Active uptake of substance P carboxy-terminal heptapeptide (5-11) into rat brain and rabbit spinal cord slices.

PubMed

Nakata, Y; Kusaka, Y; Yajima, H; Segawa, T

1981-12-01

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.

The mechanism of zinc uptake by cultured rat liver cells.

PubMed Central

Taylor, J A; Simons, T J

1994-01-01

1. The initial rate of 65Zn uptake into cultured rat hepatocytes has been measured over a range of Zn2+ concentrations from 3 x 10(-10) M to 5 x 10(-6) M. Histidine and albumin were used to buffer Zn2+ ions at concentrations below 1 x 10(-6) M. 2. The results suggest there are two mechanisms for Zn2+ uptake; a high-affinity, saturable pathway, with a maximum velocity (Vmax) of 20-30 pmol (mg protein)-1 min-1 and a Michaelis-Menten constant (Km) of about 2 x 10(-9) M Zn2+ (with histidine), and a low-affinity, linear pathway, that only makes a significant contribution to Zn2+ uptake at Zn2+ concentrations above 1 x 10(-6) M. 3. Transport via the high-affinity pathway is dependent on the concentration of Zn2+ ions and not on the concentrations of Zn(2+)-ligand complexes, suggesting that Zn2+ is the transported species. 4. The affinity of the saturable pathway for Zn2+ is slightly lower in the presence of albumin, with a Km of about 1.3 x 10(-8) M. The reason for this is uncertain. PMID:8014898

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

PubMed

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

2010-06-01

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

Regulation of Nitrate Transport in Citrus Rootstocks Depending on Nitrogen Availability

PubMed Central

Cerezo, Miguel; Camañes, Gemma; Flors, Víctor; Primo-Millo, Eduardo

2007-01-01

Previously, we reported that in Citrus plants, nitrate influx through the plasmalemma of roots cells follows a biphasic pattern, suggesting the existence of at least two different uptake systems, a high and low affinity transport system (HATS and LATS, respectively). Here, we describe a novel inducible high affinity transport system (iHATS). This new nitrate transport system has a high capacity to uptake nitrate in two different Citrus rootstocks (Cleopatra mandarin and Troyer citrange). The iHATS was saturable, showing higher affinity than constitutive high affinity transport system (cHATS) to the substrate NO3−. The Vmax for this saturable component iHATS was higher than cHATS, reaching similar values in both rootstocks. Additionally, we studied the regulation of root NO3− uptake mediated by both HATS (iHATS and cHATS) and LATS. In both rootstocks, cHATS is constitutive and independent of N-status. Concerning the regulation of iHATS, this system is upregulated by NO3− and down-regulated by the N status and by NO3− itself when plants are exposed to it for a longer period of time. LATS in Cleopatra mandarin and Troyer citrange rootstocks is repressed by the N-status. The use of various metabolic uncouplers or inhibitors indicated that NO3− net uptake mediated by iHATS and LATS was an active transport system in both rootstocks. PMID:19516998

Expression of the high-affinity choline transporter CHT1 in rat and human arteries.

PubMed

Lips, Katrin S; Pfeil, Uwe; Reiners, Katja; Rimasch, Christoph; Kuchelmeister, Klaus; Braun-Dullaeus, Ruediger C; Haberberger, Rainer V; Schmidt, Rupert; Kummer, Wolfgang

2003-12-01

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.

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

PubMed

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

2014-11-01

The high-affinity K(+) transporter HAK5 is a key system for root K(+) uptake and, under very low external K(+), the only one capable of supplying K(+) to the plant. Functional HAK5-mediated K(+) uptake should be tightly regulated for plant adaptation to different environmental conditions. Thus, it has been described that the gene encoding the transporter is transcriptionally regulated, being highly induced under K(+) limitation. Here we show that environmental conditions, such as the lack of K(+), NO(3)(-) or P, that induced a hyperpolarization of the plasma membrane of root cells, induce HAK5 transcription. However, only the deprivation of K(+) produces functional HAK5-mediated K(+) uptake in the root. These results suggest on the one hand the existence of a posttranscriptional regulation of HAK5 elicited by the low K(+) signal and on the other that HAK5 may be involved in yet-unknown functions related to NO(3)(-) and P deficiencies. These results have been obtained here with Solanum lycopersicum (cv. Micro-Tom) as well as Arabidopsis thaliana plants, suggesting that the posttranscriptional regulation of high-affinity HAK transporters take place in all plant species. © 2014 Scandinavian Plant Physiology Society.

Functional analysis of choline transporters in rheumatoid arthritis synovial fibroblasts.

PubMed

Seki, Masayuki; Kawai, Yuiko; Ishii, Chikanao; Yamanaka, Tsuyoshi; Odawara, Masato; Inazu, Masato

2017-11-01

In this study, we examined the functional characteristics of choline uptake and sought to identify the transporters in rheumatoid arthritis synovial fibroblasts (RASFs). The expression of choline transporters was evaluated by quantitative real-time PCR, western blotting, and immunocytochemistry. Time course, Na + -dependency, and kinetics of [ 3 H]choline uptake were investigated. Effects of cationic drugs on the uptake of [ 3 H]choline, cell viability, and caspase-3/7 activity were also examined. Finally, we investigated the influence of choline uptake inhibitor, hemicholinium-3 (HC-3), and choline deficiency on cell viability and caspase-3/7 activity. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in RASFs and were localized to the plasma membrane. [ 3 H]Choline uptake occurred via a Na + -independent and pH-dependent transport system. The cells have two different [ 3 H]choline transport systems, high- and low-affinity. Various organic cations, HC-3 and choline deficiency inhibited both [ 3 H]choline uptake and cell viability, and enhanced the caspase-3/7 activity. The functional inhibition of choline transporters could promote apoptotic cell death. In RASFs, [ 3 H]choline uptake was significantly increased compared with that in OASFs without a change in gene expression. These results suggest that CTL1 (high-affinity) and CTL2 (low-affinity) are highly expressed in RASFs and choline may be transported by a choline/H +  antiport system. Identification of this CTL1- and CTL2-mediated choline transport system should provide a potential new target for RA therapy.

