-1 positively regulates osteogenesis but not proliferation in MC3T3-E1 cells. ► NELL-1 regulates inorganic phosphate transporter activity. -- Abstract: NELL-1 is a potent osteoinductive molecule that enhances bone formation in multiple animal models through currently unidentified pathways. In the present manuscript, we hypothesized that NELL-1 may regulate osteogenic differentiation accompanied by alteration of inorganic phosphate (Pi) entry into the osteoblast via sodium dependent phosphate (NaPi) transporters. To determine this, MC3T3-E1 pre-osteoblasts were cultured in the presence of recombinant human (rh)NELL-1 or rhBMP-2. Analysis was performed for intracellular Pi levels through malachite green staining, Pit-1 and Pit-2 expression, and forced upregulation of Pit-1 and Pit-2. Results showed rhNELL-1 to increase MC3T3-E1 matrix mineralization and Pi influx associated with activation of both Pit-1 and Pit-2 channels, with significantly increased Pit-2 production. In contrast, Pi transport elicited by rhBMP-2 showed to be associated with increased Pit-1 production only. Next, neutralizing antibodies against Pit-1 and Pit-2 completely abrogated the Pi influx effect of rhNELL-1, suggesting rhNELL-1 is dependent on both transporters. These results identify one potential mechanism of action for rhNELL-1 induced osteogenesis and highlight a fundamental difference between NELL-1 and BMP-2 signaling.

  9. Dopamine transporter SPECT imaging of the peroral addicts of compound codeine phosphate solution

    International Nuclear Information System (INIS)

    Sun Taotao; Hu Shu; Jia Shaowei; Chen Qing; Fan Rong

    2010-01-01

    Objective: To study the damage to striatum in patients perorally addicted to compound codeine phosphate solution by using the brain dopamine transporter SPECT imaging. Methods: Patients perorally addicted to compound codeine phosphate solution (n = 29) and addicted to heroin (n = 27), as well as healthy volunteers (n = 31) were included in the study. Each of them underwent dopamine transporter (DAT) SPECT imaging with 99 Tc m -2β-[N, N'-bis-( 2- mercaptoethyl ) ethylenediamino] methyl, 3β-(4-chlorophenyl)tropane ( 99 Tc m -TRODAT-1). The striatum volume (V, cm 3 ), mass (m, g) and radioactivity ratio (Ra) of striatum to whole brain were calculated using physio-mathematical modeling method. Results: Bilateral striatum of healthy volunteers showed typical 'panda eyes' pattern and the distribution of DAT was uniform and symmetrical. Bilateral striatum of patients addicted to compound codeine phosphate showed impaired tracer uptake, similar to those addicted to heroin. The V, m and Ra of bilateral striatum of patients addicted to compound codeine phosphate were (23.68±4.94) cm 3 , (24.87±5.19) g and (5.01±0.88) %, respectively, which were significantly lower than those of healthy controls: (35.39 ±4.42) cm 3 ,(37.16±4.64) g and (7.93±0.86)% (t =-9.69, -9.69, - 13.01, all P =0.000), but significantly higher than those addicted to heroin: (18.87±4.66) cm 3 , (19.81±4.90) g and (4.26±1.02) % (t =3.74, 3.74, 2.96, P = 0.000, 0.000, 0.005). Conclusion: Long-term peroral intake of compound codeine phosphate solution may damage the function of cerebral striatum, which is someway similar to though less severe than, the impairment caused by heroin. (authors)

  10. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    Science.gov (United States)

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-07

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

  11. Modeling of glycerol-3-phosphate transporter suggests a potential 'tilt' mechanism involved in its function.

    Science.gov (United States)

    Tsigelny, Igor F; Greenberg, Jerry; Kouznetsova, Valentina; Nigam, Sanjay K

    2008-10-01

    Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane alpha-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family--the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY)--have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational "switching" mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible "switch" mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.(23) We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a "tilt" of 9 degrees -10 degrees rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the "tilted" structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while the

  12. Divalent phosphate is a counterion for carboxyatractyloside-insensitive adenine nucleotide transport in rat liver mitochondria

    International Nuclear Information System (INIS)

    Nosek, M.T.; Aprille, J.R.

    1986-01-01

    Unidirectional, carboxyatractyloside(CAT)-insensitive adenine nucleotide (AdN) fluxes have been studied in isolated rat liver mitochondria (mito). Previous work has shown that ATP x Mg transport in one direction is coupled to ATP x Mg or P/sub i/ transport in the opposite direction. The purpose of this study was to determine whether divalent HPO 4 2- or monovalent H 2 PO 4 - is the transported phosphate species. The authors used the monofluorophosphate (PO 3 F 2- ) and difluorophosphate (PO 2 F 2 - ) analogues as potential counterions forAdN efflux. After a preincubation on ice with 14 C-ADP to label the matrix AdN, efflux was measured at 30 0 C, pH 7.4, in 225mM sucrose, 10mM KCl, 5mM MgCl 2 , 5mM glutamate, 5mM malate, 10mM Tris, 0.5mM P/sub i/, 1mM ATP, and 5μM CAT. With no other additions efflux was -0.62 +/- 0.20 nmole/minute/mg protein. The data supports the hypothesis that divalent but not monovalent phosphate can act as a counterion for ATPx Mg transport over this CAT-insensitive carrier

  13. Comprehensive Genomic Identification and Expression Analysis of the Phosphate Transporter (PHT) Gene Family in Apple.

    Science.gov (United States)

    Sun, Tingting; Li, Mingjun; Shao, Yun; Yu, Lingyan; Ma, Fengwang

    2017-01-01

    Elemental phosphorus (Pi) is essential to plant growth and development. The family of phosphate transporters (PHTs) mediates the uptake and translocation of Pi inside the plants. Members include five sub-cellular phosphate transporters that play different roles in Pi uptake and transport. We searched the Genome Database for Rosaceae and identified five clusters of phosphate transporters in apple ( Malus domestica ), including 37 putative genes. The MdPHT1 family contains 14 genes while MdPHT2 has two, MdPHT3 has seven, MdPHT4 has 11, and MdPHT5 has three. Our overview of this gene family focused on structure, chromosomal distribution and localization, phylogenies, and motifs. These genes displayed differential expression patterns in various tissues. For example, expression was high for MdPHT1;12, MdPHT3;6 , and MdPHT3;7 in the roots, and was also increased in response to low-phosphorus conditions. In contrast, MdPHT4;1, MdPHT4;4 , and MdPHT4;10 were expressed only in the leaves while transcript levels of MdPHT1;4, MdPHT1;12 , and MdPHT5;3 were highest in flowers. In general, these 37 genes were regulated significantly in either roots or leaves in response to the imposition of phosphorus and/or drought stress. The results suggest that members of the PHT family function in plant adaptations to adverse growing environments. Our study will lay a foundation for better understanding the PHT family evolution and exploring genes of interest for genetic improvement in apple.

  14. Arbuscular Mycorrhizal Symbiosis Requires a Phosphate Transceptor in the Gigaspora margarita Fungal Symbiont.

    Science.gov (United States)

    Xie, Xianan; Lin, Hui; Peng, Xiaowei; Xu, Congrui; Sun, Zhongfeng; Jiang, Kexin; Huang, Antian; Wu, Xiaohui; Tang, Nianwu; Salvioli, Alessandra; Bonfante, Paola; Zhao, Bin

    2016-12-05

    The majority of terrestrial vascular plants are capable of forming mutualistic associations with obligate biotrophic arbuscular mycorrhizal (AM) fungi from the phylum Glomeromycota. This mutualistic symbiosis provides carbohydrates to the fungus, and reciprocally improves plant phosphate uptake. AM fungal transporters can acquire phosphate from the soil through the hyphal networks. Nevertheless, the precise functions of AM fungal phosphate transporters, and whether they act as sensors or as nutrient transporters, in fungal signal transduction remain unclear. Here, we report a high-affinity phosphate transporter GigmPT from Gigaspora margarita that is required for AM symbiosis. Host-induced gene silencing of GigmPT hampers the development of G. margarita during AM symbiosis. Most importantly, GigmPT functions as a phosphate transceptor in G. margarita regarding the activation of the phosphate signaling pathway as well as the protein kinase A signaling cascade. Using the substituted-cysteine accessibility method, we identified residues A 146 (in transmembrane domain [TMD] IV) and Val 357 (in TMD VIII) of GigmPT, both of which are critical for phosphate signaling and transport in yeast during growth induction. Collectively, our results provide significant insights into the molecular functions of a phosphate transceptor from the AM fungus G. margarita. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  15. Phosphate concentration and arbuscular mycorrhizal colonisation influence the growth, yield and expression of twelve PHT1 family phosphate transporters in foxtail millet (Setaria italica.

    Directory of Open Access Journals (Sweden)

    S Antony Ceasar

    Full Text Available Phosphorus (P is an essential element which plays several key roles in all living organisms. Setaria italica (foxtail millet is a model species for panacoid grasses including several millet species widely grown in arid regions of Asia and Africa, and for the bioenergy crop switchgrass. The growth responses of S. italica to different levels of inorganic phosphate (Pi and to colonisation with the arbuscular mycorrhizal fungus Funneliformis mosseae (syn. Glomus mosseae were studied. Phosphate is taken up from the environment by the PHT1 family of plant phosphate transporters, which have been well characterized in several plant species. Bioinformatic analysis identified 12 members of the PHT1 gene family (SiPHT1;1-1;12 in S. italica, and RT and qPCR analysis showed that most of these transporters displayed specific expression patterns with respect to tissue, phosphate status and arbuscular mycorrhizal colonisation. SiPHT1;2 was found to be expressed in all tissues and in all growth conditions tested. In contrast, expression of SiPHT1;4 was induced in roots after 15 days growth in hydroponic medium of low Pi concentration. Expression of SiPHT1;8 and SiPHT1;9 in roots was selectively induced by colonisation with F. mosseae. SiPHT1;3 and SiPHT1;4 were found to be predominantly expressed in leaf and root tissues respectively. Several other transporters were expressed in shoots and leaves during growth in low Pi concentrations. This study will form the basis for the further characterization of these transporters, with the long term goal of improving the phosphate use efficiency of foxtail millet.

  16. Phosphate concentration and arbuscular mycorrhizal colonisation influence the growth, yield and expression of twelve PHT1 family phosphate transporters in foxtail millet (Setaria italica).

    Science.gov (United States)

    Ceasar, S Antony; Hodge, Angela; Baker, Alison; Baldwin, Stephen A

    2014-01-01

    Phosphorus (P) is an essential element which plays several key roles in all living organisms. Setaria italica (foxtail millet) is a model species for panacoid grasses including several millet species widely grown in arid regions of Asia and Africa, and for the bioenergy crop switchgrass. The growth responses of S. italica to different levels of inorganic phosphate (Pi) and to colonisation with the arbuscular mycorrhizal fungus Funneliformis mosseae (syn. Glomus mosseae) were studied. Phosphate is taken up from the environment by the PHT1 family of plant phosphate transporters, which have been well characterized in several plant species. Bioinformatic analysis identified 12 members of the PHT1 gene family (SiPHT1;1-1;12) in S. italica, and RT and qPCR analysis showed that most of these transporters displayed specific expression patterns with respect to tissue, phosphate status and arbuscular mycorrhizal colonisation. SiPHT1;2 was found to be expressed in all tissues and in all growth conditions tested. In contrast, expression of SiPHT1;4 was induced in roots after 15 days growth in hydroponic medium of low Pi concentration. Expression of SiPHT1;8 and SiPHT1;9 in roots was selectively induced by colonisation with F. mosseae. SiPHT1;3 and SiPHT1;4 were found to be predominantly expressed in leaf and root tissues respectively. Several other transporters were expressed in shoots and leaves during growth in low Pi concentrations. This study will form the basis for the further characterization of these transporters, with the long term goal of improving the phosphate use efficiency of foxtail millet.

  17. Computational modeling and molecular imprinting for the development of acrylic polymers with high affinity for bile salts.

    Science.gov (United States)

    Yañez, Fernando; Chianella, Iva; Piletsky, Sergey A; Concheiro, Angel; Alvarez-Lorenzo, Carmen

    2010-02-05

    This work has focused on the rational development of polymers capable of acting as traps of bile salts. Computational modeling was combined with molecular imprinting technology to obtain networks with high affinity for cholate salts in aqueous medium. The screening of a virtual library of 18 monomers, which are commonly used for imprinted networks, identified N-(3-aminopropyl)-methacrylate hydrochloride (APMA.HCl), N,N-diethylamino ethyl methacrylate (DEAEM) and ethyleneglycol methacrylate phosphate (EGMP) as suitable functional monomers with medium-to-high affinity for cholic acid. The polymers were prepared with a fix cholic acid:functional monomer mole ratio of 1:4, but with various cross-linking densities. Compared to polymers prepared without functional monomer, both imprinted and non-imprinted microparticles showed a high capability to remove sodium cholate from aqueous medium. High affinity APMA-based particles even resembled the performance of commercially available cholesterol-lowering granules. The imprinting effect was evident in most of the networks prepared, showing that computational modeling and molecular imprinting can act synergistically to improve the performance of certain polymers. Nevertheless, both the imprinted and non-imprinted networks prepared with the best monomer (APMA.HCl) identified by the modeling demonstrated such high affinity for the template that the imprinting effect was less important. The fitting of adsorption isotherms to the Freundlich model indicated that, in general, imprinting increases the population of high affinity binding sites, except when the affinity of the functional monomer for the target molecule is already very high. The cross-linking density was confirmed as a key parameter that determines the accessibility of the binding points to sodium cholate. Materials prepared with 9% mol APMA and 91% mol cross-linker showed enough affinity to achieve binding levels of up to 0.4 mmol g(-1) (i.e., 170 mg g(-1)) under flow

  18. Two differentially regulated phosphate transporters from the symbiotic fungus Hebeloma cylindrosporum and phosphorus acquisition by ectomycorrhizal Pinus pinaster.

    Science.gov (United States)

    Tatry, Marie-Violaine; El Kassis, Elie; Lambilliotte, Raphaël; Corratgé, Claire; van Aarle, Ingrid; Amenc, Laurie K; Alary, Rémi; Zimmermann, Sabine; Sentenac, Hervé; Plassard, Claude

    2009-03-01

    Ectomycorrhizal symbiosis markedly improves plant phosphate uptake, but the molecular mechanisms underlying this benefit are still poorly understood. We identified two ESTs in a cDNA library prepared from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum with significant similarities to phosphate transporters from the endomycorrhizal fungus Glomus versiforme and from non-mycorrhizal fungi. The full-length cDNAs corresponding to these two ESTs complemented a yeast phosphate transport mutant (Deltapho84). Measurements of (33)P-phosphate influx into yeast expressing either cDNA demonstrated that the encoded proteins, named HcPT1 and HcPT2, were able to mediate Pi:H(+) symport with different affinities for Pi (K(m) values of 55 and 4 mum, respectively). Real-time RT-PCR showed that Pi starvation increased the levels of HcPT1 transcripts in H. cylindrosporum hyphae grown in pure culture. Transcript levels of HcPT2 were less dependent on Pi availability. The two transporters were expressed in H. cylindrosporum associated with its natural host plant, Pinus pinaster, grown under low or high P conditions. The presence of ectomycorrhizae increased net Pi uptake rates into intact Pinus pinaster roots at low or high soil P levels. The expression patterns of HcPT1 and HcPT2 indicate that the two fungal phosphate transporters may be involved in uptake of phosphate from the soil solution under the two soil P availability conditions used.

  19. Molecular mechanism of α-tocopheryl-phosphate transport across the cell membrane

    International Nuclear Information System (INIS)

    Negis, Yesim; Meydani, Mohsen; Zingg, Jean-Marc; Azzi, Angelo

    2007-01-01

    α-Tocopheryl-phosphate (α-TP) is synthesized and hydrolyzed in animal cells and tissues where it modulates several functions. α-TP is more potent than α-T in inhibiting cell proliferation, down-regulating CD36 transcription, inhibiting atherosclerotic plaque formation. Administration of α-TP to cells or animals requires its transfer through membranes, via a transporter. We show here that α-TP is passing the plasma membrane via a system that is inhibited by glibenclamide and probenecid, inhibitors of a number of transporters. Glibenclamide and probenecid prevent dose-dependently α-TP inhibition of cell proliferation. The two inhibitors act on ATP binding cassette (ABC) and organic anion transporters (OAT). Since ABC transporters function to export solutes and α-TP is transported into cells, it may be concluded that α-TP transport may occur via an OAT family member. Due to the protection by glibenclamide and probenecid on the α-TP induced cell growth inhibition it appears that α-TP acts after its uptake inside cells

  20. Modulation of small intestinal phosphate transporter by dietary supplements of mineral phosphorus and phytase in broilers.

    Science.gov (United States)

    Huber, Korinna; Zeller, Ellen; Rodehutscord, Markus

    2015-05-01

    Dietary phosphorus (P) is known as a main modulator of phosphate (Pi) transporter expression. The effect of supplemented mineral P with or without phytase on protein expression of two sodium-dependent Pi (NaPi) transporters and a calcium channel was studied in the small intestine of broilers. Thirty-six broilers were randomly assigned to six different diets at 15 days of age. Two levels of total P (tP, adjusted by monocalcium phosphate (MCP) supplementation), 0.39% (BD-) and 0.47% (BD+) were fed until day 25; and at each tP level, three levels of phytase were used with 0, 500, and 12,500 FTU/kg of an E. coli phytase. Mucosa samples from jejunum and ileum were taken and apical membranes were isolated by MgCl2 precipitation. Protein expression of NaPi IIb, NaPi type III (PiT1) and the calcium channel TRPV6 were semiquantitatively measured by Western blotting and jejunal mucosal phytase activity by measurement of Pi release. The jejunal NaPi IIb transporter was expressed with two distinct bands, which were modulated differently by diet. NaPi IIb Band1 increased (P phytase supplementation but was not affected by MCP supplementation. This inverse modulation of Band1 and Band2 was significantly related to the amount of net absorbed P with higher expression of Band1 at higher amounts of net absorbed P. In addition, a second Pi transporter, PiT1, was detected in which ileal expression decreased (P phytase supplementation. The expression of the calcium channel TRPV6 was increased in BD+ groups. A trend for an interaction between MCP and phytase supplementation on mucosal phytase activity was observed (P = 0.079) with a decrease in activity when BD+ with 12,500 FTU/kg phytase was fed. Chicken intestinal epithelial cells responded to dietary supplemented phytase and MCP by changing the Pi transporter expression in apical membranes. In conclusion, availability of Pi is most likely the key modulator of transporter protein expression. However, a contribution of lower inositol

  1. Characterization of inorganic phosphate transport in the triple-negative breast cancer cell line, MDA-MB-231.

    Science.gov (United States)

    Russo-Abrahão, Thais; Lacerda-Abreu, Marco Antônio; Gomes, Tainá; Cosentino-Gomes, Daniela; Carvalho-de-Araújo, Ayra Diandra; Rodrigues, Mariana Figueiredo; Oliveira, Ana Carolina Leal de; Rumjanek, Franklin David; Monteiro, Robson de Queiroz; Meyer-Fernandes, José Roberto

    2018-01-01

    Recent studies demonstrate that interstitial inorganic phosphate is significantly elevated in the breast cancer microenvironment as compared to normal tissue. In addition it has been shown that breast cancer cells express high levels of the NaPi-IIb carrier (SLC34A2), suggesting that this carrier may play a role in breast cancer progression. However, the biochemical behavior of inorganic phosphate (Pi) transporter in this cancer type remains elusive. In this work, we characterize the kinetic parameters of Pi transport in the aggressive human breast cancer cell line, MDA-MB-231, and correlated Pi transport with cell migration and adhesion. We determined the influence of sodium concentration, pH, metabolic inhibitors, as well as the affinity for inorganic phosphate in Pi transport. We observed that the inorganic phosphate is dependent on sodium transport (K0,5 value = 21.98 mM for NaCl). Furthermore, the transport is modulated by different pH values and increasing concentrations of Pi, following the Michaelis-Menten kinetics (K0,5 = 0.08 mM Pi). PFA, monensin, furosemide and ouabain inhibited Pi transport, cell migration and adhesion. Taken together, these results showed that the uptake of Pi in MDA-MB-231 cells is modulated by sodium and by regulatory mechanisms of intracellular sodium gradient. General Significance: Pi transport might be regarded as a potential target for therapy against tumor progression.

  2. A new human NHERF1 mutation decreases renal phosphate transporter NPT2a expression by a PTH-independent mechanism.

    Directory of Open Access Journals (Sweden)

    Marie Courbebaisse

    Full Text Available BACKGROUND: The sodium-hydrogen exchanger regulatory factor 1 (NHERF1 binds to the main renal phosphate transporter NPT2a and to the parathyroid hormone (PTH receptor. We have recently identified mutations in NHERF1 that decrease renal phosphate reabsorption by increasing PTH-induced cAMP production in the renal proximal tubule. METHODS: We compared relevant parameters of phosphate homeostasis in a patient with a previously undescribed mutation in NHERF1 and in control subjects. We expressed the mutant NHERF1 protein in Xenopus Oocytes and in cultured cells to study its effects on phosphate transport and PTH-induced cAMP production. RESULTS: We identified in a patient with inappropriate renal phosphate reabsorption a previously unidentified mutation (E68A located in the PDZ1 domain of NHERF1.We report the consequences of this mutation on NHERF1 function. E68A mutation did not modify cAMP production in the patient. PTH-induced cAMP synthesis and PKC activity were not altered by E68A mutation in renal cells in culture. In contrast to wild-type NHERF1, expression of the E68A mutant in Xenopus oocytes and in human cells failed to increase phosphate transport. Pull down experiments showed that E68A mutant did not interact with NPT2a, which robustly interacted with wild type NHERF1 and previously identified mutants. Biotinylation studies revealed that E68A mutant was unable to increase cell surface expression of NPT2a. CONCLUSIONS: Our results indicate that the PDZ1 domain is critical for NHERF1-NPT2a interaction in humans and for the control of NPT2a expression at the plasma membrane. Thus we have identified a new mechanism of renal phosphate loss and shown that different mutations in NHERF1 can alter renal phosphate reabsorption via distinct mechanisms.

  3. Antioxidant mechanism of milk mineral-high-affinity iron binding.

    Science.gov (United States)

    Allen, K; Cornforth, D

    2007-01-01

    Milk mineral (MM), a by-product of whey processing, is an effective antioxidant in meat systems, but the antioxidant mechanism has not been established. MM has been postulated to chelate iron and prevent iron-catalysis of lipid oxidation. The objective of this research was to examine this putative mechanism. MM was compared to sodium tripolyphosphate (STPP), calcium phosphate monobasic (CPM), and calcium pyrophosphate (CPP) to determine iron-binding capacity, sample solubility, and eluate soluble phosphorus after treating samples with a ferrous chloride standard. Scanning electron microscopy with energy-dispersive X-ray analysis was used to localize minerals on iron-treated MM particle surfaces. Histochemical staining for calcium was performed on raw and cooked ground beef samples with added MM. MM bound more iron per gram (P compounds, and was much less soluble (P iron across the MM particle surface, directly demonstrating iron binding to MM particles. Unlike other common chelating agents, such as STPP and citrate, histochemical staining demonstrated that MM remained insoluble in ground beef, even after cooking. The ability of MM to bind iron and remain insoluble may enhance its antioxidant effect by removing iron ions from solution. However, MM particles must be small and well distributed in order to adequately bind iron throughout the food system.

  4. myo-Inositol-1-phosphate synthase is required for polar auxin transport and organ development

    KAUST Repository

    Chen, Hao

    2010-06-01

    myo-Inositol-1-phosphate synthase is a conserved enzyme that catalyzes the first committed and rate-limiting step in inositol biosynthesis. Despite its wide occurrence in all eukaryotes, the role of myo-inositol-1-phosphate synthase and de novo inositol biosynthesis in cell signaling and organism development has been unclear. In this study, we isolated loss-of-function mutants in the Arabidopsis MIPS1 gene from different ecotypes. It was found that all mips1 mutants are defective in embryogenesis, cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower rates of endocytosis. Treatment with brefeldin A induces slower PIN2 protein aggregation in mips1, indicating altered PIN2 trafficking. Our results demonstrate that MIPS1 is critical for maintaining phosphatidylinositol levels and affects pattern formation in plants likely through regulation of auxin distribution. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. The Transporter Spns2 Is Required for Secretion of Lymph but Not Plasma Sphingosine-1-Phosphate

    Directory of Open Access Journals (Sweden)

    Alejandra Mendoza

    2012-11-01

    Full Text Available Plasma sphingosine-1-phosphate (S1P regulates vascular permeability, and plasma and lymph S1P guide lymphocyte egress from lymphoid organs. S1P is made intracellularly, and little is known about how S1P is delivered into circulatory fluids. Here, we find that mice without the major facilitator superfamily transporter Spns2 have a profound reduction in lymph S1P, but only a minor decrease in plasma S1P. Spns2-deficient mice have a redistribution of lymphocytes from the spleen to lymph nodes and a loss of circulating lymphocytes, consistent with normal egress from the spleen directed by plasma S1P and blocked egress from lymph nodes directed by lymph S1P. Spns2 is needed in endothelial cells to supply lymph S1P and support lymphocyte circulation. As a differential requirement for lymph and blood S1P, Spns2 may be an attractive target for immune suppressive drugs.

  6. Transport and homeostasis of potassium and phosphate: limiting factors for sustainable crop production.

    Science.gov (United States)

    Luan, Mingda; Tang, Ren-Jie; Tang, Yumei; Tian, Wang; Hou, Congong; Zhao, Fugeng; Lan, Wenzhi; Luan, Sheng

    2017-06-01

    Potassium (K) and phosphate (Pi) are both macronutrients essential for plant growth and crop production, but the unrenewable resources of phosphorus rock and potash have become limiting factors for food security. One critical measure to help solve this problem is to improve nutrient use efficiency (NUE) in plants by understanding and engineering genetic networks for ion uptake, translocation, and storage. Plants have evolved multiple systems to adapt to various nutrient conditions for growth and production. Within the NUE networks, transport proteins and their regulators are the primary players for maintaining nutrient homeostasis and could be utilized to engineer high NUE traits in crop plants. A large number of publications have detailed K+ and Pi transport proteins in plants over the past three decades. Meanwhile, the discovery and validation of their regulatory mechanisms are fast-track topics for research. Here, we provide an overview of K+ and Pi transport proteins and their regulatory mechanisms, which participate in the uptake, translocation, storage, and recycling of these nutrients in plants. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Copper tolerance mediated by polyphosphate degradation and low-affinity inorganic phosphate transport system in Escherichia coli.

    Science.gov (United States)

    Grillo-Puertas, Mariana; Schurig-Briccio, Lici Ariane; Rodríguez-Montelongo, Luisa; Rintoul, María Regina; Rapisarda, Viviana Andrea

    2014-03-19

    Metal tolerance in bacteria has been related to polyP in a model in which heavy metals stimulate the polymer hydrolysis, forming metal-phosphate complexes that are exported. As previously described in our laboratory, Escherichia coli cells grown in media containing a phosphate concentration >37 mM maintained an unusually high polyphosphate (polyP) level in stationary phase. The aim of the present work was to evaluate the influence of polyP levels as the involvement of low-affinity inorganic phosphate transport (Pit) system in E. coli copper tolerance. PolyP levels were modulated by the media phosphate concentration and/or using mutants in polyP metabolism. Stationary phase wild-type cells grown in high phosphate medium were significantly more tolerant to copper than those grown in sufficient phosphate medium. Copper addition to tolerant cells induced polyP degradation by PPX (an exopolyphosphatase), phosphate efflux and membrane polarization. ppk-ppx- (unable to synthesize/degrade polyP), ppx- (unable to degrade polyP) and Pit system mutants were highly sensitive to metal even in high phosphate media. In exponential phase, CopA and polyP-Pit system would act simultaneously to detoxify the metal or one could be sufficient to safeguard the absence of the other. Our results support a mechanism for copper detoxification in exponential and stationary phases of E. coli, involving Pit system and degradation of polyP. Data reflect the importance of the environmental phosphate concentration in the regulation of the microbial physiological state.

  8. Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes[S

    Science.gov (United States)

    Kobayashi, Naoki; Otsuka, Masato; Yamaguchi, Akihito; Nishi, Tsuyoshi

    2016-01-01

    Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers. PMID:27655910

  9. Phosphate Salts

    Science.gov (United States)

    ... body. They are involved in cell structure, energy transport and storage, vitamin function, and numerous other processes ... Phosphate-containing foods and beverages include cola, wine, beer, whole grain cereals, nuts, dairy products and some ...

  10. Preparation, characterization, biological activity, and transport study of polystyrene based calcium–barium phosphate composite membrane

    Energy Technology Data Exchange (ETDEWEB)

    Khan, Mohammad Mujahid Ali; Rafiuddin,, E-mail: rafi_amu@rediffmail.com

    2013-10-15

    Calcium–barium phosphate (CBP) composite membrane with 25% polystyrene was prepared by co-precipitation method. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transformed infrared (FTIR), and Thermogravimetric analysis (TGA) were used to characterize the membrane. The membrane was found to be crystalline in nature with consistent arrangement of particles and no indication of visible cracks. The electrical potentials measured across the composite membrane in contact with univalent electrolytes (KCl, NaCl and LiCl), have been found to increase with decrease in concentrations. Thus the membrane was found to be cation-selective. Transport properties of developed membranes may be utilized for the efficient desalination of saline water and more importantly demineralization process. The antibacterial study of this composite membrane shows good results for killing the disease causing bacteria along with waste water treatment. Highlights: • Transport properties of composite membrane are evaluated. • The composite membrane was found to be stable in all media. • TMS method is used for electrochemical characterization. • The membrane was found to be cation selective. • The order of surface charge density was found to be LiCl < NaCl < KCl.

  11. Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9.

    Science.gov (United States)

    Lin, I Winnie; Sosso, Davide; Chen, Li-Qing; Gase, Klaus; Kim, Sang-Gyu; Kessler, Danny; Klinkenberg, Peter M; Gorder, Molly K; Hou, Bi-Huei; Qu, Xiao-Qing; Carter, Clay J; Baldwin, Ian T; Frommer, Wolf B

    2014-04-24

    Angiosperms developed floral nectaries that reward pollinating insects. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.

  12. Role of phosphate and other proton-donating anions in respiration-coupled transport of Ca2+ by mitochondria.

    Science.gov (United States)

    Lehninger, A L

    1974-04-01

    Measurements of extra oxygen consumption, (45)Ca(2+) uptake, and the osmotic expansion of the matrix compartment show that not all permeant anions are capable of supporting and accompanying the energy-dependent transport of Ca(2+) from the medium into the matrix in respiring rat-liver mitochondria. Phosphate, arsenate, acetate, butyrate, beta-hydroxybutyrate, lactate, and bicarbonate + CO(2) supported Ca(2+) uptake, whereas the permeant anions, nitrate, thiocyanate, chlorate, and perchlorate, did not. The active anions share a common denominator, the potential ability to donate a proton to the mitochondrial matrix; the inactive anions lack this capacity. Phosphate and the other active permeant anions move into the matrix in response to the alkaline-inside electrochemical gradient of protons generated across the mitochondrial membrane by electron transport, thus forming a negative-inside anion gradient. It is postulated that the latter gradient is the immediate "pulling" force for the influx of Ca(2+) on the electrogenic Ca(2+) carrier in respiring mitochondria under intracellular conditions. Since mitochondria in the cell are normally exposed to an excess of phosphate (and the bicarbonate-CO(2) system), particularly in state 4, inward transport of these proton-yielding anions probably precedes and is necessary for inward transport of Ca(2+) and other cations under biological conditions. These observations indicate that a negative-inside gradient of phosphate generated by electron transport is a common step and provides the immediate motive power not only for (a) the inward transport of dicarboxylates and tricarboxylates and (b) the energy-dependent exchange of external ADP(3-) for internal ATP(4-) during oxidative phosphorylation, as has already been established, but also for (c) the inward transport of Ca(2+), K(+), and other cations.

  13. Molecular cloning, characterization and expression analysis of two members of the Pht1 family of phosphate transporters in Glycine max.

    Directory of Open Access Journals (Sweden)

    Zhaoyun Wu

    Full Text Available BACKGROUND: Phosphorus is one of the macronutrients essential for plant growth and development. The acquisition and translocation of phosphate are pivotal processes of plant growth. In a large number of plants, phosphate uptake by roots and translocation within the plant are presumed to occur via a phosphate/proton cotransport mechanism. PRINCIPAL FINDINGS: We cloned two cDNAs from soybean (Glycine max, GmPT1 and GmPT2, which show homology to the phosphate/proton cotransporter PHO84 from the budding yeast Saccharomyces cerevisiae. The amino acid sequence of the products predicted from GmPT1 and GmPT2 share 61% and 63% identity, respectively, with the PHO84 in amino acid sequence. The deduced structure of the encoded proteins revealed 12 membrane-spanning domains with a central hydrophilic region. The molecular mass values are ∼58.7 kDa for GmPT1 and ∼58.6 kDa for GmPT2. Transiently expressed GFP-protein fusions provide direct evidence that the two Pi transporters are located in the plasma membrane. Uptake of radioactive orthophosphate by the yeast mutant MB192 showed that GmPT1 and GmPT2 are dependent on pH and uptake is reduced by the addition of uncouplers of oxidative phosphorylation. The K(m for phosphate uptake by GmPT1 and GmPT2 is 6.65 mM and 6.63 mM, respectively. A quantitative real time RT-PCR assay indicated that these two genes are expressed in the roots and shoots of seedlings whether they are phosphate-deficient or not. Deficiency of phosphorus caused a slight change of the expression levels of GmPT1 and GmPT2. CONCLUSIONS: The results of our experiments show that the two phosphate transporters have low affinity and the corresponding genes are constitutively expressed. Thereby, the two phosphate transporters can perform translocation of phosphate within the plant.

  14. Carbon-11-labelling of a novel, trishomocubane-derived, high affinity and selectivity DAT ligand

    International Nuclear Information System (INIS)

    Dolle, F.; Le Helleix, St.; Peyronneau, M.A.; Saba, W.; Tournier, N.; Valette, H.; Banister, S.; Kassiou, M.

    2011-01-01

    Complete text of publication follows: Objectives: Parkinson's disease, schizophrenia, attention deficit disorder and drug abuse are related to abnormalities within the brain's dopaminergic system. The neuronal dopamine transporter (DAT) plays a key role in regulating the synaptic concentration of dopamine and thus dopamine neurotransmission in the brain. Since the DAT can be considered as a marker of the integrity and number of the presynaptic striatal dopamine-producing neurons, considerable efforts have been spent in recent years on the design and development of DAT-selective radioligands for use in Positron Emission Tomography (PET) studies. Notably, the tropane PE2I and its fluorinated analogue LBT-999 were identified as having high affinity and selectivity for the DAT over the norepinephrine transporter (NET) and the serotonin transporter (SERT). Besides tropanes, only a few bicyclic frameworks, e.g. bicyclo[2.2.2]octanes, have delivered compounds with high affinity for the DAT. Recently, novel poly-carbocyclic DAT ligands with selectivity over the NET and the SERT were reported. The lead compound of this series (1, N-methyl-N-(3-fluoro) benzyl-pentacyclo[5.4.0.0 2, 6 .0 3, 10 .0 5, 9 ] undec-8-ylamine, Ki = 1.2 nM, ≥ 8300-fold selectivity over NET and SERT) was selected as a potential candidate for imaging the DAT with PET and isotopically labelled with carbon-11 using [ 11 C]methyl triflate. Methods: The trishomocubane derivatives 1 (reference) and 2 (precursor for labelling with carbon-11) were prepared from commercially available Cookson's diketone in 6 and 7 steps, respectively. Carbon-11 labelling of 1 was performed using a TRACERLab FX-C Pro synthesizer (GEMS) and comprises (1) trapping at -10 C of [ 11 C]MeOTf in acetone (0.4 mL) containing the nor-derivative 2 (0.6-0.9 mg, free base) and aq. 3N NaOH (8 μL); (2) heating at 110 C for 2 min; (3) concentration to dryness and taking up the residue in 1.0 mL of the HPLC mobile phase; (4) purification

  15. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

    Science.gov (United States)

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J

    2013-07-25

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. The characterization of novel mycorrhiza-specific phosphate transporters from ¤Lycopersicon esculentum¤ and ¤Solanum tuberosum¤ uncovers functional redundancy in symbiotic phosphate transport in solanaceous species

    DEFF Research Database (Denmark)

    Nagy, F.; Karandashov, V.; Chague, W.

    2005-01-01

    , is the transfer of phosphate (Pi) from the AM fungus to the plant, facilitated by plasma membrane-spanning Pi transporter proteins. The first mycorrhiza-specific plant Pi transporter to be identified, was StPT3 from potato [Nature 414 (2004) 462]. Here, we describe novel Pi transporters from the solanaceous...... species tomato, LePT4, and its orthologue StPT4 from potato, both being members of the Pht1 family of plant Pi transporters. Phylogenetic tree analysis demonstrates clustering of both LePT4 and StPT4 with the mycorrhiza-specific Pi transporter from Medicago truncatula [Plant Cell, 14 (2002) 2413] and rice......Solanaceous species are among the >200 000 plant species worldwide forming a mycorrhiza, that is, a root living in symbiosis with soil-borne arbuscular-mycorrhizal (AM) fungi. An important parameter of this symbiosis, which is vital for ecosystem productivity, agriculture, and horticulture...

  17. Down-Regulation of the Na+-Coupled Phosphate Transporter NaPi-IIa by AMP-Activated Protein Kinase

    Directory of Open Access Journals (Sweden)

    Miribane Dërmaku-Sopjani

    2013-11-01

    Full Text Available Background/Aims: The Na+-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na+ gradient across the apical cell membrane, which is maintained by Na+ extrusion across the basolateral cell membrane through the Na+/K+ ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na+ accumulation and K+ loss with eventual decrease of cell membrane potential, Cl- entry and cell swelling. Upon energy depletion, early inhibition of Na+-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK, a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. Methods: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1-HA, of inactive AMPKαK45R (AMPKα1K45R+AMPKβ1-Flag+AMPKγ1-HA or of constitutively active AMPKγR70Q (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1R70Q. NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. Results: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM to the extracellular bath solution generated a current (Ip, which was significantly decreased by coexpression of wild-type AMPK and of AMPKγR70Q but not of AMPKαK45R. The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM. Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. Conclusion: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport.

  18. N-Oxide analogs of WAY-100635 : new high affinity 5-HT (1A) receptor antagonists

    NARCIS (Netherlands)

    Oberwinkler - Marchais, Sandrine; Nowicki, B; Pike, VW; Halldin, C; Sandell, J; Chou, YH; Gulyas, B; Brennum, LT; Farde, L; Wikstrom, H V

    2005-01-01

    WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] 1 and its O-des-methyl derivative DWAY 2 are well-known high affinity 5-HT1A receptor antagonists. which when labeled with carbon-II (beta(+): t(1/2) 20.4min) in the carbonyl group are effective

  19. ISOLATION AND CHARACTERIZATION OF THE HIGH-AFFINITY K+-TRANSLOCATING ATPASE FROM RHODOBACTER-SPHAEROIDES

    NARCIS (Netherlands)

    ABEE, T; SIEBERS, A; ALTENDORF, K; KONINGS, WN

    1992-01-01

    Cells of the purple nonsulfur bacterium Rhodobacter sphaeroides express a high-affinity K+ uptake system when grown in media with low K+ concentrations. A vanadate-sensitive, K+-stimulated and Mg2+-stimulated ATPase was purified from membranes of these cells by solubilization with

  20. High affinity calmodulin target sequence in the signalling molecule PI 3-kinase

    DEFF Research Database (Denmark)

    Fischer, R; Julsgart, J; Berchtold, M W

    1998-01-01

    M-binding peptide derived from the p110gamma isoform interacts with CaM in a calcium-dependent way. Using gel shift analysis and fluorescence spectrophotometry we discovered that the peptide forms a high affinity complex with CaM. Titration experiments using dansylated CaM gave an affinity constant of 5 n...

  1. The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

    Science.gov (United States)

    Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

    2017-08-01

    The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Modeling of Glycerol-3-Phosphate Transporter Suggests a Potential ‘Tilt’ Mechanism involved in its Function

    Science.gov (United States)

    Tsigelny, Igor F.; Greenberg, Jerry; Kouznetsova, Valentina; Nigam, Sanjay K.

    2009-01-01

    Many major facilitator superfamily (MFS) transporters have similar 12-transmembrane α-helical topologies with two six-helix halves connected by a long loop. In humans, these transporters participate in key physiological processes and are also, as in the case of members of the organic anion transporter (OAT) family, of pharmaceutical interest. Recently, crystal structures of two bacterial representatives of the MFS family — the glycerol-3-phosphate transporter (GlpT) and lac-permease (LacY) — have been solved and, because of assumptions regarding the high structural conservation of this family, there is hope that the results can be applied to mammalian transporters as well. Based on crystallography, it has been suggested that a major conformational “switching” mechanism accounts for ligand transport by MFS proteins. This conformational switch would then allow periodic changes in the overall transporter configuration, resulting in its cyclic opening to the periplasm or cytoplasm. Following this lead, we have modeled a possible “switch” mechanism in GlpT, using the concept of rotation of protein domains as in the DynDom program17 and membranephilic constraints predicted by the MAPAS program.23 We found that the minima of energies of intersubunit interactions support two alternate positions consistent with their transport properties. Thus, for GlpT, a “tilt” of 9°–10° rotation had the most favorable energetics of electrostatic interaction between the two halves of the transporter; moreover, this confirmation was sufficient to suggest transport of the ligand across the membrane. We conducted steered molecular dynamics simulations of the GlpT-ligand system to explore how glycerol-3-phosphate would be handled by the “tilted” structure, and obtained results generally consistent with experimental mutagenesis data. While biochemical data remain most consistent with a single-site alternating access model, our results raise the possibility that, while

  3. ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

    Directory of Open Access Journals (Sweden)

    Maria José Leandro

    Full Text Available Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+ symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1 (gdw(-1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2% and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

  4. Phosphate transport in pig proximal small intestines during postnatal development: lack of modulation by calcitriol.

    Science.gov (United States)

    Schröder, B; Hattenhauer, O; Breves, G

    1998-04-01

    The role of calcitriol in the intestinal absorption of inorganic phosphate (Pi) during postnatal development was studied in newborn [6 weeks pp) control piglets (con) and piglets suffering from inherited calcitriol deficiency (def). In addition, a number of def piglets were treated with vitamin D3 (def-D3). Regardless of age, plasma calcitriol concentrations in def piglets were unphysiologically low (16-21 pg/ml) and differed significantly from those in respective con animals (60-69 pg/ml) and vitamin D3-treated def piglets (50-56 pg/ml). However, newborn and suckling def piglets had normal Ca (approximately 3.0 mmol/liter) and Pi (approximately 2.8 mmol/liter) plasma levels. Def piglets became hypocalcemic (1.9 mmol/liter) and hypophosphatemic (1.9 mmol/liter) between 4-6 weeks pp. Treatment with vitamin D3 significantly increased plasma Ca (3.2 mmol/liter) and Pi (2.7 mmol/liter) levels in weaned def animals. Regardless of calcitriol status, net Pi flux rates (active Pi absorption, as determined with the in vitro Ussing-chamber technique) from the upper small intestines was maximal at birth [170-224 nmol/(cm2 x h)] and decreased by approximately 80% during the first week of life before remaining constant [30-50 nmol/(cm2 x h)] during the following development. In weaned def piglets, net Pi flux rates were significantly lower by about 80% compared with those in con animals. Treatment of def piglets with vitamin D3 had no effect in newborn and suckling animals but reconstituted net Pi flux rates to normal values at weaning age. Age-dependent and calcitriol-mediated changes in net Pi flux rates were paralleled by respective maximum velocity values of Na+-dependent Pi uptake across the brush border membrane of the enterocytes (newborn piglets, 1.9-2.2 nmol/(mg protein 10 sec); suckling piglets, 0.4-0.6 nmol/(mg protein x 10 sec); weaned piglets, 0.7, 0.3, and 0.7 nmol/(mg protein x 10 sec) in con, def, and def-D3 animals, respectively). These findings suggest that

  5. Phosphate-binding protein from Polaromonas JS666: purification, characterization, crystallization and sulfur SAD phasing

    Energy Technology Data Exchange (ETDEWEB)

    Pegos, Vanessa R.; Hey, Louis; LaMirande, Jacob; Pfeffer, Rachel; Lipsh, Rosalie; Amitay, Moshe; Gonzalez, Daniel; Elias, Mikael (JCT-Israel); (UMM); (CNRS-UMR)

    2017-05-25

    Phosphate-binding proteins (PBPs) are key proteins that belong to the bacterial ABC-type phosphate transporters. PBPs are periplasmic (or membrane-anchored) proteins that capture phosphate anions from the environment and release them to the transmembrane transporter. Recent work has suggested that PBPs have evolved for high affinity as well as high selectivity. In particular, a short, unique hydrogen bond between the phosphate anion and an aspartate residue has been shown to be critical for selectivity, yet is not strictly conserved in PBPs. Here, the PBP fromPolaromonasJS666 is focused on. Interestingly, this PBP is predicted to harbor different phosphate-binding residues to currently known PBPs. Here, it is shown that the PBP fromPolaromonasJS666 is capable of binding phosphate, with a maximal binding activity at pH 8. Its structure is expected to reveal its binding-cleft configuration as well as its phosphate-binding mode. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.35 Å resolution of the PBP fromPolaromonasJS666 are reported.

  6. Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells.

    Science.gov (United States)

    Balestrini, Raffaella; Gómez-Ariza, Jorge; Lanfranco, Luisa; Bonfante, Paola

    2007-09-01

    The establishment of a symbiotic interaction between plant roots and arbuscular mycorrhizal (AM) fungi requires both partners to undergo significant morphological and physiological modifications which eventually lead to reciprocal beneficial effects. Extensive changes in gene expression profiles recently have been described in transcriptomic studies that have analyzed the whole mycorrhizal root. However, because root colonization by AM fungi involves different cell types, a cell-specific gene expression pattern is likely to occur. We have applied the laser microdissection (LMD) technology to investigate expression profiles of both plant and fungal genes in Lycopersicon esculentum roots colonized by Glomus mosseae. A protocol to harvest arbuscule-containing cells from paraffin sections of mycorrhizal roots has been developed using a Leica AS LMD system. RNA of satisfactory quantity and quality has been extracted for molecular analysis. Transcripts for plant phosphate transporters (LePTs), selected as molecular markers for a functional symbiosis, have been detected by reverse-transcriptase polymerase chain reaction assays and associated to distinct cell types, leading to novel insights into the distribution of LePT mRNAs. In fact, the transcripts of the five phosphate transporters (PTs) have been detected contemporaneously in the same arbusculated cell population, unlike from the neighboring noncolonized cells. In addition, fungal H(+)ATPase (GmHA5) and phosphate transporter (GmosPT) mRNAs were found exclusively in arbusculated cells. The discovery that five plant and one fungal PT genes are consistently expressed inside the arbusculated cells provides a new scenario for plant-fungus nutrient exchanges.

  7. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    Science.gov (United States)

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses. © 2012 John Wiley & Sons A/S.

  8. Selective high-affinity polydentate ligands and methods of making such

    Energy Technology Data Exchange (ETDEWEB)

    Denardo, Sally J.; Denardo, Gerald L.; Balhorn, Rodney L.

    2018-02-06

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  9. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  10. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    OpenAIRE

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. CatalaseAbeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro.

  11. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death

    DEFF Research Database (Denmark)

    Nørholm, Morten Helge Hauberg; Nour-Eldin, Hussam H; Brodersen, Peter

    2006-01-01

    GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP...

  12. Reconstitution of high-affinity opioid agonist binding in brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Remmers, A.E.; Medzihradsky, F. (Univ. of Michigan Medical School, Ann Arbor (United States))

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  13. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    Science.gov (United States)

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    Directory of Open Access Journals (Sweden)

    Tal Noy-Porat

    2016-03-01

    Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  15. Proadifen-sensitive high affinity binding of 3H-alaproclate to liver membranes

    International Nuclear Information System (INIS)

    Ross, S.B.

    1987-01-01

    3 H-alaproclate, a selective 5 h ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K D -=3 nM) and large capacity (B max about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the 3 H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the 3 H-alaproclate binding with the same, high affinity (K i =3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced 3 H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed. (author)

  16. Proadifen-sensitive high affinity binding of /sup 3/H-alaproclate to liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B.

    1987-01-01

    /sup 3/H-alaproclate, a selective 5/sub h/ydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (K/sub D/-=3 nM) and large capacity (B/sub max/ about 2 nmol/g liver). This binding was stereoselective since S-( - )-alaproclate was 30 times more potent than the R-( + )-enantiomer to displace the /sup 3/H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the /sup 3/H-alaproclate binding with the same, high affinity (K/sub i/=3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5x75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced /sup 3/H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed.

  17. Systemic and local regulation of phosphate and nitrogen transporter genes by arbuscular mycorrhizal fungi in roots of winter wheat (Triticum aestivum L.).

    Science.gov (United States)

    Duan, Jianfeng; Tian, Hui; Drijber, Rhae A; Gao, Yajun

    2015-11-01

    Previous studies have reported that the expression of phosphate (Pi) or nitrogen (N) transporter genes in roots of plants could be regulated by arbuscular mycorrhizal (AM) fungi, but little is known whether the regulation is systemic or not. The present study investigated the systemic and local regulation of multiple phosphate and nitrogen transporter genes by four AM fungal species belonging to four genera in the roots of winter wheat. A split-root culture system with AM inoculated (MR) and non-inoculated root compartments (NR) was used to investigate the systemic or local responses of phosphate and nitrogen transporter genes to colonization by four AM fungi in the roots of wheat. The expression of four Pi transporter, five nitrate transporter, and three ammonium transporter genes was quantified using real-time PCR. Of the four AM fungi tested, all locally increased expression of the AM-inducible Pi transporter genes, and most locally decreased expression of a Pi-starvation inducible Pi transporter gene. The addition of N in soil increased the expression of either Pi starvation inducible Pi transporters or AM inducible Pi transporters. Inoculation with AM fungi either had no effect, or could locally or systemically down-regulate expression of nitrogen transporter genes depending on gene type and AM fungal species. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  18. Transport of Th(IV) and U(VI) through barium silico-phosphate composite membrane using electric field

    International Nuclear Information System (INIS)

    Zaki, E.E.

    2002-01-01

    The present paper describes the preparation of a novel barium silico-phosphate filter paper supported membrane. It is based on precipitation reaction of barium silico-phosphate on the outer surface and in the interstices of a filter paper by means of electrodialysis. The main physical and electrical properties of the membrane are given and its electrodialysis behaviour is assessed for Th(IV) and U(VI). The transport of Th(IV) in presence of U(VI) was studied. The cationic fluxes of Th(IV) and U(VI) were found to be 1.2 x 10 -8 and 6.5 x 10 -9 g eq cm -2 s -1 , respectively. Transport of Th(IV) and U(VI) in presence of EDTA was investigated. The cationic flux of U(VI) is found to be 9.8 x 10 -9 g eq cm -2 s -1 at a current density of 25 mA/cm 2 . A comparative study on the electro osmotic effect was carried out using the developed membrane and commercially available Nafion membranes. In this context, different parameters like current density, electrolyte concentration, etc. were investigated. The electro-osmotic permeability coefficient, D e , of Th(IV) through barium silico-phosphate and Nafion membranes were 6.9 x 10 -2 and 1.0 x 10 -2 cm 3 /As, respectively. It can be concluded that inorganic membranes have very marked electro-osmotic properties unlike their organic counterparts. (orig.)

  19. Transcriptome Characterization of the Chinese Fir (Cunninghamia lanceolata (Lamb. Hook. and Expression Analysis of Candidate Phosphate Transporter Genes

    Directory of Open Access Journals (Sweden)

    Ming Li

    2017-11-01

    Full Text Available Chinese fir (Cunninghamia lanceolata (Lamb. Hook. is the most important afforestation tree species in China because of its excellent timber quality and high yield. However, the limited availability of phosphorus in forest soils is widespread and has become an important factor in the declining productivity of Chinese fir plantations. Here we used the Illumina HiSeq™ 2000 DNA sequencing platform to sequence root, stem, and leaf transcriptomes of one-year old Chinese fir clones with phosphorus treatment. Approximately 236,529,278 clean reads were obtained and generated 35.47 G of sequencing data. These reads were assembled into 413,806 unigenes with a mean length of 520 bp. In total, 109,596 unigenes were annotated in the NR (NCBI non-redundant database, 727,287 genes were assigned for GO (Gene Ontology terms, information for 92,001 classified unigenes was assigned to 26 KOG (Karyotic Orthologous Groups categories, and 57,042 unigenes were significantly matched with 132 KEGG (Kyoto Encyclopedia of Genes and Genomes predicted pathways. In total, 49 unigenes were identified as exhibiting inorganic phosphate transporter activity, and 14 positive genes’ expression patterns in different phosphorus deficiency treatments were analyzed by qRT-PCR to explore their putative functions. This study provides a basic foundation for functional genomic studies of the phosphate transporter in Chinese fir, and also presents an extensive annotated sequence resource for molecular research.

  20. The mammalian phosphate carrier SLC25A3 is a mitochondrial copper transporter required for cytochrome c oxidase biogenesis.

    Science.gov (United States)

    Boulet, Aren; Vest, Katherine E; Maynard, Margaret K; Gammon, Micah G; Russell, Antoinette C; Mathews, Alexander T; Cole, Shelbie E; Zhu, Xinyu; Phillips, Casey B; Kwong, Jennifer Q; Dodani, Sheel C; Leary, Scot C; Cobine, Paul A

    2018-02-09

    Copper is required for the activity of cytochrome c oxidase (COX), the terminal electron-accepting complex of the mitochondrial respiratory chain. The likely source of copper used for COX biogenesis is a labile pool found in the mitochondrial matrix. In mammals, the proteins that transport copper across the inner mitochondrial membrane remain unknown. We previously reported that the mitochondrial carrier family protein Pic2 in budding yeast is a copper importer. The closest Pic2 ortholog in mammalian cells is the mitochondrial phosphate carrier SLC25A3. Here, to investigate whether SLC25A3 also transports copper, we manipulated its expression in several murine and human cell lines. SLC25A3 knockdown or deletion consistently resulted in an isolated COX deficiency in these cells, and copper addition to the culture medium suppressed these biochemical defects. Consistent with a conserved role for SLC25A3 in copper transport, its heterologous expression in yeast complemented copper-specific defects observed upon deletion of PIC2 Additionally, assays in Lactococcus lactis and in reconstituted liposomes directly demonstrated that SLC25A3 functions as a copper transporter. Taken together, these data indicate that SLC25A3 can transport copper both in vitro and in vivo . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. The transport characteristics of {sup 238}U, {sup 232}Th, {sup 226}Ra, and {sup 40}K in the production cycle of phosphate rock

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Yoon Hee; Lim, Jong Myoung; Ji, Young Yong; Chung, Kun Ho; Kang, Mun Ja [Environmental Radioactivity Assessment Team, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2017-03-15

    Phosphate rock and its by-product are widely used in various industries to produce phosphoric acid, gypsum, gypsum board, and fertilizer. Owing to its high level of natural radioactive nuclides (e.g., 238U and 226Ra), the radiological safety of workers who work with phosphate rock should be systematically managed. In this study, 238U, 232Th, 226Ra, and 40K levels were measured to analyze the transport characteristics of these radionuclides in the production cycle of phosphate rock. Energy dispersive X-ray fluorescence and gamma spectrometry were used to determine the activity of 238U, 232Th, 226Ra, and 40K. To evaluate the extent of secular disequilibrium, the analytical results were compared using statistical methods. Finally, the distribution of radioactivity across different stages of the phosphate rock production cycle was evaluated. The concentration ratios of 226Ra and 238U in phosphate rock were close to 1.0, while those found in gypsum and fertilizer were extremely different, reflecting disequilibrium after the chemical reaction process. The nuclide with the highest activity level in the production cycle of phosphate rock was 40K, and the median 40K activity was 8.972 Bq·g−1 and 1.496 Bq·g−1, respectively. For the 238U series, the activity of 238U and 226Ra was greatest in phosphate rock, and the distribution of activity values clearly showed the transport characteristics of the radionuclides, both for the byproducts of the decay sequences and for their final products. Although the activity of 40K in k-related fertilizer was relatively high, it made a relatively low contribution to the total radiological effect. However, the activity levels of 226Ra and 238U in phosphate rock were found to be relatively high, near the upper end of the acceptable limits. Therefore, it is necessary to systematically manage the radiological safety of workers engaged in phosphate rock processing.

  2. Effect of quinolinic acid in the nucleus basalis magnocellularis on cortical high-affinity choline uptake

    Energy Technology Data Exchange (ETDEWEB)

    Metcalf, R.H.; Boegman, R.J.; Quirion, R.; Riopelle, R.J.; Ludwin, S.K.

    1987-08-01

    A transient 45% increase in cortical high-affinity choline uptake (HACU) was observed after an injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM) of the rat. This was followed by a steady decline in choline uptake, which resulted in a 46% decrease by day 7. Specific (/sup 3/H)hemicholinium-3 binding to coronal brain sections showed a similar pattern following injections of QUIN into the nbM. The increase in cortical HACU elicited by QUIN appeared to be dose dependent.

  3. Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex

    International Nuclear Information System (INIS)

    John, D.; Fox, I.V.

    1986-01-01

    The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A 1 and A 2 adenosine binding sites. The ligands used were 2-chloro(8- 3 H) adenosine and N 6 -(adenine-2, 8- 3 H) cyclohexayladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The kinetic constant determinations from binding studies of the adenosine receptor are presented. Utilizing tritium-cyclohexyladenosine as ligand the authors observed evidence for a high affinity binding site in human brain cortical membranes with a kd of 5 nM

  4. High affinity binding of [3H]cocaine to rat liver microsomes

    International Nuclear Information System (INIS)

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    ] 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 [ 3 H]cocaine binding. On the other hand, chronic administration of cocaine reduced [ 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 [ 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 [ 3 H]cocaine binding to liver with a different rank order of potency than their displacement of [ 3 H]cocaine binding to striatum. This high affinity [ 3 H]cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  5. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    Science.gov (United States)

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  6. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides.

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-17

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a 'piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (K d =39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  7. High affinity, ligand specific uptake of complexed copper-67 by brain tissue incubated in vitro

    International Nuclear Information System (INIS)

    Barnea, A.; Hartter, D.E.

    1987-01-01

    Copper is an essential metal that is highly concentrated in the brain. The blood, the sole source of tissue Cu, contains 16-20 μM Cu, of which >95% is complexed to proteins and 2 was 10 times greater than that of CuAlbumin or Cu(II). Within the range of 0.2-150μM Cu, multiple uptake sites for CuHis were apparent. Increasing the molar ratio of His:Cu had a differential effect on Cu uptake: enhancing uptake at [Cu] 1 μM. Thus, using a His:Cu ratio of 1000, they observed a high affinity process exhibiting saturating and half saturating values of 5 μM and 1.5 μM Cu, respectively; using a His:Cu ratio of 2, they observed a low affinity process exhibiting saturating and half-saturating values of 100 μM and 40 μM Cu, respectively. Both processes required thermic but not metabolic energy, suggestive of facilitated diffusion. Considering the blood brain barrier for proteins, CuHis appears to be the major substrate for Cu uptake by neuronal tissue. They demonstrate the existence of a ligand specific, high affinity (apparent Km about 1.5 μM Cu) uptake process for CuHis in the brain, operative at the physiological concentration range of CuHis and histidine

  8. High-Affinity Accumulation of Chloroquine by Mouse Erythrocytes Infected with Plasmodium berghei

    Science.gov (United States)

    Fitch, Coy D.; Yunis, Norman G.; Chevli, Rekha; Gonzalez, Yolanda

    1974-01-01

    Washed erythrocytes infected with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) P. berghei were used in model systems in vitro to study the accumulation of chloroquine with high affinity. The CS model could achieve distribution ratios (chloroquine in cells: chloroquine in medium) of 100 in the absence of substrate. 200—300 in the presence of 10 mM pyruvate or lactate, and over 600 in the presence of 1 mM glucose or glycerol. In comparable studies of the CR model, the distribution ratios were 100 in the absence of substrate and 300 or less in the presence of glucose or glycerol. The presence of lactate stimulated chloroquine accumulation in the CR model, whereas the presence of pyruvate did not. Lactate production from glucose and glycerol was undiminished in the CR model, and ATP concentrations were higher than in the CS model. Cold, iodoacetate, 2,4-dinitrophenol, or decreasing pH inhibited chloroquine accumulation in both models. These findings demonstrate substrate involvement in the accumulation of chloroquine with high affinity. In studies of the CS model, certain compounds competitively inhibited chloroquine accumulation, while others did not. This finding is attributable to a specific receptor that imposes structural constraints on the process of accumulation. For chloroquine analogues, the position and length of the side chain, the terminal nitrogen atom of the side chain, and the nitrogen atom in the quinoline ring are important determinants of binding to this receptor. PMID:4600044

  9. High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes

    International Nuclear Information System (INIS)

    Lach, E.; Trifilieff, A.; Landry, Y.; Gies, J.P.

    1991-01-01

    The binding of the radiolabeled bombesin analogue [ 125 I-Tyr 4 ]bombesin to guinea-pig lung membranes was investigated. Binding of [ 125 I-Tyr 4 ]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25C indicated the presence of a single class of non-interacting binding sites for bombesin (B max = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (K D = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [ 125 I-Tyr 4 ]bombesin, neuromedin B and neuromedin C inhibited the binding of [ 125 I-Tyr 4 ]bombesin in an order of potencies as follows: [ 125 I-Tyr 4 ]bombesin > bombesin ≥ neuromedin C much-gt neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B

  10. Reconstitution of high affinity α2 adrenergic agonist binding by fusion with a pertussis toxin substrate

    International Nuclear Information System (INIS)

    Kim, M.H.; Neubig, R.R.

    1986-01-01

    High affinity α 2 adrenergic agonist binding is thought to occur via a coupling of the α 2 receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 μM phenoxybenzamine to block α 2 receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the α 2 agonist [ 3 H]UK 14,304 (UK) and the antagonist [ 3 H] yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain α 2 receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance [ 3 H] UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity α 2 agonist binding

  11. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    Science.gov (United States)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  12. Molecular cloning and functional analysis of a H(+)-dependent phosphate transporter gene from the ectomycorrhizal fungus Boletus edulis in southwest China.

    Science.gov (United States)

    Wang, Junling; Li, Tao; Wu, Xiaogang; Zhao, Zhiwei

    2014-01-01

    Phosphate transporters (PTs), as entry points for phosphorus (P) in organisms, are involved in a number of P nutrition processes such as phosphate uptake, transport, and transfer. In the study, a PT gene 1632 bp long (named BePT) was cloned, identified, and functionally characterized from Boletus edulis. BePT was expected to encode a polypeptide with 543 amino acid residues. The BePT polypeptide belonged to the major facilitator superfamily and showed a high degree of sequence identity to the Pht1 family. A topology model revealed that BePT exhibited 12 transmembrane helices, divided into two halves, and connected by a large hydrophilic loop in the middle. A yeast mutant complementation analysis suggested that BePT was a functional PT which mediated orthophosphate uptake of yeast at micromolar concentrations. Green fluorescent protein-BePT fusion proteins expressed were extensively restricted to the plasma membrane in BePT transformed yeast, and its activity was dependent on electrochemical membrane potential. In vitro, quantitative PCR confirmed that the expression of BePT was significantly upregulated at lower phosphorus availability, which may enhance phosphate uptake and transport under phosphate starvation. Our results suggest that BePT plays a key role in phosphate acquisition in the ectomycorrhizal fungus B. edulis. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  13. Changes in medium radioactivity and composition accompany high-affinity uptake of glutamate and aspartate by mouse brain slices

    International Nuclear Information System (INIS)

    Latzkovits, L.; Neidle, A.; Lajtha, A.

    1984-01-01

    In measurements of high affinity transport in tissue slices, the incubation medium is often treated as an ''infinitely large pool''. External substrate concentrations, even at the micromolar level, are assumed to be constant and metabolic interactions between tissue and medium are neglected. In the present report we describe experiments in which glutamic and aspartic acid uptake by mouse brain slices were studied using techniques that could test these assumptions. Cerebral hemispheres were cut into 0.1 mm sections and about 90 mg of tissue incubated in 10 ml of oxygenated medium. After 45 minutes of equilibration, radioactive substrates were added and the concentrations and specific activities of the amino acids and their metabolites in the medium were determined. During the first 10 min following substrate addition, rapid decreases in glutamic and aspartic acid concentrations in the medium were accompanied by large decreases in specific activity caused by the continuous release of these amino acids from the tissue. In addition, extensive conversion of both substrates to glutamine and the preferential accumulation of this metabolite, in the medium, was found. These results demonstrate that metabolism and release occur simultaneously with uptake during transport experiments in vitro and that these processes can take place in specific tissue compartments. It is therefore necessary to measure the tissue and medium concentration levels of amino acids along with their radioactivity in such experiments, since all three processes (transport, metabolism, and compartmentation) are interrelated in the clearance of amino acids from the incubation medium and probably from the extracellular spaces in vivo as well

  14. Nonredundant Regulation of Rice Arbuscular Mycorrhizal Symbiosis by Two Members of the Phosphate Transporter 1 Gene Family

    DEFF Research Database (Denmark)

    Yang, Shu-Yi; Grønlund, Mette; Jakobsen, Iver

    2012-01-01

    Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited highgrade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via...... or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands...... the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage...

  15. Effect of ionophores on phosphate and arsenate transport in Micrococcus lysodeikticus

    Energy Technology Data Exchange (ETDEWEB)

    Friedberg, I

    1977-09-01

    The effects of ionophores on P/sub i/ and arsenate transport, at acid and alkaline environment, were investigated in whole cells of Micrococcus lysodeikticus, a Gram positive obligatory aerobic bacterium. The results suggest that both ..delta.. Psi and ..delta.. pH contribute to the driving force of P/sub i/ transport; ..delta.. Psi seems to be predominant at pH 7.8, whereas at pH 5.5, the transport is primarily driven by ..delta.. pH. 12 references, 1 figure.

  16. myo-Inositol-1-phosphate synthase is required for polar auxin transport and organ development

    KAUST Repository

    Chen, Hao; Xiong, Liming

    2010-01-01

    , cotyledon venation patterning, root growth, and root cap development. The mutant roots are also agravitropic and have reduced basipetal auxin transport. mips1 mutants have significantly reduced levels of major phosphatidylinositols and exhibit much slower

  17. Functional interaction between CFTR and the sodium-phosphate co-transport type 2a in Xenopus laevis oocytes.

    Directory of Open Access Journals (Sweden)

    Naziha Bakouh

    Full Text Available A growing number of proteins, including ion transporters, have been shown to interact with Cystic Fibrosis Transmembrane conductance Regulator (CFTR. CFTR is an epithelial chloride channel that is involved in Cystic Fibrosis (CF when mutated; thus a better knowledge of its functional interactome may help to understand the pathophysiology of this complex disease. In the present study, we investigated if CFTR and the sodium-phosphate co-transporter type 2a (NPT2a functionally interact after heterologous expression of both proteins in Xenopus laevis oocytes.NPT2a was expressed alone or in combination with CFTR in X. laevis oocytes. Using the two-electrode voltage-clamp technique, the inorganic phosphate-induced current (IPi was measured and taken as an index of NPT2a activity. The maximal IPi for NPT2a substrates was reduced when CFTR was co-expressed with NPT2a, suggesting a decrease in its expression at the oolemna. This was consistent with Western blot analysis showing reduced NPT2a plasma membrane expression in oocytes co-expressing both proteins, whereas NPT2a protein level in total cell lysate was the same in NPT2a- and NPT2a+CFTR-oocytes. In NPT2a+CFTR- but not in NPT2a-oocytes, IPi and NPT2a surface expression were increased upon PKA stimulation, whereas stimulation of Exchange Protein directly Activated by cAMP (EPAC had no effect. When NPT2a-oocytes were injected with NEG2, a short amino-acid sequence from the CFTR regulatory domain that regulates PKA-dependent CFTR trafficking to the plasma membrane, IPi values and NPT2a membrane expression were diminished, and could be enhanced by PKA stimulation, thereby mimicking the effects of CFTR co-expression.We conclude that when both CFTR and NPT2a are expressed in X. laevis oocytes, CFTR confers to NPT2a a cAMPi-dependent trafficking to the membrane. This functional interaction raises the hypothesis that CFTR may play a role in phosphate homeostasis.

  18. Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters

    DEFF Research Database (Denmark)

    Sawers, Ruairidh J H; Svane, Simon F; Quan, Clement

    2016-01-01

    content, abundance of intra-radical and root-external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Larger growth responses in Oh43 were correlated with extensive development of root-external hyphae...

  19. Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters

    DEFF Research Database (Denmark)

    Sawers, Ruairidh J. H.; Svane, Simon; Quan, Clement

    2017-01-01

    -internal and -external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Superior response in Oh43 is correlated with extensive development of root-external hyphae, accumulation of specific Pht1 transcripts and high...

  20. Isolation and characterization of high affinity aptamers against DNA polymerase iota.

    Science.gov (United States)

    Lakhin, Andrei V; Kazakov, Andrei A; Makarova, Alena V; Pavlov, Yuri I; Efremova, Anna S; Shram, Stanislav I; Tarantul, Viacheslav Z; Gening, Leonid V

    2012-02-01

    Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.

  1. Haemoglobin Pierre-Benite--a high affinity variant associated with relative polycythaemia.

    Science.gov (United States)

    Beard, M E; Potter, H C; Spearing, R L; Brennan, S O

    2001-12-01

    This is the second reported example of Hb Pierre--Benite (beta90 Glu-->Asp). This mutation is associated with increased oxygen affinity and polycythaemia. No instability was found and there was no charge shift detected by cellulose acetate electrophoresis at pH 8.3. The mutation was however, clearly indicated by electrospray ionization mass spectrometry (ESI MS), which showed an abnormal beta chain with a 14 Da decrease in mass. Blood volume studies documented a relative rather than a true polycythaemia and this finding has been reported in at least two other high affinity haemoglobin variants--Hb Heathrow and Hb Rahere. This finding led to delay in diagnosis because high oxygen affinity variants are conventionally considered to cause a true polycythaemia.

  2. New immunogenic form for vasopressin: production of high-affinity antiserum and RIA for plasmatic AVP

    International Nuclear Information System (INIS)

    Rougon-Rappuzi, G.; Delaage, M.A.; Conte-Devolx, B.; Millet, Y.

    1977-01-01

    A highly sensitive and specific radioimmunoassay (RIA) for arginine-vasopressin (AVP) was developped and applied to the measurement of AVP in human plasma. High-affinity antivasopressin antibodies with limited association constant heterogeneity have been induced by immunizing rabbits with Lysine-vasopressine (LVP) coupled to a human immunoglobulin (IgA). Replacing air drying of acetone-petroleum ether extracts by lyophilisation increased significantly the yields of AVP. Equilibrium dialysis was used for separating bound and free antigen, thus reducing the total time required for the assay to 48 hours. Only 1 ml of plasma was required for routine determinations due to a sensitivity threshold better than 0.5 pg/ml. Plasma AVP levels of normal subjects and of patients with inappropriate ADH secretion (SIADH) were determined during different hydratation states and following nicotin of ethanol infusions. (orig.) [de

  3. Effects of anticonvulsants in vivo on high affinity choline uptake in vitro in mouse hippocampal synaptosomes.

    Science.gov (United States)

    Miller, J. A.; Richter, J. A.

    1985-01-01

    The effects of several anticonvulsant drugs on sodium-dependent high affinity choline uptake (HACU) in mouse hippocampal synaptosomes was investigated. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by drugs which have in common the ability to facilitate gamma-aminobutyric acid (GABA) transmission, pentobarbitone, phenobarbitone, barbitone, diazepam, chloridiazepoxide, and valproic acid. Dose-response relationships were determined for these drugs and the drugs' potencies at inhibiting HACU correlated well with their anticonvulsant potencies. Clonazepam, ethosuximide, carbamazepine, and barbituric acid had no effect on HACU in the doses used while phenytoin and trimethadione stimulated HACU. These results suggest that certain anticonvulsants may elicit a part of their anticonvulsant activity by modulating cholinergic neurones. This effect may be mediated through a GABA mechanism. PMID:3978310

  4. Exploiting the high-affinity phosphonate-hydroxyapatite nanoparticle interaction for delivery of radiation and drugs

    International Nuclear Information System (INIS)

    Ong, Hooi Tin; Loo, Joachim S. C.; Boey, Freddy Y. C.; Russell, Stephen J.; Ma Jan; Peng, Kah-Whye

    2008-01-01

    Hydroxyapatite is biocompatible and used in various biomedical applications. Here, we generated hydroxyapatite nanoparticles (HNPs) of various sizes (40-200 nm) and demonstrated that they can be stably loaded with drugs or radioisotopes by exploiting the high-affinity HA-(poly)phosphonate interaction. Clinically available phosphonates, clodronate, and Tc-99m-methylene-diphosphonate (Tc-99m-MDP), were efficiently loaded onto HNPs within 15 min. Biodistribution of radiolabeled HNP-MDP-Tc99m in mice was monitored non-invasively using microSPECT-CT. Imaging and dosimetry studies indicated that the HNPs, regardless of size, were quickly taken up by Kupffer cells in the liver after systemic administration into mice. Clodronate loaded onto HNPs remained biologically active and were able to result in selective depletion of Kupffer cells. This method of drug or isotope loading on HA is fast and easy as it eliminates the need for additional surface modifications of the nanoparticles

  5. Production and Identification of High Affinity Monoclonal Antibodies Against Pesticide Carbofuran

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To produce high-affinity monoclonal antibodies against pesticide carbofuran, and the develop immunochemical assays for people's health and environmental protection, the hapten 4-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]-amino]-butanoic acid (BFNB) of carbofuran was synthesized and Balb/c mice were immunized by the hapten-carrier (BFNB-bovine serum albumin, BFNB-BSA) conjugates. The splenocytes of immunized mice were fused with Sp2/0 cells and the cultural supernatants of hybridoma cells were screened by the indirect enzyme-linked immunoabsorbent assay (ELISA), based on BFNB-ovoalbumin conjugates (BFNB-OVA). Purified monoclonal antibody (McAb) was obtained from fluids of ascites, deposited by octanoic acid and ammonium sulfate. The affinity and the specificity of McAb were characterized by ELISA or indirect competitive ELISA. A hybridoma cell line (5D3) secreting anti-carbofuran McAb had been established. The titer of culture medium and ascites was up to 1:2.048 × 103 and 1:1.024 × 106, respectively, and the subtype of the McAb was IgG1. The affinity constant of the McAb was about 2.54 × 109 L mol-1, with an IC50 value of 1.18 ng mL-1 and a detection limit of 0.01 ng mL-1. Cross-reactivity studies showed that the McAb was quiet specific for carbofuran, as among the four analogous compounds, they were all hardly recognized (4.59 × 10-4% for 2,3-dihydro-2,2-dimethyl-7-benzofuranol and less than 3.0 × 10-4% for others). The prepared McAb had a very high affinity and specificity,and it could be used to develop ELISA for rapid determination of carbofuran.

  6. High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

    International Nuclear Information System (INIS)

    Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

    2014-01-01

    Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

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

    International Nuclear Information System (INIS)

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

    1985-01-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; [ 3 H]-5-HT). At concentrations in the range of 0.01 to 0.7 microM [ 3 H]-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM [ 3 H]-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of [ 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 [ 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 [ 3 H]-5-HT up to 44-fold at an external [ 3 H]-5-HT concentration of 10(-7) M. Inhibition of [ 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 [ 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 [ 3 H]-5-HT uptake

  8. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    International Nuclear Information System (INIS)

    Wang, Deng-Liang; Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan; Yang, Hai-Tao; Wang, Jiang-Jie; Yao, Pei-Sen; Pan, Ru-Jun; Yang, Chaoyong James; Kang, De-Zhi

    2014-01-01

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K d 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K d 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy

  9. Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition.

    Directory of Open Access Journals (Sweden)

    Yves Nominé

    Full Text Available Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15 derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F was equally well tolerated as the wild type glutamine (Q at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv-peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology. Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes.

  10. Selection of DNA aptamers against epidermal growth factor receptor with high affinity and specificity

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Deng-Liang [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Song, Yan-Ling; Zhu, Zhi; Li, Xi-Lan; Zou, Yuan [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Yang, Hai-Tao; Wang, Jiang-Jie [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yao, Pei-Sen [Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China); Pan, Ru-Jun [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Yang, Chaoyong James, E-mail: cyyang@xmu.edu.cn [State Key Laboratory for Physical Chemistry of Solid Surfaces, Key Laboratory for Chemical Biology of Fujian Province, Key Laboratory of Analytical Chemistry, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005 (China); Kang, De-Zhi, E-mail: kdzy99988@163.com [The First Clinical Medical College of Fujian Medical University, Fuzhou (China); Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou (China)

    2014-10-31

    Highlights: • This is the first report of DNA aptamer against EGFR in vitro. • Aptamer can bind targets with high affinity and selectivity. • DNA aptamers are more stable, cheap and efficient than RNA aptamers. • Our selected DNA aptamer against EGFR has high affinity with K{sub d} 56 ± 7.3 nM. • Our selected DNA aptamer against EGFR has high selectivity. - Abstract: Epidermal growth factor receptor (EGFR/HER1/c-ErbB1), is overexpressed in many solid cancers, such as epidermoid carcinomas, malignant gliomas, etc. EGFR plays roles in proliferation, invasion, angiogenesis and metastasis of malignant cancer cells and is the ideal antigen for clinical applications in cancer detection, imaging and therapy. Aptamers, the output of the systematic evolution of ligands by exponential enrichment (SELEX), are DNA/RNA oligonucleotides which can bind protein and other substances with specificity. RNA aptamers are undesirable due to their instability and high cost of production. Conversely, DNA aptamers have aroused researcher’s attention because they are easily synthesized, stable, selective, have high binding affinity and are cost-effective to produce. In this study, we have successfully identified DNA aptamers with high binding affinity and selectivity to EGFR. The aptamer named TuTu22 with K{sub d} 56 ± 7.3 nM was chosen from the identified DNA aptamers for further study. Flow cytometry analysis results indicated that the TuTu22 aptamer was able to specifically recognize a variety of cancer cells expressing EGFR but did not bind to the EGFR-negative cells. With all of the aforementioned advantages, the DNA aptamers reported here against cancer biomarker EGFR will facilitate the development of novel targeted cancer detection, imaging and therapy.

  11. A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

    Science.gov (United States)

    Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

    2003-01-01

    A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

  12. Uranyl ion transport across tri-n-butyl phosphate/n-dodecane liquid membranes

    International Nuclear Information System (INIS)

    Shukla, J.P.; Misra, S.K.

    1991-01-01

    Carrier-facilitated transport of uranium (VI) against its concentration gradient from aqueous nitrate acidic solutions across organic bulk liquid membranes (BLM) and supported liquid membranes (SLM) containing TBP as the mobile carrier and n-dodecane as the membrane solvent was investigated. Extremely dilute uranyl nitrate solutions in about 2.5 M nitric acid generally constituted as the source phase. Uranyl transport appreciably increased with both stirring of the receiving phase and the carrier concentration in the organic membrane, while enhanced acidity of the strip side adversely affected the partioning of the cation into this phase. Among the several reagents tested, diluted ammonium carbonate (∼1M) solutions served efficiently as the stripant. Besides Accurel polypropylene (PP) film as the solid support for SLM, some silicon flat-sheet membranes with different inorganic fillers like silica, calcium silicate, calcium carbonate, chromium oxide, zinc oxide etc. and teflon membranes transported about 70% of uranium in nearly 7-8 hr employing 1 M ammonium carbonate as the strippant. Specifically, 30% TBP supported on Accurel flat-sheet supports transfered better than 70% of uranium from moderate acid feeds (2.5M) under similar conditions. Membranes supporting Aliquat-336, TLA, TOPO etc. yielded somewhat poor uranium recoveries. The feed : strip volume ratio showed an inverse relationship to the fraction of cation transported. (author). 9 refs., 2 tab s

  13. Expression of four phosphate transporter genes from Finger millet (Eleusine coracana L.) in response to mycorrhizal colonization and Pi stress.

    Science.gov (United States)

    Pudake, Ramesh Namdeo; Mehta, Chandra Mohan; Mohanta, Tapan Kumar; Sharma, Suvigya; Varma, Ajit; Sharma, Anil Kumar

    2017-05-01

    Phosphorus (P) is a vital nutrient for plant growth and development, and is absorbed in cells with the help of membrane-spanning inorganic phosphate transporter (Pht) protein. Symbiosis with arbuscular mycorrhiza (AM) also helps in transporting P from the soil to plant and Pht proteins play an important role in it. To understand this phenomenon in Finger Mille plant, we have cloned four Pht genes from Finger millet, which shares the homology with Pht1 protein family of cereals. Expression pattern analysis during the AM infection indicated that EcPT4 gene was AM specific, and its expression was higher in roots where AM colonization percentage was high. The expression level of EcPT1-4 gene under the phosphorous (Pi) stress in seedlings was found to be consistent with its role in acquisition of phosphorus. Homology study of the EcPt proteins with Pht proteins of cereals shows close relationship. The findings of the study indicate that Pht1 family genes from finger millet can serve to be an important resource for the better understanding of phosphorus use efficiency.

  14. Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.

    Science.gov (United States)

    Birchfield, N B; Casida, J E

    1996-01-01

    Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.

  15. The ketamine analogue methoxetamine and 3- and 4-methoxy analogues of phencyclidine are high affinity and selective ligands for the glutamate NMDA receptor.

    Directory of Open Access Journals (Sweden)

    Bryan L Roth

    Full Text Available In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as 'designer drugs' and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS-2-(ethylamino-2-(3-methoxyphenylcyclohexanone and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenylcyclohexanamine and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenylcyclohexyl]piperidine and 1-[1-(4-methoxyphenylcyclohexyl]piperidine, were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.

  16. Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

    Science.gov (United States)

    Lai, H; Carino, M A

    1992-07-01

    Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

  17. Modeling cell membrane transport: interaction of guanidinylated poly(propylene imine) dendrimers with a liposomal membrane consisting of phosphate-based lipids.

    Science.gov (United States)

    Tsogas, Ioannis; Tsiourvas, Dimitris; Nounesis, George; Paleos, Constantinos M

    2006-12-19

    Mixed anionic liposomes consisting of dihexadecyl phosphate, phosphatidylcholine, and cholesterol were employed as model systems for assessing the ability of a series of functionalized dendrimers, bearing a varying number of guanidinium groups at their surface, to translocate across the liposomal bilayers. At low guanidinium/phosphate molar ratios or when weakly guanidinylated dendrimeric derivatives were employed, the dendrimeric derivative acted as a kind of "molecular glue" leading to a simple adhesion of the liposomes. Liposomal fusion occurred to a certain extent at high guanidinium/phosphate molar ratios or when highly guanidinylated dendrimeric derivatives were employed. Furthermore, translocation of these dendrimeric derivatives to the liposomal core was observed for low to medium guanidinylation and at low guanidinium/phosphate molar ratios which was, however, enhanced when the lipid bilayer was in its fluid liquid-crystalline phase. Thus, an optimum balance is required between the binding strength of guanidinium with the phosphate groups and the degree of hydrophilicity of the guanidinylated dendrimers for the transport of the latter to the liposomal core to occur.

  18. Solubility and transport measurements as tools for the speciation of f-elements at tracer-scale amounts; application to Eu and Th in phosphate media

    International Nuclear Information System (INIS)

    Fourest, B.; David, F.; Lagarde, G.; Lindecker, C.; Du, J.F. Le; Tarapcik, P.; Trskova, R.

    1998-01-01

    The speciation of f-elements can be examined by methods which are based either on the distribution of the radionuclide of interest between two phases (solubility measurements) or on its moving in solution (capillary diffusion and migration). Predictive curves giving the variation of the mobility and the concentration of Europium as a function of pH and/or phosphate concentration can be proposed from selected literature data. Capillary electrophoresis experiments show that the mobility decrease due to hydrolysis occurs at a pH value lower than expected. The peak intensity is correspondingly smaller, but this observation cannot be simply related to a change in the charge of the migrating species because of sorption and precipitation phenomena on the capillary walls. Diffusion coefficient measurements by the 'open end capillary' method confirm the formation of larger and/or less charged species starting at a similar pH value. In the presence of phosphate anions, both transport methods should indicate the presence in solution of species having a larger size than expected, which could be polynuclear. Solubility measurements give more information but require the synthesis of a well-defined labelled phosphate compound. The total concentration of f-elements detected in the solutions equilibrated with such compounds allow to deduce, by varying only one parameter in the solution (pH or phosphate concentration), the form and the charge of the different species prevailing in the solution. The solubility method appears particularly interesting in the case of concentrated phosphate media

  19. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    OpenAIRE

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This mo...

  20. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    Science.gov (United States)

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  1. The involvement of mitochondrial phosphate transporter in accelerating bud dormancy release during chilling treatment of tree peony (Paeonia suffruticosa).

    Science.gov (United States)

    Huang, Xin; Zhu, Wei; Dai, Silan; Gai, Shupeng; Zheng, Guosheng; Zheng, Chengchao

    2008-09-01

    A cDNA clone was isolated from tree peony (Paeonia suffruticosa) subtractive cDNA library of burst buds and characterized with regard to its sequence, expression in response to chilling treatment during the release of bud dormancy, and its function in transgenic Arabidopsis thaliana. The clone, designated as PsMPT, contains 1,615 nucleotides with an open reading frame of 1,119 nucleotides, and the deduced amino acid sequence shows high homology with mitochondrial phosphate transporters (MPTs) from various organisms. The mRNA accumulation of PsMPT in tree peony was strongly induced by chilling treatment during the release of bud dormancy. When the treated plants were transferred to normal growth conditions, the level of PsMPT transcripts induced by sufficient chilling could be maintained high, whereas that induced by insufficient chilling decreased sharply. The transgenic Arabidopsis plants that overexpress PsMPT showed rapid growth and earlier flowering than wild-type plants. ATP contents in the transgenic plants were much higher than that in wild-type plants through various developmental stages. Together, these results suggest that the product of PsMPT is a MPT and might play an important role during the release of bud dormancy in tree peony.

  2. The mitochondrial phosphate transporters modulate plant responses to salt stress via affecting ATP and gibberellin metabolism in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Wei Zhu

    Full Text Available The mitochondrial phosphate transporter (MPT plays crucial roles in ATP production in plant cells. Three MPT genes have been identified in Arabidopsis thaliana. Here we report that the mRNA accumulations of AtMPTs were up-regulated by high salinity stress in A. thaliana seedlings. And the transgenic lines overexpressing AtMPTs displayed increased sensitivity to salt stress compared with the wild-type plants during seed germination and seedling establishment stages. ATP content and energy charge was higher in overexpressing plants than those in wild-type A. thaliana under salt stress. Accordingly, the salt-sensitive phenotype of overexpressing plants was recovered after the exogenous application of atractyloside due to the change of ATP content. Interestingly, Genevestigator survey and qRT-PCR analysis indicated a large number of genes, including those related to gibberellin synthesis could be regulated by the energy availability change under stress conditions in A. thaliana. Moreover, the exogenous application of uniconazole to overexpressing lines showed that gibberellin homeostasis was disturbed in the overexpressors. Our studies reveal a possible link between the ATP content mediated by AtMPTs and gibberellin metabolism in responses to high salinity stress in A. thaliana.

  3. Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

    Directory of Open Access Journals (Sweden)

    Weonu Choe

    2016-12-01

    Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

  4. Structural insights into a high affinity nanobody:antigen complex by homology modelling.

    Science.gov (United States)

    Skottrup, Peter Durand

    2017-09-01

    Porphyromonas gingivalis is a major periodontitis-causing pathogens. P. gingivalis secrete a cysteine protease termed RgpB, which is specific for Arg-Xaa bonds in substrates. Recently, a nanobody-based assay was used to demonstrate that RgpB could represent a novel diagnostic target, thereby simplifying. P. gingivalis detection. The nanobody, VHH7, had a high binding affinity and was specific for RgpB, when tested towards the highly identical RgpA. In this study a homology model of VHH7 was build. The complementarity determining regions (CDR) comprising the paratope residues responsible for RgpB binding were identified and used as input to the docking. Furthermore, residues likely involved in the RgpB epitope was identified based upon RgpB:RgpA alignment and analysis of residue surface accessibility. CDR residues and putitative RgpB epitope residues were used as input to an information-driven flexible docking approach using the HADDOCK server. Analysis of the VHH7:RgpB model demonstrated that the epitope was found in the immunoglobulin-like domain and residue pairs located at the molecular paratope:epitope interface important for complex stability was identified. Collectively, the VHH7 homology model and VHH7:RgpB docking supplies knowledge of the residues involved in the high affinity interaction. This information could prove valuable in the design of an antibody-drug conjugate for specific RgpB targeting. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Characterization of a high affinity cocaine binding site in rat brain

    International Nuclear Information System (INIS)

    Calligaro, D.; Eldefrawi, M.

    1986-01-01

    Binding of [ 3 H]cocaine to synaptic membranes from whole rat brain was reversible and saturable. Nonlinear regression analysis of binding isotherms indicated two binding affinities: one with k/sub d/ = 16 nM, B/sub max/ = 0.65 pmoles/mg protein and the other with K/sub d/ = 660 nM, B/sub max/ = 5.1 pmoles/mg protein. The high-affinity binding of [ 3 H]cocaine was sensitive to the actions of trypsin and chymotrypsin but not carboxypeptidase, and was eliminated by exposure of the membranes to 95 0 C for 5 min. Specific binding at 2 nM was higher at pH 8.8 than at pH 7.0. Binding of [ 3 H]cocaine (15 nM) was inhibited by increasing concentrations of Na + ions. Several cocaine analogues, neurotransmitter uptake inhibitors and local anesthetics displaced specific [ 3 H]cocaine binding at 2 nM with various potencies. The cocaine analogue (-)-norcocaine was the most potent (IC 50 = 10 nM), while the local anesthetic tetracaine was the least potent in inhibiting [ 3 H]cocaine binding. Several biogenic amine uptake inhibitors, including tricyclic antidepressants and phencyclidine, had IC 50 values below μM concentrations

  6. Carbonate-sensitive phytotransferrin controls high-affinity iron uptake in diatoms

    Science.gov (United States)

    McQuaid, Jeffrey B.; Kustka, Adam B.; Oborník, Miroslav; Horák, Aleš; McCrow, John P.; Karas, Bogumil J.; Zheng, Hong; Kindeberg, Theodor; Andersson, Andreas J.; Barbeau, Katherine A.; Allen, Andrew E.

    2018-03-01

    In vast areas of the ocean, the scarcity of iron controls the growth and productivity of phytoplankton. Although most dissolved iron in the marine environment is complexed with organic molecules, picomolar amounts of labile inorganic iron species (labile iron) are maintained within the euphotic zone and serve as an important source of iron for eukaryotic phytoplankton and particularly for diatoms. Genome-enabled studies of labile iron utilization by diatoms have previously revealed novel iron-responsive transcripts, including the ferric iron-concentrating protein ISIP2A, but the mechanism behind the acquisition of picomolar labile iron remains unknown. Here we show that ISIP2A is a phytotransferrin that independently and convergently evolved carbonate ion-coordinated ferric iron binding. Deletion of ISIP2A disrupts high-affinity iron uptake in the diatom Phaeodactylum tricornutum, and uptake is restored by complementation with human transferrin. ISIP2A is internalized by endocytosis, and manipulation of the seawater carbonic acid system reveals a second-order dependence on the concentrations of labile iron and carbonate ions. In P. tricornutum, the synergistic interaction of labile iron and carbonate ions occurs at environmentally relevant concentrations, revealing that carbonate availability co-limits iron uptake. Phytotransferrin sequences have a broad taxonomic distribution and are abundant in marine environmental genomic datasets, suggesting that acidification-driven declines in the concentration of seawater carbonate ions will have a negative effect on this globally important eukaryotic iron acquisition mechanism.

  7. High-affinity binding of two molecules of cysteine proteinases to low-molecular-weight kininogen.

    Science.gov (United States)

    Turk, B.; Stoka, V.; Björk, I.; Boudier, C.; Johansson, G.; Dolenc, I.; Colic, A.; Bieth, J. G.; Turk, V.

    1995-01-01

    Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain. PMID:8528085

  8. Identification of a High Affinity Nucleocapsid Protein Binding Element from The Bovine Leukemia Virus Genome

    Science.gov (United States)

    Yildiz, F. Zehra; Babalola, Kathleen; Summers, Michael F.

    2012-01-01

    Retroviral genome recognition is mediated by interactions between the nucleocapsid (NC) domain of the virally encoded Gag polyprotein and cognate RNA packaging elements that, for most retroviruses, appear to reside primarily within the 5′-untranslated region (5′-UTR) of the genome. Recent studies suggest that a major packaging determinant of Bovine Leukemia Virus (BLV), a member of the human T-cell leukemia virus (HTLV)/BLV family and a non-primate animal model for HTLV-induced leukemogenesis, resides within the gag open reading frame. We have prepared and purified the recombinant BLV NC protein and conducted electrophoretic mobility shift and isothermal titration calorimetry studies with RNA fragments corresponding to these proposed packaging elements. The gag-derived RNAs did not exhibit significant affinity for NC, suggesting an alternate role in packaging. However, an 83-nucleotide fragment of the 5′-UTR that resides just upstream of the gag start codon binds NC stoichiometrically and with high affinity (Kd = 136 ± 21 nM). These nucleotides were predicted to form tandem hairpin structures, and studies with smaller fragments indicate that the NC binding site resides exclusively within the distal hairpin (residues G369- U399, Kd = 67 ± 8 nM at physiological ionic strength). Unlike all other structurally characterized retroviral NC binding RNAs, this fragment is not expected to contain exposed guanosines, suggesting that RNA binding may be mediated by a previously uncharacterized mechanism. PMID:22846919

  9. Identification of high-affinity calmodulin-binding proteins in rat liver

    International Nuclear Information System (INIS)

    Hanley, R.M.; Dedman, J.R.; Shenolikar, S.

    1987-01-01

    The Ca 2+ -dependent binding of [ 125 I] calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or acceptor proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised > 80% of the Ca 2+ -dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding subunit of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca 2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver

  10. Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Christopher J Arnusch

    Full Text Available Many cationic antimicrobial peptides (AMPs target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted.

  11. Early signs of pathological cognitive aging in mice lacking high-affinity nicotinic receptors.

    Directory of Open Access Journals (Sweden)

    Eleni eKonsolaki

    2016-04-01

    Full Text Available In order to address pathological cognitive decline effectively, it is critical to adopt early preventive measures in individuals considered at risk. It is therefore essential to develop approaches that identify such individuals before the onset of irreversible dementia. Α deficient cholinergic system has been consistently implicated as one of the main factors associated with a heightened vulnerability to the aging process. In the present study we used mice lacking high affinity nicotinic receptors (β2-/-, which have been proposed as an animal model of accelerated/premature cognitive aging. Our aim was to identify behavioural signs that could serve as indicators or predictors of impending cognitive decline. We used test batteries in order to assess cognitive functions and additional tasks to investigate spontaneous behaviours, such as species-specific activities and exploration/locomotion in a novel environment. Our data confirm and extend the hypothesis that β2-/- animals exhibit age-related cognitive impairments, manifested in both spatial learning and recognition memory tasks. In addition, we reveal deficits in spontaneous behaviour and habituation processes earlier in life. To our knowledge, this is the first study to perform an extensive behavioural examination of an animal model of premature cognitive aging, and our results suggest that β2-nAChR dependent cognitive deterioration progressively evolves from initial subtle behavioural changes to global dementia due to the combined effect of the neuropathology and aging.

  12. High affinity soluble ILT2 receptor: a potent inhibitor of CD8(+) T cell activation.

    Science.gov (United States)

    Moysey, Ruth K; Li, Yi; Paston, Samantha J; Baston, Emma E; Sami, Malkit S; Cameron, Brian J; Gavarret, Jessie; Todorov, Penio; Vuidepot, Annelise; Dunn, Steven M; Pumphrey, Nicholas J; Adams, Katherine J; Yuan, Fang; Dennis, Rebecca E; Sutton, Deborah H; Johnson, Andy D; Brewer, Joanna E; Ashfield, Rebecca; Lissin, Nikolai M; Jakobsen, Bent K

    2010-12-01

    Using directed mutagenesis and phage display on a soluble fragment of the human immunoglobulin super-family receptor ILT2 (synonyms: LIR1, MIR7, CD85j), we have selected a range of mutants with binding affinities enhanced by up to 168,000-fold towards the conserved region of major histocompatibility complex (MHC) class I molecules. Produced in a dimeric form, either by chemical cross-linking with bivalent polyethylene glycol (PEG) derivatives or as a genetic fusion with human IgG Fc-fragment, the mutants exhibited a further increase in ligand-binding strength due to the avidity effect, with resident half-times (t(1/2)) on the surface of MHC I-positive cells of many hours. The novel compounds antagonized the interaction of CD8 co-receptor with MHC I in vitro without affecting the peptide-specific binding of T-cell receptors (TCRs). In both cytokine-release assays and cell-killing experiments the engineered receptors inhibited the activation of CD8(+) cytotoxic T lymphocytes (CTLs) in the presence of their target cells, with subnanomolar potency and in a dose-dependent manner. As a selective inhibitor of CD8(+) CTL responses, the engineered high affinity ILT2 receptor presents a new tool for studying the activation mechanism of different subsets of CTLs and could have potential for the development of novel autoimmunity therapies.

  13. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Directory of Open Access Journals (Sweden)

    Béatrice Marquèze-Pouey

    Full Text Available Signaling mediated by the epidermal growth factor (EGF is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer. In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  14. Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

    Science.gov (United States)

    Marquèze-Pouey, Béatrice; Mailfert, Sébastien; Rouger, Vincent; Goaillard, Jean-Marc; Marguet, Didier

    2014-01-01

    Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

  15. Specific, high affinity receptors for insulin-like growth factor II in the rat kidney glomerulus

    International Nuclear Information System (INIS)

    Haskell, J.F.; Pillion, D.J.; Meezan, E.

    1988-01-01

    Rat renal glomeruli were isolated by a technique involving kidney perfusion with a solution containing magnetic iron oxide particles, followed by homogenization, sieving, and concentration over a strong magnet. Isolated glomeruli were treated with 1% Triton X-100 to solubilize plasma membrane components, while insoluble basement membrane components were removed by centrifugation. [ 125 I]Insulin-like growth factor II (IGF-II) binding to this preparation was competitively inhibited by increasing amounts of unlabeled IGF-II, with 50% inhibition at an IGF-II concentration of 1 ng/ml. [ 125 I]IGF-II was covalently cross-linked with disuccinimidyl suberate to its receptor in rat renal glomeruli and a specific high mol wt (255,000) band could be identified on autoradiograms of dodecyl sulfate-polyacrylamide gels. [ 125 I]IGF-II binding and cross-linking to this band was inhibited by a polyclonal antibody against the type II IGF receptor. These results demonstrate for the first time that the isolated rat renal glomerulus contains a high affinity receptor for IGF-II

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

    Science.gov (United States)

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

    2016-08-26

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

  17. High-affinity cannabinoid binding site in brain: A possible marijuana receptor

    International Nuclear Information System (INIS)

    Nye, J.S.

    1988-01-01

    The mechanism by which delta 9 tetrahydrocannabinol (delta 9 THC), the major psychoactive component of marijuana or hashish, produces its potent psychological and physiological effects is unknown. To find receptor binding sites for THC, we designed a water-soluble analog for use as a radioligand. 5'-Trimethylammonium-delta 8 THC (TMA) is a positively charged analog of delta- 8 THC modified on the 5' carbon, a portion of the molecule not important for its psychoactivity. We have studied the binding of [ 3 H]-5'-trimethylammonium-delta- 8 THC ([ 3 H]TMA) to rat neuronal membranes. [ 3 H]TMA binds saturably and reversibly to brain membranes with high affinity to apparently one class of sites. Highest binding site density occurs in brain, but several peripheral organs also display specific binding. Detergent solubilizes the sites without affecting their pharmacologial properties. Molecular sieve chromatography reveals a bimodal peak of [ 3 H]TMA binding activity of approximately 60,000 daltons apparent molecular weight

  18. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    International Nuclear Information System (INIS)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A.