Molecular Cloning and Functional Analysis of a Na+-Insensitive K+ Transporter of Capsicum chinense Jacq

PubMed Central

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

UPTAKE OF RADIONUCLIDE METALS BY SPME FIBERS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duff, M; S Crump, S; Robert02 Ray, R

2006-08-28

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

Galactose transport in Kluyveromyces lactis: major role of the glucose permease Hgt1.

PubMed

Baruffini, Enrico; Goffrini, Paola; Donnini, Claudia; Lodi, Tiziana

2006-12-01

In Kluyveromyces lactis, galactose transport has been thought to be mediated by the lactose permease encoded by LAC12. In fact, a lac12 mutant unable to grow on lactose did not grow on galactose either and showed low and uninducible galactose uptake activity. The existence of other galactose transport systems, at low and at high affinity, had, however, been hypothesized on the basis of galactose uptake kinetics studies. Here we confirmed the existence of a second galactose transporter and we isolated its structural gene. It turned out to be HGT1, previously identified as encoding the high-affinity glucose carrier. Analysis of galactose transporter mutants, hgt1 and lac12, and the double mutant hgt1lac12, suggested that Hgt1 was the high-affinity and Lac12 was the low-affinity galactose transporter. HGT1 expression was strongly induced by galactose and insensitive to glucose repression. This could explain the rapid adaptation to galactose observed in K. lactis after a shift from glucose to galactose medium.

Rhodamine Inhibitors of P-glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

PubMed Central

Gannon, Michael K.; Holt, Jason J.; Bennett, Stephanie M.; Wetzel, Bryan R.; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.; Sawada, Geri A.; Higgins, J. William; Tombline, Gregory; Raub, Thomas J.; Detty, Michael R.

2012-01-01

We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His10, for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave KM of 0.087 μM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC50’s of ~2 μM in MDCKII-MDR1 cells. PMID:19402665

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1999-11-01

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

( sup 14 C)-Sucrose uptake by guard cell protoplasts of pisum sativum, argenteum mutant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rohrig, K.; Raschke, K.

1991-05-01

Guard cells rely on import for their supply with reduced carbon. The authors tested by silicone oil centrifugation the ability of guard cell protoplasts to accumulated ({sup 14}C)-sucrose. Uptake rates were corrected after measurement of {sup 14}C-sorbitol and {sup 3}H{sub 2}O spaces. Sucrose uptake followed biphasic kinetics, with a high-affinity component below 1 mM external sucrose (apparent K{sub m} 0.8 mM at 25C) and a low-affinity nonsaturable component above. Uptake depended on pH (optimum at pH 5.0). Variations in the concentrations of external KCl, CCCP, and valinomycin indicated that about one-half of the sucrose uptake rate could be related tomore » an electrochemical gradient across the plasmalemma. Total uptake rates measured at 5 mM external sucrose seem to be sufficient to replenish emptied plastids with starch within a few hours.« less

Impact of bubble size on growth and CO2 uptake of Arthrospira (Spirulina) platensis KMMCC CY-007.

PubMed

Kim, Kisok; Choi, Jaeho; Ji, Yosep; Park, Soyoung; Do, Hyungki; Hwang, Cherwon; Lee, Bongju; Holzapfel, Wilhelm

2014-10-01

Optimisation of cyanobacterial cell productivity should consider the key factors light cycle and carbon source. We studied the influence of CO2 bubble size on carbon uptake and fixation, on basis of mRNA expression levels in Arthrospira platensis KMMCC CY-007 at 30°C (light intensity: 40μmolm(-2)s(-1); 1% CO2). Growth rate, carbon fixation and lipid accumulation were examined over 7days under fine bubble (FB) (100μm Ø) bulk bubble (BB) (5000μm Ø) and non-CO2 (NB) aeration. The low affinity CO2 uptake mRNA (NDH-I4 complex) was stronger expressed than the high affinity NDH-I3 complex (bicA and sbtA) under 1% CO2 and FB conditions, with no expression of bicA1 and sbtA1 after 4days. The high affinity CO2 uptake mRNA levels corresponded to biomass, carbon content and lipid accumulation, and increase in NDH-I3 complex (9.72-fold), bicA (5.69-fold), and sbtA (10.61-fold), compared to NB, or BB conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Carrier-mediated γ-aminobutyric acid transport across the basolateral membrane of human intestinal Caco-2 cell monolayers.

PubMed

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.

Origins of serotonin innervation of forebrain structures

NASA Technical Reports Server (NTRS)

Kellar, K. J.; Brown, P. A.; Madrid, J.; Bernstein, M.; Vernikos-Danellis, J.; Mehler, W. R.

1977-01-01

The tryptophan hydroxylase activity and high-affinity uptake of (3H) serotonin ((3H)5-HT) were measured in five discrete brain regions of rats following lesions of the dorsal or median raphe nuclei. Dorsal raphe lesions reduced enzyme and uptake activity in the striatum only. Median raphe lesions reduced activities in the hippocampus, septal area, frontal cortex, and, to a lesser extent, in the hypothalamus. These data are consistent with the suggestion that the dorsal and median raphe nuclei are the origins of two separate ascending serotonergic systems - one innervating striatal structures and the other mesolimbic structures, predominantly. In addition, the data suggest that measurements of high-affinity uptake of (3H)5-HT may be a more reliable index of innervation than either 5-HT content or tryptophan hydroxylase activity.

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

PubMed Central

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

2015-01-01

The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization. PMID:25901755

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xinxin; Zhang, Min; Takano, Tetsuo

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

Choline Uptake in Agrobacterium tumefaciens by the High-Affinity ChoXWV Transporter▿

PubMed Central

Aktas, Meriyem; Jost, Kathinka A.; Fritz, Christiane; Narberhaus, Franz

2011-01-01

Agrobacterium tumefaciens is a facultative phytopathogen that causes crown gall disease. For successful plant transformation A. tumefaciens requires the membrane lipid phosphatidylcholine (PC), which is produced via the methylation and the PC synthase (Pcs) pathways. The latter route is dependent on choline. Although choline uptake has been demonstrated in A. tumefaciens, the responsible transporter(s) remained elusive. In this study, we identified the first choline transport system in A. tumefaciens. The ABC-type choline transporter is encoded by the chromosomally located choXWV operon (ChoX, binding protein; ChoW, permease; and ChoV, ATPase). The Cho system is not critical for growth and PC synthesis. However, [14C]choline uptake is severely reduced in A. tumefaciens choX mutants. Recombinant ChoX is able to bind choline with high affinity (equilibrium dissociation constant [KD] of ≈2 μM). Since other quaternary amines are bound by ChoX with much lower affinities (acetylcholine, KD of ≈80 μM; betaine, KD of ≈470 μM), the ChoXWV system functions as a high-affinity transporter with a preference for choline. Two tryptophan residues (W40 and W87) located in the predicted ligand-binding pocket are essential for choline binding. The structural model of ChoX built on Sinorhizobium meliloti ChoX resembles the typical structure of substrate binding proteins with a so-called “Venus flytrap mechanism” of substrate binding. PMID:21803998

Differential Uptake Mechanisms of Fluorescent Substrates into Stem-Cell-Derived Serotonergic Neurons.

PubMed

Matthaeus, Friederike; Schloss, Patrick; Lau, Thorsten

2015-12-16

The actions of the neurotransmitters serotonin, dopamine, and norepinephrine are partly terminated by diffusion and in part by their uptake into neurons via the selective, high-affinity transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET), respectively. There is also growing evidence that all three monoamines are taken up into neurons by low-affinity, high-capacity organic cation transporters (OCT) and the plasma membrane monoamine transporter (PMAT). Pharmacological characterization of these low-affinity recombinant transporter proteins in heterologous expression systems has revealed that they are not antagonized by classical inhibitors of SERT, DAT, or NET but that decynium-22 (D22) antagonizes OCT3 and PMAT, whereas corticosterone and progesterone selectively inhibit OCT3. Here, we show that SERT, PMAT, and OCT3, but not OCT1 and OCT2, are coexpressed in murine stem cell-derived serotonergic neurons. Using selective antagonists, we provide evidence that uptake of the fluorescent substrates FFN511, ASP+, and 5-HT into stem cell-derived serotonergic neurons is mediated differentially by these transporters and also involves an as yet unknown transport mechanism.

Integration of biotic ligand models (BLM) and bioaccumulation kinetics into a mechanistic framework for metal uptake in aquatic organisms.

PubMed

Veltman, Karin; Huijbregts, Mark A J; Hendriks, A Jan

2010-07-01

Both biotic ligand models (BLM) and bioaccumulation models aim to quantify metal exposure based on mechanistic knowledge, but key factors included in the description of metal uptake differ between the two approaches. Here, we present a quantitative comparison of both approaches and show that BLM and bioaccumulation kinetics can be merged into a common mechanistic framework for metal uptake in aquatic organisms. Our results show that metal-specific absorption efficiencies calculated from BLM-parameters for freshwater fish are highly comparable, i.e. within a factor of 2.4 for silver, cadmium, copper, and zinc, to bioaccumulation-absorption efficiencies for predominantly marine fish. Conditional affinity constants are significantly related to the metal-specific covalent index. Additionally, the affinity constants of calcium, cadmium, copper, sodium, and zinc are significantly comparable across aquatic species, including molluscs, daphnids, and fish. This suggests that affinity constants can be estimated from the covalent index, and constants can be extrapolated across species. A new model is proposed that integrates the combined effect of metal chemodynamics, as speciation, competition, and ligand affinity, and species characteristics, as size, on metal uptake by aquatic organisms. An important direction for further research is the quantitative comparison of the proposed model with acute toxicity values for organisms belonging to different size classes.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1988-01-01