    1990-01-01

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite

  19. A high affinity Ca2(+)-ATPase on the surface membrane of Leishmania donovani promastigote

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, J.; Ray, M.; Sarkar, S.; Bhaduri, A. (Indian Institute of Chemical Biology, Calcutta (India))

    1990-07-05

    A Ca2(+)-dependent ATP-hydrolytic activity was detected in the crude membrane ghost of the promastigote or vector form of the protozoal parasite Leishmania donovani, the pathogen responsible for kala azar. The Ca2(+)-ATPase was purified to apparent homogeneity after solubilization with deoxycholate. The enzyme consists of two subunits of Mr = 51,000 and 57,000 and has an apparent molecular weight of 215,000 +/- 12,000. The enzyme activity is exclusively dependent on Ca2+, and the pure enzyme can hydrolyze 1.6 mumol of ATP/min/mg of protein. The apparent Km for Ca2+ is 35 nM, which is further reduced to 12 nM in the presence of heterologous calmodulin. The enzyme is sensitive to vanadate, but is insensitive to oligomycin and ouabain. The enzyme is strongly associated with the plasma membrane and has its catalytic site oriented toward the cytoplasmic face. The enzyme spans across the plasma membrane as surface labeling with radioiodine shows considerable radioactivity in the completely purified enzyme. The localization and orientation of this high affinity, calmodulin-sensitive Ca2(+)-ATPase suggest some role of this enzyme in Ca2+ movement in the life cycle of this protozoal parasite.

  20. Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

    International Nuclear Information System (INIS)

    Asermely, K.E.; O'Neill, J.J.

    1986-01-01

    The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37 0 C in buffers containing tritium choline (5 μM 0.124 μC/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10 -5 M compared with 50% inhibition on brain synaptosomes. At 5 x 10 -4 M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K + -ion (25 mM) were unsuccessful; tissue 3 H-choline uptake appeared to oscillate over a 30 min period

  1. Inhibition of high affinity choline uptake by N-allyl-3-quinuclidinol

    Energy Technology Data Exchange (ETDEWEB)

    Asermely, K.E.; O' Neill, J.J.

    1986-03-01

    The peripheral actions of N-allyl-3-quinuclidinol (N-Al-3-OHQ) on high affinity choline uptake (HAChU) on rat phrenic nerve diaphragm are described. Endplate regions (EPA) identified by the Koelle histochemical techniques for acetylcholinesterase, were dissected from adult rat hemidiaphragms and placed in cold Krebs solution (pH-7.35). All measurements of HAChU were at 37/sup 0/C in buffers containing tritium choline (5 ..mu..M 0.124 ..mu..C/mmole) at intervals of 1, 2, 4, 8, 15 and 30 min. Tissues were washed 3x, digested in 1N NaOH and counted for tritium in Chaikoff's solution. All data are expressed as pmole Ch/g wet weight. Comparison between EPA and non-EPA tissue demonstrate HAChU and slow choline diffusion, respectively. Steady state is observed in 15 min. N-Al-3-OHQ produces 15% inhibition at 5 x 10/sup -5/ M compared with 50% inhibition on brain synaptosomes. At 5 x 10/sup -4/ M N-Al-3-OHQ, 30% inhibition is observed. Attempts to deplete ACh by pre-stimulation with high K/sup +/-ion (25 mM) were unsuccessful; tissue /sup 3/H-choline uptake appeared to oscillate over a 30 min period.

  2. Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation.

    Science.gov (United States)

    Mercader, Josep V; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

    2011-03-07

    The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate.

  3. Effect of sulfur addition on the transport properties of semiconducting iron phosphate glasses

    Energy Technology Data Exchange (ETDEWEB)

    El-Desoky, M.M. [Physics Department, Faculty of Science, Suez Canal University, Suez (Egypt); Ibrahim, F.A. [Department of Physics, Faculty of Education, Suez Canal University, Al-Arish (Egypt); Hassaan, M.Y. [Department of Physics, Faculty of Science, Al-Azhar University, Nasr City, 11884 Cairo (Egypt)

    2011-08-15

    The present paper focuses on a quantitative analysis of the metallic and semiconducting behavior of electrical resistivity in La{sub 0.91}Rb{sub 0.06}Mn{sub 0.94}O{sub 3} manganites. The contribution of inherent low-frequency acoustic phonons as well as high-frequency optical phonons, to the electron-phonon resistivity is estimated following Bloch-Gruneisen model. The computed phonon resistivity is compared with that of reported metallic resistivity, accordingly {rho}diff. [{rho}exp. - {l_brace}{rho}0 + {rho}e-ph (={rho}ac + {rho}op){r_brace}] have been analysed through electron-electron scattering. Also, the difference can be varies linearly with T{sup 4.5} in accordance with the electron-magnon scattering in the double exchange process. The results reveal important aspects transport mechanism as well as point out that it is not only dominated by electron-phonon scattering, but also by electron-electron and electron-magnon scattering process. Alternatively, in high temperature regime (T {>=} T{sub P}) the semiconducting nature is discussed with Mott's variable range hopping (VRH) and small polaron conduction (SPC) model. (authors)

  4. Effect of sulfur addition on the transport properties of semiconducting iron phosphate glasses

    International Nuclear Information System (INIS)

    El-Desoky, M.M.; Ibrahim, F.A.; Hassaan, M.Y.

    2011-01-01

    The present paper focuses on a quantitative analysis of the metallic and semiconducting behavior of electrical resistivity in La 0.91 Rb 0.06 Mn 0.94 O 3 manganites. The contribution of inherent low-frequency acoustic phonons as well as high-frequency optical phonons, to the electron-phonon resistivity is estimated following Bloch-Gruneisen model. The computed phonon resistivity is compared with that of reported metallic resistivity, accordingly ρdiff. [ρexp. - {ρ0 + ρe-ph (=ρac + ρop)}] have been analysed through electron-electron scattering. Also, the difference can be varies linearly with T 4.5 in accordance with the electron-magnon scattering in the double exchange process. The results reveal important aspects transport mechanism as well as point out that it is not only dominated by electron-phonon scattering, but also by electron-electron and electron-magnon scattering process. Alternatively, in high temperature regime (T ≥ T P ) the semiconducting nature is discussed with Mott's variable range hopping (VRH) and small polaron conduction (SPC) model. (authors)

  5. Novel high-affinity and selective biaromatic 4-substituted ¿-hydroxybutyric acid (GHB) analogues as GHB ligands

    DEFF Research Database (Denmark)

    Høg, Signe; Wellendorph, Petrine; Nielsen, Birgitte

    2008-01-01

    Gamma-hydroxybutyrate (GHB) is a metabolite of gamma-aminobutyric acid (GABA) and has been proposed to function as a neurotransmitter or neuromodulator. GHB is used in the treatment of narcolepsy and is a drug of abuse. GHB binds to both GABA(B) receptors and specific high-affinity GHB sites...

  6. Are basophil histamine release and high affinity IgE receptor expression involved in asymptomatic skin sensitization?

    DEFF Research Database (Denmark)

    Jensen, Bettina Margrethe; Assing, K; Jensen, Lone Hummelshøj

    2006-01-01

    Immunoglobulin (Ig)E-sensitized persons with positive skin prick test, but no allergy symptoms, are classified as being asymptomatic skin sensitized (AS). The allergic type 1 disease is dependant on IgE binding to the high affinity IgE-receptor (FcepsilonRI) expressed on basophils and mast cells....

  7. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-Affinity γ-Hydroxybutyrate (GHB) Binding Sites

    DEFF Research Database (Denmark)

    Vogensen, Stine B.; Marek, Ales; Bay, Tina

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screen...

  8. The PhoBR two-component system regulates antibiotic biosynthesis in Serratia in response to phosphate

    Science.gov (United States)

    2009-01-01

    Background Secondary metabolism in Serratia sp. ATCC 39006 (Serratia 39006) is controlled via a complex network of regulators, including a LuxIR-type (SmaIR) quorum sensing (QS) system. Here we investigate the molecular mechanism by which phosphate limitation controls biosynthesis of two antibiotic secondary metabolites, prodigiosin and carbapenem, in Serratia 39006. Results We demonstrate that a mutation in the high affinity phosphate transporter pstSCAB-phoU, believed to mimic low phosphate conditions, causes upregulation of secondary metabolism and QS in Serratia 39006, via the PhoBR two-component system. Phosphate limitation also activated secondary metabolism and QS in Serratia 39006. In addition, a pstS mutation resulted in upregulation of rap. Rap, a putative SlyA/MarR-family transcriptional regulator, shares similarity with the global regulator RovA (regulator of virulence) from Yersina spp. and is an activator of secondary metabolism in Serratia 39006. We demonstrate that expression of rap, pigA-O (encoding the prodigiosin biosynthetic operon) and smaI are controlled via PhoBR in Serratia 39006. Conclusion Phosphate limitation regulates secondary metabolism in Serratia 39006 via multiple inter-linked pathways, incorporating transcriptional control mediated by three important global regulators, PhoB, SmaR and Rap. PMID:19476633

  9. The PhoBR two-component system regulates antibiotic biosynthesis in Serratia in response to phosphate

    Directory of Open Access Journals (Sweden)

    Everson Lee

    2009-05-01

    Full Text Available Abstract Background Secondary metabolism in Serratia sp. ATCC 39006 (Serratia 39006 is controlled via a complex network of regulators, including a LuxIR-type (SmaIR quorum sensing (QS system. Here we investigate the molecular mechanism by which phosphate limitation controls biosynthesis of two antibiotic secondary metabolites, prodigiosin and carbapenem, in Serratia 39006. Results We demonstrate that a mutation in the high affinity phosphate transporter pstSCAB-phoU, believed to mimic low phosphate conditions, causes upregulation of secondary metabolism and QS in Serratia 39006, via the PhoBR two-component system. Phosphate limitation also activated secondary metabolism and QS in Serratia 39006. In addition, a pstS mutation resulted in upregulation of rap. Rap, a putative SlyA/MarR-family transcriptional regulator, shares similarity with the global regulator RovA (regulator of virulence from Yersina spp. and is an activator of secondary metabolism in Serratia 39006. We demonstrate that expression of rap, pigA-O (encoding the prodigiosin biosynthetic operon and smaI are controlled via PhoBR in Serratia 39006. Conclusion Phosphate limitation regulates secondary metabolism in Serratia 39006 via multiple inter-linked pathways, incorporating transcriptional control mediated by three important global regulators, PhoB, SmaR and Rap.

  10. Endothelial targeting of high-affinity multivalent polymer nanocarriers directed to intercellular adhesion molecule 1.

    Science.gov (United States)

    Muro, Silvia; Dziubla, Thomas; Qiu, Weining; Leferovich, John; Cui, Xiumin; Berk, Erik; Muzykantov, Vladimir R

    2006-06-01

    Targeting of diagnostic and therapeutic agents to endothelial cells (ECs) provides an avenue to improve treatment of many maladies. For example, intercellular adhesion molecule 1 (ICAM-1), a constitutive endothelial cell adhesion molecule up-regulated in many diseases, is a good determinant for endothelial targeting of therapeutic enzymes and polymer nanocarriers (PNCs) conjugated with anti-ICAM (anti-ICAM/PNCs). However, intrinsic and extrinsic factors that control targeting of anti-ICAM/PNCs to ECs (e.g., anti-ICAM affinity and PNC valency and flow) have not been defined. In this study we tested in vitro and in vivo parameters of targeting to ECs of anti-ICAM/PNCs consisting of either prototype polystyrene or biodegradable poly(lactic-coglycolic) acid polymers (approximately 200 nm diameter spheres carrying approximately 200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 approximately 5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax approximately 350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd approximately 80 pM versus approximately 8 nM) in cell culture and, probably because of this factor, higher value (185.3 +/- 24.2 versus 50.5 +/- 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 +/- 10.9 versus 2.1 +/- 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.

  11. 99mTc(CO)3-DTMA bombesin conjugates having high affinity for the GRP receptor

    International Nuclear Information System (INIS)

    Lane, Stephanie R.; Veerendra, Bhadrasetty; Rold, Tammy L.; Sieckman, Gary L.; Hoffman, Timothy J.; Jurisson, Silvia S.; Smith, Charles J.

    2008-01-01

    Introduction: Targeted diagnosis of specific human cancer types continues to be of significant interest in nuclear medicine. 99m Tc is ideally suited as a diagnostic radiometal for in vivo tumor targeting due to its ideal physical characteristics and diverse labeling chemistries in numerous oxidation states. Methods: In this study, we report a synthetic approach toward design of a new tridentate amine ligand for the organometallic aqua-ion [ 99m Tc(H 2 O) 3 (CO) 3 ] + . The new chelating ligand framework, 2-(N,N'-Bis(tert-butoxycarbonyl)diethylenetriamine) acetic acid (DTMA), was synthesized from a diethylenetriamine precursor and fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy ( 1 H and 13 C). DTMA was conjugated to H 2 N-(X)-BBN(7-14)NH 2 , where X=an amino acid or aliphatic pharmacokinetic modifier and BBN=bombesin peptide, by means of solid phase peptide synthesis. DTMA-(X)-BBN(7-14)NH 2 conjugates were purified by reversed-phase high-performance chromatography and characterized by electrospray-ionization mass spectrometry. Results: The new conjugates were radiolabeled with [ 99m Tc(H 2 O) 3 (CO) 3 ] + produced via Isolink radiolabeling kits to produce [ 99m Tc(CO) 3 -DTMA-(X)-BBN(7-14)NH 2 ]. Radiolabeled conjugates were purified by reversed-phase high-performance chromatography. Effective receptor binding behavior was evaluated in vitro and in vivo. Conclusions: [ 99m Tc(CO) 3 -DTMA-(X)-BBN(7-14)NH 2 ] conjugates displayed very high affinity for the gastrin releasing peptide receptor in vitro and in vivo. Therefore, these conjugates hold some propensity to be investigated as molecular imaging agents that specifically target human cancers uniquely expressing the gastrin releasing peptide receptor subtypes

  12. Characterization of high affinity [3H]triazolam binding in rat brain

    International Nuclear Information System (INIS)

    Earle, M.; Concas, A.; Yamamura, H.I.

    1986-01-01

    The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on [ 3 H]TZ binding. Saturation studies showed a shift to lower affinity at 37 0 C (K/sub d/ = 0.25 +/- 0.01 nM at O 0 C; K/sub d/ = 1.46 +/- 0.03 nM at 37 0 C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. at 0 0 C and 1001 +/- 43 fmoles/mg prot. at 37 0 C). Inhibition studies showed that [ 3 H]TZ binding displayed no GABA shift at 0 0 C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37 0 C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37 0 C. In Ro 15-1788/[ 3 H]TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on [ 3 H]TZ binding at both temperatures. In conclusion [ 3 H]TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists

  13. Determination of High-affinity Antibody-antigen Binding Kinetics Using Four Biosensor Platforms.

    Science.gov (United States)

    Yang, Danlin; Singh, Ajit; Wu, Helen; Kroe-Barrett, Rachel

    2017-04-17

    Label-free optical biosensors are powerful tools in drug discovery for the characterization of biomolecular interactions. In this study, we describe the use of four routinely used biosensor platforms in our laboratory to evaluate the binding affinity and kinetics of ten high-affinity monoclonal antibodies (mAbs) against human proprotein convertase subtilisin kexin type 9 (PCSK9). While both Biacore T100 and ProteOn XPR36 are derived from the well-established Surface Plasmon Resonance (SPR) technology, the former has four flow cells connected by serial flow configuration, whereas the latter presents 36 reaction spots in parallel through an improvised 6 x 6 crisscross microfluidic channel configuration. The IBIS MX96 also operates based on the SPR sensor technology, with an additional imaging feature that provides detection in spatial orientation. This detection technique coupled with the Continuous Flow Microspotter (CFM) expands the throughput significantly by enabling multiplex array printing and detection of 96 reaction sports simultaneously. In contrast, the Octet RED384 is based on the BioLayer Interferometry (BLI) optical principle, with fiber-optic probes acting as the biosensor to detect interference pattern changes upon binding interactions at the tip surface. Unlike the SPR-based platforms, the BLI system does not rely on continuous flow fluidics; instead, the sensor tips collect readings while they are immersed in analyte solutions of a 384-well microplate during orbital agitation. Each of these biosensor platforms has its own advantages and disadvantages. To provide a direct comparison of these instruments' ability to provide quality kinetic data, the described protocols illustrate experiments that use the same assay format and the same high-quality reagents to characterize antibody-antigen kinetics that fit the simple 1:1 molecular interaction model.

  14. Cartilage Acidic Protein 2 a hyperthermostable, high affinity calcium-binding protein.

    Science.gov (United States)

    Anjos, Liliana; Gomes, Ana S; Melo, Eduardo P; Canário, Adelino V; Power, Deborah M

    2013-03-01

    Cartilage Acidic Protein 2 (CRTAC2) is a novel protein present from prokaryotes to vertebrates with abundant expression in the teleost fish pituitary gland and an isoform of CRTAC1, a chondrocyte marker in humans. The two proteins are non-integrins containing N-terminal integrin-like Ca(2+)-binding motifs and their structure and function remain to be assigned. Structural studies of recombinant sea bream (sb)CRTAC2 revealed it is composed of 8.8% α-helix, 33.4% β-sheet and 57.8% unordered protein. sbCRTAC2 bound Ca(2+) with high affinity (K(d)=1.46nM) and favourable Gibbs free energy (∆G=-12.4kcal/mol). The stoichiometry for Ca(2+) bound to sbCRTAC2 at saturation indicated six Ca(2+) ligand-binding sites exist per protein molecule. No conformational change in sbCRTAC2 occurred in the presence of Ca(2+). Fluorescence emission revealed that the tertiary structure of the protein is hyperthermostable between 25°C and 95°C and the fully unfolded state is only induced by chemical denaturing (4M GndCl). sbCRTAC has a widespread tissue distribution and is present as high molecular weight aggregates, although strong reducing conditions promote formation of the monomer. sbCRTAC2 promotes epithelial cell outgrowth in vitro suggesting it may share functional homology with mammalian CRTAC1, recently implicated in cell-cell and cell-matrix interactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

    Directory of Open Access Journals (Sweden)

    Johan Nilvebrant

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

  16. Aggregation and transport of rutile titanium dioxide nanoparticles with montmorillonite and diatomite in the presence of phosphate in porous sand.

    Science.gov (United States)

    Guo, Peng; Xu, Nan; Li, Duo; Huangfu, Xinxing; Li, Zuling

    2018-08-01

    Crop soil is inevitably contaminated by the excess of phosphate (P) fertilizers. A large amount of nanoparticle titanium dioxide (nTiO 2 ) entered soils as well due to the wide use of engineered nanomaterials. It is of great urgency and a high priority to investigate the mechanisms of nTiO 2 deposition with the presence of P in crop soils. This study investigated the transport behavior of (1.0 g L -1 ) rutile nTiO 2 with two representative clay particles (montmorillonite or diatomite) in the presence of P through the saturated quartz sand. In 10 mM NaCl electrolyte solution at pH 6.0, the recovery percentage of nTiO 2 was 36.3% from sand column. Nevertheless, it was reduced to 18.6% and 11.1% while montmorillonite and diatomite present in suspensions, respectively. Obviously, the improvement of nTiO 2 retention in sand was more pronounced by diatomite than montmorillonite. The likely mechanism for this result was that large aggregates were formed due to the attachment of nTiO 2 to montmorillonite and diatomite. Moreover, the surface of diatomite with the larger hydrodynamic radius was less negatively charged by comparison with montmorillonite. However, this phenomenon disappeared with the addition of P. P adsorption increases the repulsive force between particles and sand and the fast release of attached nTiO 2 -montmorillonite and diatomite from sand. The two-site kinetic retention model and the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory suggested that the combination of k 1/ k 1d , k 2 and secondary minimum energy can be used to accurately describe the attachment of nTiO 2 -montmorillonite and diatomite to sand in the presence of P. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Twins in spirit part II: DOTATATE and high-affinity DOTATATE - the clinical experience

    Energy Technology Data Exchange (ETDEWEB)

    Brogsitter, Claudia; Zoephel, Klaus; Hartmann, Holger; Kotzerke, Joerg [Technische Universitaet Dresden, Department of Nuclear Medicine, Dresden (Germany); Schottelius, Margret; Wester, Hans-Juergen [Technische Universitaet Muenchen, Pharmaceutical Radiochemistry and Department of Nuclear Medicine, Muenchen (Germany)

    2014-06-15

    Over recent decades interest in diagnosis and treatment of neuroendocrine tumours (NET) has steadily grown. The basis for diagnosis and therapy of NET with radiolabelled somatostatin (hsst) analogues is the variable overexpression of hsst receptors (hsst1-5 receptors). We hypothesized that radiometal derivatives of DOTA-iodo-Tyr{sup 3}-octreotide analogues might be excellent candidates for somatostatin receptor imaging. We therefore explored the diagnostic potential of {sup 68}Ga-DOTA-iodo-Tyr{sup 3}-octreotate [{sup 68}Ga-DOTA,3-iodo-Tyr{sup 3},Thr{sup 8}]octreotide ({sup 68}Ga-HA-DOTATATE; HA, high-affinity) compared to the established {sup 68}Ga-DOTA-Tyr{sup 3}-octreotate ({sup 68}Ga-DOTATATE) in vivo. The study included 23 patients with known somatostatin receptor-positive metastases from NETs, thyroid cancer or glomus tumours who were investigated with both {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE. A patient-based and a lesion-based comparative analysis was carried out of normal tissue distribution and lesion detectability in a qualitative and a semiquantitative manner. {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE showed comparable uptake in the liver (SUV{sub mean} 8.9 ± 2.2 vs. 9.3 ± 2.5, n.s.), renal cortex (SUV{sub mean} 13.3 ± 3.9 vs. 14.5 ± 3.7, n.s.) and spleen (SUV{sub mean} 24.0 ± 6.7 vs. 22.9 ± 7.3, n.s.). A somewhat higher pituitary uptake was found with {sup 68}Ga-HA-DOTATATE (SUV{sub mean} 6.3 ± 1.8 vs. 5.4 ± 2.1, p < 0.05). On a lesion-by-lesion basis a total of 344 lesions were detected. {sup 68}Ga-HA-DOTATATE demonstrated 328 lesions (95.3 % of total lesions seen), and {sup 68}Ga-DOTATATE demonstrated 332 lesions (96.4 %). The mean SUV{sub max} of all lesions was not significantly different between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE (17.8 ± 11.4 vs. 16.7 ± 10.7, n.s.). Our analysis demonstrated very good concordance between {sup 68}Ga-HA-DOTATATE and {sup 68}Ga-DOTATATE PET data. As the availability and use of {sup

  18. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

    International Nuclear Information System (INIS)

    Borgundvaag, B.; George, S.R.

    1985-01-01

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [ 3 H]-ATP to [ 3 H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC 50 values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC 50 values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D 2 dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table

  19. Characterization of high-affinity (/sup 3/H)ouabain binding in the rat central nervous system

    Energy Technology Data Exchange (ETDEWEB)

    Hauger, R.; Luu, H.M.; Meyer, D.K.; Goodwin, F.K.; Paul, S.M.

    1985-06-01

    The characteristics of (/sup 3/H)ouabain binding were examined in various areas of rat brain. In the striatum, Scatchard analysis revealed a single class of high-affinity binding sites with an apparent binding affinity (KD) of 10.4 +/- 0.9 nM and an estimated binding capacity (Bmax) of 7.6 +/- 1.9 pmol/mg protein. Similar monophasic Scatchard plots were found in the brainstem, cerebellum, hypothalamus, and frontal cerebral cortex. (/sup 3/H)Ouabain binding to rat brain was sodium- and ATP-dependent and strongly inhibited by potassium. Proscillariden A was the most potent cardiac glycoside tested in inhibiting specific (/sup 3/H)ouabain binding to brain membranes, and the rank order of inhibitory potencies for a series of cardiac glycosides was similar to that previously reported for inhibition of heart Na,K-ATPase. To assess whether the high-affinity binding sites for (/sup 3/H)ouabain were localized to neuronal or nonneuronal membranes, the effect of discrete kainic acid lesions on striatal (/sup 3/H)ouabain binding was examined. Kainic acid lesions of the striatum reduced (/sup 3/H)ouabain binding to striatal homogenates by 79.6 +/- 1.6%. This suggests that the high-affinity (/sup 3/H)ouabain binding sites measured in our experiments are localized to neuronal elements. Thus, the high-affinity binding of (/sup 3/H)ouabain to brain membranes may selectively label a neuronal form or conformation of Na,K-ATPase.

  20. Functional characterization of the high affinity IgG Receptor : making heads and tails of FcγRI

    NARCIS (Netherlands)

    van der Poel, C.E.

    2011-01-01

    This thesis focuses on human FcγRI, a high affinity receptor for antibodies of the IgG isotype. IgG is the most abundant antibody type in blood and all currently FDA approved therapeutic antibodies are of the IgG isotype. FcγRI, a member of the activating Fcγ receptors, exists as a complex of a

  1. Benzodiazepines have high-affinity binding sites and induce melanogenesis in B16/C3 melanoma cells.

    OpenAIRE

    Matthew, E; Laskin, J D; Zimmerman, E A; Weinstein, I B; Hsu, K C; Engelhardt, D L

    1981-01-01

    We found that two markers of differentiation, tyrosinase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) activity and melanin synthesis, are induced by diazepam in B16/C3 mouse melanoma cells. We also demonstrated high-affinity binding sites for [3H]diazepam in these cells by radioreceptor assay, and we visualized binding to the cell surface by fluorescence microscopy with a benzodiazepine analog conjugated to a fluorescein-labeled protein. Our studies also showed tha...

  2. Photoaffinity labeling of mammalian α1-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe

    International Nuclear Information System (INIS)

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.

    1984-01-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated α 1 -adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-[4-[5-(4-azido-3-[ 125 I]iodophenyl)pentanoyl]-1-piperazinyl] quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an α 1 -adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical α 1 -adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the α 1 -adrenergic receptor

  3. High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

    International Nuclear Information System (INIS)

    Orito, N; Umekage, S; Sakai, E; Tanaka, T; Kikuchi, Y; Sato, K; Kawauchi, S; Tanaka, H

    2012-01-01

    We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be K D = 2.25x10 -9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

  4. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    International Nuclear Information System (INIS)

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-01-01

    Forskolin labelled with [ 3 H] bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg 2+ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins

  5. Energy-dependent dissociation of ATP from high affinity catalytic sites of beef heart mitochondrial adenosine triphosphatase

    International Nuclear Information System (INIS)

    Penefsky, H.S.

    1985-01-01

    Incubation of [gamma- 32 P]ATP with a molar excess of the membrane-bound form of mitochondrial ATPase (F1) results in binding of the bulk of the radioactive nucleotide in high affinity catalytic sites (Ka = 10(12) M-1). Subsequent initiation of respiration by addition of succinate or NADH is accompanied by a profound decrease in the affinity for ATP. About one-third of the bound radioactive ATP appears to dissociate, that is, the [gamma- 32 P]ATP becomes accessible to hexokinase. The NADH-stimulated dissociation of [gamma- 32 P]ATP is energy-dependent since the stimulation is inhibited by uncouplers of oxidative phosphorylation and is prevented by respiratory chain inhibitors. The rate of the energy-dependent dissociation of ATP that occurs in the presence of NADH, ADP, and Pi is commensurate with the measured initial rate of ATP synthesis in NADH-supported oxidative phosphorylation catalyzed by the same submitochondrial particles. Thus, the rate of dissociation of ATP from the high affinity catalytic site of submitochondrial particles meets the criterion of kinetic competency under the conditions of oxidative phosphorylation. These experiments provide evidence in support of the argument that energy conserved during the oxidation of substrates by the respiratory chain can be utilized to reduce the very tight binding of product ATP in high affinity catalytic sites and to promote dissociation of the nucleotide

  6. Copper tolerance mediated by polyphosphate degradation and low-affinity inorganic phosphate transport system in Escherichia coli

    OpenAIRE

    Grillo-Puertas, Mariana; Schurig-Briccio, Lici Ariane; Rodríguez-Montelongo, Luisa; Rintoul, María Regina; Rapisarda, Viviana Andrea

    2014-01-01

    Background Metal tolerance in bacteria has been related to polyP in a model in which heavy metals stimulate the polymer hydrolysis, forming metal-phosphate complexes that are exported. As previously described in our laboratory, Escherichia coli cells grown in media containing a phosphate concentration >37 mM maintained an unusually high polyphosphate (polyP) level in stationary phase. The aim of the present work was to evaluate the influence of polyP levels as the involvement of low-affinity ...

  7. High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

    OpenAIRE

    Kongsfelt, Iben Boutrup; Byskov, Kristina; Pedersen, Lasse Ebdrup; Pedersen, Lene

    2014-01-01

    The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expre...

  8. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Andersen, Jacob; Hall, Lena Sørensen

    2016-01-01

    with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within...

  9. Species-scanning mutagenesis of the serotonin transporter reveals residues essential in selective, high-affinity recognition of antidepressants

    DEFF Research Database (Denmark)

    Mortensen, O.V.; Wiborg, O.; Kristensen, A.S.

    2001-01-01

    )tropane, or for 3,4-methylenedioxymethamphetamine (MDMA). Analysis of six hSERT/bSERT chimeras and subsequent species-scanning mutagenesis of each isoform revealed methionine-180, tyrosine-495, and phenylalanine-513 to be responsible for the increase in citalopram and paroxetine potencies at hSERT and methionine...

  10. Preliminary observations on high energy phosphates and metabolic pathway and transporter potentials in extensor carpi radialis brevis and trapezius muscles of women with work-related myalgia.

    Science.gov (United States)

    Green, Howard J; Ranney, Don; Burnett, Margaret; Galvin, Patti; Kyle, Natasha; Lounsbury, David; Ouyang, Jing; Smith, Ian C; Stewart, Riley; Tick, Heather; Tupling, A Russell

    2014-11-01

    This study compared both the extensor carpi radialis brevis (ECRB) and the trapezius (TRAP) muscles of women with work-related myalgia (WRM) with healthy controls (CON) to determine whether abnormalities existed in cellular energy status and the potentials of the various metabolic pathways and segments involved in energy production and substrate transport. For both the ECRB (CON, n = 6-9; WRM, n = 13) and the TRAP (CON, n = 6-7; WRM, n = 10), no differences (P > 0.05) were found for the concentrations (in millimoles per kilogram of dry mass) of ATP, PCr, lactate, and glycogen. Similarly, with one exception, the maximal activities (in moles per milligram of protein per hour) of mitochondrial enzymes representative of the citric acid cycle (CAC), the electron transport chain (ETC), and β-oxidation, as well as the cytosolic enzymes involved in high energy phosphate transfer, glycogenolysis, glycolysis, lactate oxidation, and glucose phosphorylation were not different (P > 0.05). The glucose transporters GLUT1 and GLUT4, and the monocarboxylate transporters MCT1 and MCT4, were also normal in WRM. It is concluded that, in general, abnormalities in the resting energy and substrate state, the potential of the different metabolic pathways and segments, as well as the glucose and monocarboxylate transporters do not appear to be involved in the cellular pathophysiology of WRM.

  11. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay.

    Science.gov (United States)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen

    2017-01-01

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  12. Topography of the high-affinity lysine binding site of plasminogen as defined with a specific antibody probe

    International Nuclear Information System (INIS)

    Miles, L.A.; Plow, E.F.

    1986-01-01

    An antibody population that reacted with the high-affinity lysine binding site of human plasminogen was elicited by immunizing rabbits with an elastase degradation product containing kringles 1-3 (EDP I). This antibody was immunopurified by affinity chromatography on plasminogen-Sepharose and elution with 0.2 M 6-aminohexanoic acid. The eluted antibodies bound [ 125 I]EDP I, [ 125 I]Glu-plasminogen, and [ 125 I]Lys-plasminogen in radioimmunoassays, and binding of each ligand was at least 99% inhibited by 0.2 M 6-aminohexanoic acid. The concentrations for 50% inhibition of [ 125 I]EDP I binding by tranexamic acid, 6-aminohexanoic acid, and lysine were 2.6, 46, and l730 μM, respectively. Similar values were obtained with plasminogen and suggested that an unoccupied high-affinity lysine binding site was required for antibody recognition. The antiserum reacted exclusively with plasminogen derivatives containing the EDP I region and did not react with those lacking an EDP I region, or with tissue plasminogen activator or prothrombin, which also contains kringles. By immunoblotting analyses, a chymotryptic degradation product of M/sub r/ 20,000 was derived from EDP I that retained reactivity with the antibody. α 2 -Antiplasmin inhibited the binding of radiolabeled EDP I, Glu-plasminogen, or Lys-plasminogen by the antiserum, suggesting that the recognized site is involved in the noncovalent interaction of the inhibitor with plasminogen. The binding of [ 125 I]EDP I to fibrin was also inhibited by the antiserum. The observations provide independent evidence for the role of the high-affinity lysine binding site in the functional interactions of plasminogen with its primary substrate and inhibitor

  13. Structural implications of hERG K+ channel block by a high-affinity minimally structured blocker

    Science.gov (United States)

    Helliwell, Matthew V.; Zhang, Yihong; El Harchi, Aziza; Du, Chunyun; Hancox, Jules C.; Dempsey, Christopher E.

    2018-01-01

    Cardiac potassium channels encoded by human ether-à-go-go–related gene (hERG) are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK293 cells was inhibited with an IC50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold, respectively). The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block. PMID:29545312

  14. Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Justin; Brault, Amy; Vincent, Fabien; Weng, Shawn; Wang, Hong; Dumlao, Darren; Aulabaugh, Ann; Aivazian, Dikran; Castro, Dana; Chen, Ming; Culp, Jeffrey; Dower, Ken; Gardner, Joseph; Hawrylik, Steven; Golenbock, Douglas; Hepworth, David; Horn, Mark; Jones, Lyn; Jones, Peter; Latz, Eicke; Li, Jing; Lin, Lih-Ling; Lin, Wen; Lin, David; Lovering, Frank; Niljanskul, Nootaree; Nistler, Ryan; Pierce, Betsy; Plotnikova, Olga; Schmitt, Daniel; Shanker, Suman; Smith, James; Snyder, William; Subashi, Timothy; Trujillo, John; Tyminski, Edyta; Wang, Guoxing; Wong, Jimson; Lefker, Bruce; Dakin, Leslie; Leach, Karen (UMASS, MED); (Pfizer)

    2017-09-21

    Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

  15. Autoradiographic imaging and quantification of the high-affinity GHB binding sites in rodent brain using (3)H-HOCPCA

    DEFF Research Database (Denmark)

    Klein, A B; Bay, T; Villumsen, I S

    2016-01-01

    analogue, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) as a tritiated version ((3)H-HOCPCA) to radioactively label the specific GHB high-affinity binding site and gain further insight into the density, distribution and developmental profile of this protein. We show that, in low nanomolar concentrations...... brain development. Due to the high sensitivity of this radioligand, we were able to detect low levels of specific binding already at E15 in mouse brain, which increased progressively until adulthood. Collectively, we show that (3)H-HOCPCA is a highly sensitive radioligand, offering advantages over...

  16. Inhibition of Enterococcus faecium adherence to collagen by antibodies against high-affinity binding subdomains of Acm.

    Science.gov (United States)

    Nallapareddy, Sreedhar R; Sillanpää, Jouko; Ganesh, Vannakambadi K; Höök, Magnus; Murray, Barbara E

    2007-06-01

    Strains of Enterococcus faecium express a cell wall-anchored protein, Acm, which mediates adherence to collagen. Here, we (i) identify the minimal and high-affinity binding subsegments of Acm and (ii) show that anti-Acm immunoglobulin Gs (IgGs) purified against these subsegments reduced E. faecium TX2535 strain collagen adherence up to 73 and 50%, respectively, significantly more than the total IgGs against the full-length Acm A domain (28%) (P Acm adherence with functional subsegment-specific antibodies raises the possibility of their use as therapeutic or prophylactic agents.

  17. Hijacking membrane transporters for arsenic phytoextraction

    Science.gov (United States)

    LeBlanc, Melissa S.; McKinney, Elizabeth C.; Meagher, Richard B.; Smith, Aaron P.

    2012-01-01

    Arsenic is a toxic metalloid and recognized carcinogen. Arsenate and arsenite are the most common arsenic species available for uptake by plants. As an inorganic phosphate (Pi) analog, arsenate is acquired by plant roots through endogenous Pi transport systems. Inside the cell, arsenate is reduced to the thiol-reactive form arsenite. Glutathione (GSH)-conjugates of arsenite may be extruded from the cell or sequestered in vacuoles by members of the ATP-binding cassette (ABC) family of transporters. In the present study we sought to enhance both plant arsenic uptake through Pi transporter overexpression, and plant arsenic tolerance through ABC transporter overexpression. We demonstrate that Arabidopsis thaliana plants overexpressing the high-affinity Pi transporter family members, AtPht1;1 or AtPht1;7, are hypersensitive to arsenate due to increased arsenate uptake. These plants do not exhibit increased sensitivity to arsenite. Co-overexpression of the yeast ABC transporter YCF1 in combination with AtPht1;1 or AtPht1;7 suppresses the arsenate-sensitive phenotype while further enhancing arsenic uptake. Taken together, our results support an arsenic transport mechanism in which arsenate uptake is increased through Pi transporter overexpression, and arsenic tolerance is enhanced through YCF1-mediated vacuolar sequestration. This work substantiates the viability of coupling enhanced uptake and vacuolar sequestration as a means for developing a prototypical engineered arsenic hyperaccumulator. PMID:23108027

  18. The transport characteristics of "2"3"8U, "2"3"2Th, "2"2"6Ra, and "4"0K in the production cycle of phosphate rock

    International Nuclear Information System (INIS)

    Jung, Yoon Hee; Lim, Jong Myoung; Ji, Young Yong; Chung, Kun Ho; Kang, Mun Ja

    2017-01-01

    Phosphate rock and its by-product are widely used in various industries to produce phosphoric acid, gypsum, gypsum board, and fertilizer. Owing to its high level of natural radioactive nuclides (e.g., 238U and 226Ra), the radiological safety of workers who work with phosphate rock should be systematically managed. In this study, 238U, 232Th, 226Ra, and 40K levels were measured to analyze the transport characteristics of these radionuclides in the production cycle of phosphate rock. Energy dispersive X-ray fluorescence and gamma spectrometry were used to determine the activity of 238U, 232Th, 226Ra, and 40K. To evaluate the extent of secular disequilibrium, the analytical results were compared using statistical methods. Finally, the distribution of radioactivity across different stages of the phosphate rock production cycle was evaluated. The concentration ratios of 226Ra and 238U in phosphate rock were close to 1.0, while those found in gypsum and fertilizer were extremely different, reflecting disequilibrium after the chemical reaction process. The nuclide with the highest activity level in the production cycle of phosphate rock was 40K, and the median 40K activity was 8.972 Bq·g−1 and 1.496 Bq·g−1, respectively. For the 238U series, the activity of 238U and 226Ra was greatest in phosphate rock, and the distribution of activity values clearly showed the transport characteristics of the radionuclides, both for the byproducts of the decay sequences and for their final products. Although the activity of 40K in k-related fertilizer was relatively high, it made a relatively low contribution to the total radiological effect. However, the activity levels of 226Ra and 238U in phosphate rock were found to be relatively high, near the upper end of the acceptable limits. Therefore, it is necessary to systematically manage the radiological safety of workers engaged in phosphate rock processing

  19. High-affinity DNA-binding Domains of Replication Protein A (RPA) Direct SMARCAL1-dependent Replication Fork Remodeling*

    Science.gov (United States)

    Bhat, Kamakoti P.; Bétous, Rémy; Cortez, David

    2015-01-01

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. PMID:25552480

  20. High-affinity DNA-binding domains of replication protein A (RPA) direct SMARCAL1-dependent replication fork remodeling.

    Science.gov (United States)

    Bhat, Kamakoti P; Bétous, Rémy; Cortez, David

    2015-02-13

    SMARCAL1 catalyzes replication fork remodeling to maintain genome stability. It is recruited to replication forks via an interaction with replication protein A (RPA), the major ssDNA-binding protein in eukaryotic cells. In addition to directing its localization, RPA also activates SMARCAL1 on some fork substrates but inhibits it on others, thereby conferring substrate specificity to SMARCAL1 fork-remodeling reactions. We investigated the mechanism by which RPA regulates SMARCAL1. Our results indicate that although an interaction between SMARCAL1 and RPA is essential for SMARCAL1 activation, the location of the interacting surface on RPA is not. Counterintuitively, high-affinity DNA binding of RPA DNA-binding domain (DBD) A and DBD-B near the fork junction makes it easier for SMARCAL1 to remodel the fork, which requires removing RPA. We also found that RPA DBD-C and DBD-D are not required for SMARCAL1 regulation. Thus, the orientation of the high-affinity RPA DBDs at forks dictates SMARCAL1 substrate specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

    Science.gov (United States)

    Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

    2017-05-13

    DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Lázár-Molnár, Eszter; Scandiuzzi, Lisa; Basu, Indranil; Quinn, Thomas; Sylvestre, Eliezer; Palmieri, Edith; Ramagopal, Udupi A.; Nathenson, Stanley G.; Guha, Chandan; Almo, Steven C.

    2017-03-01

    Programmed Cell Death-1 (PD-1) is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1) exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L) was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR) assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  3. Structure-guided development of a high-affinity human Programmed Cell Death-1: Implications for tumor immunotherapy

    Directory of Open Access Journals (Sweden)

    Eszter Lázár-Molnár

    2017-03-01

    Full Text Available Programmed Cell Death-1 (PD-1 is an inhibitory immune receptor, which plays critical roles in T cell co-inhibition and exhaustion upon binding to its ligands PD-L1 and PD-L2. We report the crystal structure of the human PD-1 ectodomain and the mapping of the PD-1 binding interface. Mutagenesis studies confirmed the crystallographic interface, and resulted in mutant PD-1 receptors with altered affinity and ligand-specificity. In particular, a high-affinity mutant PD-1 (HA PD-1 exhibited 45 and 30-fold increase in binding to PD-L1 and PD-L2, respectively, due to slower dissociation rates. This mutant (A132L was used to engineer a soluble chimeric Ig fusion protein for cell-based and in vivo studies. HA PD-1 Ig showed enhanced binding to human dendritic cells, and increased T cell proliferation and cytokine production in a mixed lymphocyte reaction (MLR assay. Moreover, in an experimental model of murine Lewis lung carcinoma, HA PD-1 Ig treatment synergized with radiation therapy to decrease local and metastatic tumor burden, as well as in the establishment of immunological memory responses. Our studies highlight the value of structural considerations in guiding the design of a high-affinity chimeric PD-1 Ig fusion protein with robust immune modulatory properties, and underscore the power of combination therapies to selectively manipulate the PD-1 pathway for tumor immunotherapy.

  4. Biphasic regulation of development of the high-affinity saxitoxin receptor by innervation in rat skeletal muscle

    International Nuclear Information System (INIS)

    Sherman, S.J.; Catterall, W.A.

    1982-01-01

    Specific binding of 3 H-saxitoxin (STX) was used to quantitate the density of voltage-sensitive sodium channels in developing rat skeletal muscle. In adult triceps surae, a single class of sites with a KD . 2.9 nM and a density of 21 fmol/mg wet wt was detected. The density of these high-affinity sites increased from 2.0 fmol/mg wet wt to the adult value in linear fashion during days 2-25 after birth. Denervation of the triceps surae at day 11 or 17 reduced final saxitoxin receptor site density to 10.4 or 9.2 fmol/mg wet wt, respectively, without changing KD. Denervation of the triceps surae at day 5 did not alter the subsequent development of saxitoxin receptor sites during days 5-9 and accelerated the increase of saxitoxin receptor sites during days 9-13. After day 13, saxitoxin receptor development abruptly ceased and the density of saxitoxin receptor sites declined to 11 fmol/wg wet wt. These results show that the regulation of high-affinity saxitoxin receptor site density by innervation is biphasic. During the first phase, which is independent of continuing innervation, the saxitoxin receptor density increases to 47-57% of the adult level. After day 11, the second phase of development, which is dependent on continuing innervation, gives rise to the adult density of saxitoxin receptors

  5. Specific recognition of the C-terminal end of A beta 42 by a high affinity monoclonal antibody

    DEFF Research Database (Denmark)

    Axelsen, Trine Veje; Holm, Arne; Birkelund, Svend

    2009-01-01

    The neurotoxic peptide A beta(42) is derived from the amyloid precursor protein by proteolytic cleavage and is deposited in the brain of patients suffering from Alzheimer's disease (AD). In this study we generate a high affinity monoclonal antibody that targets the C-terminal end of A beta(42......) with high specificity. By this is meant that the paratope of the antibody must enclose the C-terminal end of A beta(42) including the carboxy-group of amino acid 42, and not just recognize a linear epitope in the C-terminal part of A beta. This has been accomplished by using a unique antigen construct made...... by the Ligand Presenting Assembly technology (LPA technology). This strategy results in dimeric presentation of the free C-terminal end of A beta(42). The generated Mab A beta1.1 is indeed specific for the C-terminal end of A beta(42) to which it binds with high affinity. Mab A beta1.1 recognizes the epitope...

  6. Susceptibility to endotoxin induced uveitis is not reduced in mice deficient in BLT1, the high affinity leukotriene B4 receptor

    OpenAIRE

    Smith, J R; Subbarao, K; Franc, D T; Haribabu, B; Rosenbaum, J T

    2004-01-01

    Aim: To investigate the role of arachidonic acid derived chemotactic factor, LTB4, in the development of endotoxin induced uveitis (EIU), using mice deficient in the BLT1 gene which encodes the high affinity LTB4 receptor.

  7. Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters.

    Science.gov (United States)

    Sawers, Ruairidh J H; Svane, Simon F; Quan, Clement; Grønlund, Mette; Wozniak, Barbara; Gebreselassie, Mesfin-Nigussie; González-Muñoz, Eliécer; Chávez Montes, Ricardo A; Baxter, Ivan; Goudet, Jerome; Jakobsen, Iver; Paszkowski, Uta

    2017-04-01

    Plant interactions with arbuscular mycorrhizal fungi have long attracted interest for their potential to promote more efficient use of mineral resources in agriculture. Their use, however, remains limited by a lack of understanding of the processes that determine the outcome of the symbiosis. In this study, the impact of host genotype on growth response to mycorrhizal inoculation was investigated in a panel of diverse maize lines. A panel of 30 maize lines was evaluated with and without inoculation with arbuscular mycorrhizal fungi. The line Oh43 was identified to show superior response and, along with five other reference lines, was characterized in greater detail in a split-compartment system, using 33 P to quantify mycorrhizal phosphorus uptake. Changes in relative growth indicated variation in host capacity to profit from the symbiosis. Shoot phosphate content, abundance of root-internal and -external fungal structures, mycorrhizal phosphorus uptake, and accumulation of transcripts encoding plant PHT1 family phosphate transporters varied among lines. Superior response in Oh43 is correlated with extensive development of root-external hyphae, accumulation of specific Pht1 transcripts and high phosphorus uptake by mycorrhizal plants. The data indicate that host genetic factors influence fungal growth strategy with an impact on plant performance. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  8. Adsorption and desorption of phosphate on limestone in experiments simulating seawater intrusion

    Science.gov (United States)

    The absorption and desorption of phosphorus on a large block of limestone was investigated using deionized water (DIW) and seawater. The limestone had a high affinity to adsorb phosphorus in DIW. Phosphate adsorption was significantly less in seawater, and more phosphorus was desorbed in the seawate...

  9. High-affinity hemoglobin and blood oxygen saturation in diving emperor penguins.

    Science.gov (United States)

    Meir, Jessica U; Ponganis, Paul J

    2009-10-01

    The emperor penguin (Aptenodytes forsteri) thrives in the Antarctic underwater environment, diving to depths greater than 500 m and for durations longer than 23 min. To examine mechanisms underlying the exceptional diving ability of this species and further describe blood oxygen (O2) transport and depletion while diving, we characterized the O2-hemoglobin (Hb) dissociation curve of the emperor penguin in whole blood. This allowed us to (1) investigate the biochemical adaptation of Hb in this species, and (2) address blood O2 depletion during diving, by applying the dissociation curve to previously collected partial pressure of O2 (PO2) profiles to estimate in vivo Hb saturation (SO2) changes during dives. This investigation revealed enhanced Hb-O2 affinity (P50=28 mmHg, pH 7.5) in the emperor penguin, similar to high-altitude birds and other penguin species. This allows for increased O2 at low blood PO2 levels during diving and more complete depletion of the respiratory O2 store. SO2 profiles during diving demonstrated that arterial SO2 levels are maintained near 100% throughout much of the dive, not decreasing significantly until the final ascent phase. End-of-dive venous SO2 values were widely distributed and optimization of the venous blood O2 store resulted from arterialization and near complete depletion of venous blood O2 during longer dives. The estimated contribution of the blood O2 store to diving metabolic rate was low and highly variable. This pattern is due, in part, to the influx of O2 from the lungs into the blood during diving, and variable rates of tissue O2 uptake.

  10. Topical Application of Trisodium Ascorbyl 6-Palmitate 2-Phosphate Actively Supplies Ascorbate to Skin Cells in an Ascorbate Transporter-Independent Manner

    Directory of Open Access Journals (Sweden)

    Shuichi Shibuya

    2017-06-01

    Full Text Available Ascorbic acid (AA possesses multiple beneficial functions, such as regulating collagen biosynthesis and redox balance in the skin. AA derivatives have been developed to overcome this compound’s high fragility and to assist with AA supplementation to the skin. However, how AA derivatives are transferred into cells and converted to AA in the skin remains unclear. In the present study, we showed that AA treatment failed to increase the cellular AA level in the presence of AA transporter inhibitors, indicating an AA transporter-dependent action. In contrast, torisodium ascorbyl 6-palmitate 2-phosphate (APPS treatment significantly enhanced the cellular AA level in skin cells despite the presence of inhibitors. In ex vivo experiments, APPS treatment also increased the AA content in a human epidermis model. Interestingly, APPS was readily metabolized and converted to AA in keratinocyte lysates via an intrinsic mechanism. Furthermore, APPS markedly repressed the intracellular superoxide generation and promoted viability associated with an enhanced AA level in Sod1-deficient skin cells. These findings indicate that APPS effectively restores the AA level and normalizes the redox balance in skin cells in an AA transporter-independent manner. Topical treatment of APPS is a beneficial strategy for supplying AA and improving the physiology of damaged skin.