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

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

PubMed

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

2016-01-01

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

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

PubMed Central

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

2016-01-01

K+ is an essential macronutrient for plants. It is acquired by specific uptake systems located in roots. Although the concentrations of K+ in the soil solution are widely variable, K+ nutrition is secured by uptake systems that exhibit different affinities for K+. Two main systems have been described for root K+ uptake in several species: the high-affinity HAK5-like transporter and the inward-rectifier AKT1-like channel. Other unidentified systems may be also involved in root K+ uptake, although they only seem to operate when K+ is not limiting. The use of knock-out lines has allowed demonstrating their role in root K+ uptake in Arabidopsis and rice. Plant adaptation to the different K+ supplies relies on the finely tuned regulation of these systems. Low K+-induced transcriptional up-regulation of the genes encoding HAK5-like transporters occurs through a signal cascade that includes changes in the membrane potential of root cells and increases in ethylene and reactive oxygen species concentrations. Activation of AKT1 channels occurs through phosphorylation by the CIPK23/CBL1 complex. Recently, activation of the Arabidopsis HAK5 by the same complex has been reported, pointing to CIPK23/CBL as a central regulator of the plant’s adaptation to low K+. Na+ is not an essential plant nutrient but it may be beneficial for some plants. At low concentrations, Na+ improves growth, especially under K+ deficiency. Thus, high-affinity Na+ uptake systems have been described that belong to the HKT and HAK families of transporters. At high concentrations, typical of saline environments, Na+ accumulates in plant tissues at high concentrations, producing alterations that include toxicity, water deficit and K+ deficiency. Data concerning pathways for Na+ uptake into roots under saline conditions are still scarce, although several possibilities have been proposed. The apoplast is a significant pathway for Na+ uptake in rice grown under salinity conditions, but in other plant species different mechanisms involving non-selective cation channels or transporters are under discussion. PMID:27458473

GZ-793A, a lobelane analog, interacts with the vesicular monoamine transporter-2 to inhibit the effect of methamphetamine

PubMed Central

Horton, David B.; Nickell, Justin R.; Zheng, Guangrong; Crooks, Peter A.; Dwoskin, Linda P.

2013-01-01

GZ-793A inhibits methamphetamine-evoked dopamine release from striatal slices and methamphetamine self-administration in rats. GZ-793A potently and selectively inhibits dopamine uptake at the vesicular monoamine transporter-2 (VMAT2). The present study determined GZ-793A’s ability to evoke [3H]dopamine release and inhibit methamphetamine-evoked [3H]dopamine release from isolated striatal synaptic vesicles. Results show GZ-793A concentration-dependent [3H]dopamine release; nonlinear regression revealed a two-site model of interaction with VMAT2 (High- and Low-EC50 = 15.5 nM and 29.3 µM, respectively). Tetrabenazine and reserpine completely inhibited the GZ-793A-evoked [3H]dopamine release, however, only at the High-affinity site. Low concentrations of GZ-793A that interact with the extravesicular dopamine uptake site and the High-affinity intravesicular DA release site also inhibited methamphetamine-evoked [3H]dopamine release from synaptic vesicles. A rightward shift in the methamphetamine concentration-response was evident with increasing concentrations of GZ-793A, and the Schild regression slope was 0.49±0.08, consistent with surmountable allosteric inhibition. These results support a hypothetical model of GZ-793A interaction at more than one site on VMAT2 protein, which explains its potent inhibition of dopamine uptake, dopamine release via a High-affinity tetrabenazine- and reserpine-sensitive site, dopamine release via a Low-affinity tetrabenazine- and reserpine-insensitive site, and low-affinity interaction with the dihydrotetrabenazine binding site on VMAT2. GZ-793A-inhibition of the effects of methamphetamine supports its potential as a therapeutic agent for the treatment of methamphetamine abuse. PMID:23875622

Fluoroethoxy-1,4-diphenethylpiperidine and piperazine derivatives: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2.

PubMed

Hankosky, Emily R; Joolakanti, Shyam R; Nickell, Justin R; Janganati, Venumadhav; Dwoskin, Linda P; Crooks, Peter A

2017-12-15

A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine derivatives were designed, synthesized and evaluated for their ability to inhibit [ 3 H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [ 3 H]serotonin (5-HT) uptake at the serotonin transporter (SERT), and [ 3 H]dofetilide binding at the human-ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition of [ 3 H]DA uptake at VMAT2, Ki changes in the nanomolar range (K i  = 0.014-0.073 µM). Compound 15d exhibited the highest affinity (K i  = 0.014 µM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (K i  = 0.073 µM). Compound 15b was considered the lead compound from this analog series due to its high affinity and selectivity for VMAT2. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular regulation of aluminum resistance and sulfur nutrition during root growth.

PubMed

Alarcón-Poblete, Edith; Inostroza-Blancheteau, Claudio; Alberdi, Miren; Rengel, Zed; Reyes-Díaz, Marjorie

2018-01-01

Aluminum toxicity and sulfate deprivation both regulate microRNA395 expression, repressing its low-affinity sulfate transporter ( SULTR2;1 ) target. Sulfate deprivation also induces the high-affinity sulfate transporter gene ( SULTR12 ), allowing enhanced sulfate uptake. Few studies about the relationships between sulfate, a plant nutrient, and aluminum, a toxic ion, are available; hence, the molecular and physiological processes underpinning this interaction are poorly understood. The Al-sulfate interaction occurs in acidic soils, whereby relatively high concentrations of trivalent toxic aluminum (Al 3+ ) may hamper root growth, limiting uptake of nutrients, including sulfur (S). On the other side, Al 3+ may be detoxified by complexation with sulfate in the acid soil solution as well as in the root-cell vacuoles. In this review, we focus on recent insights into the mechanisms governing plant responses to Al toxicity and its relationship with sulfur nutrition, emphasizing the role of phytohormones, microRNAs, and ion transporters in higher plants. It is known that Al 3+ disturbs gene expression and enzymes involved in biosynthesis of S-containing cysteine in root cells. On the other hand, Al 3+ may induce ethylene biosynthesis, enhance reactive oxygen species production, alter phytohormone transport, trigger root growth inhibition and promote sulfate uptake under S deficiency. MicroRNA395, regulated by both Al toxicity and sulfate deprivation, represses its low-affinity Sulfate Transporter 2;1 (SULTR2;1) target. In addition, sulfate deprivation induces High Affinity Sulfate Transporters (HAST; SULTR1;2), improving sulfate uptake from low-sulfate soil solutions. Identification of new microRNAs and cloning of their target genes are necessary for a better understanding of the role of molecular regulation of plant resistance to Al stress and sulfate deprivation.