  11. Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate*

    Science.gov (United States)

    Jutabha, Promsuk; Anzai, Naohiko; Kitamura, Kenichiro; Taniguchi, Atsuo; Kaneko, Shuji; Yan, Kunimasa; Yamada, Hideomi; Shimada, Hidetaka; Kimura, Toru; Katada, Tomohisa; Fukutomi, Toshiyuki; Tomita, Kimio; Urano, Wako; Yamanaka, Hisashi; Seki, George; Fujita, Toshiro; Moriyama, Yoshinori; Yamada, Akira; Uchida, Shunya; Wempe, Michael F.; Endou, Hitoshi; Sakurai, Hiroyuki

    2010-01-01

    The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4. PMID:20810651

  12. SNF3 as high affinity glucose sensor and its function in supporting the viability of Candida glabrata under glucose-limited environment

    Directory of Open Access Journals (Sweden)

    Tzu Shan eNg

    2015-12-01

    Full Text Available Candida glabrata is an emerging human fungal pathogen that has efficacious nutrient sensing and responsiveness ability. It can be seen through its ability to thrive in diverse range of nutrient limited-human anatomical sites. Therefore, nutrient sensing particularly glucose sensing is thought to be crucial in contributing to the development and fitness of the pathogen. This study aimed to elucidate the role of SNF3 (Sucrose Non Fermenting 3 as a glucose sensor and its possible role in contributing to the fitness and survivability of C. glabrata in glucose-limited environment. The SNF3 knockout strain was constructed and subjected to different glucose concentrations to evaluate its growth, biofilm formation, amphotericin B susceptibility, ex vivo survivability and effects on the transcriptional profiling of the sugar receptor repressor (SRR pathway-related genes. The SNF3Δ strain showed a retarded growth in low glucose environments (0.01% and 0.1% in both fermentation and respiration-preferred conditions but grew well in high glucose concentration environments (1% and 2%. It was also found to be more susceptible to amphotericin B in low glucose environment (0.1% and macrophage engulfment but showed no difference in the biofilm formation capability. The deletion of SNF3 also resulted in the down-regulation of about half of hexose transporters genes (4 out of 9. Overall, the deletion of SNF3 causes significant reduction in the ability of C. glabrata to sense limited surrounding glucose and consequently disrupts its competency to transport and perform the uptake of this critical nutrient. This study highlighted the role of SNF3 as a high affinity glucose sensor and its role in aiding the survivability of C. glabrata particularly in glucose limited environment.

  13. Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

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    Guy Caljon

    Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

  14. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...... that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of (beta)arr interaction between family A and B receptors also at the molecular level....

  15. Phosphate transport by hyphae of field communities of arbuscular mycorrhizal fungi at two levels of P fertilization

    DEFF Research Database (Denmark)

    Thingstrup, I.; Kahiluoto, H.; Jakobsen, I.

    2000-01-01

    at the higher P level (77 and 49% of total P uptake, respectively). The AMF in P-fertilized soil transported less P-32 from the root-free compartment to the plant after 23 days than the AMF in unfertilized soil, but this difference disappeared in plants harvested after 27 and 32 days. The production of hyphae...... was largely similar in both fertilization treatments, indicating that the capacity for P uptake and transport by hyphae of the two AMF communities was similar....

  16. High-affinity α4β2 nicotinic receptors mediate the impairing effects of acute nicotine on contextual fear extinction.

    Science.gov (United States)

    Kutlu, Munir Gunes; Holliday, Erica; Gould, Thomas J

    2016-02-01

    Previously, studies from our lab have shown that while acute nicotine administered prior to training and testing enhances contextual fear conditioning, acute nicotine injections prior to extinction sessions impair extinction of contextual fear. Although there is also strong evidence showing that the acute nicotine's enhancing effects on contextual fear conditioning require high-affinity α4β2 nicotinic acetylcholine receptors (nAChRs), it is unknown which nAChR subtypes are involved in the acute nicotine-induced impairment of contextual fear extinction. In this study, we investigated the effects of acute nicotine administration on contextual fear extinction in knock-out (KO) mice lacking α4, β2 or α7 subtypes of nAChRs and their wild-type (WT) littermates. Both KO and WT mice were first trained and tested for contextual fear conditioning and received a daily contextual extinction session for 4 days. Subjects received intraperitoneal injections of nicotine (0.18 mg/kg) or saline 2-4 min prior to each extinction session. Our results showed that the mice that lack α4 and β2 subtypes of nAChRs showed normal contextual fear extinction but not the acute nicotine-induced impairment while the mice that lack the α7 subtype showed both normal contextual extinction and nicotine-induced impairment of contextual extinction. In addition, control experiments showed that acute nicotine-induced impairment of contextual fear extinction persisted when nicotine administration was ceased and repeated acute nicotine administrations alone did not induce freezing behavior in the absence of context-shock learning. These results clearly demonstrate that high-affinity α4β2 nAChRs are necessary for the effects of acute nicotine on contextual fear extinction. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    International Nuclear Information System (INIS)

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-01-01

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 0 C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17β-[ 3 H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins

  18. Cyr61/CCN1 displays high-affinity binding to the somatomedin B(1-44 domain of vitronectin.

    Directory of Open Access Journals (Sweden)

    Ivo M B Francischetti

    2010-02-01

    Full Text Available Cyr61 is a member of the CCN (Cyr61, connective tissue growth, NOV family of extracellular-associated (matricellular proteins that present four distinct functional modules, namely insulin-like growth factor binding protein (IGFBP, von Willebrand factor type C (vWF, thrombospondin type 1 (TSP, and C-terminal growth factor cysteine knot (CT domain. While heparin sulphate proteoglycans reportedly mediate the interaction of Cyr61 with the matrix and cell surface, the role of other extracellular associated proteins has not been revealed.In this report, surface plasmon resonance (SPR experiments and solid-phase binding assays demonstrate that recombinant Cyr61 interacts with immobilized monomeric or multimeric vitronectin (VTNC with K(D in the nanomolar range. Notably, the binding site for Cyr61 was identified as the somatomedin B domain (SMTB(1-44 of VTNC, which mediates its interaction with PAI-1, uPAR, and integrin alphav beta3. Accordingly, PAI-1 outcompetes Cyr61 for binding to immobilized SMTB(1-44, and Cyr61 attenuates uPAR-mediated U937 adhesion to VTNC. In contrast, isothermal titration calorimetry shows that Cyr61 does not display high-affinity binding for SMTB(1-44 in solution. Nevertheless, competitive ELISA revealed that multimeric VTNC, heat-modified monomeric VTNC, or SMTB(1-44 at high concentrations attenuate Cyr61 binding to immobilized VTNC, while monomeric VTNC was ineffective. Therefore, immobilization of VTNC exposes cryptic epitopes that recognize Cyr61 with high affinity, as reported for a number of antibodies, beta-endorphin, and other molecules.The finding that Cyr61 interacts with the SMTB(1-44 domain suggests that VTNC represent a point of anchorage for CCN family members to the matrix. Results are discussed in the context of the role of CCN and VTNC in matrix biology and angiogenesis.

  19. High affinity antigen recognition of the dual specific variants of herceptin is entropy-driven in spite of structural plasticity.

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    Jenny Bostrom

    Full Text Available The antigen-binding site of Herceptin, an anti-human Epidermal Growth Factor Receptor 2 (HER2 antibody, was engineered to add a second specificity toward Vascular Endothelial Growth Factor (VEGF to create a high affinity two-in-one antibody bH1. Crystal structures of bH1 in complex with either antigen showed that, in comparison to Herceptin, this antibody exhibited greater conformational variability, also called "structural plasticity". Here, we analyzed the biophysical and thermodynamic properties of the dual specific variants of Herceptin to understand how a single antibody binds two unrelated protein antigens. We showed that while bH1 and the affinity-improved bH1-44, in particular, maintained many properties of Herceptin including binding affinity, kinetics and the use of residues for antigen recognition, they differed in the binding thermodynamics. The interactions of bH1 and its variants with both antigens were characterized by large favorable entropy changes whereas the Herceptin/HER2 interaction involved a large favorable enthalpy change. By dissecting the total entropy change and the energy barrier for dual interaction, we determined that the significant structural plasticity of the bH1 antibodies demanded by the dual specificity did not translate into the expected increase of entropic penalty relative to Herceptin. Clearly, dual antigen recognition of the Herceptin variants involves divergent antibody conformations of nearly equivalent energetic states. Hence, increasing the structural plasticity of an antigen-binding site without increasing the entropic cost may play a role for antibodies to evolve multi-specificity. Our report represents the first comprehensive biophysical analysis of a high affinity dual specific antibody binding two unrelated protein antigens, furthering our understanding of the thermodynamics that drive the vast antigen recognition capacity of the antibody repertoire.

  20. Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

    Science.gov (United States)

    Honey, Denise M.; Best, Annie; Qiu, Huawei

    2018-01-01

    ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

  1. Influence of nutrient signals and carbon allocation on the expression of phosphate and nitrogen transporter genes in winter wheat (Triticum aestivum L.) roots colonized by arbuscular mycorrhizal fungi.

    Science.gov (United States)

    Tian, Hui; Yuan, Xiaolei; Duan, Jianfeng; Li, Wenhu; Zhai, Bingnian; Gao, Yajun

    2017-01-01

    Arbuscular mycorrhizal (AM) colonization of plant roots causes the down-regulation of expression of phosphate (Pi) or nitrogen (N) transporter genes involved in direct nutrient uptake pathways. The mechanism of this effect remains unknown. In the present study, we sought to determine whether the expression of Pi or N transporter genes in roots of winter wheat colonized by AM fungus responded to (1) Pi or N nutrient signals transferred from the AM extra-radical hyphae, or (2) carbon allocation changes in the AM association. A three-compartment culture system, comprising a root compartment (RC), a root and AM hyphae compartment (RHC), and an AM hyphae compartment (HC), was used to test whether the expression of Pi or N transporter genes responded to nutrients (Pi, NH4+ and NO3-) added only to the HC. Different AM inoculation density treatments (roots were inoculated with 0, 20, 50 and 200 g AM inoculum) and light regime treatments (6 hours light and 18 hours light) were established to test the effects of carbon allocation on the expression of Pi or N transporter genes in wheat roots. The expression of two Pi transporter genes (TaPT4 and TaPHT1.2), five nitrate transporter genes (TaNRT1.1, TaNRT1.2, TaNRT2.1, TaNRT2.2, and TaNRT2.3), and an ammonium transporter gene (TaAMT1.2) was quantified using real-time polymerase chain reaction. The expression of TaPT4, TaNRT2.2, and TaAMT1.2 was down-regulated by AM colonization only when roots of host plants received Pi or N nutrient signals. However, the expression of TaPHT1.2, TaNRT2.1, and TaNRT2.3 was down-regulated by AM colonization, regardless of whether there was nutrient transfer from AM hyphae. The expression of TaNRT1.2 was also down-regulated by AM colonization even when there was no nutrient transfer from AM hyphae. The present study showed that an increase in carbon consumption by the AM fungi did not necessarily result in greater down-regulation of expression of Pi or N transporter genes.

  2. Regional characterization of energy metabolism in the brain of normal and MPTP-intoxicated mice using new markers of glucose and phosphate transport

    Directory of Open Access Journals (Sweden)

    Touhami Jawida

    2010-12-01

    Full Text Available Abstract The gibbon ape leukemia virus (GALV, the amphotropic murine leukemia virus (AMLV and the human T-cell leukemia virus (HTLV are retroviruses that specifically bind nutrient transporters with their envelope glycoproteins (Env when entering host cells. Here, we used tagged ligands derived from GALV, AMLV, and HTLV Env to monitor the distribution of their cognate receptors, the inorganic phosphate transporters PiT1 and PiT2, and the glucose transporter GLUT1, respectively, in basal conditions and after acute energy deficiency. For this purpose, we monitored changes in the distribution of PiT1, PiT2 and GLUT1 in the cerebellum, the frontal cortex, the corpus callosum, the striatum and the substantia nigra (SN of C57/BL6 mice after administration of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridinium (MPTP, a mitochondrial complex I inhibitor which induces neuronal degeneration in the striato-nigral network. The PiT1 ligand stained oligodendrocytes in the corpus callosum and showed a reticular pattern in the SN. The PiT2 ligand stained particularly the cerebellar Purkinje cells, while GLUT1 labelling was mainly observed throughout the cortex, basal ganglia and cerebellar gray matter. Interestingly, unlike GLUT1 and PiT2 distributions which did not appear to be modified by MPTP intoxication, PiT1 immunostaining seemed to be more extended in the SN. The plausible reasons for this change following acute energy stress are discussed. These new ligands therefore constitute new metabolic markers which should help to unravel cellular adaptations to a wide variety of normal and pathologic conditions and to determine the role of specific nutrient transporters in tissue homeostasis.

  3. Expression of a xanthine permease and phosphate transporter in cultures and field populations of the harmful alga Aureococcus anophagefferens: tracking nutritional deficiency during brown tides.

    Science.gov (United States)

    Wurch, Louie L; Gobler, Christopher J; Dyhrman, Sonya T

    2014-08-01

    Targeted gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) was employed to track patterns in the expression of genes indicative of nitrogen or phosphorus deficiency in the brown tide-forming alga Aureococcus anophagefferens. During culture experiments, a xanthine/uracil/vitamin C permease (XUV) was upregulated ∼20-fold under nitrogen-deficient conditions relative to a nitrogen-replete control and rapidly returned to nitrogen-replete levels after nitrogen-deficient cells were resupplied with nitrate or ammonium. It was not responsive to phosphorus deficiency. Expression of an inorganic phosphate transporter (PTA3) was enriched ∼10-fold under phosphorus-deficient conditions relative to a phosphorus-replete control, and this signal was rapidly lost upon phosphate resupply. PTA3 was not upregulated by nitrogen deficiency. Natural A. anophagefferens populations from a dense brown tide that occurred in Long Island, NY, in 2009 were assayed for XUV and PTA3 expression and compared with nutrient concentrations over the peak of a bloom. Patterns in XUV expression were consistent with nitrogen-replete growth, never reaching the values observed in N-deficient cultures. PTA3 expression was highest prior to peak bloom stages, reaching expression levels within the range of P-deficient cultures. These data highlight the value of molecular-level assessments of nutrient deficiency and suggest that phosphorus deficiency could play a role in the dynamics of destructive A. anophagefferens blooms. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  4. Comparison of high affinity binding of 3H-proadifen and 3H-(-)-cocaine t rat liver membranes

    International Nuclear Information System (INIS)

    Ross, S.B.

    1995-01-01

    The characteristics of the binding of 3 H-proadifen to rat liver membranes were studied and compared to those of 3 H-cocaine. It was found that 3 H-proadifen was bound reversibly with high affinity (K D =1.8±0.5 nM) and large capacity (B max =2010±340 pmol/g wet tissue) to liver membranes. The corresponding values for the 3 H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of 3 H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 μM CdCl 2 in the incubation buffer it was possible to differentiate between two 3 H-cocaine binding sites with K d values of 1.6 and 7.7 nM and B max values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of 3 H-proadifen and 3 H-cocaine inhibited the binding of 3 H-proadifen (IC 50 =10 nM) and proadifen that of 3 H-cocaine (IC 50 =1 nM). There was a high correlation coefficient (r r =0.972; P 50 =100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl 2 , ZnCl 2 and CuCl 2 inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd 2+ on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15μM. The similarity of the characteristics of the binding of these ligands with that of 3 H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.)

  5. Peptides in headlock – a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy

    Science.gov (United States)

    Braun, Michael B.; Traenkle, Bjoern; Koch, Philipp A.; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-01

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies. PMID:26791954

  6. Peptides in headlock--a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy.

    Science.gov (United States)

    Braun, Michael B; Traenkle, Bjoern; Koch, Philipp A; Emele, Felix; Weiss, Frederik; Poetz, Oliver; Stehle, Thilo; Rothbauer, Ulrich

    2016-01-21

    Nanobodies are highly valuable tools for numerous bioanalytical and biotechnical applications. Here, we report the characterization of a nanobody that binds a short peptide epitope with extraordinary affinity. Structural analysis reveals an unusual binding mode where the extended peptide becomes part of a β-sheet structure in the nanobody. This interaction relies on sequence-independent backbone interactions augmented by a small number of specificity-determining side chain contacts. Once bound, the peptide is fastened by two nanobody side chains that clamp it in a headlock fashion. Exploiting this unusual binding mode, we generated a novel nanobody-derived capture and detection system. Matrix-coupled nanobody enables the fast and efficient isolation of epitope-tagged proteins from prokaryotic and eukaryotic expression systems. Additionally, the fluorescently labeled nanobody visualizes subcellular structures in different cellular compartments. The high-affinity-binding and modifiable peptide tag of this system renders it a versatile and robust tool to combine biochemical analysis with microscopic studies.

  7. High affinity [3H]glibenclamide binding sites in rat neuronal and cardiac tissue: Localization and developmental characteristics

    International Nuclear Information System (INIS)

    Miller, J.A.; Velayo, N.L.; Dage, R.C.; Rampe, D.

    1991-01-01

    We examined the binding of the antidiabetic sulfonylurea [3H] glibenclamide to rat brain and heart membranes. High affinity binding was observed in adult rat forebrain (Kd = 137.3 pM, maximal binding site density = 91.8 fmol/mg of protein) and ventricle (Kd = 77.1 pM, maximal binding site density = 65.1 fmol/mg of protein). Binding site density increased approximately 250% in forebrain membranes during postnatal development but was constant in ventricular membranes. Quantitative autoradiography was used to examine the regional distribution of [3H] glibenclamide binding sites in sections from rat brain, spinal cord and heart. The greatest density of binding in adult brain was found in the substantia nigra and globus pallidus, whereas the other areas displayed heterogenous binding. In agreement with the membrane binding studies, 1-day-old rat brain had significantly fewer [3H]glibenclamide binding sites than adult brain. Additionally, the pattern of distribution of these sites was qualitatively different from that of the adult. In adult rat spinal cord, moderate binding densities were observed in spinal cord gray and displayed a rostral to caudal gradient. In adult rat heart, moderate binding densities were observed and the sites were distributed homogeneously. In conclusion, significant development of [3H]glibenclamide binding sites was seen in the brain but not the heart during postnatal maturation. Furthermore, a heterogeneous distribution of binding sites was observed in both the brain and spinal cord of adult rats

  8. ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

    Directory of Open Access Journals (Sweden)

    Anuradha Kuchibhatla

    Full Text Available A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0 pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.

  9. A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

    Science.gov (United States)

    Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

    2018-04-30

    Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

  10. A soluble form of the high affinity IgE receptor, Fc-epsilon-RI, circulates in human serum.

    Directory of Open Access Journals (Sweden)

    Eleonora Dehlink

    Full Text Available Soluble IgE receptors are potential in vivo modulators of IgE-mediated immune responses and are thus important for our basic understanding of allergic responses. We here characterize a novel soluble version of the IgE-binding alpha-chain of Fc-epsilon-RI (sFcεRI, the high affinity receptor for IgE. sFcεRI immunoprecipitates as a protein of ∼40 kDa and contains an intact IgE-binding site. In human serum, sFcεRI is found as a soluble free IgE receptor as well as a complex with IgE. Using a newly established ELISA, we show that serum sFcεRI levels correlate with serum IgE in patients with elevated IgE. We also show that serum of individuals with normal IgE levels can be found to contain high levels of sFcεRI. After IgE-antigen-mediated crosslinking of surface FcεRI, we detect sFcεRI in the exosome-depleted, soluble fraction of cell culture supernatants. We further show that sFcεRI can block binding of IgE to FcεRI expressed at the cell surface. In summary, we here describe the alpha-chain of FcεRI as a circulating soluble IgE receptor isoform in human serum.

  11. Design, Synthesis, and Biological Evaluation of Small, High-Affinity Siglec-7 Ligands: Toward Novel Inhibitors of Cancer Immune Evasion.

    Science.gov (United States)

    Prescher, Horst; Frank, Martin; Gütgemann, Stephan; Kuhfeldt, Elena; Schweizer, Astrid; Nitschke, Lars; Watzl, Carsten; Brossmer, Reinhard

    2017-02-09

    Natural killer cells are able to directly lyse tumor cells, thereby participating in the immune surveillance against cancer. Unfortunately, many cancer cells use immune evasion strategies to avoid their eradication by the immune system. A prominent escape strategy of malignant cells is to camouflage themselves with Siglec-7 ligands, thereby recruiting the inhibitory receptor Siglec-7 expressed on the NK cell surface which subsequently inhibits NK-cell-mediated lysis. Here we describe the synthesis and evaluation of the first, high-affinity low molecular weight Siglec-7 ligands to interfere with cancer cell immune evasion. The compounds are Sialic acid derivatives and bind with low micromolar K d values to Siglec-7. They display up to a 5000-fold enhanced affinity over the unmodified sialic acid scaffold αMe Neu5Ac, the smallest known natural Siglec-7 ligand. Our results provide a novel immuno-oncology strategy employing natural immunity in the fight against cancers, in particular blocking Siglec-7 with low molecular weight compounds.

  12. High affinity γPNA sandwich hybridization assay for rapid detection of short nucleic acid targets with single mismatch discrimination.

    Science.gov (United States)

    Goldman, Johnathan M; Zhang, Li Ang; Manna, Arunava; Armitage, Bruce A; Ly, Danith H; Schneider, James W

    2013-07-08

    Hybridization analysis of short DNA and RNA targets presents many challenges for detection. The commonly employed sandwich hybridization approach cannot be implemented for these short targets due to insufficient probe-target binding strengths for unmodified DNA probes. Here, we present a method capable of rapid and stable sandwich hybridization detection for 22 nucleotide DNA and RNA targets. Stable hybridization is achieved using an n-alkylated, polyethylene glycol γ-carbon modified peptide nucleic acid (γPNA) amphiphile. The γPNA's exceptionally high affinity enables stable hybridization of a second DNA-based probe to the remaining bases of the short target. Upon hybridization of both probes, an electrophoretic mobility shift is measured via interaction of the n-alkane modification on the γPNA with capillary electrophoresis running buffer containing nonionic surfactant micelles. We find that sandwich hybridization of both probes is stable under multiple binding configurations and demonstrate single base mismatch discrimination. The binding strength of both probes is also stabilized via coaxial stacking on adjacent hybridization to targets. We conclude with a discussion on the implementation of the proposed sandwich hybridization assay as a high-throughput microRNA detection method.

  13. High-Affinity Interaction of the K-Ras4B Hypervariable Region with the Ras Active Site

    Science.gov (United States)

    Chavan, Tanmay S.; Jang, Hyunbum; Khavrutskii, Lyuba; Abraham, Sherwin J.; Banerjee, Avik; Freed, Benjamin C.; Johannessen, Liv; Tarasov, Sergey G.; Gaponenko, Vadim; Nussinov, Ruth; Tarasova, Nadya I.

    2015-01-01

    Ras proteins are small GTPases that act as signal transducers between cell surface receptors and several intracellular signaling cascades. They contain highly homologous catalytic domains and flexible C-terminal hypervariable regions (HVRs) that differ across Ras isoforms. KRAS is among the most frequently mutated oncogenes in human tumors. Surprisingly, we found that the C-terminal HVR of K-Ras4B, thought to minimally impact the catalytic domain, directly interacts with the active site of the protein. The interaction is almost 100-fold tighter with the GDP-bound than the GTP-bound protein. HVR binding interferes with Ras-Raf interaction, modulates binding to phospholipids, and slightly slows down nucleotide exchange. The data indicate that contrary to previously suggested models of K-Ras4B signaling, HVR plays essential roles in regulation of signaling. High affinity binding of short peptide analogs of HVR to K-Ras active site suggests that targeting this surface with inhibitory synthetic molecules for the therapy of KRAS-dependent tumors is feasible. PMID:26682817

  14. The RCK1 high-affinity Ca2+ sensor confers carbon monoxide sensitivity to Slo1 BK channels.

    Science.gov (United States)

    Hou, Shangwei; Xu, Rong; Heinemann, Stefan H; Hoshi, Toshinori

    2008-03-11

    Carbon monoxide (CO) is a lethal gas, but it is also increasingly recognized as a physiological signaling molecule capable of regulating a variety of proteins. Among them, large-conductance Ca(2+)- and voltage-gated K(+) (Slo1 BK) channels, important in vasodilation and neuronal firing, have been suggested to be directly stimulated by CO. However, the molecular mechanism of the stimulatory action of CO on the Slo1 BK channel has not been clearly elucidated. We report here that CO reliably and repeatedly activates Slo1 BK channels in excised membrane patches in the absence of Ca(2+) in a voltage-sensor-independent manner. The stimulatory action of CO on the Slo1 BK channel requires an aspartic acid and two histidine residues located in the cytoplasmic RCK1 domain, and the effect persists under the conditions known to inhibit the conventional interaction between CO and heme in other proteins. We propose that CO acts as a partial agonist for the high-affinity divalent cation sensor in the RCK1 domain of the Slo1 BK channel.

  15. VNARs: An Ancient and Unique Repertoire of Molecules That Deliver Small, Soluble, Stable and High Affinity Binders of Proteins

    Directory of Open Access Journals (Sweden)

    Caroline Barelle

    2015-09-01

    Full Text Available At 420 million years, the variable domain of New Antigen Receptors or VNARs are undoubtedly the oldest (and smallest antigen binding single domains identified in the vertebrate kingdom. Their role as an integral part of the adaptive immune system of sharks has been well established and has served to provide a greater understanding of the evolution of humoral immunity; their cellular components and processes as well as the underlying genetic organization and molecular control mechanisms. Intriguingly, unlike the variable domain of the camelid heavy chain antibodies or VHH, VNARs do not conform to all of the characteristic properties of classical antibodies with an ancestral origin that clearly distinguishes them from true immunoglobulin antibodies. However, this uniqueness of their origin only adds to their potential as next generation therapeutic biologics with their structural and functional attributes and commercial freedom all enhancing their profile and current success. In fact their small size, remarkable stability, molecular flexibility and solubility, together with their high affinity and selectivity for target, all reinforce the potential of these domains as drug candidates. The purpose of this review is to provide an overview of the existing basic biology of these unique domains, to highlight the drug-like properties of VNARs and describe current progress in their journey towards the clinic.

  16. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    Energy Technology Data Exchange (ETDEWEB)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M. (Centre de Biochimie, Nice (France))

    1989-07-05

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of {sup 125}I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.

  17. Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom

    International Nuclear Information System (INIS)

    Lambeau, G.; Barhanin, J.; Schweitz, H.; Qar, J.; Lazdunski, M.

    1989-01-01

    Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of 125 I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity

  18. High levels of the type III inorganic phosphate transporter PiT1 (SLC20A1) can confer faster cell adhesion

    DEFF Research Database (Denmark)

    Kongsfelt, Iben Boutrup; Byskov, Kristina; Pedersen, Lasse Ebdrup

    2014-01-01

    overexpression led to faster cell spreading. The final total numbers of attached cells did, however, not differ between cultures of PiT1 overexpressing cells and control cells of neither cell type. We suggest that the PiT1-mediated fast adhesion potentials allow the cells to go faster out of G0/G1 and thereby......The inorganic phosphate transporter PiT1 (SLC20A1) is ubiquitously expressed in mammalian cells. We recently showed that overexpression of human PiT1 was sufficient to increase proliferation of two strict density-inhibited cell lines, murine fibroblastic NIH3T3 and pre-osteoblastic MC3T3-E1 cells......, and allowed the cultures to grow to higher cell densities. In addition, upon transformation NIH3T3 cells showed increased ability to form colonies in soft agar. The cellular regulation of PiT1 expression supports that cells utilize the PiT1 levels to control proliferation, with non-proliferating cells showing...

  19. Temporal variation of nitrate and phosphate transport in headwater catchments: the hydrological controls and land use alteration

    Directory of Open Access Journals (Sweden)

    T.-Y. Lee

    2013-04-01

    Full Text Available Oceania rivers are hotspots of DIN (dissolved inorganic nitrogen and DIP (dissolved inorganic phosphorus transport due to humid/warm climate, typhoon-induced episodic rainfall and high tectonic activity that create an environment favorable for high/rapid runoff and soil erosion. In spite of its uniqueness, effects of hydrologic controls and land use on the transport behaviors of DIN and DIP are rarely documented. A 2 yr monitoring study for DIN and DIP from three headwater catchments with different cultivation gradient (0 To 8.9% was implemented during a ~ 3 day interval with an additional monitoring campaign at a 3 h interval during typhoon periods. Results showed the DIN yields in the pristine, moderately cultivated (2.7%, and intensively cultivated (8.9% watersheds were 8.3, 26, and 37 kg N ha−1 yr−1, respectively. For the DIP yields, they were 0.36, 0.35, and 0.56 kg P ha−1 yr−1, respectively. Higher year-round DIN concentrations and five times larger in DIN yields in intensively cultivated watersheds indicate DIN is more sensitive to land use changes. The high background DIN yield from the relatively pristine watershed was likely due to high atmospheric nitrogen deposition and large subterranean N pool. The correlations between runoff and concentration reveals that typhoon floods purge out more DIN from the subterranean reservoir, i.e., soil, by contrast, runoff washes off surface soil resulting in higher suspended sediment with higher DIP. Collectively, typhoon runoff contributes 20–70% and 47–80%, respectively, to the annual DIN and DIP exports. The DIN yield to DIP yield ratio varied from 97 to 410, which is higher than the global mean of ~ 18. Such a high ratio indicates a P-limiting condition in stream and the downstream aquatic environment. Based on our field observation, we constructed a conceptual model illustrating different remobilization mechanisms for DIN and DIP from headwaters in a mountainous river, which is

  20. High-affinity human leucocyte antigen class I binding variola-derived peptides induce CD4(+) T cell responses more than 30 years post-vaccinia virus vaccination

    DEFF Research Database (Denmark)

    Wang, M.; Tang, Sheila Tuyet; Lund, Ole

    2009-01-01

    Interferon-gamma secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human leucoc...

  1. High-Affinity Sites Form an Interaction Network to Facilitate Spreading of the MSL Complex across the X Chromosome in Drosophila

    NARCIS (Netherlands)

    Ramírez, Fidel; Lingg, Thomas; Toscano, Sarah; Lam, Kin Chung; Georgiev, Plamen; Chung, Ho-Ryun; Lajoie, Bryan R; de Wit, Elzo; Zhan, Ye; de Laat, Wouter; Dekker, Job; Manke, Thomas; Akhtar, Asifa

    2015-01-01

    Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation toward targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses, we show that high-affinity sites (HAS), landing platforms of the male-specific

  2. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity

    Science.gov (United States)

    Algimantas P. Valaitis; Jeremy L. Jenkins; Mi Kyong Lee; Donald H. Dean; Karen J. Garner

    2001-01-01

    BTR-270, a gypsy moth (Lymantria dispar) brush border membrane molecule that binds Bacillus thuringiensis (Bt) Cry1A toxins with high affinity, was purified by preparative gel electrophoresis. Rabbit antibodies specific for the Bt toxin-binding molecule were raised. Attempts to label BTR-270 by protein-directed techniques were...

  3. Contrast-enhanced CT with a High-Affinity Cationic Contrast Agent for Imaging ex Vivo Bovine, Intact ex Vivo Rabbit, and in Vivo Rabbit Cartilage

    OpenAIRE

    Stewart, Rachel C.; Bansal, Prashant N.; Entezari, Vahid; Lusic, Hrvoje; Nazarian, Rosalynn M.; Snyder, Brian D.; Grinstaff, Mark W.

    2013-01-01

    The high affinity of a cationic iodinated contrast agent for cartilage provides better tissue visualization, easier segmentation, higher contrast-to-noise ratios, and longer usable imaging windows and requires a lower dose of injected contrast agent compared with an anionic contrast agent.

  4. Soil carbon content and relative abundance of high affinity H2-oxidizing bacteria predict atmospheric H2 soil uptake activity better than soil microbial community composition

    NARCIS (Netherlands)

    Khdhiri, Mondher; Hesse, Laura; Popa, Maria Elena; Quiza, Liliana; Lalonde, Isabelle; Meredith, Laura K.; Röckmann, Thomas; Constant, Philippe

    2015-01-01

    Soil-atmosphere exchange of H2 is controlled by gas diffusion and the microbial production and oxidation activities in soil. Among these parameters, the H2 oxidation activity catalyzed by soil microorganisms harboring high affinity hydrogenase is the most difficult variable to parameterize because

  5. Amino propynyl benzoic acid building block in rigid spacers of divalent ligands binding to the Syk SH2 domains with equally high affinity as the natural ligand

    NARCIS (Netherlands)

    Dekker, Frank J; de Mol, Nico J; Fischer, Marcel J E; Liskamp, Rob M J; Dekker, Frank

    2003-01-01

    The construction of rigid spacers composed of amino propynyl benzoic acid building blocks is described. These spacers were used to link two phosphopeptide ligand sites towards obtaining divalent ligands with a high affinity for Syk tandem SH2 domains, which are important in signal transduction. The

  6. Transportation Safety of Lithium Iron Phosphate Batteries - A Feasibility Study of Storing at Very Low States of Charge.

    Science.gov (United States)

    Barai, Anup; Uddin, Kotub; Chevalier, Julie; Chouchelamane, Gael H; McGordon, Andrew; Low, John; Jennings, Paul

    2017-07-11

    In freight classification, lithium-ion batteries are classed as dangerous goods and are therefore subject to stringent regulations and guidelines for certification for safe transport. One such guideline is the requirement for batteries to be at a state of charge of 30%. Under such conditions, a significant amount of the battery's energy is stored; in the event of mismanagement, or indeed an airside incident, this energy can lead to ignition and a fire. In this work, we investigate the effect on the battery of removing 99.1% of the total stored energy. The performance of 8Ah C 6 /LiFePO 4 pouch cells were measured following periods of calendar ageing at low voltages, at and well below the manufacturer's recommended value. Battery degradation was monitored using impedance spectroscopy and capacity tests; the results show that the cells stored at 2.3 V exhibited no change in cell capacity after 90 days; resistance rise was negligible. Energy-dispersive X-ray spectroscopy results indicate that there was no significant copper dissolution. To test the safety of the batteries at low voltages, external short-circuit tests were performed on the cells. While the cells discharged to 2.3 V only exhibited a surface temperature rise of 6 °C, cells at higher voltages exhibited sparks, fumes and fire.

  7. High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

    Directory of Open Access Journals (Sweden)

    Stefan Ryser

    Full Text Available We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs from peripheral blood mononuclear cells (PBMC of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii hemagglutinin (HA of influenza virus and (iii the extracellular domain of anaplastic lymphoma kinase (ALK. One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

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

    Science.gov (United States)

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

    2014-07-01

    Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. NK1 receptor fused to beta-arrestin displays a single-component, high-affinity molecular phenotype.

    Science.gov (United States)

    Martini, Lene; Hastrup, Hanne; Holst, Birgitte; Fraile-Ramos, Alberto; Marsh, Mark; Schwartz, Thue W

    2002-07-01

    Arrestins are cytosolic proteins that, upon stimulation of seven transmembrane (7TM) receptors, terminate signaling by binding to the receptor, displacing the G protein and targeting the receptor to clathrin-coated pits. Fusion of beta-arrestin1 to the C-terminal end of the neurokinin NK1 receptor resulted in a chimeric protein that was expressed to some extent on the cell surface but also accumulated in transferrin-labeled recycling endosomes independently of agonist stimulation. As expected, the fusion protein was almost totally silenced with respect to agonist-induced signaling through the normal Gq/G11 and Gs pathways. The NK1-beta-arrestin1 fusion construct bound nonpeptide antagonists with increased affinity but surprisingly also bound two types of agonists, substance P and neurokinin A, with high, normal affinity. In the wild-type NK1 receptor, neurokinin A (NKA) competes for binding against substance P and especially against antagonists with up to 1000-fold lower apparent affinity than determined in functional assays and in homologous binding assays. When the NK1 receptor was closely fused to G proteins, this phenomenon was eliminated among agonists, but the agonists still competed with low affinity against antagonists. In contrast, in the NK1-beta-arrestin1 fusion protein, all ligands bound with similar affinity independent of the choice of radioligand and with Hill coefficients near unity. We conclude that the NK1 receptor in complex with arrestin is in a high-affinity, stable, agonist-binding form probably best suited to structural analysis and that the receptor can display binding properties that are nearly theoretically ideal when it is forced to complex with only a single intracellular protein partner.

  10. Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

    Science.gov (United States)

    Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

    2014-10-01

    Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

  11. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    Energy Technology Data Exchange (ETDEWEB)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T. [Samsung Medical Center, Seoul (Korea, Republic of)

    2005-07-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2{alpha} of 5.2 min and late T1/2{beta} of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8{+-}0.5 ml/min, T1/2 of 4.1{+-}0.4 hr, and volume of distribution of 69.7{+-}4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors.

  12. Pharmacokinetics and biodistribution of a radioiodine labeled peptidomimetic ligand for high-affinity nerve growth factor receptors

    International Nuclear Information System (INIS)

    Jung, K. H.; Kim, D. H.; Paik, J. Y.; Koh, B. H.; Bae, J. S.; Choe, Y. S.; Lee, K. H.; Kim, B. T.

    2005-01-01

    Some of the obstacles for the clinical application of whole nerve growth factor (NGF) may be overcome by utilizing small molecule mimetics. We thus investigated the in vivo pharmacokinetics and biodistribution of a small cyclic peptide derived from NGF-[C(92-96)] with high receptor binding affinity. I-125 C(92-96) was labeled with the Bolton-Hunter method, and binding to TrkA/IgG chimeric protein was confirmed on a polyacrylamide gel after cross-linking. Pharmacokinetic analysis was performed in normal ICR mice intravenously injected with 0.5 MBq I-125 C(92-96) containing varying doses of C(92-96). Biodistribution studies were done at 6 h after injection. Cross-linkage analysis confirmed binding of I-125 C(92-96) to the high affinity NGF receptor, TrkA. Intravenously injected I-125 C(92-96) was cleared from the blood in a biexponential manner with an early T1/2α of 5.2 min and late T1/2β of 121.3 min. Log blood-concentration decreased over time with a k-slope of 0.0025, clearance of 11.8±0.5 ml/min, T1/2 of 4.1±0.4 hr, and volume of distribution of 69.7±4.6 ml. The pattern of elimination from the blood remained essentially unchanged regardless of the dose of added C(92-96), with dose-proportionate increases in AUCs and peak concentrations consistent with linear pharmacokinetics. Biodistribution studies demonstrated high kidney activity suggesting renal excretion of I-125 C(92-96). There were moderate levels of accumulation in the spleen, lungs and liver, followed by the myocardium and skeletal muscle, whereas brain uptake was low (< 0.2 %ID/gm). Intravenously administered C(92-96) follows linear pharmacokinetics, and is cleared from the circulation at a rate comparable to whole NGF despite its substantially smaller size. Although intravenous C(92-96) does not adequately reach brain tissue, clinically relevant doses can achieve major organ accumulation levels that may be sufficient to elicit biologic responses through NGF receptors

  13. Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

    Science.gov (United States)

    Sioud, Mouldy; Westby, Phuong; Vasovic, Vlada; Fløisand, Yngvar; Peng, Qian

    2018-04-16

    mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and

  14. High-level expression of a novel chromoplast phosphate transporter ClPHT4;2 is required for flesh color development in watermelon.

    Science.gov (United States)

    Zhang, Jie; Guo, Shaogui; Ren, Yi; Zhang, Haiying; Gong, Guoyi; Zhou, Ming; Wang, Guizhang; Zong, Mei; He, Hongju; Liu, Fan; Xu, Yong

    2017-02-01

    Chromoplast development plays a crucial role in controlling carotenoid content in watermelon flesh. Modern cultivated watermelons with colorful flesh are believed to originate from pale-colored and no-sweet progenitors. But the molecular basis of flesh color formation and regulation is poorly understood. More chromoplasts and released carotenoid globules were observed in the red-fleshed fruit of the 97103 cultivar than in the pale-colored fruits of the PI296341-FR line. Transcriptome profiles of these two materials identified Cla017962, predicted as ClPHT4;2, was dramatically up-regulated during flesh color formation. High ClPHT4;2 expression levels were closely correlated with increased flesh carotenoid contents among 198 representative watermelon accessions. Down-regulation of ClPHT4;2 expression in transgenic watermelons reduced the fruit carotenoid accumulation. ClPHT4;2 as a function of chromoplast-localized phosophate transporter was tested by heterologous expression into a yeast phosphate-uptake-defective mutant, western blotting, subcellular localization, and immunogold electron microscopy analysis. Two transcription factors, ClbZIP1 and ClbZIP2, were identified, which responded to ABA and sugar signaling to regulate ClPHT4;2 transcription only in cultivated watermelon species. Our findings suggest that elevated ClPHT4;2 gene expression is necessary for carotenoid accumulation, and may help to characterize the co-development of flesh color and sweetness during watermelon development and domestication. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  15. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    International Nuclear Information System (INIS)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [ 99m Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise

  16. Control of plant phosphate homeostasis by inositol pyrophosphates and the SPX domain.

    Science.gov (United States)

    Jung, Ji-Yul; Ried, Martina K; Hothorn, Michael; Poirier, Yves

    2018-02-01

    Proteins containing a SPX domain are involved in phosphate (Pi) homeostasis, including Pi transport and adaptation to Pi deficiency. The SPX domain harbors a basic surface binding Pi at low affinity and inositol pyrophosphates (PP-InsPs) at high affinity. Genetic and biochemical studies revealed that PP-InsPs serve as ligands for the SPX domain. Residues in the PHO1 SPX domain involved in PP-InsPs binding are critical for its Pi export activity, and the interaction between SPX proteins and the PHR1 transcription factor, which results in PHR1 inactivation, is promoted by PP-InsPs. Changes in PP-InsPs levels in response to Pi deficiency may thus contribute to the adaptation of plants to stress via the modulation of the activity of SPX-containing proteins and their interactors. Modulating PP-InsP levels or the affinity/specificity of the SPX domain for PP-InsP could potentially be used to engineer crops to maintain high yield under reduced Pi fertilizer input. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation.

    Science.gov (United States)

    López-Bucio, José; Hernández-Abreu, Esmeralda; Sánchez-Calderón, Lenin; Pérez-Torres, Anahí; Rampey, Rebekah A; Bartel, Bonnie; Herrera-Estrella, Luis

    2005-02-01

    Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.

  18. In vitro and in vivo evaluation of new radiolabeled neurotensin(8-13) analogues with high affinity for NT1 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Garayoa, Elisa Garcia-; Allemann-Tannahill, Lesley; Blaeuenstein, Peter; Willmann, Martine; Carrel-Remy, Nathalie; Tourwe, Dirk; Iterbeke, Koen; Conrath, Peter; Schubiger, P. August E-mail: schubiger@psi.ch

    2001-01-01

    The potential utility of neurotensin (NT) in cancer diagnosis and therapy is limited by its rapid degradation. New stabilized analogues were synthesized, labeled with [{sup 99m}Tc] and screened in vitro and in vivo. High affinity and rapid internalization were obtained in binding assays. Despite their longer human plasma half-lives, a rapid degradation was observed with low concentrations as used in biodistribution tests. The tumor uptake rates were rather low but tumor/blood ratios increased according to the stability raise.

  19. A potential therapy for chordoma via antibody-dependent cell-mediated cytotoxicity employing NK or high-affinity NK cells in combination with cetuximab.

    Science.gov (United States)

    Fujii, Rika; Schlom, Jeffrey; Hodge, James W

    2018-05-01

    OBJECTIVE Chordoma is a rare bone tumor derived from the notochord and is resistant to conventional therapies such as chemotherapy, radiotherapy, and targeting therapeutics. Expression of epidermal growth factor receptor (EGFR) in a large proportion of chordoma specimens indicates a potential target for therapeutic intervention. In this study the authors investigated the potential role of the anti-EGFR antibody cetuximab in immunotherapy for chordoma. METHODS Since cetuximab is a monoclonal antibody of the IgG1 isotype, it has the potential to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) employing natural killer (NK) cells as effectors. Polymorphisms in the CD16 allele expressed on NK cells have been shown to influence the degree of ADCC of tumor cells, with the high-affinity valine (V)/V allele being responsible for more lysis than the V/phenylalanine (F) or FF allele. Unfortunately, however, only approximately 10% of the population expresses the VV allele on NK cells. An NK cell line, NK-92, has now been engineered to endogenously express IL-2 and the high-affinity CD16 allele. These irradiated high-affinity (ha)NK cells were analyzed for lysis of chordoma cells with and without cetuximab, and the levels of lysis observed in ADCC were compared with those of NK cells from donors expressing the VV, VF, and FF alleles. RESULTS Here the authors demonstrate for the first time 1) that cetuximab in combination with NK cells can mediate ADCC of chordoma cells; 2) the influence of the NK CD16 polymorphism in cetuximab-mediated ADCC for chordoma cell lysis; 3) that engineered haNK cells-that is, cells transduced to express the CD16 V158 FcγRIIIa receptor-bind cetuximab with similar affinity to normal NK cells expressing the high-affinity VV allele; and 4) that irradiated haNK cells induce ADCC with cetuximab in chordoma cells. CONCLUSIONS These studies provide rationale for the use of cetuximab in combination with irradiated haNK cells for therapy for

  20. High-Affinity Methanotrophy Informed by Genome-Wide Analysis of Upland Soil Cluster Alpha (USCα) from Axel Heiberg Island, Canadian High Arctic

    Science.gov (United States)

    Rusley, C.; Onstott, T. C.; Lau, M.

    2017-12-01

    Methane (CH4) is a potent greenhouse gas whose proper budgeting is vital to climate predictions. Recent studies have identified upland Arctic mineral cryosols as consistent CH4 sinks, drawing CH4 from both the atmosphere and underlying anaerobic soil layers. Global atmospheric CH4 uptake is proposed to be mediated by high-affinity methanotrophs based on the detection of the marker gene pmoA (particulate methane monooxygenase beta subunit). However, a lack of pure cultures and scarcity of genomic information have hindered our understanding of their metabolic capabilities and versatility. Together with the missing genetic linkage between its pmoA and 16S ribosomal RNA (rRNA) gene, the factors that control the distribution and magnitude of high-affinity methanotrophy in the Arctic permafrost-affected region have remained elusive. Using 21 metagenomic datasets of surface soils obtained from long-term core incubation experiments,1 this bioinformatics study aimed to reconstruct the draft genome of the Upland Soil Cluster α-proteobacteria (USCα), the high-affinity methanotroph previously detected in the samples,2 and to determine its phylogeny and metabolic requirements. We obtained a genome bin containing the high-affinity form of the USCα-like pmoA gene. The 3.03 Mbp assembly is 91.6% complete with a unique set of single-copy marker genes. The 16S rRNA gene fragment of USCα belongs to the α-proteobacterial family Beijerinckiaceae. Genome annotation indicates possible formaldehyde oxidation via tetrahydromethanopterin-linked C1 transfer pathways, acetate utilization, carbon fixation via the Calvin-Benson-Bassham cycle, and glycogen production. Notably, the key enzymes for formaldehyde assimilation via the serine and ribulose monophosphate pathways are missing. The presence of genes encoding nitrate reductase and hemoglobin suggests adaptation to low O2 under water-logged conditions. Since USCα has versatile carbon metabolisms, it may not be an obligate methanotroph

  1. A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage

    DEFF Research Database (Denmark)

    Bach, Anders*; Clausen, Bettina H; Møller, Magda

    2012-01-01

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-d-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are ...... of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke....

  2. Mapping of the minimal inorganic phosphate transporting unit of human PiT2 suggests a structure universal to PiT-related proteins from all kingdoms of life

    DEFF Research Database (Denmark)

    Bøttger, Pernille; Pedersen, Lene

    2011-01-01

    -keeping functions. Alignment of protein sequences representing PiT family members from all kingdoms reveals the presence of conserved amino acids and that bacterial phosphate permeases and putative phosphate permeases from archaea lack substantial parts of the protein sequence when compared to the mammalian Pi...... PiT2 histidine, H502, and the human PiT1 glutamate, E70, - both conserved in eukaryotic PiT family members - are critical for Pi transport function. Noticeably, human PiT2 H502 is located in the C-terminal PiT family signature sequence, and human PiT1 E70 is located in ProDom domains characteristic....... Conclusions The results suggest that the overall structure of the Pi-transporting unit of the PiT family proteins has remained unchanged during evolution. Moreover, in combination, our studies of the gene structure of the human PiT1 and PiT2 genes (SLC20A1 and SLC20A2, respectively) and alignment of protein...

  3. A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

    Science.gov (United States)

    Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

    2016-01-01

    Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

  4. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    International Nuclear Information System (INIS)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H.

    1988-01-01

    Binding studies were performed with two 125 I-labeled Bacillus thuringiensis δ-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One δ-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other δ-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis δ-endotoxins active against M. sexta compete for binding of 125 I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles

  5. Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis.

    Science.gov (United States)

    Hastrup, H; Schwartz, T W

    1996-12-16

    The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.

  6. BIOCHEMICAL AND ULTRASTRUCTURAL ASPECTS OF CA2+ TRANSPORT BY MITOCHONDRIA OF THE HEPATOPANCREAS OF THE BLUE CRAB CALLINECTES SAPIDUS

    Science.gov (United States)

    Chen, Chung-Ho; Greenawalt, John W.; Lehninger, Albert L.