Functional activity of L-carnitine transporters in human airway epithelial cells.

PubMed

Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

2016-02-01

Carnitine plays a physiologically important role in the β-oxidation of fatty acids, facilitating the transport of long-chain fatty acids across the inner mitochondrial membrane. Distribution of carnitine within the body tissues is mainly performed by novel organic cation transporter (OCTN) family, including the isoforms OCTN1 (SLC22A4) and OCTN2 (SLC22A5) expressed in human. We performed here a characterization of carnitine transport in human airway epithelial cells A549, Calu-3, NCl-H441, and BEAS-2B, by means of an integrated approach combining data of mRNA/protein expression with the kinetic and inhibition analyses of L-[(3)H]carnitine transport. Carnitine uptake was strictly Na(+)-dependent in all cell models. In A549 and BEAS-2B cells, carnitine uptake was mediated by one high-affinity component (Km<2 μM) identifiable with OCTN2. In both these cell models, indeed, carnitine uptake was maximally inhibited by betaine and strongly reduced by SLC22A5/OCTN2 silencing. Conversely, Calu-3 and NCl-H441 exhibited both a high (Km~20 μM) and a low affinity (Km>1 mM) transport component. While the high affinity component is identifiable with OCTN2, the low affinity uptake is mediated by ATB(0,+), a Na(+), and Cl(-)-coupled transport system for neutral and cationic amino acids, as demonstrated by the inhibition by leucine and arginine, as well as by SLC6A14/ATB(0,+) silencing. The presence of this transporter leads to a massive accumulation of carnitine inside the cells and may be of peculiar relevance in pathologic conditions of carnitine deficiency, such as those associated to OCTN2 defects. Copyright © 2015 Elsevier B.V. All rights reserved.

SOLID PHASE MICROEXTRACTION SAMPLING OF HIGH EXPLOSIVE RESIDUES IN THE PRESENCE OF RADIONUCLIDES AND RADIONUCLIDE SURROGATE METALS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duff, M; S Crump, S; Robert02 Ray, R

2007-04-13

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

Transport mechanism for lovastatin acid in bovine kidney NBL-1 cells: kinetic evidences imply involvement of monocarboxylate transporter 4.

PubMed

Nagasawa, Kazuki; Nagai, Katsuhito; Ishimoto, Atsushi; Fujimoto, Sadaki

2003-08-27

We previously indicated that lovastatin acid, a 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, was transported by a monocarboxylate transporter (MCT) in cultured rat mesangial cells. In this study, to identify the MCT isoform(s) responsible for the lovastatin acid uptake, the transport mechanism was investigated using bovine kidney NBL-1 cells, which have been reported to express only MCT4 at the protein level. On RT-PCR analysis, the message of mRNAs for MCT1 and MCT4 was detected in the NBL-1 cells used in this study, which was confirmed by kinetic analysis of [14C]L-lactic acid uptake, consisting of high- and low-affinity components corresponding to MCT1 and MCT4, respectively. The lovastatin acid uptake depended on an inwardly directed H+-gradient, and was inhibited by representative monocarboxylates, but not by inhibitors/substrates for organic anion transporting polypeptides and organic anion transporters. In addition, L-lactic acid competitively inhibited the uptake of lovastatin acid and lovastatin acid inhibited the low affinity component of [14C]L-lactic acid uptake dose dependently. The inhibition constant of L-lactic acid for lovastatin acid uptake was almost the same as the Michaelis constant for [14C]L-lactic acid uptake by the low-affinity component. These kinetic evidences imply that lovastatin acid was taken up into NBL-1 cells via MCT4.

Quantitating Antibody Uptake In Vivo: Conditional Dependence on Antigen Expression Levels

PubMed Central

Thurber, Greg M.; Weissleder, Ralph

2010-01-01

Purpose Antibodies form an important class of cancer therapeutics, and there is intense interest in using them for imaging applications in diagnosis and monitoring of cancer treatment. Despite the expanding body of knowledge describing pharmacokinetic and pharmacodynamic interactions of antibodies in vivo, discrepancies remain over the effect of antigen expression level on tumoral uptake with some reports indicating a relationship between uptake and expression and others showing no correlation. Procedures Using a cell line with high EpCAM expression and moderate EGFR expression, fluorescent antibodies with similar plasma clearance were imaged in vivo. A mathematical model and mouse xenograft experiments were used to describe the effect of antigen expression on uptake of these high affinity antibodies. Results As predicted by the theoretical model, under subsaturating conditions, uptake of the antibodies in such tumors is similar because localization of both probes is limited by delivery from the vasculature. In a separate experiment, when the tumor is saturated, the uptake becomes dependent on the number of available binding sites. In addition, targeting of small micrometastases is shown to be higher than larger vascularized tumors. Conclusions These results are consistent with the prediction that high affinity antibody uptake is dependent on antigen expression levels for saturating doses and delivery for subsaturating doses. It is imperative for any probe to understand whether quantitative uptake is a measure of biomarker expression or transport to the region of interest. The data provide support for a predictive theoretical model of antibody uptake, enabling it to be used as a starting point for the design of more efficacious therapies and timely quantitative imaging probes. PMID:20809210

Quantitating antibody uptake in vivo: conditional dependence on antigen expression levels.

PubMed

Thurber, Greg M; Weissleder, Ralph

2011-08-01

Antibodies form an important class of cancer therapeutics, and there is intense interest in using them for imaging applications in diagnosis and monitoring of cancer treatment. Despite the expanding body of knowledge describing pharmacokinetic and pharmacodynamic interactions of antibodies in vivo, discrepancies remain over the effect of antigen expression level on tumoral uptake with some reports indicating a relationship between uptake and expression and others showing no correlation. Using a cell line with high epithelial cell adhesion molecule expression and moderate epidermal growth factor receptor expression, fluorescent antibodies with similar plasma clearance were imaged in vivo. A mathematical model and mouse xenograft experiments were used to describe the effect of antigen expression on uptake of these high-affinity antibodies. As predicted by the theoretical model, under subsaturating conditions, uptake of the antibodies in such tumors is similar because localization of both probes is limited by delivery from the vasculature. In a separate experiment, when the tumor is saturated, the uptake becomes dependent on the number of available binding sites. In addition, targeting of small micrometastases is shown to be higher than larger vascularized tumors. These results are consistent with the prediction that high affinity antibody uptake is dependent on antigen expression levels for saturating doses and delivery for subsaturating doses. It is imperative for any probe to understand whether quantitative uptake is a measure of biomarker expression or transport to the region of interest. The data provide support for a predictive theoretical model of antibody uptake, enabling it to be used as a starting point for the design of more efficacious therapies and timely quantitative imaging probes.

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

PubMed

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

2016-07-01

Dietary stilbenoids are associated with many benefits for human health, which depend on their bioavailability and bioaccessibility. The stilbenoid-human serum albumin (HSA) interactions are investigated to explore the structure-affinity relationship and influence on the stability, free radical scavenging activity and cell uptake of stilbenoids. The structure-affinity relationship of the stilbenoids-HSA interaction was found as: (1) the methoxylation enhanced the affinity, (2) an additional hydroxyl group increases the affinity and (3) the glycosylation significantly weakened the affinity. HSA obviously masked the free radical scavenging potential of stilbenoids. The stabilities of stilbenoids in different medium were determined as: HSA solution>human plasma>Dulbecco's modified Eagle's medium. It appears that the milk enhanced the cell uptake of stilbenoids with multi-hydroxyl groups and weakened the cell uptake of stilbenoids with methoxyl group on EA.hy 926 endothelial cells. The stilbenoids are hardly absorbed by human umbilical vein endothelial cells in the presence of milk. Copyright © 2016 Elsevier Ltd. All rights reserved.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei.

PubMed

Mathieu, Christoph; Macêdo, Juan P; Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

2017-01-01

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.

Arginine and Lysine Transporters Are Essential for Trypanosoma brucei

PubMed Central

Hürlimann, Daniel; Wirdnam, Corina; Haindrich, Alexander C.; Suter Grotemeyer, Marianne; González-Salgado, Amaia; Schmidt, Remo S.; Inbar, Ehud; Mäser, Pascal; Bütikofer, Peter; Zilberstein, Dan; Rentsch, Doris

2017-01-01

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei. PMID:28045943

Uptake Kinetics of Arsenic Species in Rice Plants

PubMed Central

Abedin, Mohammed Joinal; Feldmann, Jörg; Meharg, Andy A.

2002-01-01

Arsenic (As) finds its way into soils used for rice (Oryza sativa) cultivation through polluted irrigation water, and through historic contamination with As-based pesticides. As is known to be present as a number of chemical species in such soils, so we wished to investigate how these species were accumulated by rice. As species found in soil solution from a greenhouse experiment where rice was irrigated with arsenate contaminated water were arsenite, arsenate, dimethylarsinic acid, and monomethylarsonic acid. The short-term uptake kinetics for these four As species were determined in 7-d-old excised rice roots. High-affinity uptake (0–0.0532 mm) for arsenite and arsenate with eight rice varieties, covering two growing seasons, rice var. Boro (dry season) and rice var. Aman (wet season), showed that uptake of both arsenite and arsenate by Boro varieties was less than that of Aman varieties. Arsenite uptake was active, and was taken up at approximately the same rate as arsenate. Greater uptake of arsenite, compared with arsenate, was found at higher substrate concentration (low-affinity uptake system). Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly suppressed in the presence of phosphate, whereas arsenite transport was not affected by phosphate. At a slow rate, there was a hyperbolic uptake of monomethylarsonic acid, and limited uptake of dimethylarsinic acid. PMID:11891266

Transporters, channels, or simple diffusion? Dogmas, atypical roles and complexity in transport systems.

PubMed

Conde, Artur; Diallinas, George; Chaumont, François; Chaves, Manuela; Gerós, Hernâni

2010-06-01

The recent breakthrough discoveries of transport systems assigned with atypical functions provide evidence for complexity in membrane transport biochemistry. Some channels are far from being simple pores creating hydrophilic passages for solutes and can, unexpectedly, act as enzymes, or mediate high-affinity uptake, and some transporters are surprisingly able to function as sensors, channels or even enzymes. Furthermore, numerous transport studies have demonstrated complex multiphasic uptake kinetics for organic and mineral nutrients. The biphasic kinetics of glucose uptake in Saccharomyces cerevisiae, a result of several genetically distinct uptake systems operating simultaneously, is a classical example that is a subject of continuous debate. In contrast, some transporters display biphasic kinetics, being bona fidae dual-affinity transporters, their kinetic properties often modulated by post-translational regulation. Also, aquaporins have recently been reported to exhibit diverse transport properties and can behave as highly adapted, multifunctional channels, transporting solutes such as CO(2), hydrogen peroxide, urea, ammonia, glycerol, polyols, carbamides, purines and pyrimidines, metalloids, glycine, and lactic acid, rather than being simple water pores. The present review provides an overview on some atypical functions displayed by transporter proteins and discusses how this novel knowledge on cellular uptake systems may be related to complex multiphasic uptake kinetics often seen in a wide variety of living organisms and the intriguing diffusive uptake of sugars and other solutes. Copyright 2009 Elsevier Ltd. All rights reserved.