    1974-01-01

    Mitochondria isolated from the hepatopancreas of the blue crab Callinectes sapidus show up to 12-fold stimulation of respiration on addition of Ca2+, which is accompanied by Ca2+ accumulation (Ca2+:site = 1.9) and H+ ejection (H+:Ca2+ = 0.85). Sr2+ and Mn2+ are also accumulated; Mg2+ is not. A strongly hypertonic medium (383 mosM), Mg2+, and phosphate are required for maximal Ca2+ uptake. Ca2+ uptake takes precedence over oxidative phosphorylation of ADP for respiratory energy. Once Ca2+ is accumulated by the crab mitochondria, it is stable and only very slowly released, even by uncoupling agents. ATP hydrolysis also supports Ca2+ uptake. Respiration-inhibited crab hepatopancreas mitochondria show both high-affinity and low-affinity Ca2+-binding sites, which are inactive in the presence of uncoupling agents. Crab hepatopancreas mitochondria have an enormous capacity for accumulation of Ca2+, up to 5,500 ng-atoms Ca2+ per mg protein, with an equivalent amount of phosphate. Freshly isolated mitochondria contain very large amounts of Ca2+, Mg2+, phosphate, K+, and Na+; their high Ca2+ content is a reflection of the vary large amount of extra-mitochondrial Ca2+ in the whole tissue. Electron microscopy of crab mitochondria loaded with Ca2+ and phosphate showed large electron-dense deposits, presumably of precipitated calcium phosphate. They consisted of bundles of needle-like crystals, whereas Ca2+-loaded rat liver mitochondria show only amorphous deposits of calcium phosphate under similar conditions. The very pronounced capacity of crab hepatopancreas mitochondria for transport of Ca2+ appears to be adapted to a role in the storage and release of Ca2+ during the molting cycle of this crustacean. PMID:4827906

  7. Pivotal role of α2 Na+ pumps and their high affinity ouabain binding site in cardiovascular health and disease

    Science.gov (United States)

    Chen, Ling; Hamlyn, John M.; Leenen, Frans H. H.; Lingrel, Jerry B.; Wier, W. Gil; Zhang, Jin

    2016-01-01

    Abstract Reduced smooth muscle (SM)‐specific α2 Na+ pump expression elevates basal blood pressure (BP) and increases BP sensitivity to angiotensin II (Ang II) and dietary NaCl, whilst SM‐α2 overexpression lowers basal BP and decreases Ang II/salt sensitivity. Prolonged ouabain infusion induces hypertension in rodents, and ouabain‐resistant mutation of the α2 ouabain binding site (α2R/R mice) confers resistance to several forms of hypertension. Pressure overload‐induced heart hypertrophy and failure are attenuated in cardio‐specific α2 knockout, cardio‐specific α2 overexpression and α2R/R mice. We propose a unifying hypothesis that reconciles these apparently disparate findings: brain mechanisms, activated by Ang II and high NaCl, regulate sympathetic drive and a novel neurohumoral pathway mediated by both brain and circulating endogenous ouabain (EO). Circulating EO modulates ouabain‐sensitive α2 Na+ pump activity and Ca2+ transporter expression and, via Na+/Ca2+ exchange, Ca2+ homeostasis. This regulates sensitivity to sympathetic activity, Ca2+ signalling and arterial and cardiac contraction. PMID:27350568

  8. Ferric reductase genes involved in high-affinity iron uptake are differentially regulated in yeast and hyphae of Candida albicans.

    Science.gov (United States)

    Jeeves, Rose E; Mason, Robert P; Woodacre, Alexandra; Cashmore, Annette M

    2011-09-01

    The pathogenic yeast Candida albicans possesses a reductive iron uptake system which is active in iron-restricted conditions. The sequestration of iron by this mechanism initially requires the reduction of free iron to the soluble ferrous form, which is catalysed by ferric reductase proteins. Reduced iron is then taken up into the cell by a complex of a multicopper oxidase protein and an iron transport protein. Multicopper oxidase proteins require copper to function and so reductive iron and copper uptake are inextricably linked. It has previously been established that Fre10 is the major cell surface ferric reductase in C. albicans and that transcription of FRE10 is regulated in response to iron levels. We demonstrate here that Fre10 is also a cupric reductase and that Fre7 also makes a significant contribution to cell surface ferric and cupric reductase activity. It is also shown, for the first time, that transcription of FRE10 and FRE7 is lower in hyphae compared to yeast and that this leads to a corresponding decrease in cell surface ferric, but not cupric, reductase activity. This demonstrates that the regulation of two virulence determinants, the reductive iron uptake system and the morphological form of C. albicans, are linked. Copyright © 2011 John Wiley & Sons, Ltd.

  9. International symposium on cellular and molecular biology of phosphate and phosphorylated compounds in microorganisms: Proceedings

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1993-12-31

    This report contains the abstracts of papers presented at the conference. Attention is focused on the following topics: regulation of phosphate metabolism in bacteria; structure-function of alkaline phosphatase; regulation of phosphate metabolism in yeast; transport of phosphate and phosphorylated compounds; and phosphate regulation in pathogenesis and secondary metabolism.

  10. The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake

    DEFF Research Database (Denmark)

    Hvidberg, Vibeke; Jacobsen, Christian; Strong, Roland K

    2005-01-01

    Neutrophil-gelatinase-associated lipocalin (NGAL) is a prominent protein of specific granules of human neutrophils also synthesized by epithelial cells during inflammation. NGAL binds bacterial siderophores preventing bacteria from retrieving iron from this source. Also, NGAL may be important in ...... by surface plasmon resonance analysis. Furthermore, a rat yolk sac cell line known to express high levels of megalin, endocytosed NGAL by a mechanism completely blocked by an antibody against megalin.......Neutrophil-gelatinase-associated lipocalin (NGAL) is a prominent protein of specific granules of human neutrophils also synthesized by epithelial cells during inflammation. NGAL binds bacterial siderophores preventing bacteria from retrieving iron from this source. Also, NGAL may be important...

  11. Rhodamine-labeled 2beta-carbomethoxy-3beta-(3,4-dichlorophenyl)tropane analogues as high-affinity fluorescent probes for the dopamine transporter

    DEFF Research Database (Denmark)

    Cha, Joo Hwan; Zou, Mu-Fa; Adkins, Erika M

    2005-01-01

    linker. The resulting 2-substituted (5) and N-substituted (9) rhodamine-labeled ligands provided the highest DAT binding affinities expressed in COS-7 cells (Ki= 27 and 18 nM, respectively) in the series. Visualization of the DAT with 5 and 9 was demonstrated by confocal fluorescence laser scanning...

  12. Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype.

    Science.gov (United States)

    Decker, T; Schneller, F; Kronschnabl, M; Dechow, T; Lipford, G B; Wagner, H; Peschel, C

    2000-05-01

    CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B

  13. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    International Nuclear Information System (INIS)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N.; Moran, Jeffery H.; Prather, Paul L.

    2013-01-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB 1 Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB 2 Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB 2 Rs (hCB 2 Rs). The affinity of cannabinoids for hCB 2 Rs was determined by competition binding studies employing CHO-hCB 2 membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB 2 cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB 2 Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB 2 Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ 9 -tetrahydrocannabinol (Δ 9 -THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB 2 R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB 2 Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB 2 Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB 1 and CB 2 Rs. - Highlights: • JWH-018 and JWH-073 are synthetic cannabinoids present in abused K2

  14. Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Rajasekaran, Maheswari; Brents, Lisa K.; Franks, Lirit N. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Moran, Jeffery H. [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Arkansas Department of Public Health, Public Health Laboratory, Little Rock, AR 72205 (United States); Prather, Paul L., E-mail: pratherpaull@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States)

    2013-06-01

    K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB{sub 1}Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB{sub 2}Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB{sub 2}Rs (hCB{sub 2}Rs). The affinity of cannabinoids for hCB{sub 2}Rs was determined by competition binding studies employing CHO-hCB{sub 2} membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB{sub 2} cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB{sub 2}Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB{sub 2}Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ{sup 9}-tetrahydrocannabinol (Δ{sup 9}-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB{sub 2}R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB{sub 2}Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB{sub 2}Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB{sub 1} and CB{sub 2}Rs. - Highlights: • JWH-018

  15. Comparison of high affinity binding of {sup 3}H-proadifen and {sup 3}H-(-)-cocaine t rat liver membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S.B. [Astra Arcus AB, Dept. of Neuropharmacology, Soedertaelje (Sweden)

    1995-06-01

    The characteristics of the binding of {sup 3}H-proadifen to rat liver membranes were studied and compared to those of {sup 3}H-cocaine. It was found that {sup 3}H-proadifen was bound reversibly with high affinity (K{sub D}=1.8{+-}0.5 nM) and large capacity (B{sub max}=2010{+-}340 pmol/g wet tissue) to liver membranes. The corresponding values for the {sup 3}H-cocaine binding were 3.5 nM and 1000 pmol/g wet tissue. The binding of {sup 3}H-proadifen was mainly localised to the microsomal fraction. The number of binding sites was not increased by treatment of rats with phenobarbitone. With 1 {mu}M CdCl{sub 2} in the incubation buffer it was possible to differentiate between two {sup 3}H-cocaine binding sites with K{sub d} values of 1.6 and 7.7 nM and B{sub max} values of 280 and 940 pmol/g wet liver tissue. S-(-)-Alaproclate inhibited the binding of {sup 3}H-proadifen and {sup 3}H-cocaine inhibited the binding of {sup 3}H-proadifen (IC{sub 50}=10 nM) and proadifen that of {sup 3}H-cocaine (IC{sub 50}=1 nM). There was a high correlation coefficient (r{sub r}=0.972; P<0.01; n=12) in the Spearman rank test between the inhibitory potencies of compounds examined in both systems. Beside some potent alaproclate analogues a couple of compounds had moderately high affinity (IC{sub 50}=100-500 nM): chloroquine, phenoxybenzamine, amitriptyline, ajmaline, remoxipride, imipramine and (-)-alaprenolol. CdCl{sub 2}, ZnCl{sub 2} and CuCl{sub 2} inhibited the binding of both ligands with low Hill coefficients, indicating heterogeneous binding sites. The inhibition curve of Cd{sup 2+} on the cocaine binding was biphasic with a high affinity part around 50 nM and a low affinity part at 15{mu}M. The similarity of the characteristics of the binding of these ligands with that of {sup 3}H-alaproclate is discussed. It is suggested that all three compounds bind to the same sites, although additional binding sites seem to exist for proadifen. (au) (9 refs.).

  16. Mapping of barley alpha-amylases and outer subsite mutants reveals dynamic high-affinity subsites and barriers in the long substrate binding cleft

    DEFF Research Database (Denmark)

    Kandra, L.; Abou Hachem, Maher; Gyemant, G.

    2006-01-01

    Subsite affinity maps of long substrate binding clefts in barley alpha-amylases, obtained using a series of maltooligosaccharides of degree of polymerization of 3-12, revealed unfavorable binding energies at the internal subsites -3 and -5 and at subsites -8 and +3/+4 defining these subsites...... as binding barriers. Barley a-amylase I mutants Y105A and T212Y at subsite -6 and +4 resulted in release or anchoring of bound substrate, thus modifying the affinities of other high-affinity subsites (-2 and +2) and barriers. The double mutant Y105A-T212Y displayed a hybrid subsite affinity profile......, converting barriers to binding areas. These findings highlight the dynamic binding energy distribution and the versatility of long maltooligosaccharide derivatives in mapping extended binding clefts in a-amylases....

  17. Two classes of astrocytes in the adult human and pig retina in terms of their expression of high affinity NGF receptor (TrkA).

    Science.gov (United States)

    Ruiz-Ederra, Javier; Hitchcock, Peter F; Vecino, Elena

    2003-02-13

    Astrocytes have been implicated in axon guidance and synaptic regeneration in the retina and these processes involve activation of the high affinity nerve growth factor receptor, known as the tyrosine kinase A (TrkA) receptor. The purpose of the present study was to characterize the expression of TrkA in astrocytes of the adult pig and human retina. To this end, sections of human and pig retinas were immunolabeled with a combination of antibodies to glial fibrillary acidic protein (GFAP) and TrkA. Our study revealed that most of the GFAP-positive cells express TrkA, whereas a rare, novel subpopulation of astrocytes was found to be devoid of TrkA. Our results support the idea that astrocytes play an important neurotrophic role in the retina.

  18. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

    Science.gov (United States)

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 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 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. ADCC employing an NK cell line (haNK) expressing the high affinity CD16 allele with avelumab, an anti-PD-L1 antibody.

    Science.gov (United States)

    Jochems, Caroline; Hodge, James W; Fantini, Massimo; Tsang, Kwong Y; Vandeveer, Amanda J; Gulley, James L; Schlom, Jeffrey

    2017-08-01

    NK-92 cells, and their derivative, designated aNK, were obtained from a patient with non-Hodgkin lymphoma. Prior clinical studies employing adoptively transferred irradiated aNK cells have provided evidence of clinical benefit and an acceptable safety profile. aNK cells have now been engineered to express IL-2 and the high affinity (ha) CD16 allele (designated haNK). Avelumab is a human IgG1 anti-PD-L1 monoclonal antibody, which has shown evidence of clinical activity in a range of human tumors. Prior in vitro studies have shown that avelumab has the ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) of human tumor cells when combined with NK cells. In the studies reported here, the ability of avelumab to enhance the lysis of a range of human carcinoma cells by irradiated haNK cells via the ADCC mechanism is demonstrated; this ADCC is shown to be inhibited by anti-CD16 blocking antibody and by concanamycin A, indicating the use of the granzyme/perforin pathway in tumor cell lysis. Studies also show that while NK cells have the ability to lyse aNK or haNK cells, the addition of NK cells to irradiated haNK cells does not inhibit haNK-mediated lysis of human tumor cells, with or without the addition of avelumab. Avelumab-mediated lysis of tumor cells by irradiated haNK cells is also shown to be similar to that of NK cells bearing the V/V Fc receptor high affinity allele. These studies thus provide the rationale for the clinical evaluation of the combined use of avelumab with that of irradiated adoptively transferred haNK cells. © 2017 UICC.

  1. Pentose phosphates in nucleoside interconversion and catabolism.

    Science.gov (United States)

    Tozzi, Maria G; Camici, Marcella; Mascia, Laura; Sgarrella, Francesco; Ipata, Piero L

    2006-03-01

    Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.

  2. Uranium from phosphate ores

    International Nuclear Information System (INIS)

    Hurst, F.J.

    1983-01-01

    The following topics are described briefly: the way phosphate fertilizers are made; how uranium is recovered in the phosphate industry; and how to detect covert uranium recovery operations in a phsophate plant

  3. Studies on ion transport of Eu (III) and Gd (III) through supported liquid membranes containing Di-(2-ethylhexyl) phosphate, as a carrier

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez, L.; Bolarin, A.M.; Sanchez, F.; Patino, F.; Rivera, I.; Salinas, E. [Area de Investigaciones en Materiales y Metalurgia, Universidad Autonoma del Estado de Hidalgo, Carretera Pachuca-Tulancingo Km. 4.5, 42072 Pachuca, Hidalgo (Mexico)]. e-mail: hcruz@uaeh.reduaeh.mx

    2007-07-01

    In this work, permeation of Eu (III) and Gd (III) between aqueous oxide solutions through supported membrane containing di-(2-ethylhexyl) phosphate as a carrier and kerosene has been studied. Mass transfer of these cations has been determined in terms of permeability coefficients (P) by measurement of metal concentrations in strip solution. The permeability coefficient increases with increase in pH acidity of feed solution ranged from pH 0.5 to 2.5 while that of strip solution was kept constant at 0.1 mol L{sup -1} HNO{sub 3}. Thus, the results obtained show that it is possible to get up to 93% of percentage of extraction. (Author)

  4. Studies on ion transport of Eu (III) and Gd (III) through supported liquid membranes containing Di-(2-ethylhexyl) phosphate, as a carrier

    International Nuclear Information System (INIS)

    Hernandez, L.; Bolarin, A.M.; Sanchez, F.; Patino, F.; Rivera, I.; Salinas, E.

    2007-01-01

    In this work, permeation of Eu (III) and Gd (III) between aqueous oxide solutions through supported membrane containing di-(2-ethylhexyl) phosphate as a carrier and kerosene has been studied. Mass transfer of these cations has been determined in terms of permeability coefficients (P) by measurement of metal concentrations in strip solution. The permeability coefficient increases with increase in pH acidity of feed solution ranged from pH 0.5 to 2.5 while that of strip solution was kept constant at 0.1 mol L -1 HNO 3 . Thus, the results obtained show that it is possible to get up to 93% of percentage of extraction. (Author)

  5. Characterization of Phosphate Transporters BdPT4 and BdPT8 in Mycorrhizal and Non-Mychorrhizal Brachypodium distachyon

    DEFF Research Database (Denmark)

    Clausen, Signe Sandbech

    PT proteins were expressed in cells where direct PTs are believed to perform their function, as well as in the vascular tissue. In NM plants, BdPT4 was localized to the plasma membrane whereas BdPT8 accumulated in the endoplasmic reticulum (ER) in the secretory pathway. A similar expression pattern...... was observed in non-arbusculated cells of AM plants. In arbusculated cells, BdPT8 was detected in the ER while BdPT4 was localized to the plasma membrane and the periarbuscular membrane (PAM) surrounding the arbuscular trunk and thick branches, which further supported a role of both BdPTs in direct Pi uptake......Phosphorus (P) is an essential plant nutrient in agricultural production but the P rock reserves used for production of fertilizers are currently being depleted. One approach to reduce the demand on P fertilizers is to optimize the phosphate (Pi) uptake and utilization efficiency of crop plants...

  6. Molecular analysis of phosphate limitation in Geobacteraceae during the bioremediation of a uranium-contaminated aquifer

    Energy Technology Data Exchange (ETDEWEB)

    N' Guessan, L.A.; Elifantz, H.; Nevin, K.P.; Mouser, P.J.; Methe, B.; Woodard, T. L.; Manley, K.; Williams, K. H.; Wilkins, M. J.; Larsen, J.T.; Long, P. E.; Lovley, D. R.

    2009-09-01

    Nutrient limitation is an environmental stress that may reduce the effectiveness of bioremediation strategies, especially when the contaminants are organic compounds or when organic compounds are added to promote microbial activities such as metal reduction. Genes indicative of phosphate-limitation were identified via microarray analysis of chemostat cultures of Geobacter sulfureducens. This analysis revealed that genes in the pst-pho operon, which is associated with a high affinity phosphate uptake system in other microorganisms, had significantly higher transcript abundance under phosphate-limiting conditions, with the genes pstB and phoU the most up-regulated. Quantitative PCR analysis of pstB and phoU transcript levels in G. sulfurreducens grown in chemostats demonstrated that the expression of these genes increased when phosphate was removed from the culture medium. Transcripts of pstB and phoU within the subsurface Geobacter species predominating during an in situ uranium bioremediation field experiment were more abundant than in chemostat cultures of G. sulfurreducens that were not limited for phosphate. Addition of phosphate to incubations of subsurface sediments did not stimulate dissimilatory metal reduction. The added phosphate was rapidly adsorbed onto the sediments. The results demonstrate that Geobacter species can effectively reduce U(VI) even when experiencing suboptimal phosphate concentrations and that increasing phosphate availability with phosphate additions is difficult to achieve due to the high reactivity of this compound. This transcript-based approach developed for diagnosing phosphate limitation should be applicable to assessing the potential need for additional phosphate in other bioremediation processes.

  7. Transportation

    National Research Council Canada - National Science Library

    Adams, James; Carr, Ron; Chebl, Maroun; Coleman, Robert; Costantini, William; Cox, Robert; Dial, William; Jenkins, Robert; McGovern, James; Mueller, Peter

    2006-01-01

    ...., trains, ships, etc.) and maximizing intermodal efficiency. A healthy balance must be achieved between the flow of international commerce and security requirements regardless of transportation mode...

  8. Transportation

    International Nuclear Information System (INIS)

    Anon.

    1998-01-01

    Here is the decree of the thirtieth of July 1998 relative to road transportation, to trade and brokerage of wastes. It requires to firms which carry out a road transportation as well as to traders and to brokers of wastes to declare their operations to the prefect. The declaration has to be renewed every five years. (O.M.)

  9. An HIV-1 encoded peptide mimics the DNA binding loop of NF-κB and binds thioredoxin with high affinity

    International Nuclear Information System (INIS)

    Su Guoping; Wang Min; Taylor, Ethan Will

    2005-01-01

    Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-κB), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-κB via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-κB/Rel family of transcription factors by the putative HIV-1 pro-fs protein

  10. Synthesis and characterization of [{sup 76}Br]-labeled high-affinity A{sub 3} adenosine receptor ligands for positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Kiesewetter, Dale O. [Positron Emission Tomography Radiochemistry Group, NIBIB, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States)], E-mail: dk7k@nih.gov; Lang Lixin; Ma Ying; Bhattacharjee, Abesh Kumar [Positron Emission Tomography Radiochemistry Group, NIBIB, Clinical Center, National Institutes of Health, Bethesda, MD 20892 (United States); Gao, Zhan-Guo; Joshi, Bhalchandra V.; Melman, Artem; Castro, Sonia de; Jacobson, Kenneth A. [Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)

    2009-01-15

    Introduction: Bromine-76-radiolabeled analogues of previously reported high-affinity A{sub 3} adenosine receptor (A{sub 3}AR) nucleoside ligands have been prepared as potential radiotracers for positron emission tomography. Methods: The radiosyntheses were accomplished by oxidative radiobromination on the N{sup 6}-benzyl moiety of trimethyltin precursors. Biodistribution studies of the kinetics of uptake were conducted in awake rats. Results: We prepared an agonist ligand {l_brace}[{sup 76}Br](1'S,2'R,3'S,4'R,5'S)-4'-{l_brace}2-chloro-6-[(3-bromophenylmethyl)amino] purin-9-yl{r_brace}-1'-(methylaminocarbonyl)bicyclo[3.1.0]hexane-2',3'-diol (MRS3581){r_brace} in 59% radiochemical yield with a specific activity of 19.5 GBq/{mu}mol and an antagonist ligand {l_brace}[{sup 76}Br](1'R,2'R,3'S,4'R,5'S)-4'-(6-(3-bromobenzylamino) -2-chloro-9H-purin-9-yl)bicyclo[3.1.0]hexane-2',3'-diol (MRS5147){r_brace} in 65% radiochemical yield with a specific activity of 22 GBq/{mu}mol. The resultant products exhibited the expected high affinity (K{sub i}{approx}0.6 nM) and specific binding at the human A{sub 3}AR in vitro. Biodistribution studies in the rat showed uptake in the organs of excretion and metabolism. The antagonist MRS5147 exhibited increasing uptake in testes, an organ that contains significant quantities of A{sub 3}AR, over a 2-h time course, which suggests the presence of a specific A{sub 3}AR retention mechanism. Conclusion: We were able to compare uptake of the [{sup 76}Br]-labeled antagonist MRS5147 to [{sup 76}Br]agonist MRS3581. The antagonist MRS5147 shows increasing uptake in the testes, an A{sub 3}AR-rich tissue, suggesting that this ligand may have promise as a molecular imaging agent.

  11. Phosphate acquisition efficiency and phosphate starvation tolerance ...

    Indian Academy of Sciences (India)

    3Department of Genetics and Plant Breeding, College of Agriculture, Lembucherra, Tripura 799 ... vated in soil like red and lateritic or acid, with low soluble phosphate content. ..... activation of genes involved in the adaptation of Arabidopsis to.

  12. N- and C-terminally truncated forms of glucose-dependent insulinotropic polypeptide are high-affinity competitive antagonists of the human GIP receptor

    DEFF Research Database (Denmark)

    Hansen, L S; Sparre-Ulrich, A H; Christensen, M.

    2016-01-01

    functions and pharmacological potential. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here we characterize eight N-terminal trrncations of human GIP(1-30)NH2 : GIP(2- to 9-30)NH2 . EXPERIMENTAL APPROACH: COS-7 cells were transiently transfected with the human GIP receptor and assessed...... displayed lower affinities (Ki 2.3-347 nM) with highest affinities of GIP(3-30)NH2 and (5-30)NH2 . Agonism was only observed for GIP(1-30)NH2 with an Emax on 100% of GIP(1-42) and GIP(2-30)NH2 (Emax 20%). GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and right-shifts of the GIP(1-42)-response......, but superior antagonist GIP(3-30)NH2 , that together with GIP(5-30)NH2 were high-affinity competitive antagonist and thus may be suitable tool compounds for basic GIP research and future pharmacological interventions....

  13. Premature Aging Phenotype in Mice Lacking High-Affinity Nicotinic Receptors: Region-Specific Changes in Layer V Pyramidal Cell Morphology.

    Science.gov (United States)

    Konsolaki, Eleni; Skaliora, Irini

    2015-08-01

    The mechanisms by which aging leads to alterations in brain structure and cognitive deficits are unclear. Α deficient cholinergic system has been implicated as one of the main factors that could confer a heightened vulnerability to the aging process, and mice lacking high-affinity nicotinic receptors (β2(-/-)) have been proposed as an animal model of accelerated cognitive aging. To date, however, age-related changes in neuronal microanatomy have not been studied in these mice. In the present study, we examine the neuronal structure of yellow fluorescent protein (YFP(+)) layer V neurons in 2 cytoarchitectonically distinct cortical regions in wild-type (WT) and β2(-/-) animals. We find that (1) substantial morphological differences exist between YFP(+) cells of the anterior cingulate cortex (ACC) and primary visual cortex (V1), in both genotypes; (2) in WT animals, ACC cells are more susceptible to aging compared with cells in V1; and (3) β2 deletion is associated with a regionally and temporally specific increase in vulnerability to aging. ACC cells exhibit a prematurely aged phenotype already at 4-6 months, whereas V1 cells are spared in adulthood but strongly affected in old animals. Collectively, our data reveal region-specific synergistic effects of aging and genotype and suggest distinct vulnerabilities in V1 and ACC neurons. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer's disease: an autoradiographic study using 3H-hemicholinium-3

    International Nuclear Information System (INIS)

    Rodriguez-Puertas, R.; Pazos, A.; Zarranz, J.J.; Pascual, J.

    1994-01-01

    H-hemicholinium-3 (H-HC-3) binding, a marker of the presynaptic high-affinity choline uptake carrier (HACU), was measured by autoradiography in several brain regions of 17 Alzheimer's disease (AD) patients and of 11 matched controls. A significant decrease in the density of H-HC-3 binding sites was found in entorhinal cortex, hippocampus and layers I-III of the frontal cortex. By contrast, in the caudate-putamen the number of H-HC-3 binding sites in AD cases was comparable to that of control striata. These data concur with previous results using classical presynaptic markers and reflect the loss in the activity of HACU, and, hence, in the synthesis of acetylcholine, that selectively occurs in cortical areas of AD brains due to the degeneration of presynaptic cholinergic terminals arising from the basal forebrain. However, the relatively low mean reduction in HACU in cortical areas (-40 %), together with the apparent indemnity of this marker in certain severely demented AD cases, suggest that AD dementia cannot be explained simply by the loss of presynaptic terminals originating in the basal forebrain. These data seem to be a good explanation for the poor response to cholinergic replacement in AD. (author)

  15. High Affinity vs. Native Fibronectin in the Modulation of αvβ3 Integrin Conformational Dynamics: Insights from Computational Analyses and Implications for Molecular Design.

    Directory of Open Access Journals (Sweden)

    Antonella Paladino

    2017-01-01

    Full Text Available Understanding how binding events modulate functional motions of multidomain proteins is a major issue in chemical biology. We address several aspects of this problem by analyzing the differential dynamics of αvβ3 integrin bound to wild type (wtFN10, agonist or high affinity (hFN10, antagonist mutants of fibronectin. We compare the dynamics of complexes from large-scale domain motions to inter-residue coordinated fluctuations to characterize the distinctive traits of conformational evolution and shed light on the determinants of differential αvβ3 activation induced by different FN sequences. We propose an allosteric model for ligand-based integrin modulation: the conserved integrin binding pocket anchors the ligand, while different residues on the two FN10's act as the drivers that reorganize relevant interaction networks, guiding the shift towards inactive (hFN10-bound or active states (wtFN10-bound. We discuss the implications of results for the design of integrin inhibitors.

  16. High affinity radiopharmaceuticals based upon lansoprazole for PET imaging of aggregated tau in Alzheimer's disease and progressive supranuclear palsy: synthesis, preclinical evaluation, and lead selection.

    Science.gov (United States)

    Fawaz, Maria V; Brooks, Allen F; Rodnick, Melissa E; Carpenter, Garrett M; Shao, Xia; Desmond, Timothy J; Sherman, Phillip; Quesada, Carole A; Hockley, Brian G; Kilbourn, Michael R; Albin, Roger L; Frey, Kirk A; Scott, Peter J H

    2014-08-20

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [(18)F]lansoprazole, [(11)C]N-methyl lansoprazole, and [(18)F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [(18)F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials.

  17. High Affinity Radiopharmaceuticals Based Upon Lansoprazole for PET Imaging of Aggregated Tau in Alzheimer’s Disease and Progressive Supranuclear Palsy: Synthesis, Preclinical Evaluation, and Lead Selection

    Science.gov (United States)

    2014-01-01

    Abnormally aggregated tau is the hallmark pathology of tauopathy neurodegenerative disorders and is a target for development of both diagnostic tools and therapeutic strategies across the tauopathy disease spectrum. Development of carbon-11- or fluorine-18-labeled radiotracers with appropriate affinity and specificity for tau would allow noninvasive quantification of tau burden using positron emission tomography (PET) imaging. We have synthesized [18F]lansoprazole, [11C]N-methyl lansoprazole, and [18F]N-methyl lansoprazole and identified them as high affinity radiotracers for tau with low to subnanomolar binding affinities. Herein, we report radiosyntheses and extensive preclinical evaluation with the aim of selecting a lead radiotracer for translation into human PET imaging trials. We demonstrate that [18F]N-methyl lansoprazole, on account of the favorable half-life of fluorine-18 and its rapid brain entry in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau over amyloid, is the lead compound for progression into clinical trials. PMID:24896980

  18. Carbon-11 labelling of eticlopride in two different positions - a selective high-affinity ligand for the study of dopamine D-2 receptors using PET

    International Nuclear Information System (INIS)

    Halldin, Christer; Hall, Haakan

    1990-01-01

    A new highly selective high-affinity dopamine D-2 receptor antagonist, eticlopride ((-)-(S)-5-chloro-3-ethyl-N-(1-ethyl-2-pyrrolidinyl)methyl)-6-methoxysalicylamide), was labelled with 11 C in two different positions ([N-ethyl- 11 C]eticlopride (I) and ([methyl- 11 C]eticlopride (II)). Product I was prepared by N-alkylation of the N-desethyl compound with [ 11 C]ethyl iodide. II was prepared by O-alkylation of the diphenolic precursor with [ 11 C]methyl iodide followed by separation of the two methylated products. The radiochemical yields were 15-20% (EOB) with an overall synthesis time of 45-60 min. Both compounds were isolated by semi-preparative HPLC and the radiochemical purity was in both cases > 99%. I was injected i.v. in a Cynomolgus monkey and brain radioactivity was measured by positron emission tomography (PET). The specific activity was 70 Ci/mmol at time of injection. There was a marked accumulation of radioactivity in the basal ganglia, regions known to have a high density of dopamine D-2 receptors. (author)

  19. Selective cortical decrease of high-affinity choline uptake carrier in Alzheimer`s disease: an autoradiographic study using {sup 3}H-hemicholinium-3

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Puertas, R; Pazos, A [Dept. of Physiology and Pharmacology, Unit of Pharmacology, Univ. of Cantabria, Santander (Spain); Zarranz, J J [Dept. of Neuroscience, Univ. of the Basque Country, Leioa (Spain); Pascual, J [Dept. of Medicine, Service of Neurology, Univ. Hospital ` Marques de Valdecilla` , Univ. of Cantabria, Santander (Spain)

    1994-12-31

    H-hemicholinium-3 (H-HC-3) binding, a marker of the presynaptic high-affinity choline uptake carrier (HACU), was measured by autoradiography in several brain regions of 17 Alzheimer`s disease (AD) patients and of 11 matched controls. A significant decrease in the density of H-HC-3 binding sites was found in entorhinal cortex, hippocampus and layers I-III of the frontal cortex. By contrast, in the caudate-putamen the number of H-HC-3 binding sites in AD cases was comparable to that of control striata. These data concur with previous results using classical presynaptic markers and reflect the loss in the activity of HACU, and, hence, in the synthesis of acetylcholine, that selectively occurs in cortical areas of AD brains due to the degeneration of presynaptic cholinergic terminals arising from the basal forebrain. However, the relatively low mean reduction in HACU in cortical areas (-40 %), together with the apparent indemnity of this marker in certain severely demented AD cases, suggest that AD dementia cannot be explained simply by the loss of presynaptic terminals originating in the basal forebrain. These data seem to be a good explanation for the poor response to cholinergic replacement in AD. (author).

  20. An in vitro-identified high-affinity nucleosome-positioning signal is capable of transiently positioning a nucleosome in vivo

    Directory of Open Access Journals (Sweden)

    Gracey Lia E

    2010-07-01

    Full Text Available Abstract Background The physiological function of eukaryotic DNA occurs in the context of nucleosomal arrays that can expose or obscure defined segments of the genome. Certain DNA sequences are capable of strongly positioning a nucleosome in vitro, suggesting the possibility that favorable intrinsic signals might reproducibly structure chromatin segments. As high-throughput sequencing analyses of nucleosome coverage in vitro and in vivo have become possible, a vigorous debate has arisen over the degree to which intrinsic DNA:nucleosome affinities orchestrate the in vivo positions of nucleosomes, thereby controlling physical accessibility of specific sequences in DNA. Results We describe here the in vivo consequences of placing a synthetic high-affinity nucleosome-positioning signal, the 601 sequence, into a DNA plasmid vector in mice. Strikingly, the 601 sequence was sufficient to position nucleosomes during an early phase after introduction of the DNA into the mice (when the plasmid vector transgene was active. This positioning capability was transient, with a loss of strong positioning at a later time point when the transgenes had become silent. Conclusions These results demonstrate an ability of DNA sequences selected solely for nucleosome affinity to organize chromatin in vivo, and the ability of other mechanisms to overcome these interactions in a dynamic nuclear environment.

  1. SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of [3H]dextromethorphan and σligands to guinea pig brain

    International Nuclear Information System (INIS)

    Klein, M.; Canoll, P.D.; Musacchio, J.M.

    1991-01-01

    The DM 1 /σ 1 site binds dextromethorphan (DM) and σ receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of [ 3 H]dextromethorphan, [ 3 H]3-(3-Hydroxyphenyl)-N-(1-propyl)piperidine and (+)-[ 3 H]1,3-Di-o-tolyl-guanidine ([ 3 H]DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM K i values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM 1 σ 1 site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K i values of 9-13 and 3-4 μM respectively against the three labeled ligands. These results, the broad specificity of the DM 1 /σ 1 binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor

  2. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors

    International Nuclear Information System (INIS)

    Amagai, Yosuke; Tanaka, Akane; Ohmori, Keitaro; Matsuda, Hiroshi

    2008-01-01

    Much is known regarding participations of mast cells with innate and acquired immunity by secreting various cytokines and chemical mediators. However, details of mast cell biology still remain unclear. In this study, we successfully established a novel growth factor-independent mast cell line (MPT-1) derived from canine mast cell tumor. MPT-1 cells manifested factor-independent proliferation as floating cells containing a large amount of histamine, as well as chymase-like dog mast cell protease 3, in cytosolic granules. Particularly, MPT-1 cells expressed high-affinity IgE receptors (FcεRI) and wild-type c-kit receptors. Degranulation of MPT-1 cells was induced not only by stimulation with calcium ionophore but also by cross-linkage of the surface IgE. Given that MPT-1 is the first mast cell line with FcεRI which has no c-kit mutations, MPT-1 cells may provide great contribution for investigation of IgE-mediated activation mechanisms of mast cells, leading to development of effective treatment for allergic disorders

  3. High affinity (3H) β-Alanine uptake by scar margins of ferric chloride-induced epileptogenic foci in rat isocortex

    International Nuclear Information System (INIS)

    Robitaille, Y.; Sherwin, A.

    1984-01-01

    Cortical astrocytes of normal mammalian brain are endowed with a high affinity uptake system for β-Alanine which is competitively inhibited by gamma aminobutyric acid (GABA), a neurotransmitter strongly implicated in epileptogenesis. The authors evaluated ( 3 H) β-Alanine uptake by reactive astrocytes proliferating within scar of epileptogenic foci induced in rat motor cortex by microinjections of 100 mM ferric chloride. Following in vitro incubation of scar tissue with ( 3 H) β-Alanine, ultrastructural morphometry of grain patterns at 5, 30 and 120 days post injection revealed early and significant grain count increases over astroglial processes, predominantly those related to perivascular glial end-feet. Astrocytic cell body and endothelial cell counts showed a more gradual and stepwise increase. Similar data were obtained by comparing visual and edited mean astrocytic grain counts. These results suggest that the enhanced uptake of reactive astrocytes may reflect a marked decrease of inhibitory GABAergic neurons within ferric chloride-induced scars. 7 figures, 1 table

  4. Transportation

    National Research Council Canada - National Science Library

    Allshouse, Michael; Armstrong, Frederick Henry; Burns, Stephen; Courts, Michael; Denn, Douglas; Fortunato, Paul; Gettings, Daniel; Hansen, David; Hoffman, D. W; Jones, Robert

    2007-01-01

    .... The ability of the global transportation industry to rapidly move passengers and products from one corner of the globe to another continues to amaze even those wise to the dynamics of such operations...

  5. Zinc phosphate conversion coatings

    Science.gov (United States)

    Sugama, Toshifumi

    1997-01-01

    Zinc phosphate conversion coatings for producing metals which exhibit enhanced corrosion prevention characteristics are prepared by the addition of a transition-metal-compound promoter comprising a manganese, iron, cobalt, nickel, or copper compound and an electrolyte such as polyacrylic acid, polymethacrylic acid, polyitaconic acid and poly-L-glutamic acid to a phosphating solution. These coatings are further improved by the incorporation of Fe ions. Thermal treatment of zinc phosphate coatings to generate .alpha.-phase anhydrous zinc phosphate improves the corrosion prevention qualities of the resulting coated metal.

  6. Design, Synthesis, and in Vitro Pharmacology of New Radiolabeled γ-Hydroxybutyric Acid Analogues Including Photolabile Analogues with Irreversible Binding to the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Sabbatini, Paola; Wellendorph, Petrine; Høg, Signe

    2010-01-01

    γ-Hydroxybutyric acid (GHB) is a psychotropic compound endogenous to the brain. Despite its potential physiological significance, the complete molecular mechanisms of action remain unexplained. To facilitate the isolation and identification of the high-affinity GHB binding site, we herein report ...

  7. Aluminium fluoride and magnesium, activators of heterotrimeric GTP-binding proteins, affect high-affinity binding of the fungal toxin fusicoccin to the fusicoccin-binding protein in oat root plasma membranes.

    NARCIS (Netherlands)

    de Boer, A.H.; Van der Molen, G.W.; Prins, H.B.A.; Korthout, H.A.A.J.; van der Hoeven, P.C.J.

    1994-01-01

    The fusicoccin-binding protein was solubilised from purified oat root plasma membranes. The solubilised protein retained full binding activity, provided that protease inhibitors were included. Sodium fluoride reduced the high-affinity [H-3]fusicoccin binding to almost zero in a

  8. Astrocytic GABA Transporters

    DEFF Research Database (Denmark)

    Schousboe, Arne; Wellendorph, Petrine; Frølund, Bente

    2017-01-01

    , and several of these compounds have been shown to exhibit pronounced anticonvulsant activity in a variety of animal seizure models. As proof of concept of the validity of this drug development approach, one GABA-transport inhibitor, tiagabine, has been developed as a clinically active antiepileptic drug......Inactivation of GABA-mediated neurotransmission is achieved by high-affinity transporters located at both GABAergic neurons and the surrounding astrocytes. Early studies of the pharmacological properties of neuronal and glial GABA transporters suggested that different types of transporters might...... be expressed in the two cell types, and such a scenario was confirmed by the cloning of four distinctly different GABA transporters from a number of different species. These GABA-transport entities have been extensively characterized using a large number of GABA analogues of restricted conformation...

  9. Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema.

    Science.gov (United States)

    Ozawa, Chihiro; Horiguchi, Michiko; Akita, Tomomi; Oiso, Yuki; Abe, Kaori; Motomura, Tomoki; Yamashita, Chikamasa

    2016-01-01

    Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) β/δ. A selective PPARβ/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARβ/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARβ/δ agonists are potential therapeutic agents for pulmonary emphysema.

  10. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma

    Directory of Open Access Journals (Sweden)

    Darwish Ibrahim A

    2012-10-01

    Full Text Available Abstract Background For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND, the potent drug for treatment of multiple myeloma (MM, a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. Results In this study, a hapten of LND (N-glutaryl-LND was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA and keyhole limpet hemocyanin (KLH proteins by ethyl-3-(3-dimethylaminopropyl carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. Conclusions The high affinity of the antibody (IC50 = 10 ng/mL will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  11. Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma.

    Science.gov (United States)

    Darwish, Ibrahim A; Alzoman, Nourh Z; Abuhejail, Reem M; El-Samani, Tilal E

    2012-10-26

    For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM), a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. In this study, a hapten of LND (N-glutaryl-LND) was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND) was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA) using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. The high affinity of the antibody (IC50 = 10 ng/mL) will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

  12. Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

    Science.gov (United States)

    Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

    2018-01-01

    Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

  13. High-affinity binding of [3H]estradiol-17 beta by an estrogen receptor in the liver of the turtle

    International Nuclear Information System (INIS)

    Ho, S.M.; Fehrer, S.; Yu, M.; Liang, L.C.; Press, D.

    1988-01-01

    Specific [3H]estradiol-17 beta ([3H]E2) binding activity (EBA) with characteristics of an estrogen receptor (ER) was demonstrated in cytosols and nuclear extracts of the female turtle, Chrysemys picta. Three different receptor assays (dextran-coated charcoal assay, hydroxylapatite batch procedure, and DNA-cellulose chromatography) were evaluated in terms of their applicability in analyzing large numbers of samples. For the measurement of cytosolic EBA, the hydroxylapatite batch procedure was found to be the most reliable assay. On the other hand, the dextran-coated charcoal assay was found to be the most appropriate method for the measurement of nuclear EBA. Turtle hepatic EBA binds [3H]E2 with high affinity (cytosolic, 17.4 +/- 2.8 X 10(9) M-1; nuclear, 17.7 +/- 1.9 X 10(9) M-1), limited capacity (cytosolic, 133.7 +/- 4.6 fmol/g tissue; nuclear, 81.1 +/- 9.0 fmol/g tissue), and strict steroid specificity. The EBA bound natural estrogens (E2, estrone, estriol) as well as the nonsteroidal estrogen, diethylstilbestrol, but exhibited little affinity for androgens, progesterone, or corticosterone. The turtle hepatic EBA resembled mammalian and avian ERs in terms of binding characteristics; however, unlike mammalian and avian ERs it was shown to be heat-labile. Incubation at 30 degrees caused rapid loss of [3H]E2 binding activity in both cytosolic and nuclear fractions. The exchange between [3H]E2 and the endogenously bound estrogen was slow at 4 and 15 degrees, but the exchange process was facilitated in the presence of the chaotropic salt, NaSCN. Establishment of quantitation methods for both cytosolic and nuclear forms of EBA will enable future investigation of the mechanism and regulation of estrogen action in the liver of this turtle species

  14. (3H)leukotriene B4 binding to the guinea pig spleen membranes: a rich tissue source for a high affinity leukotriene B4 receptor site

    International Nuclear Information System (INIS)

    Cheng, J.B.; Kohi, F.; Townley, R.G.

    1986-01-01

    To select a tissue rich for the high affinity leukotriene (LT)B 4 receptor site, they compared binding of 1 nM ( 3 H)LTB 4 (180 Ci/mmol) to the crude membrane preparations of guinea pig spleen, thymus, lung, uterus, bladder, brain, adrenal gland, small intestine, liver, kidney and heart. They found that the membrane preparations from spleen contained the highest binding activity per mg protein. They characterized the LTB 4 binding to the spleen preparation in detail. LTB 4 binding was rapid, reversible, stereoselective and saturable. The data from equilibrium experiments showed a linear Scatchard plot with a K/sub d/ of 1.6 nM and a binding site density of 259 fmol/mg prot. The rank order of agents competing for spleen ( 3 H)LTB 4 binding at 25 0 C was: LTB 4 (K/sub i/ = 2.8 nM) > 20-OH-LTB 4 (23 nM) > LTA 4 (48 nM) > LTA 4 methyl ester (0.13 μM) > 20-COOH-LTB 4 (> 6.6 μM) ≥ arachidonic acid (0.15 mM) similarly ordered FPL-55,712 (0.11 mM). At 4 0 C, LTB 4 (2.3 nM) competed at least 10x more effectively than 20-OH-LTB 4 (29 nM) and 20-COOH-LTB 4 (> 6.6 μM). HPLC analysis indicated that incubation of 84 ng LTB 4 with the spleen membrane at 25 0 C did not result in the formation of 20-OH-LTB 4 ( 3 H)LTB 4 receptor binding sites

  15. Preliminary assessment of extrastriatal dopamine d-2 receptor binding in the rodent and nonhuman primate brains using the high affinity radioligand, {sup 18}F-fallypride

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Jogeshwar E-mail: jogeshwar-mukherjee@ketthealth.com; Yang, Z.-Y.; Brown, Terry; Lew, Robert; Wernick, Miles; Ouyang Xiaohu; Yasillo, Nicholas; Chen, C.-T.; Mintzer, Robert; Cooper, Malcolm

    1999-07-01

    We have identified the value of {sup 18}F-fallypride {l_brace}(S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[{sup 18}F]fluoropropyl)-2,3-dim= ethoxybenzamide{r_brace}, as a dopamine D-2 receptor radiotracer for the study of striatal and extrastriatal receptors. Fallypride exhibits high affinities for D-2 and D-3 subtypes and low affinity for D-4 ({sup 3}H-spiperone IC{sub 50}s: D-2=0.05 nM [rat striata], D-3=0.30 nM [SF9 cell lines, rat recombinant], and D-4=240 nM [CHO cell lines, human recombinant]). Biodistribution in the rat brain showed localization of {sup 18}F-fallypride in striata and extrastriatal regions such as the frontal cortex, parietal cortex, amygdala, hippocampus, thalamus, and hypothalamus. In vitro autoradiographic studies in sagittal slices of the rat brain showed localization of {sup 18}F-fallypride in striatal and several extrastriatal regions, including the medulla. Positron emission tomography (PET) experiments with {sup 18}F-fallypride in male rhesus monkeys were carried out in a PET VI scanner. In several PET experiments, apart from the specific binding seen in the striatum, specific binding of {sup 18}F-fallypride was also identified in extracellular regions (in a lower brain slice, possibly the thalamus). Specific binding in the extrastriata was, however, significantly lower compared with that observed in the striata of the monkeys (extrastriata/cerebellum = 2, striata/cerebellum = 10). Postmortem analysis of the monkey brain revealed significant {sup 18}F-fallypride binding in the striata, whereas binding was also observed in extrastriatal regions such as the thalamus, cortical areas, and brain stem.

  16. A conserved motif in the linker domain of STAT1 transcription factor is required for both recognition and release from high-affinity DNA-binding sites.

    Science.gov (United States)

    Hüntelmann, Bettina; Staab, Julia; Herrmann-Lingen, Christoph; Meyer, Thomas

    2014-01-01

    Binding to specific palindromic sequences termed gamma-activated sites (GAS) is a hallmark of gene activation by members of the STAT (signal transducer and activator of transcription) family of cytokine-inducible transcription factors. However, the precise molecular mechanisms involved in the signal-dependent finding of target genes by STAT dimers have not yet been very well studied. In this study, we have characterized a sequence motif in the STAT1 linker domain which is highly conserved among the seven human STAT proteins and includes surface-exposed residues in close proximity to the bound DNA. Using site-directed mutagenesis, we have demonstrated that a lysine residue in position 567 of the full-length molecule is required for GAS recognition. The substitution of alanine for this residue completely abolished both binding to high-affinity GAS elements and transcriptional activation of endogenous target genes in cells stimulated with interferon-γ (IFNγ), while the time course of transient nuclear accumulation and tyrosine phosphorylation were virtually unchanged. In contrast, two glutamic acid residues (E559 and E563) on each monomer are important for the dissociation of dimeric STAT1 from DNA and, when mutated to alanine, result in elevated levels of tyrosine-phosphorylated STAT1 as well as prolonged IFNγ-stimulated nuclear accumulation. In conclusion, our data indicate that the kinetics of signal-dependent GAS binding is determined by an array of glutamic acid residues located at the interior surface of the STAT1 dimer. These negatively charged residues appear to align the long axis of the STAT1 dimer in a position perpendicular to the DNA, thereby facilitating the interaction between lysine 567 and the phosphodiester backbone of a bound GAS element, which is a prerequisite for transient gene induction.

  17. Murine CMV Expressing the High Affinity NKG2D Ligand MULT-1: A Model for the Development of Cytomegalovirus-Based Vaccines

    Directory of Open Access Journals (Sweden)

    Lea Hiršl

    2018-05-01

    Full Text Available The development of a vaccine against human cytomegalovirus (CMV has been a subject of long-term medical interest. The research during recent years identified CMV as an attractive vaccine vector against infectious diseases and tumors. The immune response to CMV persists over a lifetime and its unique feature is the inflationary T cell response to certain viral epitopes. CMV encodes numerous genes involved in immunoevasion, which are non-essential for virus growth in vitro. The deletion of those genes results in virus attenuation in vivo, which enables us to dramatically manipulate its virulence and the immune response. We have previously shown that the murine CMV (MCMV expressing RAE-1γ, one of the cellular ligands for the NKG2D receptor, is highly attenuated in vivo but retains the ability to induce a strong CD8+ T cell response. Here, we demonstrate that recombinant MCMV expressing high affinity NKG2D ligand murine UL16 binding protein-like transcript (MULT-1 (MULT-1MCMV inserted in the place of its viral inhibitor is dramatically attenuated in vivo in a NK cell-dependent manner, both in immunocompetent adult mice and in immunologically immature newborns. MULT-1MCMV was more attenuated than the recombinant virus expressing RAE-1γ. Despite the drastic sensitivity to innate immune control, MULT-1MCMV induced an efficient CD8+ T cell response to viral and vectored antigens. By using in vitro assay, we showed that similar to RAE-1γMCMV, MULT-1 expressing virus provided strong priming of CD8+ T cells. Moreover, MULT-1MCMV was able to induce anti-viral antibodies, which after passing the transplacental barrier protect offspring of immunized mothers from challenge infection. Altogether, this study further supports the concept that CMV expressing NKG2D ligand possesses excellent characteristics to serve as a vaccine or vaccine vector.

  18. High-affinity interaction of hnRNP A1 with conserved RNA structural elements is required for translation and replication of enterovirus 71.