Third system for neutral amino acid transport in a marine pseudomonad.

PubMed Central

Pearce, S M; Hildebrandt, V A; Lee, T

1977-01-01

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system. PMID:856786

A nitrogen-dependent switch in the high affinity ammonium transport in Medicago truncatula.

PubMed

Straub, Daniel; Ludewig, Uwe; Neuhäuser, Benjamin

2014-11-01

Ammonium transporters (AMTs) are crucial for the high affinity primary uptake and translocation of ammonium in plants. In the model legume Medicago truncatula, the genomic set of AMT-type ammonium transporters comprises eight members. Only four genes were abundantly expressed in young seedlings, both in roots and shoots. While the expression of all AMTs in the shoot was not affected by the nitrogen availability, the dominating MtAMT1;1 gene was repressed by nitrogen in roots, despite that cellular nitrogen concentrations were far above deficiency levels. A contrasting de-repression by nitrogen was observed for MtAMT1;4 and MtAMT2;1, which were both expressed at intermediate level. Weak expression was found for MtAMT1;2 and MtAMT2;3, while the other AMTs were not detected in young seedlings. When expressed from their endogenous promoters, translational fusion proteins of MtAMT1;1 and MtAMT2;1 with green fluorescent protein were co-localized in the plasma membrane of rhizodermal cells, but also detected in cortical root layers. Both transporter proteins similarly functionally complemented a yeast strain that is deficient in high affinity ammonium transport, both at acidic and neutral pH. The uptake into yeast mediated by these transporters saturated with Km AMT1;1 = 89 µM and Km AMT2;1 = 123 µM, respectively. When expressed in oocytes, MtAMT1;1 mediated much larger (15)N-ammonium uptake than MtAMT2;1, but NH4 (+) currents were only recorded for MtAMT1;1. These currents saturated with a voltage-dependent Km = 90 µM at -80 mV. The cellular localization and regulation of the AMTs suggests that MtAMT1;1 encodes the major high affinity ammonium transporter gene in low nitrogen grown young M. truncatula roots and despite the similar localization and substrate affinity, MtAMT2;1 appears functionally distinct and more important at higher nitrogen supply.

Iron restriction and the growth of Salmonella enteritidis.

PubMed Central

Chart, H.; Rowe, B.

1993-01-01

Strains of Salmonella enteritidis were examined for their ability to remove ferric-ions from the iron chelating agents ovotransferrin, Desferal and EDDA. Growth of S. enteritidis phage type (PT) 4 (SE4) in trypticase soy broth containing ovotransferrin resulted in the expression of iron regulated outer membrane proteins (OMPs) of 74, 78 and 81 kDa, and unexpectedly the repression of expression of OMP C. The 38 MDa 'mouse virulence' plasmid was not required for the expression of the iron-regulated OMPs (IROMPs). SE4 was able to obtain iron bound to the iron chelator Desferal and EDDA without expressing a high-affinity iron uptake system. Strains of S. enteritidis belonging to PTs 7, 8, 13a, 23, 24 and 30 were also able to remove ferric ions from Desferal and EDDA without expressing a high-affinity iron uptake system. We conclude that strains of SE4 possess a high-affinity iron sequestering mechanism that can readily remove iron from ovotransferrin. It is likely that iron limitation, and not iron restriction, is responsible for the bacteriostatic properties of fresh egg whites. Images Fig. 2 PMID:8432322

Interaction between phloretin and the red blood cell membrane

PubMed Central

1976-01-01

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. PMID:5575

Characterization of loxoprofen transport in Caco-2 cells: the involvement of a proton-dependent transport system in the intestinal transport of loxoprofen.

PubMed

Narumi, Katsuya; Kobayashi, Masaki; Kondo, Ayuko; Furugen, Ayako; Yamada, Takehiro; Takahashi, Natsuko; Iseki, Ken

2016-11-01

Loxoprofen, a propionate non-steroidal anti-inflammatory drug (NSAID), is used widely in East Asian countries. However, little is known about the transport mechanisms contributing to its intestinal absorption. The objectives of this study were to characterize the intestinal transport of loxoprofen using the human intestinal Caco-2 cell model. The transport of loxoprofen was investigated in cellular uptake studies. The uptake of loxoprofen into Caco-2 cells was pH- and concentration-dependent, and was described by a Michaelis-Menten equation with passive diffusion (K m : 4.8 mm, V max : 142 nmol/mg protein/30 s, and K d : 2.2 μl/mg protein/30 s). Moreover, the uptake of loxoprofen was inhibited by a typical monocarboxylate transporter (MCT) inhibitor as well as by various monocarboxylates. The uptake of [ 14 C] l-lactic acid, a typical MCT substrate, in Caco-2 cells was saturable with relatively high affinity for MCT. Because loxoprofen inhibited the uptake of [ 14 C] l-lactic acid in a noncompetitive manner, it was unlikely that loxoprofen uptake was mediated by high-affinity MCT(s). Our results suggest that transport of loxoprofen in Caco-2 cells is, at least in part, mediated by a proton-dependent transport system. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

AtCHX13 is a plasma membrane K+ transporter.