    Science.gov (United States)

    Levengood, Jeffrey D; Tolbert, Michele; Li, Mei-Ling; Tolbert, Blanton S

    2013-07-01

    Human Enterovirus 71 (EV71) is an emerging pathogen of infectious disease and a serious threat to public health. Currently, there are no antivirals or vaccines to slow down or prevent EV71 infections, thus underscoring the urgency to better understand mechanisms of host-enterovirus interactions. EV71 uses a type I internal ribosome entry site (IRES) to recruit the 40S ribosomal subunit via a pathway that requires the cytoplasmic localization of hnRNP A1, which acts as an IRES trans-activating factor. The mechanism of how hnRNP A1 trans activates EV71 RNA translation is unknown, however. Here, we report that the UP1 domain of hnRNP A1 interacts specifically with stem loop II (SLII) of the IRES, via a thermodynamically well-defined biphasic transition that involves conserved bulge 5'-AYAGY-3' and hairpin 5'-RY(U/A)CCA-3' loops. Calorimetric titrations of wild-type and mutant SLII constructs reveal these structural elements are essential to form a high-affinity UP1-SLII complex. Mutations that alter the bulge and hairpin primary or secondary structures abrogate the biphasic transition and destabilize the complex. Notably, mutations within the bulge that destabilize the complex correlate with a large reduction in IRES-dependent translational activity and impair EV71 replication. Taken together, this study shows that a conserved SLII structure is necessary to form a functional hnRNP A1-IRES complex, suggesting that small molecules that target this stem loop may have novel antiviral properties.

  19. Interactions of [14C]phosphonoformic acid with renal cortical brush-border membranes. Relationship to the Na+-phosphate co-transporter

    International Nuclear Information System (INIS)

    Szczepanska-Konkel, M.; Yusufi, A.N.; Dousa, T.P.

    1987-01-01

    Since phosphonoformic acid (PFA) acts as a specific competitive inhibitor of Na+-Pi co-transport across renal brush-border membrane (BBM), we employed the [ 14 C]PFA as a probe to determine the mechanism of its interaction with rat renal BBM. The binding of [ 14 C]PFA to BBM vesicles (BBMV), with Na+ present in extravesicular medium (Na+o), was time- and temperature-dependent. The replacement of Na+o with other monovalent cations reduced the PFA binding by -80%. Cl- was the most effective accompanying monovalent anion as NaCl for maximum PFA binding. The Na+o increased the apparent affinity of BBMV for [ 14 C]PFA binding, but it did not change the maximum binding capacity. The maximum [ 14 C]PFA binding was achieved at Na+o approximately equal to 50 mM. The extent of Na+-dependent [ 14 C]PFA binding correlated with percent inhibition by an equimolar dose of PFA of the dependent BBMV uptake of 32Pi. Intravesicular Na+ (Na+i) decreased [ 14 C]PFA binding, on BBMV, and this inhibition by Na+i was dependent on the presence of Na+o. The increase in Na+i, at constant [Na+]o, decreased the Vmax, but not the Km, for [ 14 C]PFA binding on BBMV. Bound [ 14 C]PFA was displaced from BBMV by phosphonocarboxylic acids proportionally to their ability to inhibit gradient-dependent Pi transport, whereas other monophosphonates, diphosphonates, L-proline, or D-glucose did not influence the [ 14 C]PFA binding. The Na+-dependent binding of [ 14 C]PFA and of [ 3 H]phlorizin by BBMV was 10 times higher than binding of these ligands to renal basolateral membranes and to mitochondria. [ 14 C]PFA probably binds onto the same locus on the luminal surface of BBM, where Pi and Na+ form a ternary complex with the Na+-Pi co-transporter

  20. Transportation

    Science.gov (United States)

    2007-01-01

    Faculty ii INDUSTRY TRAVEL Domestic Assistant Deputy Under Secretary of Defense (Transportation Policy), Washington, DC Department of...developed between the railroad and trucking industries. Railroads: Today’s seven Class I freight railroad systems move 42% of the nation’s intercity ...has been successfully employed in London to reduce congestion and observed by this industry study during its travels . It is currently being

  1. Occupational radiation risks in conveyance of bulk phosphate and potash

    International Nuclear Information System (INIS)

    Grof, Y.; Even, O.; Schlesinger, T.; Margaliot, M.

    1996-01-01

    The issue of occupational ionizing radiation risks encountered in the conveyance and storage of Phosphates and Potash as loose cargo got very minor attention from the national health and occupational safety authorities in the world. In Israel, the Phosphates include an average 100- 150 ppm of Uranium in equilibrium with its daughters, while in Phosphates produced in most other countries the inaction reaches regularly only few ppm up to 50 ppm. Because of the high content of the Uranium in the Phosphate in Israel we must take into consideration the radiological implications involved in the handling of this mineral. The radiological implications of handling Potash are less significant but can not be neglected as we demonstrate bellow In this presentation we will estimate the occupational radiological risks involved in the storing and transportation of Phosphate and Potash. Note, that the main risk in working with Phosphate and Potash is the risk from the dust itself (authors)

  2. Occupational radiation risks in conveyance of bulk phosphate and potash

    Energy Technology Data Exchange (ETDEWEB)

    Grof, Y; Even, O; Schlesinger, T; Margaliot, M [Israel Atomic Energy Commission, Yavne (Israel). Soreq Nuclear Research Center

    1996-12-01

    The issue of occupational ionizing radiation risks encountered in the conveyance and storage of Phosphates and Potash as loose cargo got very minor attention from the national health and occupational safety authorities in the world. In Israel, the Phosphates include an average 100- 150 ppm of Uranium in equilibrium with its daughters, while in Phosphates produced in most other countries the inaction reaches regularly only few ppm up to 50 ppm. Because of the high content of the Uranium in the Phosphate in Israel we must take into consideration the radiological implications involved in the handling of this mineral. The radiological implications of handling Potash are less significant but can not be neglected as we demonstrate bellow In this presentation we will estimate the occupational radiological risks involved in the storing and transportation of Phosphate and Potash. Note, that the main risk in working with Phosphate and Potash is the risk from the dust itself (authors).

  3. PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    2010-05-01

    Full Text Available Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases and hydrolysis (by cAMP phosphodiesterases. We functionally characterized gene-deletion mutants of a high-affinity (PdeH and a low-affinity (PdeL cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHDelta showed enhanced conidiation (2-3 fold, precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHDelta defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling

  4. Preclinical evaluation of multistep targeting of diasialoganglioside GD2 using a IgG-scFv bispecific antibody with high affinity for GD2 and DOTA metal complex

    Science.gov (United States)

    Cheal, Sarah M.; Xu, Hong; Guo, Hong-fen; Zanzonico, Pat B.; Larson, Steven M.; Cheung, Nai-Kong

    2014-01-01

    Bispecific antibodies (BsAb) have proven to be useful targeting vectors for pretargeted radioimmunotherapy (PRIT). We sought to overcome key PRIT limitations such as high renal radiation exposure and immunogenicity (e.g. of streptavidin-antibody fusions), to advance clinical translation of this PRIT strategy for diasialoganglioside GD2-positive (GD2(+)) tumors. For this purpose, a IgG-scFv BsAb was engineered using the sequences for the anti-GD2 humanized monoclonal antibody hu3F8 (1) and C825, a murine scFv antibody with high affinity for the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) complexed with beta-particle emitting radiometals such as 177Lu and 90Y (2, 3). A three-step regimen including hu3F8-C825, a dextran-based clearing agent, and p-aminobenzyl-DOTA radiolabeled with 177Lu (as 177Lu-DOTA-Bn; t1/2 = 6.71 days (d)) was optimized in immunocompromised mice carrying subcutaneous (s.c.) human GD2(+) neuroblastoma (NB) xenografts. Absorbed doses for tumor and normal tissues were ∼85 cGy/MBq and ≤3.7 cGy/MBq, respectively, with therapeutic indicies (TI) of 142 for blood and 23 for kidney. A therapy study (n = 5 per group; tumor volume: 240 ± 160 mm3) with three successive PRIT cycles (total 177Lu: ∼33 MBq; tumor dose ∼3400 cGy), revealed complete tumor response in 5/5 animals, with no recurrence up to 28 d post-treatment. Tumor ablation was confirmed histologically in 4/5 mice, and normal organs showed minimal overall toxicities. All non-treated mice required sacrifice within 12 d (>1.0 cm3 tumor volume). We conclude that this novel anti-GD2 PRIT approach has sufficient TI to successfully ablate s.c. GD2(+)–NB in mice while sparing kidney and bone marrow. PMID:24944121

  5. Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

    Directory of Open Access Journals (Sweden)

    Candolfi Ermanno

    2011-07-01

    Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

  6. Radionuclide containment in soil by phosphate treatment

    International Nuclear Information System (INIS)

    Lee, S.Y.; Francis, C.W.; Timpson, M.E.; Elless, M.P.

    1995-01-01

    Radionuclide transport from a contaminant source to groundwater and surface water is a common problem faced by most US Department of Energy (DOE) facilities. Containment of the radionuclide plume, including strontium-90 and uranium, is possible using phosphate treatment as a chemical stabilizer. Such a chemical process occurs in soils under natural environmental conditions. Therefore, the concept of phosphate amendment for radiostrontium and uranium immobilization is already a proven principle. In this presentation, results of bench-scale experiments and the concept of a field-scale demonstration are discussed. The phosphate treatment is possible at the source or near the advancing contaminant plume. Cleanup is still the ideal concept; however, containment through stabilization is a more practical and costeffective concept that should be examined by DOE Environmental Restoration programs

  7. Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    Energy Technology Data Exchange (ETDEWEB)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C. (National Institute of Mental Health, Bethesda, MD (USA))

    1991-02-01

    Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.

  8. Labeling by [3H]1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

    International Nuclear Information System (INIS)

    Rothman, R.B.; Reid, A.; Mahboubi, A.; Kim, C.H.; De Costa, B.R.; Jacobson, A.E.; Rice, K.C.

    1991-01-01

    Equilibrium binding studies with the sigma receptor ligand [ 3 H]1,3-di(2-tolyl)guanidine ([ 3 H]DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. [Life Sci. 45:1721-1732 (1989)]. Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and low affinity for most other sigma ligands. Kinetic experiments demonstrated that [ 3 H]DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of [ 3 H]DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of [ 3 H]DTG from site 2, suggesting an association of this binding site with calcium channels

  9. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    International Nuclear Information System (INIS)

    Leff, S.E.; Creese, I.

    1985-01-01

    The interactions of dopaminergic agonists and antagonists with 3 H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [ 3 H]dopamine and [ 3 H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/[ 3 H]dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [ 3 H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [ 3 H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [ 3 H]dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/[ 3 H]flupentixol competition curves. Both D3 specific [ 3 H] dopamine binding and the high affinity agonist-binding component of dopamine/[ 3 H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor

  10. Effect of submarine groundwater discharge containing phosphate on coral calcification

    Science.gov (United States)

    Yasumoto, J.; Yasumoto, K.; Iijima, M.; Nozaki, M.; Asai, K.; Yasumoto, M. H.

    2017-12-01

    It is well known that the anthropogenic eutrophication enriched with various substances including phosphate in coastal waters has resulted in coral degradation. However, to the best of our knowledge, the phosphate threshold value to inhibit the coral calcification has been unclear, due to the unknown mechanisms involved in the inhibition of the calcification by phosphate. In island regions, groundwater is one of the most important clues to transport the nutrients contained in livestock or agricultural wastewaters. However, the actual conditions of coastal pollution with such nutrients have not been understood because of unperceived submarine groundwater discharge (SGD). In this study, to quantify of extremely rapid and localized SGD from Ryukyu limestone aquifer, we investigated the rate and concentration of phosphate of SGD using automated seepage mater in Yoron Island, which is located southern part of Japan. And, to elucidate the inhibition mechanisms for phosphate against coral calcification, we examined its effect on the bottom skeleton formation in primary polyps of Acropora digitifera by using the fluorescence derivatizing reagent having phosphate group (FITC-AA). As a result, the SGD was found to contain 1 to 2 µM of phosphate as much as the concentration in the coastal ground water under agricultural land. Moreover, the amount of phosphate contained in the surface layers of bottom calcareous sands close to the region of SGD were about 5 µmol/g. When the primary polyps were treated with 50 µM of FITC-AA, the bottom skeleton of the primary polyps showed the fluorescence from FITC-AA within a few minutes, suggesting the phosphate binding. Furthermore, when the polyps were treated with 10 µM of FITC-AA, irregular patterns of the elongated skeleton were observed. These results led us to conclude that phosphate is transported via a paracellular pathway to the subcalicoblastic extracellular calcifying medium. These results indicate that the phosphate adsorbed

  11. Radiosynthesis and Evaluation of [(11)C]3-Hydroxycyclopent-1-enecarboxylic Acid as Potential PET Ligand for the High-Affinity γ-Hydroxybutyric Acid Binding Sites

    DEFF Research Database (Denmark)

    Jensen, Claus H; Hansen, Hanne D; Bay, Tina

    2017-01-01

    understanding of this population of binding sites. With its high specific affinity and monocarboxylate transporter (MCT1) mediated transport across the blood-brain barrier in pharmacological doses, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) seems like a suitable PET radiotracer candidate. Here, we report...... autoradiography on sections of pig brain was performed using [(3)H]HOCPCA. In vivo evaluation of [(11)C]HOCPCA showed no brain uptake, possibly due to a limited uptake of HOCPCA by the MCT1 transporter at tracer doses of [(11)C]HOCPCA....

  12. Phosphate control in dialysis

    Directory of Open Access Journals (Sweden)

    Cupisti A

    2013-10-01

    Full Text Available Adamasco Cupisti,1 Maurizio Gallieni,2 Maria Antonietta Rizzo,2 Stefania Caria,3 Mario Meola,4 Piergiorgio Bolasco31Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy; 2Nephrology and Dialysis Unit, San Carlo Borromeo Hospital, Milan, Italy; 3Territorial Department of Nephrology and Dialysis, ASL Cagliari, Italy; 4Sant'Anna School of Advanced Studies, University of Pisa, Pisa, ItalyAbstract: Prevention and correction of hyperphosphatemia is a major goal of chronic kidney disease–mineral and bone disorder (CKD–MBD management, achievable through avoidance of a positive phosphate balance. To this aim, optimal dialysis removal, careful use of phosphate binders, and dietary phosphate control are needed to optimize the control of phosphate balance in well-nourished patients on a standard three-times-a-week hemodialysis schedule. Using a mixed diffusive–convective hemodialysis tecniques, and increasing the number and/or the duration of dialysis tecniques are all measures able to enhance phosphorus (P mass removal through dialysis. However, dialytic removal does not equal the high P intake linked to the high dietary protein requirement of dialysis patients; hence, the use of intestinal P binders is mandatory to reduce P net intestinal absorption. Unfortunately, even a large dose of P binders is able to bind approximately 200–300 mg of P on a daily basis, so it is evident that their efficacy is limited in the case of an uncontrolled dietary P load. Hence, limitation of dietary P intake is needed to reach the goal of neutral phosphate balance in dialysis, coupled to an adequate protein intake. To this aim, patients should be informed and educated to avoid foods that are naturally rich in phosphate and also processed food with P-containing preservatives. In addition, patients should preferentially choose food with a low P-to-protein ratio. For example, patients could choose egg white or protein from a vegetable source

  13. Uranium production from phosphates

    International Nuclear Information System (INIS)

    Ketzinel, Z.; Folkman, Y.

    1979-05-01

    According to estimates of the world's uranium consumption, exploitation of most rich sources is expected by the 1980's. Forecasts show that the rate of uranium consumption will increase towards the end of the century. It is therefore desirable to exploit poor sources not yet in use. In the near future, the most reasonable source for developing uranium is phosphate rock. Uranium reserves in phosphates are estimated at a few million tons. Production of uranium from phosphates is as a by-product of phosphate rock processing and phosphoric acid production; it will then be possible to save the costs incurred in crushing and dissolving the rock when calculating uranium production costs. Estimates show that the U.S. wastes about 3,000 tons of uranium per annum in phosphoric acid based fertilisers. Studies have also been carried out in France, Yugoslavia and India. In Israel, during the 1950's, a small plant was operated in Haifa by 'Chemical and Phosphates'. Uranium processes have also been developed by linking with the extraction processes at Arad. Currently there is almost no activity on this subject because there are no large phosphoric acid plants which would enable production to take place on a reasonable scale. Discussions are taking place about the installation of a plant for phosphoric acid production utilising the 'wet process', producing 200 to 250,000 tons P 2 O 5 per annum. It is necessary to combine these facilities with uranium production plant. (author)

  14. Recovering uranium from phosphates

    Energy Technology Data Exchange (ETDEWEB)

    Bergeret, M [Compagnie de Produits Chimiques et Electrometallurgiques Pechiney-Ugine Kuhlmann, 75 - Paris (France)

    1981-06-01

    Processes for the recovery of the uranium contained in phosphates have today become competitive with traditional methods of working uranium sources. These new possibilities will make it possible to meet more rapidly any increases in the demand for uranium: it takes ten years to start working a new uranium deposit, but only two years to build a recovery plant.

  15. Ascorbic acid transport and accumulation in human neutrophils

    International Nuclear Information System (INIS)

    Washko, P.; Rotrosen, D.; Levine, M.

    1989-01-01

    The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake

  16. Molecular cloning of a second subunit of the receptor for human granulocyte - macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor

    International Nuclear Information System (INIS)

    Hayashida, Kazuhiro; Kitamura, Toshio; Gorman, D.M.; Miyajima, Atsushi; Arai, Kenichi; Yokota, Takashi

    1990-01-01

    Using the mouse interleukin 3 (IL-3) receptor cDNA as a probe, the authors obtained a monologous cDNA (KH97) from a cDNA library of a human hemopoietic cell line, TF-1. The protein encoded by the KH97 cDNA has 56% amino acid sequence identity with the mouse IL-3 receptor and retains features common to the family of cytokine receptors. Fibroblasts transfected with the KH97 cDNA expressed a protein of 120 kDa but did not bind any human cytokines, including IL-3 and granulocyte - macrophage colony-stimulating factor (GM-CSF). Interestingly, cotransfection of cDNAs for KH97 and the low-affinity human GM-CSF receptor in fibroblasts resulted in formation of a high-affinity receptor for GM-CSF. The dissociation rate of GM-CSF from the reconstituted high-affinity receptor was slower than that from the low-affinity site, whereas the association rate was unchanged. Cross-linking of 125 I-labeled GM-CSF to fibroblasts cotransfected with both cDNAs revealed the same cross-linking patterns as in TF-1 cells - i.e., two major proteins of 80 and 120 kDa which correspond to the low-affinity GM-CSF receptor and the KH97 protein, respectively. These results indicate that the high-affinity GM-CSF receptor is composed of at least two components in a manner analogous to the IL-2 receptor. They therefore propose to designate the low-affinity GM-CSF receptor and the KH97 protein as the α and β subunits of the GM-CSF receptor, respectively

  17. Crystal structure of the high-affinity Na+,K+-ATPase–ouabain complex with Mg2+ bound in the cation binding site

    DEFF Research Database (Denmark)

    Laursen, Mette; Yatime, Laure; Nissen, Poul

    2013-01-01

    of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg2+, and crystallographic studies indicate that Rb+ and Mn2+, but not Na+, bind to this site. Comparison with the low-affinity [K2]E2–MgFx–ouabain structure [Ogawa et al...

  18. A high-affinity inhibitor of yeast carboxypeptidase Y is encoded by TFS1 and shows homology to a family of lipid binding proteins

    DEFF Research Database (Denmark)

    Bruun, A W; Svendsen, I; Sørensen, S O

    1998-01-01

    signals for transport into the endoplasmic reticulum. Surprisingly, Ic is encoded by TFS1, which has previously been isolated as a high-copy suppressor of cdc25-1. CDC25 encodes the putative GTP exchange factor for Ras1p/Ras2p in yeast. In an attempt to rationalize this finding, we looked...... degree of specificity, showing a 200-fold higher Ki toward a carboxypeptidase from Candida albicans which is highly homologous to carboxypeptidase Y. The TFS1 gene product shows extensive similarity to a class of proteins termed "21-23-kDa lipid binding proteins", members of which are found in several...

  19. High affinity and temperature sensitivity of blood oxygen binding in Pangasianodon hypophthalmus due to lack of chloride-hemoglobin allosteric interaction

    DEFF Research Database (Denmark)

    Damsgaard, Christian; Phuong, Le My; Huong, Do Thi Thanh

    2015-01-01

    Air-breathing fishes represent interesting organisms in terms of understanding the physiological changes associated with the terrestrialization of vertebrates, and, further, are of great socio-economic importance for aquaculture in Southeast Asia. To understand how environmental factors......, such as high temperature, affect O2 transport in air-breathing fishes, this study assessed the effects of temperature on O2 binding of blood and Hb in the economically important air-breathing fish Pangasianodon hypophthalmus. To determine blood O2 binding properties, blood was drawn from resting cannulated...

  20. Molecular Cloning and Functional Analysis of a Na+-Insensitive K+ Transporter of Capsicum chinense Jacq

    Science.gov (United States)

    Ruiz-Lau, Nancy; Bojórquez-Quintal, Emanuel; Benito, Begoña; Echevarría-Machado, Ileana; Sánchez-Cach, Lucila A.; Medina-Lara, María de Fátima; Martínez-Estévez, Manuel

    2016-01-01

    High-affinity K+ (HAK) transporters are encoded by a large family of genes and are ubiquitous in the plant kingdom. These HAK-type transporters participate in low- and high-affinity potassium (K+) uptake and are crucial for the maintenance of K+ homeostasis under hostile conditions. In this study, the full-length cDNA of CcHAK1 gene was isolated from roots of the habanero pepper (Capsicum chinense). CcHAK1 expression was positively regulated by K+ starvation in roots and was not inhibited in the presence of NaCl. Phylogenetic analysis placed the CcHAK1 transporter in group I of the HAK K+ transporters, showing that it is closely related to Capsicum annuum CaHAK1 and Solanum lycopersicum LeHAK5. Characterization of the protein in a yeast mutant deficient in high-affinity K+ uptake (WΔ3) suggested that CcHAK1 function is associated with high-affinity K+ uptake, with Km and Vmax for Rb of 50 μM and 0.52 nmol mg−1 min−1, respectively. K+ uptake in yeast expressing the CcHAK1 transporter was inhibited by millimolar concentrations of the cations ammonium (NH4+) and cesium (Cs+) but not by sodium (Na+). The results presented in this study suggest that the CcHAK1 transporter may contribute to the maintenance of K+ homeostasis in root cells in C. chinense plants undergoing K+-deficiency and salt stress. PMID:28083010

  1. The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor.

    Science.gov (United States)

    Klippel, A; Escobedo, J A; Fantl, W J; Williams, L T

    1992-01-01

    Upon stimulation by its ligand, the platelet-derived growth factor (PDGF) receptor associates with the 85-kDa subunit of phosphatidylinositol (PI) 3-kinase. The 85-kDa protein (p85) contains two Src homology 2 (SH2) domains and one SH3 domain. To define the part of p85 that interacts with the PDGF receptor, a series of truncated p85 mutants was analyzed for association with immobilized PDGF receptor in vitro. We found that a fragment of p85 that contains a single Src homology domain, the C-terminal SH2 domain (SH2-C), was sufficient for directing the high-affinity interaction with the receptor. Half-maximal binding of SH2-C to the receptor was observed at an SH2-C concentration of 0.06 nM. SH2-C, like full-length p85, was able to distinguish between wild-type PDGF receptor and a mutant receptor lacking the PI 3-kinase binding site. An excess of SH2-C blocked binding of full-length p85 and PI 3-kinase to the receptor but did not interfere with the binding of two other SH2-containing proteins, phospholipase C-gamma and GTPase-activating protein. These results demonstrate that a region of p85 containing a single SH2 domain accounts both for the high affinity and specificity of binding of PI 3-kinase to the PDGF receptor. Images PMID:1312663

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

    NARCIS (Netherlands)

    Soysa, R.; Venselaar, H.; Poston, J.; Ullman, B.; Hasne, M.P.

    2013-01-01

    The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural

  3. Renal phosphate handling: Physiology

    Directory of Open Access Journals (Sweden)

    Narayan Prasad

    2013-01-01

    Full Text Available Phosphorus is a common anion. It plays an important role in energy generation. Renal phosphate handling is regulated by three organs parathyroid, kidney and bone through feedback loops. These counter regulatory loops also regulate intestinal absorption and thus maintain serum phosphorus concentration in physiologic range. The parathyroid hormone, vitamin D, Fibrogenic growth factor 23 (FGF23 and klotho coreceptor are the key regulators of phosphorus balance in body.

  4. Triphenyl phosphate allergy from spectacle frames

    DEFF Research Database (Denmark)

    Carlsen, L; Andersen, K E; Egsgaard, Helge

    1986-01-01

    A case of triphenyl phosphate allergy from spectacle frames is reported. Patch tests with analytical grade triphenyl phosphate, tri-m-cresyl phosphate, and tri-p-cresyl phosphate in the concentrations 5%, 0.5% and 0.05% pet. showed positive reactions to 0.05% triphenyl phosphate and 0.5% tri......-m-cresyl phosphate, but no reaction to tri-p-cresyl phosphate. Gas chromatography of the tricresyl phosphate 5% pet. patch test material supplied from Trolab showed that it contained a mixture of a wide range of triaryl phosphates, including 0.08% triphenyl phosphate which is above the threshold for detecting...

  5. Genesis of apatite in the phosphatized limestones of the western continental shelf of India

    Digital Repository Service at National Institute of Oceanography (India)

    Rao, V.P.; Lamboy, M.

    ) and this siliciclastic flux appear to have transported episodically to the shelf for short duration at about 8300 yr B.P. This flux at places is enriched with iron (Table 1). Iron oxides have strong adsorption capacity for phosphate ions (Nriagu, 1976....C. Burnett and S.R. Riggs (Editors), Neogene to Modern Phosphorites. Phosphate Deposits of the World, 3. Cam- bridge University Press, Cambridge. Cayeux, L., 1939. Existence de nombreuses batteries dans les phosphates sedimentaires de tout age...

  6. Integrated assessment of the phosphate industry

    International Nuclear Information System (INIS)

    Ryan, M.T.; Cotter, S.J.

    1980-05-01

    The phosphate industry in the United States includes three major activities, namely, mining and milling of phosphate rock, phosphate product manufacture, and phosphate product use. Phosphatic materials contain uranium, thorium, and their decay products in greater than background amounts. This assessment of the radiological impacts associated with the redistribution of radioactive components of phosphate materials may provide insight into the effects of uranium extraction from phosphate materials for use in the nuclear fuel cycle

  7. Crystal structure of the high-affinity Na+K+-ATPase-ouabain complex with Mg2+ bound in the cation binding site.

    Science.gov (United States)

    Laursen, Mette; Yatime, Laure; Nissen, Poul; Fedosova, Natalya U

    2013-07-02

    The Na(+),K(+)-ATPase maintains electrochemical gradients for Na(+) and K(+) that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na(+),K(+)-ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na(+),K(+)-ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg(2+), and crystallographic studies indicate that Rb(+) and Mn(2+), but not Na(+), bind to this site. Comparison with the low-affinity [K2]E2-MgF(x)-ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742-13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg(2+). K(+) binding at the same site unwinds a turn of αM4, dragging residues Ile318-Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K(+)-Mg(2+) competition and explain the well-known antagonistic effect of K(+) on CTS binding.

  8. Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

    Science.gov (United States)

    De Meyer, Sofie E; Briscoe, Leah; Martínez-Hidalgo, Pilar; Agapakis, Christina M; de-Los Santos, Paulina Estrada; Seshadri, Rekha; Reeve, Wayne; Weinstock, George; O'Hara, Graham; Howieson, John G; Hirsch, Ann M

    2016-08-01

    Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

  9. Synthesis, in vitro validation and in vivo pharmacokinetics of [{sup 125}I]N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl) ethylamine: A high-affinity ligand for imaging sigma receptor positive tumors

    Energy Technology Data Exchange (ETDEWEB)

    John, Christy S; Gulden, Mary E; Vilner, Bertold J; Bowen, Wayne D

    1996-08-01

    N-[2-(4-iodophenyl)ethyl]-N-methyl-2-(1-piperidinyl)ethylamine, IPEMP, and the corresponding bromo derivative, BrPEMP, have been synthesized and characterized. Both BrPEMP and IPEMP were evaluated for sigma-1 and sigma-2 subtype receptor affinities and found to possess very high affinities for both receptor subtypes. The precursor for radioiodination n-tributylstannylphenylethylpiperidinylethylamine was prepared from its bromo derivative by palladium-catalyzed stannylation reaction. Radioiodinated 4-[{sup 125}I]PEMP was readily prepared in high yields and high specific activity by oxidative iododestannylation reaction using chloramine-T as oxidizing agent. Sites labeled by 4-[{sup 125}I]PEMP in guinea pig brain membranes showed high affinity for BD1008, haloperidol, and (+)-pentazocine (Ki = 5.06 {+-} 0.40, 32.6 {+-} 2.75, and 48.1 {+-} 8.60 nM, respectively), which is consistent with sigma receptor pharmacology. Competition binding studies of 4-[{sup 125}I]PEMP in melanoma (A375) and MCF-7 breast cancer cells showed a high affinity, dose-dependent inhibition of binding with known sigma ligand N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl) ethylamine, BD1008 (Ki = 5, 11 nM, respectively), supporting the labeling of sigma sites in these cells. Haloperidol, however showed a weaker (Ki 100-200 nM) affinity for the sites labeled by 4-[{sup 125}I]PEMP in these cells. Biodistribution studies of 4-[{sup 125}I]PEMP in rats showed a fast clearance of this radiopharmaceutical from blood, liver, lung, and other organs. A co-injection of 4-IPEMP with 4-[{sup 125}I]PEMP resulted in 37%, 69%, and 35% decrease in activity in liver, kidney, and brain (organs possessing sigma receptors), respectively at 1-h postinjection. These results suggest that 4-[{sup 125}I]PEMP is a promising radiopharmaceutical for pursuing further studies in animal models with tumors.

  10. D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

    International Nuclear Information System (INIS)

    Akana, J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

    2006-01-01

    The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (β/α) 8 -barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn 2+ which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn 2+ and inactive apoenzyme cannot be prepared, the affinity for Zn 2+ is decreased by alanine substitutions for the two histidine residues that coordinate the Zn 2+ ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn 2+ . The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn 2+ that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn 2+ and participate as acid/base catalysts are not conserved. We conclude that only the phosphate

  11. Better prospects for phosphate production

    Energy Technology Data Exchange (ETDEWEB)

    1980-06-01

    The extraction of uranium as a by product of phosphate production is discussed. Techniques being commercially developed are described. The trend towards the wet process, in which sulphuric acid is used to dissolve the phosphate, producing phosphoric acid, is also the preferred method for uranium recovery. Recovery from a wet process phosphoric acid stream, integrated with phosphate fertilizer manufacture, is becoming increasingly commercially viable for the production of yellow-cake.

  12. on association of trialkyl phosphates

    International Nuclear Information System (INIS)

    Petkovic, D.M.; Maksimovic, Z.B.

    1976-01-01

    The association constants of tri-n-butyl (TBP), tri-n-propyl (TPP) and triethyl phosphate (TEP) with chloroform, carbon tetrachloride and benzene were determined by dielectric constant, proton magnetic resonance and vapor pressure measurements. Correlation of the trialkyl phosphate-chloroform association constants, using the Hammett equation, showed their increase with the number of carbon atoms in the aliphatic radicals. The change of trialkyl phosphate reactivity with temperature was used to determine the thermodynamic quantities. (author)

  13. Water diffusion in phosphate-containing hydrogels

    International Nuclear Information System (INIS)

    George, K.A.; Wentrup-Byrne, E.; Hill, D.J.T.; Whittaker, A.K.

    2003-01-01

    An understanding of the kinetics and diffusion of liquids through polymeric hydrogels is critical for the successful design and application of these materials in biomedical field, particularly as controlled drug delivery systems. In this study, the mechanisms of water transport and parameters that describe the diffusion process in crosslinked poly(2-hydroxyethylmethacrylate-co-methyloxyethylene phosphate), poly(HEMA-co-MOEP) polymers were investigated. The copolymerisation of HEMA with MOEP was initiated by γ radiolysis with full conversion of monomer to polymer. The sorption of water into the polymers with 0 - 30 mol% MOEP was monitored gravimetrically over a period of 2 - 3 weeks. This study provided an insight into the diffusion mechanism and showed that the PHEMA hydrogel displayed concentration-independent Fickian diffusion. As the concentration of MOEP in the network increased, the diffusion rate and the rigidity of the network also increased in a linear fashion. NMR imaging was used in conjunction with the gravimetric study to elucidate the transport mechanisms, diffusion coefficients and proportionality constants governing the water diffusion in the phosphate-containing polymers. The hydrogels with 3 - 20 mol% MOEP exhibited exponential concentration-dependent Fickian diffusion and the transport mechanism in the system with 30 mol% MOEP was shown to be anomalous. The systems with greater concentrations of MOEP displayed a high degree of fracturing during water sorption and resulted in the ultimate destruction of the cylindrical geometry

  14. Sphingosine 1-phosphate and cancer.

    Science.gov (United States)

    Pyne, Nigel J; El Buri, Ashref; Adams, David R; Pyne, Susan

    2017-09-15

    The bioactive lipid, sphingosine 1-phosphate (S1P) is produced by phosphorylation of sphingosine and this is catalysed by two sphingosine kinase isoforms (SK1 and SK2). Here we discuss structural functional aspects of SK1 (which is a dimeric quaternary enzyme) that relate to coordinated coupling of membrane association with phosphorylation of Ser225 in the 'so-called' R-loop, catalytic activity and protein-protein interactions (e.g. TRAF2, PP2A and G q ). S1P formed by SK1 at the plasma-membrane is released from cells via S1P transporters to act on S1P receptors to promote tumorigenesis. We discuss here an additional novel mechanism that can operate between cancer cells and fibroblasts and which involves the release of the S1P receptor, S1P 2 in exosomes from breast cancer cells that regulates ERK-1/2 signalling in fibroblasts. This novel mechanism of signalling might provide an explanation for the role of S1P 2 in promoting metastasis of cancer cells and which is dependent on the micro-environmental niche. Copyright © 2017. Published by Elsevier Ltd.

  15. Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

    International Nuclear Information System (INIS)

    Butterworth, R.F.; Lavoie, J.; Giguere, J.F.; Pomier-Layrargues, G.

    1988-01-01

    The integrity of GABA-A receptors and of central benzodiazepine receptors was evaluated in membrane preparations from prefrontal cortex and caudate nuclei obtained at autopsy from nine cirrhotic patients who died in hepatic coma and an equal number of age-matched control subjects. Histopathological studies revealed Alzheimer Type II astrocytosis in all cases in the cirrhotic group; controls were free from neurological, psychiatric or hepatic diseases. Binding to GABA-A receptors was studied using [ 3 H]muscimol as radioligand. The integrity of central benzodiazepine receptors was evaluated using [ 3 H]flunitrazepam and [ 3 H]Ro15-1788. Data from saturation binding assays was analyzed by Scatchard plot. No modifications of either affinities (Kd) or densities (Bmax) of [ 3 H]muscimol of central benzodiazepine binding sites were observed. These findings do not support recent suggestions that alterations of either high-affinity GABA or benzodiazepine receptors play a significant role in the pathogenesis of hepatic encephalopathy

  16. Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

    International Nuclear Information System (INIS)

    Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

    1989-01-01

    The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

  17. Phosphate Recovery From Sewage Sludge Containing Iron Phosphate

    NARCIS (Netherlands)

    Wilfert, P.K.

    2018-01-01

    The scope of this thesis was to lay the basis for a phosphate recovery technology that can be applied on sewage sludge containing iron phosphate. Such a technology should come with minimal changes to the existing sludge treatment configuration while keeping the use of chemicals or energy as small as

  18. 21 CFR 137.175 - Phosphated flour.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 2 2010-04-01 2010-04-01 false Phosphated flour. 137.175 Section 137.175 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN... Related Products § 137.175 Phosphated flour. Phosphated flour, phosphated white flour, and phosphated...

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

    NARCIS (Netherlands)

    Berghe, van den P.V.E; Folmer, D.E.; Malingré, H.E.M.; Beurden, van E.; Klomp, A.E.M.; Sluis, van de B.; Merkx, M.; Berger, R.J.; Klomp, L.W.J.

    2007-01-01

    High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis,

  20. Mechanism of Osmotic Activation of the Quaternary Ammonium Compound Transporter (QacT) of Lactobacillus plantarum

    NARCIS (Netherlands)

    Glaasker, Erwin; Heuberger, Esther H.M.L.; Konings, Wil N.; Poolman, Bert

    1998-01-01

    The accumulation of quaternary ammonium compounds in Lactobacillus plantarum is mediated via a single transport system with a high affinity for glycine betaine (apparent Km of 18 μM) and carnitine and a low affinity for proline (apparent Km of 950 μM) and other analogues. Mutants defective in the

  1. Post-transcriptional regulation of the arginine transporter Cat-1 by amino acid availability

    NARCIS (Netherlands)

    Aulak, K. S.; Mishra, R.; Zhou, L.; Hyatt, S. L.; de Jonge, W.; Lamers, W.; Snider, M.; Hatzoglou, M.

    1999-01-01

    The regulation of the high affinity cationic amino acid transporter (Cat-1) by amino acid availability has been studied. In C6 glioma and NRK kidney cells, cat-1 mRNA levels increased 3.8-18-fold following 2 h of amino acid starvation. The transcription rate of the cat-1 gene remained unchanged

  2. A Mycorrhizal-Specific Ammonium Transporter from Lotus japonicus Acquires Nitrogen Released by Arbuscular Mycorrhizal Fungi1

    Science.gov (United States)

    Guether, Mike; Neuhäuser, Benjamin; Balestrini, Raffaella; Dynowski, Marek; Ludewig, Uwe; Bonfante, Paola

    2009-01-01

    In mycorrhizal associations, the fungal partner assists its plant host by providing nitrogen (N) in addition to phosphate. Arbuscular mycorrhizal (AM) fungi have access to inorganic or organic forms of N and translocate them via arginine from the extra- to the intraradical mycelium, where the N is transferred to the plant without any carbon skeleton. However, the molecular form in which N is transferred, as well as the involved mechanisms, is still under debate. NH4+ seems to be the preferential transferred molecule, but no plant ammonium transporter (AMT) has been identified so far. Here, we offer evidence of a plant AMT that is involved in N uptake during mycorrhiza symbiosis. The gene LjAMT2;2, which has been shown to be the highest up-regulated gene in a transcriptomic analysis of Lotus japonicus roots upon colonization with Gigaspora margarita, has been characterized as a high-affinity AMT belonging to the AMT2 subfamily. It is exclusively expressed in the mycorrhizal roots, but not in the nodules, and transcripts have preferentially been located in the arbusculated cells. Yeast (Saccharomyces cerevisiae) mutant complementation has confirmed its functionality and revealed its dependency on acidic pH. The transport experiments using Xenopus laevis oocytes indicated that, unlike other plant AMTs, LjAMT2;2 transports NH3 instead of NH4+. Our results suggest that the transporter binds charged ammonium in the apoplastic interfacial compartment and releases the uncharged NH3 into the plant cytoplasm. The implications of such a finding are discussed in the context of AM functioning and plant phosphorus uptake. PMID:19329566

  3. Overexpression of Thellungiella halophila H+-pyrophosphatase Gene Improves Low Phosphate Tolerance in Maize

    Science.gov (United States)

    Pei, Laming; Wang, Jiemin; Li, Kunpeng; Li, Yongjun; Li, Bei; Gao, Feng; Yang, Aifang

    2012-01-01

    Low phosphate availability is a major constraint on plant growth and agricultural productivity. Engineering a crop with enhanced low phosphate tolerance by transgenic technique could be one way of alleviating agricultural losses due to phosphate deficiency. In this study, we reported that transgenic maize plants that overexpressed the Thellungiella halophila vacuolar H+-pyrophosphatase gene (TsVP) were more tolerant to phosphate deficit stress than the wild type. Under phosphate sufficient conditions, transgenic plants showed more vigorous root growth than the wild type. When phosphate deficit stress was imposed, they also developed more robust root systems than the wild type, this advantage facilitated phosphate uptake, which meant that transgenic plants accumulated more phosphorus. So the growth and development in the transgenic maize plants were not damaged as much as in the wild type plants under phosphate limitation. Overexpression of TsVP increased the expression of genes involved in auxin transport, which indicated that the development of larger root systems in transgenic plants might be due in part to enhanced auxin transport which controls developmental events in plants. Moreover, transgenic plants showed less reproductive development retardation and a higher grain yield per plant than the wild type plants when grown in a low phosphate soil. The phenotypes of transgenic maize plants suggested that the overexpression of TsVP led to larger root systems that allowed transgenic maize plants to take up more phosphate, which led to less injury and better performance than the wild type under phosphate deficiency conditions. This study describes a feasible strategy for improving low phosphate tolerance in maize and reducing agricultural losses caused by phosphate deficit stress. PMID:22952696

  4. Phosphate glasses, containing nitrogen

    International Nuclear Information System (INIS)

    Lisitsyna, E.A.; Khalilev, V.D.; Koryavin, A.A.; Goncharova, L.N.

    1987-01-01

    Possibilities of nitrogen-containing glass synthesis by the introduction into the charge of ammonium salts, as well as aluminium nitride, are studied. Zinc alumoyttrium phosphate glass (mol. %) Zn(PO 3 ) 2 - 4O, Al(PO 3 ) 3 - 3O, Y(PO 3 ) 3 -3O is suggested as a matrix. It is shown that the effect of amide and imide groups on the properties of the glass is less noticeable than the effect of nitride groups. Direct introduction of nitride constituent was realized using AlN, but aluminium introduction was taken into account so that the oxide was subtracted. The attempt to introduce more than 2.5 mass % of nitrogen into initial matrix by aluminium nitride has failed due to repeated restoration of glass with amorphous phosphorus isolation

  5. Plant nutrient transporter regulation in arbuscular mycorrhizas

    DEFF Research Database (Denmark)

    Burleigh, Stephen; Bechmann, I.E.

    2002-01-01

    of nutrition. Their down-regulation in mycorrhizal roots, therefore, would be predicted as a result of symbiotic function. A variety of studies on Pi- Zn- and ammonium- or nitrate-transporter genes from two plant species indirectly support this model. For example, one study showed that the expression...... of the high-affinity Pi-transporter MtPT2 within mycorrhizal roots of Medicago truncatula was inversely correlated with the concentration of P within the shoots, which suggested that P supply from the fungus influenced this gene's expression. However, there is some evidence that these plant nutrient...

  6. Inorganic phosphate uptake in unicellular eukaryotes.

    Science.gov (United States)

    Dick, Claudia F; Dos-Santos, André L A; Meyer-Fernandes, José R

    2014-07-01

    Inorganic phosphate (Pi) is an essential nutrient for all organisms. The route of Pi utilization begins with Pi transport across the plasma membrane. Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of Pi transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding Pi uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum. Pi uptake is the key step of Pi homeostasis and in the subsequent signaling event in eukaryotic microorganisms. Biochemical and structural studies are important for clarifying mechanisms of Pi homeostasis, as well as Pi sensor and downstream pathways, and raise possibilities for future studies in this field. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Electrochemical Reduction of Zinc Phosphate

    International Nuclear Information System (INIS)

    Kim, Chang Hwan; Lee, Jung Hyun; Shin, Woon Sup

    2010-01-01

    We demonstrated first that the electrochemical reduction of zinc phosphate in neutral phosphate buffer is possible and potentially applicable to bio-compatible rechargeable battery. The actual redox component is Zn(s)/Zn phosphate(s) and the future research about the control of crystal formation for the better cyclability is required. In lead-acid battery, the electrochemical redox reaction of Pb (s) /PbSO 4(s) is used by reducing Pb(II) and oxidizing Pb(0) in sulfate rich solution. Since both reduced form and oxidized form are insoluble, they cannot diffuse to the opposite electrodes and react. It is a very common strategy to make a stable battery electrode that a metal element is reduced and oxidized in solution containing an abundance of anion readily precipitating with the metal ion. For the application of this strategy to construction of rechargeable battery using bio-compatible electrode materials and electrolytes, the use of phosphate ion can be considered as anion readily precipitating with metal ions. If phosphate buffer with neutral pH is used as electrolyte, the better bio-compatibility will be achieved than most of rechargeable battery using strong acid, strong base or organic solvent as electrolyte solution. There are many metal ions readily precipitating with phos-phate ion, and zinc is one of them

  8. Hydrogen permeation resistant phosphate coatings

    International Nuclear Information System (INIS)

    McGuire, J.C.

    1979-01-01

    A method for reducing hydrogen diffusion through metal wherein the metal is coated with a phosphate-radical-containing, phosphate-glass-forming material on at least one surface thereof. The coating is then heated to at least 350 0 C to form a phosphate glass. This method is especially applicable to nuclear reactors to minimize tritium diffusion. The coating is preferably formed with a solution of phosphoric acid which may also contain compounds such as MnSO 4 , SiO 2 and Na 2 Cr 2 0 7 . (author)

  9. Hydrogen permeation resistant phosphate coatings

    International Nuclear Information System (INIS)

    McGuire, J.C.

    1979-01-01

    A method for reducing hydrogen diffusion through metal is described. The metal is coated with a phosphate-radical-containing, phosphate-glass-forming material on at least one surface. The coating is then heated to at least 350 0 C to form a phosphate glass. This method is especially applicable to nuclear reactors to minimize tritium diffusion. The coating is preferably formed with a solution of phosphoric acid which may also contain compounds such as MnSO 4 , SiO 2 and Na 2 Cr 2 O 7 . (author)

  10. Calcium phosphates for biomedical applications

    Directory of Open Access Journals (Sweden)

    Maria Canillas

    2017-05-01

    Full Text Available The history of calcium phosphates in the medicine field starts in 1769 when the first evidence of its existence in the bone tissue is discovered. Since then, the interest for calcium phosphates has increased among the scientific community. Their study has been developed in parallel with new advances in materials sciences, medicine or tissue engineering areas. Bone tissue engineering is the field where calcium phosphates have had a great importance. While the first bioceramics are selected according to bioinert, biocompatibility and mechanical properties with the aim to replace bone tissue damaged, calcium phosphates open the way to the bone tissue regeneration challenge. Nowadays, they are present in the majority of commercial products directed to repair or regenerate damaged bone tissue. Finally, in the last few decades, they have been suggested and studied as drug delivering devices and as vehicles of DNA and RNA for the future generation therapies.

  11. Variability of nitrate and phosphate

    Digital Repository Service at National Institute of Oceanography (India)

    Sardessai, S.; Sundar, D.

    Nitrate and phosphate are important elements of the biogeochemical system of an estuary. Observations carried out during the dry season April-May 2002, and March 2003 and wet season September 2002, show temporal and spatial variability of these two...

  12. Preparation of calcium phosphate paste

    International Nuclear Information System (INIS)

    Mohd Reusmaazran Yusof; Norzita Yaacob; Idris Besar; Che Seman Mahmood; Rusnah Mustafa

    2010-01-01

    Calcium phosphate paste were prepared by mixing between calcium sodium potassium phosphate, Ca 2 NaK (PO 4 ) 2 (CSPP) and monocalcium phosphate monohydrate, Ca(H 2 PO 4 ) 2 .H 2 O (MCPM). CSPP were obtained by reaction between calcium hydrogen phosphate (CaHPO 4 ), potassium carbonate (K 2 CO 3 ) and sodium carbonate (Na 2 CO 3 ) in solid state sintering process followed by quenching in air at 1000 degree Celsius. The paste was aging in simulated body fluid (SBF) for 0.5, 1, 3, 6, 12, 24, 48 hrs, 3, 7 and 14 days. The morphological investigation indicated the formation of apatite crystal were first growth after 24 hours. The obvious growth of apatite crystal was shown at 3 days. The obvious growth of apatite crystal was shown in 7 and 14 days indicated the prediction of paste would have rapid reaction with bone after implantation. (author)

  13. Calcium phosphates for biomedical applications

    Energy Technology Data Exchange (ETDEWEB)

    Canillas, M.; Pena, P.; Aza, A.H. de; Rodriguez, M.A.

    2017-07-01

    The history of calcium phosphates in the medicine field starts in 1769 when the first evidence of its existence in the bone tissue is discovered. Since then, the interest for calcium phosphates has increased among the scientific community. Their study has been developed in parallel with new advances in materials sciences, medicine or tissue engineering areas. Bone tissue engineering is the field where calcium phosphates have had a great importance. While the first bioceramics are selected according to bioinert, biocompatibility and mechanical properties with the aim to replace bone tissue damaged, calcium phosphates open the way to the bone tissue regeneration challenge. Nowadays, they are present in the majority of commercial products directed to repair or regenerate damaged bone tissue. Finally, in the last few decades, they have been suggested and studied as drug delivering devices and as vehicles of DNA and RNA for the future generation therapies. (Author)

  14. Reference: 150 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ridization, Pht1;4 was found mainly expressed in inorgan...physiological characterization of Arabidopsis pht1;4 high affinity phosphate transporter mutants. Using GUS-gene trap and in situ hyb

  15. Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity

    OpenAIRE

    Vasudevan, Gayatri; Ullman, Buddy; Landfear, Scott M.

    2001-01-01

    Leishmania parasites lack a purine biosynthetic pathway and depend on surface nucleoside and nucleobase transporters to provide them with host purines. Leishmania donovani possess two closely related genes that encode high affinity adenosine-pyrimidine nucleoside transporters LdNT1.1 and LdNT1.2 and that transport the toxic adenosine analog tubercidin in addition to the natural substrates. In this study, we have characterized a drug-resistant clonal mutant of L. do...

  16. Phosphate solubilization and multiple plant growth promoting ...

    African Journals Online (AJOL)

    Phosphate solubilizing efficiencies of the strains were analyzed using different insoluble phosphorus sources and the results show that most isolates released a substantial amount of soluble phosphate from tricalcium phosphate, rock phosphate and bone meal. Screening for multiple plant growth promoting attributes ...

  17. Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and α1β2δ GABAA receptors

    DEFF Research Database (Denmark)

    Damgaard, Maria; Al-Khawaja, Anas; Nittegaard-Nielsen, Mia

    2017-01-01

    -affinity binding and is furthermore reported as an allosteric modulator selective for the α1β2δ GABAARs. Therefore, structural determinants for selectivity at the two targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing the Biginelli cyclocondensation and examined......-affinity binding. However, three analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [(3)H]NCS-382 to a higher maximal level than seen for monastrol itself. Selected compounds were further characterized as modulators at α1β2δ, α1β2γ2s and α1β2 GABAARs. Most of these modulators were shown to have...... δ-specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB high-affinity binding and α1β2δ GABAAR activity was also shown for the compounds 11, 18 and 24. Compound 29 displayed minimal modulatory effect on GABAARs and therefore appears to be a GHB high...

  18. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications.

    Science.gov (United States)

    Imaduwage, Kasun P; Go, Eden P; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have K i values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods. Graphical Abstract ᅟ.

  19. Electrical properties of phosphate glasses

    International Nuclear Information System (INIS)

    Mogus-Milankovic, A; Santic, A; Reis, S T; Day, D E

    2009-01-01

    Investigation of the electrical properties of phosphate glasses where transition metal oxide such as iron oxide is the network former and network modifier is presented. Phosphate glasses containing iron are electronically conducting glasses where the polaronic conduction is due to the electron hopping from low to high iron valence state. The identification of structural defects caused by ion/polaron migration, the analysis of dipolar states and electrical conductivity in iron phosphate glasses containing various alkali and mixed alkali ions was performed on the basis of the impedance spectroscopy (IS). The changes in electrical conductivity from as-quenched phosphate glass to fully crystallized glass (glass-ceramics) by IS are analyzed. A change in the characteristic features of IS follows the changes in glass and crystallized glass network. Using IS, the contribution of glass matrix, crystallized grains and grain boundary to the total electrical conductivity for iron phosphate glasses was analyzed. It was shown that decrease in conductivity is caused by discontinuities in the conduction pathways as a result of the disruption of crystalline network where two or more crystalline phases are formed. Also, phosphate-based glasses offer a unique range of biomaterials, as they form direct chemical bonding with hard/soft tissue. The surface charges of bioactive glasses are recognized to be the most important factors in determining biological responses. The improved bioactivity of the bioactive glasses as a result of the effects of the surface charges generated by electrical polarization is discussed.

  20. High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture.

    Science.gov (United States)

    Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

    2018-05-01

    Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4×10-6 M and BMAX[APPARENT] ≈ 6 pmol/106 cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5×10-3 M and BMAX[APPARENT] ≈ 350 pmol/106 cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

  1. In Vitro and In Vivo Evaluation of Alexa Fluor 680-Bombesin[7–14]NH2 Peptide Conjugate, a High-Affinity Fluorescent Probe with High Selectivity for the Gastrin-Releasing Peptide Receptor

    Directory of Open Access Journals (Sweden)

    Lixin Ma

    2007-05-01

    Full Text Available Gastrin-releasing peptide (GRP receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN, a 14–amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H2N-glycylglycylglycine-BBN[7–14]NH2 peptide with the following general sequence: H2N-G-G-G-Q-W-A-V-G-H-L-M-(NH2. This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7–14]NH2 conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H2N-G-G-G-BBN[7–14]NH2 in dimethylformamide (DMF. In vitro competitive binding assays, using 125I-Tyr4-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 ± 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7–14]NH2 in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

  2. Use of osmolytes during solubilization and reconstitution of phosphate: sugar phosphate antiport from bacteria

    International Nuclear Information System (INIS)

    Ambudkar, S.V.; Sonna, L.A.; Maloney, P.C.

    1986-01-01

    Phosphate:2-deoxyglucose 6-phosphate (Pi:2DG6P) antiport was extracted from Streptococcus lactis or Staphylococcus aureus with 1.1% octylglucoside in the presence of 0.37% E. coli lipid and reconstituted by detergent dilution. Because previous work suggested inactivation at an early stage, the authors introduced protein stabilants during solubilization. When 20% glycerol was used, proteoliposomes showed a 20-fold increase in 32 Pi transport. This enhanced recovery required phospholipid plus glycerol, and was found only when both were added together with the detergent. Glycerol protection yielded proteoliposomes in which antiporters retained their normal kinetic properties, and Pi exchange by the streptococcal example gave a maximal rate (200-400 nmol/min per mg protein) and a turnover number (30-50/s) which suggested that inactivation had been avoided. Further study showed that 20% glycerol could be replaced by equally high concentrations of compounds classified as osmolytes polyols (erythritol, xylitol, sorbitol), sugars (glucose, trehalose) and certain amino acids (glycine, proline, but not valine). The authors suggest that osmolytes may be used to fully stabilize chemiosmotic transporters during reconstitution

  3. Uranium endowments in phosphate rock

    International Nuclear Information System (INIS)

    Ulrich, Andrea E.; Schnug, Ewald; Prasser, Horst-Michael; Frossard, Emmanuel

    2014-01-01

    This study seeks to identify and specify the components that make up the prospects of U recovery from phosphate rock. A systems approach is taken. The assessment includes i) reviewing past recovery experience and lessons learned; ii) identifying factors that determine recovery; and iii) establishing a contemporary evaluation of U endowments in phosphate rock reserves, as well as the available and recoverable amounts from phosphate rock and phosphoric acid production. We find that in the past, recovery did not fulfill its potential and that the breakup of the Soviet Union worsened then-favorable recovery market conditions in the 1990s. We find that an estimated 5.7 million tU may be recoverable from phosphate rock reserves. In 2010, the recoverable tU from phosphate rock and phosphoric acid production may have been 15,000 tU and 11,000 tU, respectively. This could have filled the world U supply-demand gap for nuclear energy production. The results suggest that the U.S., Morocco, Tunisia, and Russia would be particularly well-suited to recover U, taking infrastructural considerations into account. We demonstrate future research needs, as well as sustainability orientations. We conclude that in order to promote investment and production, it seems necessary to establish long-term contracts at guaranteed prices, ensuring profitability for phosphoric acid producers. - Highlights: • We identify components that underlie the recovery of uranium from phosphate rock. • We estimate that 11,000 tU may have been recoverable from phosphoric acid in 2010. • Recovery is a resource conservation and environmental pollution control strategy. • To ensure investment in recovery technology, profitability needs to be secured

  4. Uranium endowments in phosphate rock

    Energy Technology Data Exchange (ETDEWEB)

    Ulrich, Andrea E., E-mail: andrea.ulrich@env.ethz.ch [Institute for Environmental Decisions (IED), Natural and Social Science Interface, ETH Zurich Universitässtrasse 22, 8092 Zurich (Switzerland); Institute for Agricultural Sciences, Plant Nutrition, ETH Zurich, Eschikon 33, 8315 Lindau (Switzerland); Schnug, Ewald, E-mail: e.schnug@tu-braunschweig.de [Department of Life Sciences, Technical University of Braunschweig, Pockelsstraße 14, D-38106 Braunschweig (Germany); Prasser, Horst-Michael, E-mail: prasser@lke.mavt.ethz.ch [Institute of Energy Technology, Laboratory of Nuclear Energy Systems, ETH Zurich, Sonneggstrasse 3, 8092 Zurich (Switzerland); Frossard, Emmanuel, E-mail: emmanuel.frossard@usys.ethz.ch [Institute for Agricultural Sciences, Plant Nutrition, ETH Zurich, Eschikon 33, 8315 Lindau (Switzerland)

    2014-04-01

    This study seeks to identify and specify the components that make up the prospects of U recovery from phosphate rock. A systems approach is taken. The assessment includes i) reviewing past recovery experience and lessons learned; ii) identifying factors that determine recovery; and iii) establishing a contemporary evaluation of U endowments in phosphate rock reserves, as well as the available and recoverable amounts from phosphate rock and phosphoric acid production. We find that in the past, recovery did not fulfill its potential and that the breakup of the Soviet Union worsened then-favorable recovery market conditions in the 1990s. We find that an estimated 5.7 million tU may be recoverable from phosphate rock reserves. In 2010, the recoverable tU from phosphate rock and phosphoric acid production may have been 15,000 tU and 11,000 tU, respectively. This could have filled the world U supply-demand gap for nuclear energy production. The results suggest that the U.S., Morocco, Tunisia, and Russia would be particularly well-suited to recover U, taking infrastructural considerations into account. We demonstrate future research needs, as well as sustainability orientations. We conclude that in order to promote investment and production, it seems necessary to establish long-term contracts at guaranteed prices, ensuring profitability for phosphoric acid producers. - Highlights: • We identify components that underlie the recovery of uranium from phosphate rock. • We estimate that 11,000 tU may have been recoverable from phosphoric acid in 2010. • Recovery is a resource conservation and environmental pollution control strategy. • To ensure investment in recovery technology, profitability needs to be secured.

  5. Deletion mutants of the Escherichia coli K-12 mannitol permease: dissection of transport-phosphorylation, phospho-exchange, and mannitol-binding activities.

    Science.gov (United States)

    Grisafi, P L; Scholle, A; Sugiyama, J; Briggs, C; Jacobson, G R; Lengeler, J W

    1989-05-01

    We have constructed a series of deletion mutations of the cloned Escherichia coli K-12 mtlA gene, which encodes the mannitol-specific enzyme II of the phosphoenolpyruvate (PEP)-dependent carbohydrate phosphotransferase system. This membrane-bound permease consists of 637 amino acid residues and is responsible for the concomitant transport and phosphorylation of D-mannitol in E. coli. Deletions into the 3' end of mtlA were constructed by exonuclease III digestion. Restriction mapping of the resultant plasmids identified several classes of deletions that lacked approximately 5% to more than 75% of the gene. Immunoblotting experiments revealed that many of these plasmids expressed proteins within the size range predicted by the restriction analyses, and all of these proteins were membrane localized, which demonstrated that none of the C-terminal half of the permease is required for membrane insertion. Functional analyses of the deletion proteins, expressed in an E. coli strain deleted for the chromosomal copy of mtlA, showed that all but one of the strains containing confirmed deletions were inactive in transport and PEP-dependent phosphorylation of mannitol, but deletions removing up to at least 117 amino acid residues from the C terminus of the permease were still active in catalyzing phospho exchange between mannitol 1-phosphate and mannitol. A deletion protein that lacked 240 residues from the C terminus of the permease was inactive in phospho exchange but still bound mannitol with high affinity. These experiments localize sites important for transport and PEP-dependent phosphorylation to the extreme C terminus of the mannitol permease, sites important for phospho exchange to between residues 377 and 519, and sites necessary for mannitol binding to the N-terminal 60% of the molecule. The results are discussed with respect to the fact that the mannitol permease consists of structurally independent N- and C-terminal domains.

  6. Phosphorus release from phosphate rock and iron phosphate by low-molecular-weight organic acids.

    Science.gov (United States)

    Xu, Ren-kou; Zhu, Yong-guan; Chittleborough, David

    2004-01-01

    Low-molecular-weight(LMW) organic acids widely exist in soils, particularly in the rhizosphere. A series of batch experiments were carried out to investigate the phosphorus release from rock phosphate and iron phosphate by low-molecular-weight organic acids. Results showed that citric acid had the highest capacity to solubilize P from both rock and iron phosphate. P solubilization from rock phosphate and iron phosphate resulted in net proton consumption. P release from rock phosphate was positively correlated with the pKa values. P release from iron phosphate was positively correlated with Fe-organic acid stability constants except for aromatic acids, but was notcorrelated with pKa. Increase in the concentrations of organic acids enhanced P solubilization from both rock and iron phosphate almost linearly. Addition of phenolic compounds further increased the P release from iron phosphate. Initial solution pH had much more substantial effect on P release from rock phosphate than from iron phosphate.

  7. SLC37A1 and SLC37A2 are phosphate-linked, glucose-6-phosphate antiporters.

    Directory of Open Access Journals (Sweden)

    Chi-Jiunn Pan

    Full Text Available Blood glucose homeostasis between meals depends upon production of glucose within the endoplasmic reticulum (ER of the liver and kidney by hydrolysis of glucose-6-phosphate (G6P into glucose and phosphate (P(i. This reaction depends on coupling the G6P transporter (G6PT with glucose-6-phosphatase-α (G6Pase-α. Only one G6PT, also known as SLC37A4, has been characterized, and it acts as a P(i-linked G6P antiporter. The other three SLC37 family members, predicted to be sugar-phosphate:P(i exchangers, have not been characterized functionally. Using reconstituted proteoliposomes, we examine the antiporter activity of the other SLC37 members along with their ability to couple with G6Pase-α. G6PT- and mock-proteoliposomes are used as positive and negative controls, respectively. We show that SLC37A1 and SLC37A2 are ER-associated, P(i-linked antiporters, that can transport G6P. Unlike G6PT, neither is sensitive to chlorogenic acid, a competitive inhibitor of physiological ER G6P transport, and neither couples to G6Pase-α. We conclude that three of the four SLC37 family members are functional sugar-phosphate antiporters. However, only G6PT/SLC37A4 matches the characteristics of the physiological ER G6P transporter, suggesting the other SLC37 proteins have roles independent of blood glucose homeostasis.

  8. Dopamine transporter-dependent and -independent striatal binding of the benztropine analog JHW 007, a cocaine antagonist with low abuse liability

    Science.gov (United States)

    The benztropine analog JHW 007 displays high affinity for the dopamine transporter (DAT), but unlike typical DAT ligands, has relatively low abuse liability and blocks effects of cocaine,including its self-administration. To determine sites responsible for the cocaine-antagonist effects of JHW 007, ...

  9. Nitric oxide induces the alternative oxidase pathway in Arabidopsis seedlings deprived of inorganic phosphate.

    Science.gov (United States)

    Royo, Beatriz; Moran, Jose F; Ratcliffe, R George; Gupta, Kapuganti J

    2015-10-01

    Phosphate starvation compromises electron flow through the cytochrome pathway of the mitochondrial electron transport chain, and plants commonly respond to phosphate deprivation by increasing flow through the alternative oxidase (AOX). To test whether this response is linked to the increase in nitric oxide (NO) production that also increases under phosphate starvation, Arabidopsis thaliana seedlings were grown for 15 d on media containing either 0 or 1mM inorganic phosphate. The effects of the phosphate supply on growth, the production of NO, respiration, the AOX level and the production of superoxide were compared for wild-type (WT) seedlings and the nitrate reductase double mutant nia. Phosphate deprivation increased NO production in WT roots, and the AOX level and the capacity of the alternative pathway to consume electrons in WT seedlings; whereas the same treatment failed to stimulate NO production and AOX expression in the nia mutant, and the plants had an altered growth phenotype. The NO donor S-nitrosoglutathione rescued the growth phenotype of the nia mutants under phosphate deprivation to some extent, and it also increased the respiratory capacity of AOX. It is concluded that NO is required for the induction of the AOX pathway when seedlings are grown under phosphate-limiting conditions. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  10. Uranium abundance in some sudanese phosphate ores

    International Nuclear Information System (INIS)

    Adam, A.A.; Eltayeb, M.A.H.

    2009-01-01

    This work was carried out mainly to analysis of some Sudanese phosphate ores, for their uranium abundance and total phosphorus content measured as P 2 O 5 %. For this purpose, 30 samples of two types of phosphate ore from Eastern Nuba Mountains, in Sudan namely, Kurun and Uro areas were examined. In addition, the relationship between uranium and major, and trace elements were obtained, also, the natural radioactivity of the phosphate samples was measured, in order to characterize and differentiate between the two types of phosphate ores. The uranium abundance in Uro phosphate with 20.3% P 2 O 5 is five time higher than in Kurun phosphate with 26.7% P 2 O 5 . The average of uranium content was found to be 56.6 and 310 mg/kg for Kurun and Uro phosphate ore, respectively. The main elements in Kurun and Uro phosphate ore are silicon, aluminum, and phosphorus, while the most abundant trace elements in these two ores are titanium, strontium and barium. Pearson correlation coefficient revealed that uranium in Kurun phosphate shows strong positive correlation with P 2 O 5 , and its distribution is essentially controlled by the variations of P2O5 concentration, whereas uranium in Uro phosphate shows strong positive correlation with strontium, and its distribution is controlled by the variations of Sr concentration. Uranium behaves in different ways in Kurun phosphate and in Uro phosphate. Uro phosphate shows higher concentrations of all the estimated radionuclides than Kurun phosphate. According to the obtained results, it can be concluded that Uro phosphate is consider as secondary uranium source, and is more suitable for uranium recovery, because it has high uranium abundance and low P 2 O 5 %, than Kurun phosphate. (authors) [es

  11. Motif III in superfamily 2 "helicases" helps convert the binding energy of ATP into a high-affinity RNA binding site in the yeast DEAD-box protein Ded1.

    Science.gov (United States)

    Banroques, Josette; Doère, Monique; Dreyfus, Marc; Linder, Patrick; Tanner, N Kyle

    2010-03-05

    Motif III in the putative helicases of superfamily 2 is highly conserved in both its sequence and its structural context. It typically consists of the sequence alcohol-alanine-alcohol (S/T-A-S/T). Historically, it was thought to link ATPase activity with a "helicase" strand displacement activity that disrupts RNA or DNA duplexes. DEAD-box proteins constitute the largest family of superfamily 2; they are RNA-dependent ATPases and ATP-dependent RNA binding proteins that, in some cases, are able to disrupt short RNA duplexes. We made mutations of motif III (S-A-T) in the yeast DEAD-box protein Ded1 and analyzed in vivo phenotypes and in vitro properties. Moreover, we made a tertiary model of Ded1 based on the solved structure of Vasa. We used Ded1 because it has relatively high ATPase and RNA binding activities; it is able to displace moderately stable duplexes at a large excess of substrate. We find that the alanine and the threonine in the second and third positions of motif III are more important than the serine, but that mutations of all three residues have strong phenotypes. We purified the wild-type and various mutants expressed in Escherichia coli. We found that motif III mutations affect the RNA-dependent hydrolysis of ATP (k(cat)), but not the affinity for ATP (K(m)). Moreover, mutations alter and reduce the affinity for single-stranded RNA and subsequently reduce the ability to disrupt duplexes. We obtained intragenic suppressors of the S-A-C mutant that compensate for the mutation by enhancing the affinity for ATP and RNA. We conclude that motif III and the binding energy of gamma-PO(4) of ATP are used to coordinate motifs I, II, and VI and the two RecA-like domains to create a high-affinity single-stranded RNA binding site. It also may help activate the beta,gamma-phosphoanhydride bond of ATP. (c) 2009 Elsevier Ltd. All rights reserved.

  12. Radiosynthesis and in vitro evaluation of 2-(N-alkyl-N-1'-11C-propyl)amino-5-hydroxytetralin analogs as high affinity agonists for dopamine D-2 receptors

    International Nuclear Information System (INIS)

    Shi Bingzhi; Narayanan, Tanjore K.; Yang, Z.-Y.; Christian, Bradley T.; Mukherjee, Jogeshwar

    1999-01-01

    We have developed radiotracers based on agonists that may potentially allow the in vivo assessment of the high affinity (HA) state of the dopamine D-2 receptors. The population of HA state, which is likely the functional state of the receptor, may be altered in certain diseases. We carried out radiosyntheses and evaluated the binding affinities, lipophilicity, and in vitro autoradiographic binding characteristics of three dopamine D-2 receptor agonists: (±)-2-(N,N-dipropyl)amino-5-hydroxytetralin (5-OH-DPAT), (±)-2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin (PPHT), and (±)-2-(N-cyclohexylethyl-N-propyl)amino-5-hydroxytetralin (ZYY-339). In 3 H-spiperone assays using rat striata, ZYY-339 exhibited subnanomolar affinity for D-2 receptor sites ( IC 50 =0.010 nM), PPHT was somewhat weaker ( IC 50 =0.65 nM), and 5-OH-DPAT exhibited the weakest affinity ( IC 50 =2.5 nM) of the three compounds. Radiosynthesis of these derivatives, 2-(N-propyl-N-1'- 11 C-propyl)amino-5-hydroxytetralin ( 11 C-5-OH-DPAT), 2-(N-phenethyl-N-1'- 11 C-propyl)amino-5-hydroxytetralin ( 11 C-PPHT), and 2-(N-cyclohexylethyl-N-1'- 11 C-propyl)amino-5-hydroxytetralin ( 11 C-ZYY-339) was achieved by first synthesizing 11 C-1-propionyl chloride and subsequent coupling with the appropriate secondary amine precursor to form the respective amide, which was then reduced to provide the desired tertiary amine products. The final products were obtained by reverse-phase high performance liquid chromatography (HPLC) purification in radiochemical yields of 5-10% after 60-75 min from the end of 11 CO 2 trapping and with specific activities in the range of 250-1,000 Ci/mmol. In vitro autoradiographs in rat brain slices with 11 C-5-OH-DPAT, 11 C-PPHT, and 11 C-ZYY-339 revealed selective binding of the three radiotracers to the dopamine D-2 receptors in the striata

  13. (D-Pen2,4 prime -125I-Phe4,D-Pen5)enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Knapp, R.J.; Sharma, S.D.; Toth, G.; Duong, M.T.; Fang, L.; Bogert, C.L.; Weber, S.J.; Hunt, M.; Davis, T.P.; Wamsley, J.K. (Department of Pharmacology, University of Arizona, College of Medicine, Tucson (United States))

    1991-09-01

    (D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.

  14. Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain*

    Science.gov (United States)

    Bhattacharya, Sarbani; Liang, Zhong; Quek, Adam J.; Ploplis, Victoria A.; Law, Ruby; Castellino, Francis J.

    2014-01-01

    A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97–125 + Tyr), interacted specifically with isolated K2hPg. However, the binding strength of VEK30 (KD = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (KD = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH2 and COOH termini of VEK30. VEK64 (PAM residues 83–145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2hPg with nearly the same affinity as PAM (KD = 1–2 nm). However, addition of two PAM residues (Arg126-His127) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg113, His114, Glu116, Arg126, His127), mutation of which reduced PAM binding affinity for K2hPg by ∼1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence. PMID:24962580

  15. Dimerization is not a determining factor for functional high affinity human plasminogen binding by the group A streptococcal virulence factor PAM and is mediated by specific residues within the PAM a1a2 domain.

    Science.gov (United States)

    Bhattacharya, Sarbani; Liang, Zhong; Quek, Adam J; Ploplis, Victoria A; Law, Ruby; Castellino, Francis J

    2014-08-01

    A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97-125 + Tyr), interacted specifically with isolated K2hPg. However, the binding strength of VEK30 (KD = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (KD = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH2 and COOH termini of VEK30. VEK64 (PAM residues 83-145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2hPg with nearly the same affinity as PAM (KD = 1-2 nm). However, addition of two PAM residues (Arg(126)-His(127)) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg(113), His(114), Glu(116), Arg(126), His(127)), mutation of which reduced PAM binding affinity for K2hPg by ∼ 1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. N-Acetyl-2-Aminofluorene (AAF) Processing in Adult Rat Hepatocytes in Primary Culture Occurs by High-Affinity Low-Velocity and Low-Affinity High-Velocity AAF Metabolite-Forming Systems.

    Science.gov (United States)

    Koch, Katherine S; Moran, Tom; Shier, W Thomas; Leffert, Hyam L

    2018-05-01

    N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression, and pharmacological properties inside living hepatocytes-the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3 H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 μg AAF/106 cells/day). Five ring-hydroxylated and one deacetylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 × 10-7  M and VMAX[APPARENT] = 0.1 nmol/106 cells/day and System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 × 10-5  M and VMAX[APPARENT] = 1000 nmol/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 × 10-5  M and 4.7 nmol/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

  17. Amphetamine Action at the Cocaine- and Antidepressant-Sensitive Serotonin Transporter Is Modulated by αCaMKII

    DEFF Research Database (Denmark)

    Steinkellner, Thomas; Montgomery, Therese R; Hofmaier, Tina

    2015-01-01

    Serotonergic neurotransmission is terminated by reuptake of extracellular serotonin (5-HT) by the high-affinity serotonin transporter (SERT). Selective 5-HT reuptake inhibitors (SSRIs) such as fluoxetine or escitalopram inhibit SERT and are currently the principal treatment for depression and anx...... and efflux at monoamine transporters are asymmetric processes that can be targeted separately. Ultimately, this may provide a molecular mechanism for putative drug developments to treat amphetamine addiction....

  18. Occurrence and functioning of phosphate solubilizing ...

    African Journals Online (AJOL)

    Occurrence and functioning of phosphate solubilizing microorganisms from oil palm tree ( Elaeis guineensis ) rhizosphere in Cameroon. ... While the use of soluble mineral phosphate fertilizers is the obvious best means to combat phosphate ... in order to improve agricultural production, using low inputs technology. Isolates ...

  19. D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (beta/alpha)8-Barrel Superfamily

    Energy Technology Data Exchange (ETDEWEB)

    Akana,J.; Federov, A.; Federov, E.; Novak, W.; Babbitt, P.; Almo, S.; Gerlt, J.

    2006-01-01

    The 'ribulose phosphate binding' superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common ({beta}/{alpha}){sub 8}-barrel ancestor. The superfamily includes D-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice and Plasmodium falciparum, the RPE from Streptococcus pyogenes is activated by Zn{sup 2+} which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn{sup 2+} and inactive apoenzyme cannot be prepared, the affinity for Zn{sup 2+} is decreased by alanine substitutions for the two histidine residues that coordinate the Zn{sup 2+} ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn{sup 2+}. The crystal structure of the RPE was solved at 1.8 Angstroms resolution in the presence of D-xylitol 5-phosphate, an inert analogue of the D-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn{sup 2+} that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn{sup 2+} and participate as acid/base catalysts

  20. Zirconium Phosphate Supported MOF Nanoplatelets.

    Science.gov (United States)

    Kan, Yuwei; Clearfield, Abraham

    2016-06-06

    We report a rare example of the preparation of HKUST-1 metal-organic framework nanoplatelets through a step-by-step seeding procedure. Sodium ion exchanged zirconium phosphate, NaZrP, nanoplatelets were judiciously selected as support for layer-by-layer (LBL) assembly of Cu(II) and benzene-1,3,5-tricarboxylic acid (H3BTC) linkers. The first layer of Cu(II) is attached to the surface of zirconium phosphate through covalent interaction. The successive LBL growth of HKUST-1 film is then realized by soaking the NaZrP nanoplatelets in ethanolic solutions of cupric acetate and H3BTC, respectively. The amount of assembled HKUST-1 can be readily controlled by varying the number of growth cycles, which was characterized by powder X-ray diffraction and gas adsorption analyses. The successful construction of HKUST-1 on NaZrP was also supported by its catalytic performance for the oxidation of cyclohexene.

  1. Phosphate-a poison for humans?

    Science.gov (United States)

    Komaba, Hirotaka; Fukagawa, Masafumi

    2016-10-01

    Maintenance of phosphate balance is essential for life, and mammals have developed a sophisticated system to regulate phosphate homeostasis over the course of evolution. However, due to the dependence of phosphate elimination on the kidney, humans with decreased kidney function are likely to be in a positive phosphate balance. Phosphate excess has been well recognized as a critical factor in the pathogenesis of mineral and bone disorders associated with chronic kidney disease, but recent investigations have also uncovered toxic effects of phosphate on the cardiovascular system and the aging process. Compelling evidence also suggests that increased fibroblastic growth factor 23 and parathyroid hormone levels in response to a positive phosphate balance contribute to adverse clinical outcomes. These insights support the current practice of managing serum phosphate in patients with advanced chronic kidney disease, although definitive evidence of these effects is lacking. Given the potential toxicity of excess phosphate, the general population may also be viewed as a target for phosphate management. However, the widespread implementation of dietary phosphate intervention in the general population may not be warranted due to the limited impact of increased phosphate intake on mineral metabolism and clinical outcomes. Nonetheless, the increasing incidence of kidney disease or injury in our aging society emphasizes the potential importance of this issue. Further work is needed to more completely characterize phosphate toxicity and to establish the optimal therapeutic strategy for managing phosphate in patients with chronic kidney disease and in the general population. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  2. Circadian Regulation of Glutamate Transporters

    Directory of Open Access Journals (Sweden)

    Donají Chi-Castañeda

    2018-06-01

    Full Text Available L-glutamate is the major excitatory amino acid in the mammalian central nervous system (CNS. This neurotransmitter is essential for higher brain functions such as learning, cognition and memory. A tight regulation of extra-synaptic glutamate levels is needed to prevent a neurotoxic insult. Glutamate removal from the synaptic cleft is carried out by a family of sodium-dependent high-affinity transporters, collectively known as excitatory amino acid transporters. Dysfunction of glutamate transporters is generally involved in acute neuronal injury and neurodegenerative diseases, so characterizing and understanding the mechanisms that lead to the development of these disorders is an important goal in the design of novel treatments for the neurodegenerative diseases. Increasing evidence indicates glutamate transporters are controlled by the circadian system in direct and indirect manners, so in this contribution we focus on the mechanisms of circadian regulation (transcriptional, translational, post-translational and post-transcriptional regulation of glutamate transport in neuronal and glial cells, and their consequence in brain function.

  3. Synthesis and pre-clinical evaluation of a new class of high-affinity {sup 18}F-labeled PSMA ligands for detection of prostate cancer by PET imaging

    Energy Technology Data Exchange (ETDEWEB)

    Kelly, James; Amor-Coarasa, Alejandro; Williams, Clarence; Ponnala, Shashikanth [Weill Cornell Medicine, Division of Radiopharmaceutical Sciences and Molecular Imaging Innovations Institute, Department of Radiology, New York, NY (United States); Nikolopoulou, Anastasia [Weill Cornell Medicine, Division of Radiopharmaceutical Sciences and Molecular Imaging Innovations Institute, Department of Radiology, New York, NY (United States); Weill Cornell Medicine, Citigroup Biomedical Imaging Center, New York, NY (United States); Kim, Dohyun [Weill Cornell Medicine, Citigroup Biomedical Imaging Center, New York, NY (United States); Babich, John W. [Weill Cornell Medicine, Division of Radiopharmaceutical Sciences and Molecular Imaging Innovations Institute, Department of Radiology, New York, NY (United States); Weill Cornell Medicine, Citigroup Biomedical Imaging Center, New York, NY (United States); Weill Cornell Medicine, Meyer Cancer Center, New York, NY (United States)

    2017-04-15

    Current clinical imaging of PSMA-positive prostate cancer by positron emission tomography (PET) mainly features {sup 68}Ga-labeled tracers, notably [{sup 68}Ga]Ga-PSMA-HBED-CC. The longer half-life of fluorine-18 offers significant advantages over Ga-68, clinically and logistically. We aimed to develop high-affinity PSMA inhibitors labeled with fluorine-18 as alternative tracers for prostate cancer. Six triazolylphenyl ureas and their alkyne precursors were synthesized from the Glu-urea-Lys PSMA binding moiety. PSMA affinity was determined in a competitive binding assay using LNCaP cells. The [{sup 18}F]triazoles were isolated following a Cu(I)-catalyzed click reaction between the alkynes and [{sup 18}F]fluoroethylazide. The {sup 18}F-labeled compounds were evaluated in nude mice bearing LNCaP tumors and compared to [{sup 68}Ga]Ga-PSMA-HBED-CC and [{sup 18}F]DCFPyL. Biodistribution studies of the two tracers with the highest imaged-derived tumor uptake and highest PSMA affinity were undertaken at 1 h, 2 h and 4 h post-injection (p.i.), and co-administration of PMPA was used to determine whether uptake was PSMA-specific. F-18-labeled triazolylphenyl ureas were prepared with a decay-corrected RCY of 20-40 %, >98 % radiochemical and chemical purity, and specific activity of up to 391 GBq/μmol. PSMA binding (IC{sub 50}) ranged from 3-36 nM. The position of the triazole influenced tumor uptake (3 > 4 > 2), and direct conjugation of the triazole with the phenylurea moiety was preferred to insertion of a spacer group. Image-derived tumor uptake ranged from 6-14 %ID/g at 2 h p.i., the time of maximum tumor uptake; uptake of [{sup 68}Ga]Ga-PSMA-HBED-CC and [{sup 18}F]DCFPyL was 5-6 %ID/g at 1-3 h p.i., the time of maximum tumor uptake. Biodistribution studies of the two most promising compounds gave maximum tumor uptakes of 10.9 ± 1.0 % and 14.3 ± 2.5 %ID/g, respectively, as compared to 6.27 ± 1.44 %ID/g for [{sup 68}Ga]Ga-PSMA-HBED-CC. Six [{sup 18}F

  4. Synthesis and pre-clinical evaluation of a new class of high-affinity "1"8F-labeled PSMA ligands for detection of prostate cancer by PET imaging

    International Nuclear Information System (INIS)

    Kelly, James; Amor-Coarasa, Alejandro; Williams, Clarence; Ponnala, Shashikanth; Nikolopoulou, Anastasia; Kim, Dohyun; Babich, John W.

    2017-01-01

    Current clinical imaging of PSMA-positive prostate cancer by positron emission tomography (PET) mainly features "6"8Ga-labeled tracers, notably ["6"8Ga]Ga-PSMA-HBED-CC. The longer half-life of fluorine-18 offers significant advantages over Ga-68, clinically and logistically. We aimed to develop high-affinity PSMA inhibitors labeled with fluorine-18 as alternative tracers for prostate cancer. Six triazolylphenyl ureas and their alkyne precursors were synthesized from the Glu-urea-Lys PSMA binding moiety. PSMA affinity was determined in a competitive binding assay using LNCaP cells. The ["1"8F]triazoles were isolated following a Cu(I)-catalyzed click reaction between the alkynes and ["1"8F]fluoroethylazide. The "1"8F-labeled compounds were evaluated in nude mice bearing LNCaP tumors and compared to ["6"8Ga]Ga-PSMA-HBED-CC and ["1"8F]DCFPyL. Biodistribution studies of the two tracers with the highest imaged-derived tumor uptake and highest PSMA affinity were undertaken at 1 h, 2 h and 4 h post-injection (p.i.), and co-administration of PMPA was used to determine whether uptake was PSMA-specific. F-18-labeled triazolylphenyl ureas were prepared with a decay-corrected RCY of 20-40 %, >98 % radiochemical and chemical purity, and specific activity of up to 391 GBq/μmol. PSMA binding (IC_5_0) ranged from 3-36 nM. The position of the triazole influenced tumor uptake (3 > 4 > 2), and direct conjugation of the triazole with the phenylurea moiety was preferred to insertion of a spacer group. Image-derived tumor uptake ranged from 6-14 %ID/g at 2 h p.i., the time of maximum tumor uptake; uptake of ["6"8Ga]Ga-PSMA-HBED-CC and ["1"8F]DCFPyL was 5-6 %ID/g at 1-3 h p.i., the time of maximum tumor uptake. Biodistribution studies of the two most promising compounds gave maximum tumor uptakes of 10.9 ± 1.0 % and 14.3 ± 2.5 %ID/g, respectively, as compared to 6.27 ± 1.44 %ID/g for ["6"8Ga]Ga-PSMA-HBED-CC. Six ["1"8F]triazolylphenyl ureas were prepared in good radiochemical yield

  5. Transport Statistics - Transport - UNECE

    Science.gov (United States)

    Sustainable Energy Statistics Trade Transport Themes UNECE and the SDGs Climate Change Gender Ideas 4 Change UNECE Weekly Videos UNECE Transport Areas of Work Transport Statistics Transport Transport Statistics About us Terms of Reference Meetings and Events Meetings Working Party on Transport Statistics (WP.6

  6. The electrical properties of semiconducting vanadium phosphate glasses

    International Nuclear Information System (INIS)

    Moridi, G.R.; Hogarth, C.A.; Hekmat Shooar, N.H.

    1984-01-01

    Vanadium phosphate glasses are a group of oxide glasses which show the semiconducting behaviour. In contrast to the conventional glasses, the conduction mechanism in these glasses is electronic, rather than being ionic. Since 1954, when the first paper appeared on the semiconducting properties of these glasses, much work has been carried out on transition-metal-oxide glasses in general, and vanadium phosphate glasses in particular. The mechanism of conduction is basicaly due to the transport of electrons between the transition-metal ions in different valency states. In the present paper, we have reviewed the previous works on the electrical characteristics of P 2 O 5 -V 2 O 5 glasses and also discussed the current theoretical ideas relevant for the interpretation of the experimental data

  7. Development of nanosensors for studying intracellular phosphate levels

    DEFF Research Database (Denmark)

    Gu, Hong

    -time monitoring of Pi metabolism in living cells, providing a new tool for fluxomics (measurement of metabolic flux), analysis of pathophysiology or changes of Pi during cell activity. Transformation of plants with FLIPs had resulted in only low expression levels. As an alternative a protein transduction domain......Abstract Inorganic phosphate (Pi) is an essential macronutrient that plays a central role in metabolism and signal transduction in plants. Uptake, compartmentation and transport are important players of cellular Pi homeostasis; however, methods to determine the cellular phosphate concentration...... of a substrate-binding protein linked to two fluorescent reporter proteins. Substrate binding changes the conformation of the nanosensor and, hence, the efficiency of fluorescence resonance energy transfer (FRET) between the reporter proteins. The aim of the present project was to develop nanosensors for Pi...

  8. Vibrio Iron Transport: Evolutionary Adaptation to Life in Multiple Environments

    Science.gov (United States)

    Mey, Alexandra R.; Wyckoff, Elizabeth E.

    2015-01-01

    SUMMARY Iron is an essential element for Vibrio spp., but the acquisition of iron is complicated by its tendency to form insoluble ferric complexes in nature and its association with high-affinity iron-binding proteins in the host. Vibrios occupy a variety of different niches, and each of these niches presents particular challenges for acquiring sufficient iron. Vibrio species have evolved a wide array of iron transport systems that allow the bacteria to compete for this essential element in each of its habitats. These systems include the secretion and uptake of high-affinity iron-binding compounds (siderophores) as well as transport systems for iron bound to host complexes. Transporters for ferric and ferrous iron not complexed to siderophores are also common to Vibrio species. Some of the genes encoding these systems show evidence of horizontal transmission, and the ability to acquire and incorporate additional iron transport systems may have allowed Vibrio species to more rapidly adapt to new environmental niches. While too little iron prevents growth of the bacteria, too much can be lethal. The appropriate balance is maintained in vibrios through complex regulatory networks involving transcriptional repressors and activators and small RNAs (sRNAs) that act posttranscriptionally. Examination of the number and variety of iron transport systems found in Vibrio spp. offers insights into how this group of bacteria has adapted to such a wide range of habitats. PMID:26658001

  9. Design and synthesis of 1-(3-(dimethylamino)propyl)-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-carbonitrile (citalopram) analogues as novel probes for the serotonin transporter S1 and S2 binding sites

    DEFF Research Database (Denmark)

    Banala, Ashwini K; Zhang, Peng; Plenge, Per

    2013-01-01

    The serotonin transporter (SERT) is the primary target for antidepressant drugs. The existence of a high affinity primary orthosteric binding site (S1) and a low affinity secondary site (S2) has been described, and their relation to antidepressant pharmacology has been debated. Herein, structural...

  10. Phosphate vibrations as reporters of DNA hydration

    Science.gov (United States)

    Corcelli, Steven

    The asymmetric phosphate stretch vibrational frequency is extraordinarily sensitive to its local solvent environment. Using density functional theory calculations on the model compound dimethyl phosphate, the asymmetric phosphate stretch vibrational frequency was found to shift linearly with the magnitude of an electric field along the symmetry axis of the PO2 moiety (i.e. the asymmetric phosphate stretch is an excellent linear vibrational Stark effect probe). With this linear relationship established, asymmetric phosphate stretch vibrational frequencies were computed during the course of a molecular dynamics simulation of fully hydrated DNA. Moreover, contributions to shifts in the frequencies from subpopulations of water molecules (e.g. backbone, minor groove, major groove, etc.) were calculated to reveal how phosphate vibrations report the onset of DNA hydration in experiments that vary the relative humidity of non-condensing (dry) DNA samples.

  11. Application of Calcium Phosphate Materials in Dentistry

    Directory of Open Access Journals (Sweden)

    Jabr S. Al-Sanabani

    2013-01-01

    Full Text Available Calcium phosphate materials are similar to bone in composition and in having bioactive and osteoconductive properties. Calcium phosphate materials in different forms, as cements, composites, and coatings, are used in many medical and dental applications. This paper reviews the applications of these materials in dentistry. It presents a brief history, dental applications, and methods for improving their mechanical properties. Notable research is highlighted regarding (1 application of calcium phosphate into various fields in dentistry; (2 improving mechanical properties of calcium phosphate; (3 biomimetic process and functionally graded materials. This paper deals with most common types of the calcium phosphate materials such as hydroxyapatite and tricalcium phosphate which are currently used in dental and medical fields.

  12. Complex formation of uranium(VI) with fructose and glucose phosphates

    International Nuclear Information System (INIS)

    Koban, A.; Geipel, G.; Bernhard, G.; Fanghaenel, T.

    2002-01-01

    The uptake of heavy metals into plants is commonly quantified by the soil-plant transfer factor. Up to now little is known about the chemical speciation of actinides in plants. To compare the obtained spectroscopic data of uranium complexes in plants with model compounds, we investigate the complexation of uranium with relevant bioligands of various functionalities. A very important class of ligands consists of phosphate esters, which serve as phosphate group and energy transmitters as well as energy storage media in biological systems. Heavy metal ions bound to the phosphate esters can be transported into living cells and then deposited. Therefore, in our study we present the results of uranium complexation with glucose-6-phosphate (G6P), and fructose-6-phosphate (F6P) obtained by time-resolved laser-induced fluorescence spectroscopy (TRLFS). The experiments were performed at a fixed uranyl concentration (10 -5 M) as a function of the ligand concentrations (10 -5 to 10 -3 M) in a pH range from 2 to 4.5. For the glucose phosphate system we observed, using increasing ligand concentrations, a decrease in the fluorescence intensity and a small red shift of the emission bands. From this we conclude that the complexed uranyl glucose phosphate species show only minor or no fluorescence properties. The TRLFS spectra of the glucose phosphate samples indicated the presence of a single species with fluorescence properties. This species has a lifetime of approximately 1.5 μs and was identified as the free uranyl ion. An opposite phenomenon was observed for the fructose phosphate system: there was no decrease in fluorescence intensity. However, a strong red shift of the spectra was observed, illustrating the fluorescence properties of the uranyl fructose phosphate complex. The TRLFS spectra of the fructose phosphate system showed a second lifetime ( 2 2+ UO 2 (lig) x (2-y)+ + y H + (lig = sugar phosphate). Applying the mass action law and transformation to the logarithmic

  13. Crystalline cerium(IV) phosphates

    International Nuclear Information System (INIS)

    Herman, R.G.; Clearfield, A.

    1976-01-01

    The ion exchange behaviour of seven crystalline cerium(IV) phosphates towards some of the alkali metal cations is described. Only two of the compounds (A and C) possess ion exchange properties in acidic solutions. Four others show some ion exchange characteristics in basic media with some of the alkali cations. Compound G does not behave as an ion exchanger in solutions of pH + , but show very little Na + uptake. Compound E undergoes ion exchange with Na + and Cs + , but not with Li+. Both Li + and Na + are sorbed by compounds A and C. The results are indicative of structures which show steric exclusion phenomena. (author)

  14. A critical evaluation of phosphate retardation and leaching in Hapludults

    Science.gov (United States)

    Dao, Thanh

    2016-04-01

    Nutrients used in production agriculture, in particular bioactive phosphorus (P), continue to present challenges in trying to reverse the degradation of fragile aquatic ecosystems. Soils treated with large amounts of nutrient-enriched animal manure have elevated P levels in regions of intensive animal agriculture and the residual effects of past large P additions were found to be long-lived. Mathematical models are increasingly used in the evaluation and development of mitigation strategies and sustainable management practices. A large number of predictive tools are currently used in the U.S. for simulating phosphorus environmental fate, including models such AGNPS (Agricultural Non-Point Source), FHANTM Field Hydrologic And Nutrient Transport Model (Field Hydrologic And Nutrient Transport Model), SWAT (Soil & Water Assessment Tool), or APEX (Agric. Policy/Environmental EXtender). The P routines in these models have had limited changes in spite of the advances in our understanding of speciation and transport of various P forms in soil and water systems that have occurred over the last three decades. We conducted soil sorption isotherm experiments that yielded basic information for estimating the Phosphorus Sorption coefficient (PSP) a key parameter used to allocate mineral P into soil labile, active, and stable pools. We compare these coefficients to parameters derived from breakthrough curves (BTC) for determining the extent of retardation and transport of phosphate supplied as KH2PO4 under a constant hydraulic head. Sigmoidal and multi-reaction rate models were observed in the BTCs of the anion, which undermine the rationale for using an overall simple partition coefficient to describe the transport and dispersal of phosphate in soil. Minimizing such generalities used in estimating nutrient availability and transport gives a more accurate picture of status of P in soil to conserve nutrients and minimize loss of excess P inputs to the environment.

  15. Radiological impact of use of phosphate fertilizers

    International Nuclear Information System (INIS)

    Shukla, V.K.; Chinnaesakki, S.; Sartandel, S.J.; Shanbhag, A.A.; Puranik, V.D.

    2003-01-01

    The paper describes the results of gamma spectrometric measurements of 238 U, 233 Th, 226 Ra and 40 K in rock phosphates and various types of phosphate fertilizers and by-products. The increase in soil natural radioactivity has been assessed for major Indian crops. No significant increase in soil natural radioactivity is expected due to the application of phosphate fertilizers for agricultural productions. (author)

  16. Uranium and heavy metals in phosphate fertilizers

    International Nuclear Information System (INIS)

    Khater, Ashraf E.M.; King Saud University, Riyadh

    2008-01-01

    Full text: Agricultural applications of chemical fertilizers are a worldwide practice. The specific activity of uranium-238 and heavy metals in phosphate fertilizers depends on the phosphate ore from which the fertilizer produced and on the chemical processing of the ore. Composite phosphate fertilizers samples were collected and the uranium-238 specific activity, in Bq/kg, and As, Cd, Cu, Pb, Se concentration were measured. The annual addition of these elements in soil due to soil fertilization were calculated and discussed. (author)

  17. Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root development.

    Science.gov (United States)

    Ticconi, Carla A; Delatorre, Carla A; Lahner, Brett; Salt, David E; Abel, Steffen

    2004-03-01

    Plants have evolved complex strategies to maintain phosphate (Pi) homeostasis and to maximize Pi acquisition when the macronutrient is limiting. Adjustment of root system architecture via changes in meristem initiation and activity is integral to the acclimation process. However, the mechanisms that monitor external Pi status and interpret the nutritional signal remain to be elucidated. Here, we present evidence that the Pi deficiency response, pdr2, mutation disrupts local Pi sensing. The sensitivity and amplitude of metabolic Pi-starvation responses, such as Pi-responsive gene expression or accumulation of anthocyanins and starch, are enhanced in pdr2 seedlings. However, the most conspicuous alteration of pdr2 is a conditional short-root phenotype that is specific for Pi deficiency and caused by selective inhibition of root cell division followed by cell death below a threshold concentration of about 0.1 mm external Pi. Measurements of general Pi uptake and of total phosphorus (P) in root tips exclude a defect in high-affinity Pi acquisition. Rescue of root meristem activity in Pi-starved pdr2 by phosphite (Phi), a non-metabolizable Pi analog, and divided-root experiments suggest that pdr2 disrupts sensing of low external Pi availability. Thus, PDR2 is proposed to function at a Pi-sensitive checkpoint in root development, which monitors environmental Pi status, maintains and fine-tunes meristematic activity, and finally adjusts root system architecture to maximize Pi acquisition.

  18. Amiloride-enhanced gene transfection of octa-arginine functionalized calcium phosphate nanoparticles.

    Directory of Open Access Journals (Sweden)

    Juan Ramón Vanegas Sáenz

    Full Text Available Nanoparticles represent promising gene delivery systems in biomedicine to facilitate prolonged gene expression with low toxicity compared to viral vectors. Specifically, nanoparticles of calcium phosphate (nCaP, the main inorganic component of human bone, exhibit high biocompatibility and good biodegradability and have been reported to have high affinity for protein or DNA, having thus been used as gene transfer vectors. On the other hand, Octa-arginine (R8, which has a high permeability to cell membrane, has been reported to improve intracellular delivery systems. Here, we present an optimized method for nCaP-mediated gene delivery using an octa-arginine (R8-functionalized nCaP vector containing a marker or functional gene construct. nCaP particle size was between 220-580 nm in diameter and all R8-functionalized nCaPs carried a positive charge. R8 concentration significantly improved nCaP transfection efficiency with high cell compatibility in human mesenchymal stem cells (hMSC and human osteoblasts (hOB in particular, suggesting nCaPs as a good option for non-viral vector gene delivery. Furthermore, pre-treatment with different endocytosis inhibitors identified that the endocytic pathway differed among cell lines and functionalized nanoparticles, with amiloride increasing transfection efficiency of R8-functionalized nCaPs in hMSC and hOB.

  19. Calcium phosphates: what is the evidence?

    Science.gov (United States)

    Larsson, Sune

    2010-03-01

    A number of different calcium phosphate compounds such as calcium phosphate cements and solid beta-tricalcium phosphate products have been introduced during the last decade. The chemical composition mimics the mineral phase of bone and as a result of this likeness, the materials seem to be remodeled as for normal bone through a cell-mediated process that involves osteoclastic activity. This is a major difference when compared with, for instance, calcium sulphate compounds that after implantation dissolve irrespective of the new bone formation rate. Calcium phosphates are highly biocompatible and in addition, they act as synthetic osteoconductive scaffolds after implantation in bone. When placed adjacent to bone, osteoid is formed directly on the surface of the calcium phosphate with no soft tissue interposed. Remodeling is slow and incomplete, but by adding more and larger pores, like in ultraporous beta-tricalcium phosphate, complete or nearly complete resorption can be achieved. The indications explored so far include filling of metaphyseal fracture voids or bone cysts, a volume expander in conjunction with inductive products, and as a carrier for various growth factors and antibiotics. Calcium phosphate compounds such as calcium phosphate cement and beta-tricalcium phosphate will most certainly be part of the future armamentarium when dealing with fracture treatment. It is reasonable to believe that we have so far only seen the beginning when it comes to clinical applications.

  20. Biosynthesis and characterization of layered iron phosphate

    International Nuclear Information System (INIS)

    Zhou Weijia; He Wen; Wang Meiting; Zhang Xudong; Yan Shunpu; Tian Xiuying; Sun Xianan; Han Xiuxiu; Li Peng

    2008-01-01

    Layered iron phosphate with uniform morphology has been synthesized by a precipitation method with yeast cells as a biosurfactant. The yeast cells are used to regulate the nucleation and growth of layered iron phosphate. The uniform layered structure is characterized by small-angle x-ray diffraction (SAXD), scanning electron microscopy (SEM) and atomic force microscopy (AFM) analyses. Fourier transform infrared spectroscopy (FT-IR) is used to analyze the chemical bond linkages in organic–inorganic hybrid iron phosphate. The likely synthetic mechanism of nucleation and oriented growth is discussed. The electrical conductivity of hybrid iron phosphate heat-treated at different temperatures is presented

  1. [Phosphate-solubilizing activity of aerobic methylobacteria].

    Science.gov (United States)

    Agafonova, N V; Kaparullina, E N; Doronina, N V; Trotsenko, Iu A

    2014-01-01

    Phosphate-solubilizing activity was found in 14 strains of plant-associated aerobic methylobacteria belonging to the genera Methylophilus, Methylobacillus, Methylovorus, Methylopila, Methylobacterium, Delftia, and Ancyclobacter. The growth of methylobacteria on medium with methanol as the carbon and energy source and insoluble tricalcium phosphate as the phosphorus source was accompanied by a decrease in pH due to the accumulation of up to 7 mM formic acid as a methanol oxidation intermediate and by release of 120-280 μM phosphate ions, which can be used by both bacteria and plants. Phosphate-solubilizing activity is a newly revealed role of methylobacteria in phytosymbiosis.

  2. Synthesis of amorphous acid iron phosphate nanoparticles

    International Nuclear Information System (INIS)

    Palacios, E.; Leret, P.; Fernández, J. F.; Aza, A. H. De; Rodríguez, M. A.

    2012-01-01

    A simple method to precipitate nanoparticles of iron phosphate with acid character has been developed in which the control of pH allows to obtain amorphous nanoparticles. The acid aging of the precipitated amorphous nanoparticles favored the P–O bond strength that contributes to the surface reordering, the surface roughness and the increase of the phosphate acid character. The thermal behavior of the acid iron phosphate nanoparticles has been also studied and the phosphate polymerization at 400 °C produces strong compacts of amorphous nanoparticles with interconnected porosity.

  3. A novel 5-enolpyruvylshikimate-3-phosphate synthase from Rahnella aquatilis with significantly reduced glyphosate sensitivity.

    Directory of Open Access Journals (Sweden)

    Ri-He Peng

    Full Text Available The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19 is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroA(R. aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroA(E. coli, while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroA(R. aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroA(E. coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R. aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R. aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P and phosphoenolpyruvate (PEP. The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.

  4. Sphingosine 1-Phosphate Receptor 1 as a Useful Target for Treatment of Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Kunitomo Adachi

    2012-05-01

    Full Text Available Sphingosine 1-phosphate (S1P, a lysophospholipid mediator, is generated from sphingosine by sphingosine kinases and binds five known cell surface receptors. S1P receptor 1 (S1P1 plays an essential role in lymphocyte egress from secondary lymphoid organs (SLO, as evinced by the inability of lymphocytes to exit from the SLO in mice lacking lymphocytic S1P1. Fingolimod hydrochloride (FTY720 is a first-in-class, orally active, S1P receptor modulator with a structure closely related to sphingosine. FTY720 was first synthesized by chemical modification of a natural product, myriocin. FTY720 is effectively converted to an active metabolite, FTY720 phosphate (FTY720-P by sphingosine kinases. FTY720-P shows high affinity to 4 of the S1P receptors (S1P1, S1P3, S1P4, and S1P5. In particular, FTY720-P strongly induces internalization and degradation of S1P1, inhibits S1P responsiveness of lymphocytes in the SLO, and acts as a functional antagonist at lymphocytic S1P1. Consequently, FTY720 inhibits S1P1-dependent lymphocyte egress from the SLO to decrease circulation of lymphocytes including autoreactive Th17 cells and is highly effective in experimental autoimmune encephalomyelitis (EAE, an animal model of multiple sclerosis (MS. Because FTY720 shows a superior efficacy in relapsing remitting MS patients compared to intramuscular interferon-β-1a (Avonex®, S1P1 is presumed to be a useful target for the therapy of MS.

  5. Method of stripping plutonium from tributyl phosphate solution which contains dibutyl phosphate-plutonium stable complexes

    International Nuclear Information System (INIS)

    Ochsenfeld, W.; Schmieder, H.

    1976-01-01

    Fast breeder fuel elements which have been highly burnt-up are reprocessed by extracting uranium and plutonium into an organic solution containing tributyl phosphate. The tributyl phosphate degenerates at least partially into dibutyl phosphate and monobutyl phosphate, which form stable complexes with tetravalent plutonium in the organic solution. This tetravalent plutonium is released from its complexed state and stripped into aqueous phase by contacting the organic solution with an aqueous phase containing tetravalent uranium. 6 claims, 1 drawing figure

  6. Structural features of PhoX, one of the phosphate-binding proteins from Pho regulon of Xanthomonas citri

    Science.gov (United States)

    Pegos, Vanessa R.; Santos, Rodrigo M. L.; Medrano, Francisco J.

    2017-01-01

    In Escherichia coli, the ATP-Binding Cassette transporter for phosphate is encoded by the pstSCAB operon. PstS is the periplasmic component responsible for affinity and specificity of the system and has also been related to a regulatory role and chemotaxis during depletion of phosphate. Xanthomonas citri has two phosphate-binding proteins: PstS and PhoX, which are differentially expressed under phosphate limitation. In this work, we focused on PhoX characterization and comparison with PstS. The PhoX three-dimensional structure was solved in a closed conformation with a phosphate engulfed in the binding site pocket between two domains. Comparison between PhoX and PstS revealed that they originated from gene duplication, but despite their similarities they show significant differences in the region that interacts with the permeases. PMID:28542513

  7. NOVEL FLUORESCENT PROBES FOR THE DOPAMINE TRANSPORTER

    DEFF Research Database (Denmark)

    Cha, J; Vægter, Christian Bjerggaard; Adkins, Erica

    -reactive rhodamine red derivatives. The resulting N-substituted (JHC 1-64) and 2-substituted (JHC 1-53) ligands showed high affinity binding to DAT expressed in HEK 293 cells (Ki= 6.4 and 29 nM, respectively). Their ability to selectively label the DAT was demonstrated by confocal laser scanning microscopy of HEK......To enable visualization of the dopamine transporter (DAT) through fluorescence technologies we have synthesized a novel series of fluorescently tagged analogs of cocaine. Previous structure-activity relationship (SAR) studies have demonstrated that the dopamine transporter (DAT) can tolerate...... in untransfected control cells. The possibility of using these ligands for direct labeling of the DAT in living cells represents a new and important approach for understanding cellular targeting and trafficking of the DAT. Moreover, these fluorescent ligands might also provide the molecular tools...

  8. Exposure of Postnatal Rats to a Static Magnetic Field of 0.14 T Influences Functional Laterality of the Hippocampal High-Affinity Choline Uptake System in Adulthood; In Vitro Test With Magnetic Nanoparticles

    Czech Academy of Sciences Publication Activity Database

    Krištofíková, Z.; Čermák, M.; Benešová, O.; Klaschka, Jan; Zach, P.

    2005-01-01

    Roč. 30, č. 2 (2005), s. 253-262 ISSN 0364-3190 R&D Projects: GA MZd NF7576 Keywords : magnetic nanoparticles * choline transport * cholinergic * functional impairment * hippocampus * laterality * magnetoreception * static magnetic field Subject RIV: BB - Applied Statistics, Operational Research Impact factor: 2.187, year: 2005

  9. Bio-treatment of phosphate from synthetic wastewater using ...

    African Journals Online (AJOL)

    In this study, the efficient phosphate utilizing isolates were used to remove phosphate from synthetic phosphate wastewater was tested using batch scale process. Hence the objective of the present study was to examine the efficiency of bacterial species individually for the removal of phosphate from synthetic phosphate ...

  10. Deregulated Renal Calcium and Phosphate Transport during Experimental Kidney Failure

    NARCIS (Netherlands)

    Pulskens, W.P.C.; Verkaik, M.; Sheedfar, F.; Loon, E.P.M. van; Sluis, B. van de; Vervloet, M.G.; Hoenderop, J.G.J.; Bindels, R.J.M.

    2015-01-01

    Impaired mineral homeostasis and inflammation are hallmarks of chronic kidney disease (CKD), yet the underlying mechanisms of electrolyte regulation during CKD are still unclear. Here, we applied two different murine models, partial nephrectomy and adenine-enriched dietary intervention, to induce

  11. Structure and transport investigations on lithium-iron-phosphate glasses

    International Nuclear Information System (INIS)

    Banday, Azeem; Sharma, Monika; Murugavel, Sevi

    2016-01-01

    Cathode materials for Lithium Ion Batteries (LIB’s) are being constantly studied and reviewed especially in the past few decades. LiFePO_4 (LFP) is one of the most potential candidates in the pedigree of cathode materials and has been under extensive study ever since. In this work, we report the synthesis of amorphous analogs of crystallite LFP by conventional melt quenching method. Thermal study by using differential scanning calorimetry (DSC) was used to determine the glass transition T_g and crystallization T_c temperatures on the obtained glass sample Fourier transform infrared (FTIR) absorption spectroscopy is being used to investigate the structural properties of the glass sample. The intrinsic electrical conductivity measurements were done using broad-band impedance spectroscopy with wide different temperature ranges. The conduction mechanism is described by non-adiabatic small polaron hopping between nearest neighbors. Based on the obtained results, we suggest that the glassy LFP is more suitable cathode material as compared to its crystalline counterpart.

  12. Calcitonin, phosphate, and the osteocyte--osteoblast bone cell unit

    Energy Technology Data Exchange (ETDEWEB)

    Talmage, R.V.; Matthews, J.L.; Martin, J.H.; Kennedy, J.W. III; Davis, W.L.; Roycroft, J.H. Jr.

    1974-01-01

    In this report we have attempted to correlate the morphological and chemical changes that occur in the long bone (tibia) of rats with the hypocalcemia that is produced following calcitonin injection or release from its gland of origin. By varying the supply of phosphate available to the rat, it has been possible to demonstrate that changes produced by CT both in bone and in plasma calcium concentrations were dependent upon an adequate supply of this ion. It is, therefore, postulated that the hypocalcemia produced by calcitonin is secondary to the formation of a calcium phosphate complex in and around osteocytes and lining cells. It is suggested that this complex, which is normally prevented from transforming to apatite crystal by the presence of an inhibitor, reduces the availability of calcium for rapid transport to the ECF. The reduction in calcium flux from bone to ECF results in a rapid and transient hypocalcemia. Regardless of the status of this postulate, we have at least demonstrated that the osteocyte-osteoblast unit of compact bone reacts rapidly to calcitonin in a process requiring phosphate in a sequence of events which can be closely correlated to the hypocalcemic action of the hormone.

  13. Review of Phosphate in soils: Interaction with micronutrients, radionuclides, and heavy metals

    Science.gov (United States)

    Phosphate-phosphorus present in the vadose zone of soil as native, added, or residual fertilizer influences the retention, transport, and bioavailability of heavy metals, metalloids, or metallic radionuclides to aboveground vegetation, soil microorganisms, and fauna that browse that vegetation, or d...

  14. Removal of phosphate from solution by adsorption and precipitation of calcium phosphate onto monohydrocalcite.

    Science.gov (United States)

    Yagi, Shintaro; Fukushi, Keisuke

    2012-10-15

    The sorption behavior and mechanism of phosphate on monohydrocalcite (CaCO(3)·H(2)O: MHC) were examined using batch sorption experiments as a function of phosphate concentrations, ionic strengths, temperatures, and reaction times. The mode of PO(4) sorption is divisible into three processes depending on the phosphate loading. At low phosphate concentrations, phosphate is removed by coprecipitation of phosphate during the transformation of MHC to calcite. The sorption mode at the low-to-moderate phosphate concentrations is most likely an adsorption process because the sorption isotherm at the conditions can be fitted reasonably with the Langmuir equation. The rapid sorption kinetics at the conditions is also consistent with the adsorption reaction. The adsorption of phosphate on MHC depends strongly on ionic strength, but slightly on temperature. The maximum adsorption capacities of MHC obtained from the regression of the experimental data to the Langmuir equation are higher than those reported for stable calcium carbonate (calcite or aragonite) in any conditions. At high phosphate concentrations, the amount of sorption deviates from the Langmuir isotherm, which can fit the low-to-moderate phosphate concentrations. Speciation-saturation analyses of the reacted solutions at the conditions indicated that the solution compositions which deviate from the Langmuir equation are supersaturated with respect to a certain calcium phosphate. The obtained calcium phosphate is most likely amorphous calcium phosphate (Ca(3)(PO(4))(2)·xH(2)O). The formation of the calcium phosphate depends strongly on ionic strength, temperature, and reaction times. The solubility of MHC is higher than calcite and aragonite because of its metastability. Therefore, the higher solubility of MHC facilitates the formation of the calcium phosphates more than with calcite and aragonite. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

    OpenAIRE

    de Saint-Jean, Maud; Delfosse, Vanessa; Douguet, Dominique; Chicanne, Gaetan; Payrastre, Bernard; Bourguet, William; Antonny, Bruno; Drin, Guillaume

    2011-01-01

    Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Os...

  16. Cyanotoxins: a poison that frees phosphate.

    Science.gov (United States)

    Raven, John A

    2010-10-12

    Autotrophic organisms obtain phosphorus from the environment by secreting alkaline phosphatases that act on esters, resulting in inorganic phosphate that is then taken up. New work shows that the cyanobacterium Aphanizomenon ovalisporum obtains inorganic phosphate by secreting the cyanotoxin cylindrospermopsin, which induces alkaline phosphatase in other phytoplankton species. Copyright © 2010 Elsevier Ltd. All rights reserved.

  17. Phosphate recycling in the phosphorus industry

    NARCIS (Netherlands)

    Schipper, W.J.; Klapwijk, A.; Potjer, A.; Rulkens, W.H.; Temmink, B.G.; Kiestra, F.D.G.; Lijmbach, A.C.M.

    2001-01-01

    The feasibility of phosphate recycling in the white phosphorus production process is discussed. Several types of materials may be recycled, provided they are dry inorganic materials, low in iron, copper and zinc. Sewage sludge ash may be used if no iron is used for phosphate precipitation in the

  18. Photoelectron spectroscopy of phosphites and phosphates

    Energy Technology Data Exchange (ETDEWEB)

    Chattopadhyay, S.; Findley, G.L.; McGlynn, S.P.

    1981-01-01

    The ultraviolet photoelectron spectra (UPS) of trimethyl and triethyl phosphite, trimethyl and triethyl phosphate and four substituted phosphates are presented. Assignments are based on analogies to the UPS of phosphorus trichloride and phosphoryl trichloride and are substantiated by CNDO/2 computations. The mechanisms of P-O (axial) bond formation is discussed.

  19. Phosphate recycling in the phosphorus industry

    NARCIS (Netherlands)

    Schipper, W.J.; Klapwijk, A.; Potjer, B.; Rulkens, W.H.; Temmink, B.G.; Kiestra, F.D.G.; Lijmbach, A.C.M.

    2004-01-01

    The article describes the potential and limitations for recovery of phosphate from secondary materials in the production process for white phosphorus. This thermal process involves the feeding of phosphate rock, cokes and pebbles to a furnace. The reducing conditions in the furnace promote the

  20. Rock phosphate solubilization by the ectomycorrhizal fungus ...

    African Journals Online (AJOL)

    SAM

    2014-06-18

    Jun 18, 2014 ... To evaluate phosphate solubilization of ... and MHB had the potential to solubilize these phosphates by decreasing the pH and confirmed that ... Minerals like N, P, K, Ca, S, Zn, Cu and Sr are ... sterile distilled water, chopped, homogenized in 10 ml sterile .... The role of carbon source is important in mineral.

  1. Genetics Home Reference: glucose phosphate isomerase deficiency

    Science.gov (United States)

    ... glycolytic pathway; in this step, a molecule called glucose-6-phosphate is converted to another molecule called fructose-6-phosphate. When GPI remains a single molecule (a monomer) it is involved in the development and maintenance of nerve cells ( neurons ). In this context, it is often known as ...

  2. Carrier-mediated γ-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers.

    Science.gov (United States)

    Nielsen, Carsten Uhd; Carstensen, Mette; Brodin, Birger

    2012-06-01

    The aim of the present study was to investigate the transport of γ-aminobutyric acid (GABA) across the basolateral membrane of intestinal cells. The proton-coupled amino acid transporter, hPAT1, mediates the influx of GABA and GABA mimetic drug substances such as vigabatrin and gaboxadol and the anticancer prodrug δ-aminolevulinic acid across the apical membrane of small intestinal enterocytes. Little is however known about the basolateral transport of these substances. We investigated basolateral transport of GABA in mature Caco-2 cell monolayers using isotope studies. Here we report that, at least two transporters seem to be involved in the basolateral transport of GABA. The basolateral uptake consisted of a high-affinity system with a K(m) of 290 μM and V(max) of 75 pmol cm(-2) min(-1) and a low affinity system with a K(m) of approximately 64 mM and V(max) of 1.6 nmol cm(-2) min(-1). The high-affinity transporter is Na(+) and Cl(-) dependent. The substrate specificity of the high-affinity transporter was further studied and Gly-Sar, Leucine, gaboxadol, sarcosine, lysine, betaine, 5-hydroxythryptophan, proline and glycine reduced the GABA uptake to approximately 44-70% of the GABA uptake in the absence of inhibitor. Other substances such as β-alanine, GABA, 5-aminovaleric acid, taurine and δ-aminolevulinic acid reduced the basolateral GABA uptake to 6-25% of the uptake in the absence of inhibitor. Our results indicate that the distance between the charged amino- and acid-groups is particular important for inhibition of basolateral GABA uptake. Thus, there seems to be a partial substrate overlap between the basolateral GABA transporter and hPAT1, which may prove important for understanding drug interactions at the level of intestinal transport. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Mineral phosphate solubilizing bacterial community in agro-ecosystem

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... patterns. Four insoluble phosphate sources; purulia rock phosphate (PRP), mussourie rock phosphate. (MRP) ... community composition analysis (Garland, 1996a) and ..... the threshold level that enabled only a few species to.

  4. Sonochemical precipitation of amorphous uranium phosphates from trialkyl phosphate solutions and their thermal conversion to UP2O7

    Czech Academy of Sciences Publication Activity Database

    Doroshenko, I.; Žurková, J.; Moravec, Z.; Bezdička, Petr; Pinkas, J.

    2015-01-01

    Roč. 26, SEP (2015), s. 157-162 ISSN 1350-4177 Institutional support: RVO:61388980 Keywords : Uranium * Phosphates * Sonochemistry * Nuclear waste * Trimethyl phosphate * Triethyl phosphate Subject RIV: CA - Inorganic Chemistry Impact factor: 4.556, year: 2015

  5. High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

    Energy Technology Data Exchange (ETDEWEB)

    Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.; Marceau, François, E-mail: francois.marceau@crchul.ulaval.ca

    2013-07-15

    Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent K{sub M} 1.1 vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥ 2.5 μM, ≥ 2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake V{sub max}. PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes). - Highlights: • Quinacrine is concentrated in acidic organelles via V-ATPase-mediated ion

  6. High affinity capture and concentration of quinacrine in polymorphonuclear neutrophils via vacuolar ATPase-mediated ion trapping: Comparison with other peripheral blood leukocytes and implications for the distribution of cationic drugs

    International Nuclear Information System (INIS)

    Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.; Marceau, François

    2013-01-01

    Many cationic drugs are concentrated in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping), with an ensuing vacuolar and autophagic cytopathology. In solid tissues, there is evidence that phagocytic cells, e.g., histiocytes, preferentially concentrate cationic drugs. We hypothesized that peripheral blood leukocytes could differentially take up a fluorescent model cation, quinacrine, depending on their phagocytic competence. Quinacrine transport parameters were determined in purified or total leukocyte suspensions at 37 °C. Purified polymorphonuclear leukocytes (PMNLs, essentially neutrophils) exhibited a quinacrine uptake velocity inferior to that of lymphocytes, but a consistently higher affinity (apparent K M 1.1 vs. 6.3 μM, respectively). However, the vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine transport or initiated its release in either cell type. PMNLs capture most of the quinacrine added at low concentrations to fresh peripheral blood leukocytes compared with lymphocytes and monocytes (cytofluorometry). Accumulation of the autophagy marker LC3-II occurred rapidly and at low drug concentrations in quinacrine-treated PMNLs (significant at ≥ 2.5 μM, ≥ 2 h). Lymphocytes contained more LAMP1 than PMNLs, suggesting that the mass of lysosomes and late endosomes is a determinant of quinacrine uptake V max . PMNLs, however, exhibited the highest capacity for pinocytosis (uptake of fluorescent dextran into endosomes). The selectivity of quinacrine distribution in peripheral blood leukocytes may be determined by the collaboration of a non-concentrating plasma membrane transport mechanism, tentatively identified as pinocytosis in PMNLs, with V-ATPase-mediated concentration. Intracellular reservoirs of cationic drugs are a potential source of toxicity (e.g., loss of lysosomal function in phagocytes). - Highlights: • Quinacrine is concentrated in acidic organelles via V-ATPase-mediated ion trapping

  7. Are Polyphosphates or Phosphate Esters Prebiotic Reagents?

    Science.gov (United States)

    Keefe, Anthony D.; Miller, Stanley L.

    1995-01-01

    It is widely held that there was a phosphate compound in prebiotic chemistry that played the role of adenosine triphosphate and that the first living organisms had ribose-phosphate in the backbone of their genetic material. However, there are no known efficient prebiotic synthesis of high-energy phosphates or phosphate esters. We review the occurrence of phosphates in nature, the efficiency of the volcanic synthesis of P4O10, the efficiency of polyphosphate synthesis by heating phosphate minerals under geological conditions, and the use of high-energy organic compounds such as cyanamide or hydrogen cyanide. These are shown to be inefficient processes especially when the hydrolysis of the polyphosphates is taken into account. For example, if a whole atmosphere of methane or carbon monoxide were converted to cyanide which somehow synthesized polyphosphates quantitatively, the polyphosphate concentration in the ocean would still have been insignificant. We also attempted to find more efficient high-energy polymerizing agents by spark discharge syntheses, but without success. There may still be undiscovered robust prebiotic syntheses of polyphosphates, or mechanisms for concentrating them, but we conclude that phosphate esters may not have been constituents of the first genetic material. Phosphoanhydrides are also unlikely as prebiotic energy sources.

  8. Enhancement of 210Po and 210Pb arising from phosphate industry in the Syrian coast

    International Nuclear Information System (INIS)

    Al-Masri, M.S.; Mamish, S.; Budeir, Y.

    1999-01-01

    Phosphate industry is considered to be one of the potential sources of natural radionuclide in Syria. Most of the phosphate processed ore is exported in large quantities via one of the Syrian main ports (Tartous) situated on the east part of the Mediterranean Sea (34 deg. 54 N, 35 deg. 52 E). Loading activities into ships have been carried out for more than 20 years. Dust carrying radioactivity is elevated and transported to the surroundings; most of the port area is affected. The impact of these loading activities on the marine environment has been evaluated. 210 Po and other natural radionuclides in seawater, sediment and marine organisms have been determined

  9. How do arbuscular mycorrhizal fungi handle phosphate? New insight into fine-tuning of phosphate metabolism.

    Science.gov (United States)

    Ezawa, Tatsuhiro; Saito, Katsuharu

    2018-04-27

    Contents Summary I. Introduction II. Foraging for phosphate III. Fine-tuning of phosphate homeostasis IV. The frontiers: phosphate translocation and export V. Conclusions and outlook Acknowledgements References SUMMARY: Arbuscular mycorrhizal fungi form symbiotic associations with most land plants and deliver mineral nutrients, in particular phosphate, to the host. Therefore, understanding the mechanisms of phosphate acquisition and delivery in the fungi is critical for full appreciation of the mutualism in this association. Here, we provide updates on physical, chemical, and biological strategies of the fungi for phosphate acquisition, including interactions with phosphate-solubilizing bacteria, and those on the regulatory mechanisms of phosphate homeostasis based on resurveys of published genome sequences and a transcriptome with reference to the latest findings in a model fungus. For the mechanisms underlying phosphate translocation and export to the host, which are major research frontiers in this field, not only recent advances but also testable hypotheses are proposed. Lastly, we briefly discuss applicability of the latest tools to gene silencing in the fungi, which will be breakthrough techniques for comprehensive understanding of the molecular basis of fungal phosphate metabolism. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

  10. Titrimetric determination of uranium in tributyl phosphate

    International Nuclear Information System (INIS)

    Sobkowska, A.

    1978-01-01

    The titrimetric method involving the reduction of U(VI) to uranium(IV) by iron(II) in phosphoric acid, selective oxidation of the excess of iron(II) and potentiometric titration with dichromate was directly used for the determination of uranium in tributyl phosphate mixtures. The procedure was applied to solutions containing more than 2 mg of uranium in the sample but the highest precision and accuracy were obtained in the range from 20 to 200 mg of uranium. Dibutyl phosphate and monobutyl phosphate as well as the other radiolysis products of TBP had no influence on the results of determinations. (author)

  11. Uranium and heavy metals in phosphate fertilizers

    International Nuclear Information System (INIS)

    Khater, A.E.M.

    2008-01-01

    Agricultural applications of chemical fertilizers are a worldwide practice. The specific activity of uranium-238 and heavy metals in phosphate fertilizers depends on the phosphate ore from which the fertilizer produced and on the chemical processing of the ore. Composite phosphate fertilizers samples where collected and the uranium-238 specific activity, in Bq/kg, and As, Cd, Cu, Pb, Se concentration, in ppm, were measured. The annual addition of these elements in soil due to fertilization were calculated and discussed. (author)(tk)

  12. Global radiological impact of the phosphate fertilizers

    International Nuclear Information System (INIS)

    Morales, Rudnei Karam; Alves, Rex Nazare

    1996-01-01

    About ninety percent of the products obtained in the phosphate industry are directly used in agriculture as fertilizers. The uranium, thorium and radium content in phosphate fertilizers pollute the soil, water and air, creating risks due to associated natural radiation. This work shows the concentration of radionuclides present in various products of the national and American phosphate fertilizers industry, and compared them with worldwide mean values. The radiological impact of the products on the environment is evaluated and suggestions are presented in order to minimize the risks due to radioactivity. (author)

  13. Heat Stress Affects Pi-related Genes Expression and Inorganic Phosphate Deposition/Accumulation in Barley

    DEFF Research Database (Denmark)

    Pacak, Andrzej; Barciszewska-Pacak, Maria; Swida-Barteczka, Aleksandra

    2016-01-01

    Phosphorus (P) in plants is taken from soil as an inorganic phosphate (Pi) and is one of the most important macroelements in growth and development. Plants actively react to Pi starvation by the induced expression of Pi transporters, MIR399, MIR827, and miR399 molecular sponge - IPS1 genes...... and by the decreased expression of the ubiquitin-conjugating enzyme E2 (PHOSPHATE2 - PHO2) and Pi sensing and transport SPX-MFS genes. The PHO2 protein is involved in the degradation of Pi transporters PHT1;1 (from soil to roots) and PHO1 (from roots to shoots). The decreased expression of PHO2 leads to Pi....... In shoots, the PHO2 mRNA level is decreased, leading to an increased Pi level. We concluded that Pi homeostasis in barley during heat stress is maintained by dynamic changes in Pi-related genes expression....

  14. Rare Autism-Associated Variants Implicate Syntaxin 1 (STX1 R26Q) Phosphorylation and the Dopamine Transporter (hDAT R51W) in Dopamine Neurotransmission and Behaviors

    OpenAIRE

    Cartier, Etienne; Hamilton, Peter J.; Belovich, Andrea N.; Shekar, Aparna; Campbell, Nicholas G.; Saunders, Christine; Andreassen, Thorvald F.; Gether, Ulrik; Veenstra-Vanderweele, Jeremy; Sutcliffe, James S.; Ulery-Reynolds, Paula G.; Erreger, Kevin; Matthies, Heinrich J.G.; Galli, Aurelio

    2015-01-01

    Background: Syntaxin 1 (STX1) is a presynaptic plasma membrane protein that coordinates synaptic vesicle fusion. STX1 also regulates the function of neurotransmitter transporters, including the dopamine (DA) transporter (DAT). The DAT is a membrane protein that controls DA homeostasis through the high-affinity re-uptake of synaptically released DA. Methods: We adopt newly developed animal models and state-of-the-art biophysical techniques to determine the contribution of the identified gen...

  15. Radiation doses from phosphate fertilizers

    International Nuclear Information System (INIS)

    Anon.

    1975-01-01

    The activity concentrations determined of 226 Ra, 232 Th and 40 K in nCi/kg P 2 O 5 for the five most important kinds of fertilizer as well as their percent share in the economy year 1973/74 in the FRG are compiled in a table. From these values, the consumption of 0.917 million tons P 2 O 5 and from an average annual fertilizer coverage of 68.3 kg/ha, one can calculate a distribution of 32 Ci 226 Ra, 1 Ci 232 Th and 543 Ci 40 K over the total agriculturally used area, in other words, a deposit of 2.4 μCi 226 Ra, 0.07 μCi 232 Th and 40.5 μCi 40 K per ha. Taking a pessimistic view, an external radiation exposure of 0.11 mrad/a was calculated for gonads and bone marrow. If the total accumulation of 226 Ra (38% of the radiation exposure) from phosphate fertilizers from the ground during the last 80 years is assumed, then there is an exposure of 1.7 mrad/a for individual members of the population and 2.0 mrad/a for those occupied in agriculture. (HP/LH) [de

  16. Natural radioactivity in phosphate fertilizers

    International Nuclear Information System (INIS)

    Arndt, J.; Aurand, K.; Ruehle, H.; Schmier, H.; Wolter, R.

    1974-12-01

    The activity concentrations determined of 226 Ra, 232 Th and 40 K in nCi/kg P 2 O 5 for the five most important kinds of fertilizer as well as their percent share in the economy year 1973/74 in the FRG are compiled in a table. From these values, the consumption of 0.917 million tons P 2 O 5 and from an average annual fertilizer covering of 68.3 kg/ha, one can calculate a distribution of 32 Ci 226 Ra, 1 Ci 232 Th and 543 Ci 40 K over the total agriculturally used area, in other words a deposit of 2.4 μCi 226 Ra, 0.07 μCi 232 Th and 40.5 μCi 40 K per ha. Taking a pessimistic view, an external radiation exposure of 0.11 mrad/a was calculated for gonads and bone marrow. If the total accumulation of 226 Ra (38% of the radiation exposure) from phosphate fertilizers from the ground during the last 80 years is assumed, then there is an exposure of 1.7 mrad/a for individual members of the population and 2.0 mrad/a for those occupied in agriculture. (HP/LH) [de

  17. Hemodialysis for near-fatal sodium phosphate toxicity in a child receiving sodium phosphate enemas.

    Science.gov (United States)

    Becknell, Brian; Smoyer, William E; O'Brien, Nicole F

    2014-11-01

    This study aimed to demonstrate the importance of considering hemodialysis as a treatment option in the management of sodium phosphate toxicity. This is a case report of a 4-year-old who presented to the emergency department with shock, decreased mental status, seizures, and tetany due to sodium phosphate toxicity from sodium phosphate enemas. Traditional management of hyperphosphatemia with aggressive hydration and diuretics was insufficient to reverse the hemodynamic and neurological abnormalities in this child. This is the first report of the use of hemodialysis in a child without preexisting renal failure for the successful management of near-fatal sodium phosphate toxicity. Hemodialysis can safely be used as an adjunctive therapy in sodium phosphate toxicity to rapidly reduce serum phosphate levels and increase serum calcium levels in children not responding to conventional management.

  18. Performance of pineapple slips inoculated with diazotrophic phosphate-solubilizing bacteria and rock phosphate

    OpenAIRE

    Lílian Estrela Borges Baldotto; Marihus Altoé Baldotto; Fábio Lopes Olivares; Adriane Nunes de Souza

    2014-01-01

    Besides fixing N2, some diazotrophic bacteria or diazotrophs, also synthesize organic acids and are able to solubilize rock phosphates, increasing the availability of P for plants. The application of these bacteria to pineapple leaf axils in combination with rock phosphate could increase N and P availability for the crop, due to the bacterial activity of biological nitrogen fixation and phosphate solubilization. The objectives of this study were: (i) to select and characterize diazotrophs abl...

  19. Uranium recovery from phosphate rocks concentrated

    International Nuclear Information System (INIS)

    Azevedo, M.F. de.

    1986-01-01

    The reserves, geological data, chemical data and technical flowsheet from COPEBRAS and Goiasfertil ores are described, including the process of mining ore concentration. Samples of Goiasfertil ores are analysed by gravimetric analysis, for phosphate, and spectrofluorimetry for uranium. (author)

  20. Hanford phosphate precipitation filtration process evaluation

    International Nuclear Information System (INIS)

    Walker, B.W.; McCabe, D.J.

    1997-01-01

    The purpose of this filter study was to evaluate cross-flow filtration as effective solid-liquid separation technology for treating Hanford wastes, outline operating conditions for equipment, examine the expected filter flow rates, and determine proper cleaning. A proposed Hanford waste pre-treatment process uses sodium hydroxide at high temperature to remove aluminum from sludge. This process also dissolves phosphates. Upon cooling to 40 degrees centigrade the phosphates form a Na7(PO4)2F9H2O precipitate which must be removed prior to further treatment. Filter studies were conducted with a phosphate slurry simulant to evaluate whether 0.5 micron cross-flow sintered metal Mott filters can separate the phosphate precipitate from the wash solutions. The simulant was recirculated through the filters at room temperature and filtration performance data was collected

  1. short communication agronomic effectiveness of novel phosphate

    African Journals Online (AJOL)

    igneous Dorowa (Zimbabwe) phosphate rock was investigated in a greenhouse ... are higher than the levels considered desirable for acidulation was selected based on proportions ... The analysis for dry matter yield of and P uptake by maize ...

  2. Phosphate coating on stainless steel 304 sensitized

    International Nuclear Information System (INIS)

    Cruz V, J. P.; Vite T, J.; Castillo S, M.; Vite T, M.

    2009-01-01

    The stainless steel 304 can be sensitized when welding processes are applied, that causes the precipitation of chromium carbide in the grain limits, being promoted in this way the formation of galvanic cells and consequently the corrosion process. Using a phosphate coating is possible to retard the physiochemical damages that can to happen in the corrosion process. The stainless steel 304 substrate sensitized it is phosphate to base of Zn-Mn, in a immersion cell very hot. During the process was considered optimization values, for the characterization equipment of X-rays diffraction and scanning electron microscopy was used. The XRD technique confirmed the presence of the phases of manganese phosphate, zinc phosphate, as well as the phase of the stainless steel 304. When increasing the temperature from 60 to 90 C in the immersion process a homogeneous coating is obtained. (Author)

  3. Issues of natural radioactivity in phosphates

    International Nuclear Information System (INIS)

    Schnug, E.; Haneklaus, S.; Schnier, C.; Scholten, L.C.

    1996-01-01

    The fertilization of phosphorus (P) fertilizers is essential in agricultural production, but phosphates contain in dependence on their origin different amounts of trace elements. The problem of cadmium (Cd) loads and other heavy metals is well known. However, only a limited number of investigations examined the contamination of phosphates with the two heaviest metals, uranium (U) and thorium (Th), which are radioactive. Also potassium (K) is lightly radioactive. Measurements are done n the radioactivity content of phosphates, P fertilizers and soils. The radiation doses to workers and public as well as possible contamination of soils from phosphate rock or fertilizer caused by these elements or their daughter products is of interest with regard to radiation protection. The use of P fertilizers is necessary for a sustainable agriculture, but it involves radioactive contamination of soils. The consequences of the use of P fertilizers is discussed, also with regard to existing and proposed legislation. 11 refs., 2 figs., 7 tabs

  4. Geochemistry and mineralogy of Ogun phosphate rock

    African Journals Online (AJOL)

    user

    m deep) within the same location. The pellets ... diffracted rays are collected by a detector and the information relayed to a ... Ogun phosphate deposits were formed in shallow marine ... process thus confirming the earlier work done by Jones.

  5. Rock phosphate solubilization by the ectomycorrhizal fungus ...

    African Journals Online (AJOL)

    SAM

    2014-06-18

    Jun 18, 2014 ... phosphate solubilization is accompanied by acid production. Thus, the evidence ..... of organic acids. (Khan et al., 2010) such as acetate, lactate, oxalate, ... (2014) also observed that oxalic acid was secreted by L. fraterna to ...

  6. Thermal stability of phosphate coatings on steel

    Czech Academy of Sciences Publication Activity Database

    Pokorný, P.; Szelag, P.; Novák, M.; Mastný, L.; Brožek, Vlastimil

    2015-01-01

    Roč. 54, č. 3 (2015), s. 489-492 ISSN 0543-5846 Institutional support: RVO:61389021 Keywords : Steel * phosphates * coatings * structure Subject RIV: CA - Inorganic Chemistry Impact factor: 0.959, year: 2014

  7. Corrosion inhibition by lithium zinc phosphate pigment

    International Nuclear Information System (INIS)

    Alibakhshi, E.; Ghasemi, E.; Mahdavian, M.

    2013-01-01

    Highlights: •Synthesis of lithium zinc phosphate (LZP) by chemical co-precipitation method. •Corrosion inhibition activity of pigments compare with zinc phosphate (ZP). •LZP showed superior corrosion inhibition effect in EIS measurements. •Evaluation of adhesion strength and dispersion stability. -- Abstract: Lithium zinc phosphate (LZP) has been synthesized through a co-precipitation process and characterized by XRD and IR spectroscopy. The inhibitive performances of this pigment for corrosion of mild steel have been discussed in comparison with the zinc phosphate (ZP) in the pigment extract solution by means of EIS and in the epoxy coating by means of salt spray. The EIS and salt spray results revealed the superior corrosion inhibitive effect of LZP compared to ZP. Moreover, adhesion strength and dispersion stability of the pigmented epoxy coating showed the advantage of LZP compared to ZP

  8. Synthesis and characterization of porous calcium phosphate

    International Nuclear Information System (INIS)

    Granados C, F.; Serrano G, J.; Bonifacio M, J.

    2007-01-01

    The porous calcium phosphate was prepared by the continuous precipitation method using Ca(NO 3 ) 2 .4H 2 O and NH 4 H 2 PO 4 salts. The synthesized material was structurally and superficially characterized using the XRD, BET, IR TGA and SEM techniques. The obtained inorganic material was identified as calcium phosphate that presents a great specific area for what can be efficiently used as adsorbent material for adsorption studies in the radioactive wastes treatment present in aqueous solution. (Author)

  9. Removal of organic wastes containing tributyl phosphate

    International Nuclear Information System (INIS)

    Drobnik, S.

    TBP in dodecane and kerosene is one of the waste solutions from the reprocessing of spent nuclear fuels by the Purex process. The following methods were investigated for removing the organic solvents: adsorption on suitable solids, extraction, reaction with neutral salts, and saponification with acids or alkalis. Results showed that the best method of TBP removal is saponification with alkali hydroxides, either with dibutyl phosphate or with ortho-phosphate

  10. Kinetics of strontium sorption in calcium phosphate

    International Nuclear Information System (INIS)

    Bacic, S.; Komarov, V.F.; Vukovic, Z.

    1989-01-01

    Kinetics of strontium sorption by highly dispersed solids: tricalcium phosphate (Ca 3 (PO 4 ) 2 , TCP) and hydroxyapatite (Ca 5 (PO 4 ) 3 )H, HAP) were investigated. Analysis of sorption data was made taking into consideration composition and morphology of ultra micro particles. Conclusion is that the isomorphous strontium impurity is structurally sensitive element for calcium phosphate. It was determined that the beginning of strontium desorption corresponds to the beginning of transformation of the TCP - HAP (author)

  11. Fine-tuning by strigolactones of root response to low phosphate.

    Science.gov (United States)

    Kapulnik, Yoram; Koltai, Hinanit

    2016-03-01

    Strigolactones are plant hormones that regulate the development of different plant parts. In the shoot, they regulate axillary bud outgrowth and in the root, root architecture and root-hair length and density. Strigolactones are also involved with communication in the rhizosphere, including enhancement of hyphal branching of arbuscular mycorrhizal fungi. Here we present the role and activity of strigolactones under conditions of phosphate deprivation. Under these conditions, their levels of biosynthesis and exudation increase, leading to changes in shoot and root development. At least for the latter, these changes are likely to be associated with alterations in auxin transport and sensitivity. On the other hand, strigolactones may positively affect plant-mycorrhiza interactions and thereby promote phosphate acquisition by the plant. Strigolactones may be a way for plants to fine-tune their growth pattern under phosphate deprivation. © 2015 Institute of Botany, Chinese Academy of Sciences.

  12. Phosphate rock costs, prices and resources interaction.

    Science.gov (United States)

    Mew, M C

    2016-01-15

    This article gives the author's views and opinions as someone who has spent his working life analyzing the international phosphate sector as an independent consultant. His career spanned two price hike events in the mid-1970's and in 2008, both of which sparked considerable popular and academic interest concerning adequacy of phosphate rock resources, the impact of rising mining costs and the ability of mankind to feed future populations. An analysis of phosphate rock production costs derived from two major industry studies performed in 1983 and 2013 shows that in nominal terms, global average cash production costs increased by 27% to $38 per tonne fob mine in the 30 year period. In real terms, the global average cost of production has fallen. Despite the lack of upward pressure from increasing costs, phosphate rock market prices have shown two major spikes in the 30 years to 2013, with periods of less volatility in between. These price spike events can be seen to be related to the escalating investment cost required by new mine capacity, and as such can be expected to be repeated in future. As such, phosphate rock price volatility is likely to have more impact on food prices than rising phosphate rock production costs. However, as mining costs rise, recycling of P will also become increasingly driven by economics rather than legislation. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Sphingosine 1-Phosphate and Atherosclerosis

    Science.gov (United States)

    Yatomi, Yutaka

    2018-01-01

    Sphingosine 1-phosphate (S1P) is a potent lipid mediator that works on five kinds of S1P receptors located on the cell membrane. In the circulation, S1P is distributed to HDL, followed by albumin. Since S1P and HDL share several bioactivities, S1P is believed to be responsible for the pleiotropic effects of HDL. Plasma S1P levels are reportedly lower in subjects with coronary artery disease, suggesting that S1P might be deeply involved in the pathogenesis of atherosclerosis. In basic experiments, however, S1P appears to possess both pro-atherosclerotic and anti-atherosclerotic properties; for example, S1P possesses anti-apoptosis, anti-inflammation, and vaso-relaxation properties and maintains the barrier function of endothelial cells, while S1P also promotes the egress and activation of lymphocytes and exhibits pro-thrombotic properties. Recently, the mechanism for the biased distribution of S1P on HDL has been elucidated; apolipoprotein M (apoM) carries S1P on HDL. ApoM is also a modulator of S1P, and the metabolism of apoM-containing lipoproteins largely affects the plasma S1P level. Moreover, apoM modulates the biological properties of S1P. S1P bound to albumin exerts both beneficial and harmful effects in the pathogenesis of atherosclerosis, while S1P bound to apoM strengthens anti-atherosclerotic properties and might weaken the pro-atherosclerotic properties of S1P. Although the detailed mechanisms remain to be elucidated, apoM and S1P might be novel targets for the alleviation of atherosclerotic diseases in the future. PMID:28724841

  14. miRNA778 and SUVH6 are involved in phosphate homeostasis in Arabidopsis.

    Science.gov (United States)

    Wang, Lei; ZengJ, Hou Qing; Song, Jun; Feng, Sheng Jun; Yang, Zhi Min

    2015-09-01

    microRNAs (miRNAs) play an important role in plant adaptation to phosphate (Pi) starvation. Histone methylation can remodel chromatin structure and mediate gene expression. This study identified Arabidopsis miR778, a Pi-responsive miRNA, and its target gene Su(var) 3-9 homologs 6 (SUVH6) encoding a histone H3 lysine 9 (H3K9) methyltransferase. Overexpression of miR778 moderately enhanced primary and lateral root growth, free phosphate accumulation in shoots, and accumulation of anthocyanin under Pi deficient conditions. miR778 overexpression relieved the arrest of columella cell development under Pi starvation. Conversely, transgenic plants overexpressing a miR778-target mimic (35S::MIM778), that act as a sponge and sequesters miR778, showed opposite phenotypes of 35S::miR778 plants under Pi deficiency. Expression of several Pi deficiency-responsive genes such as miR399, Phosphate Transporter (PHT1;4), Low Phosphate-Resistant1 (LPR1) and Production of Anthocyanin Pigment 1 (PAP1) were elevated in the miR778 overexpressing plants, suggesting that both miR778 and SUVH6 are involved in phosphate homeostasis in plants. This study has provided a basis for further investigation on how SUVH6 regulates its downstream genes through chromatin remodeling and DNA methylation in plants stressed by Pi deficiency. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Ternary complex formation of lanthanides and radiolanthanides with phosphate and serum proteins

    International Nuclear Information System (INIS)

    Neumaier, B.; Roesch, F.

    1999-01-01

    Radioyttrium was recently reported to form ternary complexes with phosphate and serum proteins in blood. In the present work it was investigated whether the trivalent radiolanthanides react in a chemically similar way. In systematic binding studies using gel filtration a ternary complex formation between different lanthanides, phosphate and serum proteins could be identified. The tendency to build a ternary compound of the type Ln III - phosphate - serum protein, however, is dependent on the ionic radii of the lanthanides. Whereas the light and transition lanthanides have a strong inclination to build a ternary complex, this tendency is weaker for the heavier ones. Taking into account the high content of phosphate in human blood, the corresponding ternary complexes of radiolanthanides represent an important transport form of these elements in blood. This finding may contribute to an understanding of the nuclear medical observation on the biodistribution of radiolanthanides. The heavy radiolanthanides can be classified as bone seeking metals, whereas the light and transition lanthanide elements accumulate mainly in the liver and the spleen. For the lighter radiolanthanides the corresponding ternary complexes thus represent an important transport form in blood. This physicochemical form of lanthanides mainly results in reticulo endothelial accumulation; on the other hand, the lower tendency of heavier lanthanides leads to preferential skeletal deposition. (orig.)

  16. Structural and In Vivo Studies on Trehalose-6-Phosphate Synthase from Pathogenic Fungi Provide Insights into Its Catalytic Mechanism, Biological Necessity, and Potential for Novel Antifungal Drug Design

    Directory of Open Access Journals (Sweden)

    Yi Miao

    2017-07-01

    Full Text Available The disaccharide trehalose is critical to the survival of pathogenic fungi in their human host. Trehalose-6-phosphate synthase (Tps1 catalyzes the first step of trehalose biosynthesis in fungi. Here, we report the first structures of eukaryotic Tps1s in complex with substrates or substrate analogues. The overall structures of Tps1 from Candida albicans and Aspergillus fumigatus are essentially identical and reveal N- and C-terminal Rossmann fold domains that form the glucose-6-phosphate and UDP-glucose substrate binding sites, respectively. These Tps1 structures with substrates or substrate analogues reveal key residues involved in recognition and catalysis. Disruption of these key residues severely impaired Tps1 enzymatic activity. Subsequent cellular analyses also highlight the enzymatic function of Tps1 in thermotolerance, yeast-hypha transition, and biofilm development. These results suggest that Tps1 enzymatic functionality is essential for the fungal stress response and virulence. Furthermore, structures of Tps1 in complex with the nonhydrolyzable inhibitor, validoxylamine A, visualize the transition state and support an internal return-like catalytic mechanism that is generalizable to other GT-B-fold retaining glycosyltransferases. Collectively, our results depict key Tps1-substrate interactions, unveil the enzymatic mechanism of these fungal proteins, and pave the way for high-throughput inhibitor screening buttressed and guided by the current structures and those of high-affinity ligand-Tps1 complexes.

  17. Determination of tricresyl phosphate air contamination in aircraft.

    Science.gov (United States)

    Denola, G; Hanhela, P J; Mazurek, W

    2011-08-01

    Monitoring of tricresyl phosphate (TCP) contamination of cockpit air was undertaken in three types of military aircraft [fighter trainer (FT), fighter bomber (FB), and cargo transport (CT) aircraft]. The aircraft had a previous history of pilot complaints about cockpit air contamination suspected to originate from the engine bleed air supply through the entry of aircraft turbine engine oil (ATO) into the engine compressor. Air samples were collected in flight and on the ground during engine runs using sorbent tubes packed with Porapak Q and cellulose filters. A total of 78 air samples were analysed, from 46 different aircraft, and 48 samples were found to be below the limit of detection. Nine incidents of smoke/odour were identified during the study. The concentrations of toxic o-cresyl phosphate isomers were below the level of detection in all samples. The highest total TCP concentration was 51.3 μg m(-3), while most were generally found to be contamination of cabin/cockpit air has been the subject of much concern in aviation, quantitative data are sparse.

  18. Fc receptor-γ subunits with both polar or non-polar amino acids at position of T22 are capable of restoring surface expression of the high-affinity IgE receptor and degranulation in γ subunit-deficient rat basophilic leukemia cells

    Czech Academy of Sciences Publication Activity Database

    Rashid, A.; Housden, J.E.; Helm, B.A.; Dráber, Petr

    2013-01-01

    Roč. 53, č. 3 (2013), s. 270-273 ISSN 0161-5890 R&D Projects: GA MŠk LD12073; GA ČR GA301/09/1826; GA ČR GAP302/10/1759; GA ČR(CZ) GBP302/12/G101; GA MŠk 1M0506 Grant - others:European Cooperation in Science and Technology(XE) BM1007; AV ČR(CZ) M200520901 Institutional research plan: CEZ:AV0Z50520514 Keywords : allergy * high-affinity IgE receptor * plasma membrane * transmembrane signaling * 3-helix assembly model Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.003, year: 2013

  19. [(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors.

    Science.gov (United States)

    Müller, Christa E; Diekmann, Martina; Thorand, Mark; Ozola, Vita

    2002-02-11

    This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.

  20. Agp2, a Member of the Yeast Amino Acid Permease Family, Positively Regulates Polyamine Transport at the Transcriptional Level

    KAUST Repository

    Aouida, Mustapha

    2013-06-03

    Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3? nor sam3? single mutant is defective in polyamine transport, while the dur3? sam3? double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2? mutant. Studies of Agp2 localization indicate that in the double mutant dur3? sam3?, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport. © 2013 Aouida et al.