PubMed

Zhao, Jian; Cheng, Ning-Hui; Motes, Christy M; Blancaflor, Elison B; Moore, Miranda; Gonzales, Naomi; Padmanaban, Senthilkumar; Sze, Heven; Ward, John M; Hirschi, Kendal D

2008-10-01

Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (CHX), which may mediate K+ transport; however, the vast majority of these transporters remain uncharacterized. Here, we show that AtCHX13 (At2g30240) has a role in K+ acquisition. AtCHX13 suppressed the sensitivity of yeast (Saccharomyces cerevisiae) mutant cells defective in K+ uptake. Uptake experiments using (86)Rb+ as a tracer for K+ demonstrated that AtCHX13 mediated high-affinity K+ uptake in yeast and in plant cells with a K(m) of 136 and 196 microm, respectively. Functional green fluorescent protein-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K+ deficiency conditions, whereas overexpression of AtCHX13 reduced the sensitivity to K+ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K+ uptake, although the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K+-deficient conditions. These results indicate that one role of AtCHX13 is to promote K+ uptake into plants when K+ is limiting in the environment.

In vivo evaluation of radiotracers targeting the melanin-concentrating hormone receptor 1: [11C]SNAP-7941 and [18F]FE@SNAP reveal specific uptake in the ventricular system.

PubMed

Zeilinger, Markus; Dumanic, Monika; Pichler, Florian; Budinsky, Lubos; Wadsak, Wolfgang; Pallitsch, Katharina; Spreitzer, Helmut; Lanzenberger, Rupert; Hacker, Marcus; Mitterhauser, Markus; Philippe, Cécile

2017-08-14

The MCHR1 is involved in the regulation of energy homeostasis and changes of the expression are linked to a variety of associated diseases, such as diabetes and adiposity. The study aimed at the in vitro and in vivo evaluation of [ 11 C]SNAP-7941 and [ 18 F]FE@SNAP as potential PET-tracers for the MCHR1. Competitive binding studies with non-radioactive derivatives and small-animal PET/CT and MRI brain studies were performed under baseline conditions and tracer displacement with the unlabelled MCHR1 antagonist (±)-SNAP-7941. Binding studies evinced high binding affinity of the non-radioactive derivatives. Small-animal imaging of [ 11 C]SNAP-7941 and [ 18 F]FE@SNAP evinced high tracer uptake in MCHR1-rich regions of the ventricular system. Quantitative analysis depicted a significant tracer reduction after displacement with (±)-SNAP-7941. Due to the high binding affinity of the non-labelled derivatives and the high specific tracer uptake of [ 11 C]SNAP-7941 and [ 18 F]FE@SNAP, there is strong evidence that both radiotracers may serve as highly suitable agents for specific MCHR1 imaging.

Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Amato, R.J.; Largent, B.L.; Snowman, A.M.

1987-07-01

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine bindingmore » reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.« less

Evaluation of 111In-labeled EPep and FibPep as tracers for fibrin SPECT imaging.

PubMed

Starmans, Lucas W E; van Duijnhoven, Sander M J; Rossin, Raffaella; Berben, Monique; Aime, Silvio; Daemen, Mat J A P; Nicolay, Klaas; Grüll, Holger

2013-11-04

Fibrin targeting is an attractive strategy for nuclear imaging of thrombosis, atherosclerosis and cancer. Recently, FibPep, an (111)In-labeled fibrin-binding peptide, was established as a tracer for fibrin SPECT imaging and was reported to allow sensitive detection of minute thrombi in mice using SPECT. In this study, we developed EPep, a novel (111)In-labeled fibrin-binding peptide containing the fibrin-binding domain of the clinically verified EP-2104R peptide, and sought to compare the potential of EPep and FibPep as tracers for fibrin SPECT imaging. In vitro, both EPep and FibPep showed high stability in serum, but were less stable in liver and kidney homogenate assays. Both peptide probes displayed comparable affinities toward human and mouse derived fibrin (Kd ≈ 1 μM), and similarly to FibPep, EPep showed fast blood clearance, low nontarget uptake and high thrombus uptake (6.8 ± 1.2% ID g(-1)) in a mouse carotid artery thrombosis model. Furthermore, EPep showed a similar affinity toward rat derived fibrin (Kd ≈ 1 μM), displayed high thrombus uptake in a rat carotid artery thrombosis model (0.74 ± 0.39% ID g(-1)), and allowed sensitive detection of thrombosis in rats using SPECT. In contrast, FibPep displayed a significantly lower affinity toward rat derived fibrin (Kd ≈ 14 μM) and low uptake in rat thrombi (0.06 ± 0.02% ID g(-1)) and did not allow clear visualization of carotid artery thrombosis in rats using SPECT. These results were confirmed ex vivo by autoradiography, which showed a 7-fold higher ratio of activity in the thrombus over the contralateral carotid artery for EPep in comparison to FibPep. These findings suggest that the FibPep binding fibrin epitope is not fully homologous between humans and rats, and that preclinical rat models of disease should not be employed to gauge the clinical potential of FibPep. In conclusion, both peptides showed approximately similar metabolic stability and affinity toward human and mouse derived fibrin, and displayed high thrombus uptake in a mouse carotid artery thrombosis model. Therefore, both EPep and FibPep are promising fibrin targeted tracers for translation into clinical settings to serve as novel tools for molecular imaging of fibrin.

The levels of boron-uptake proteins in roots are correlated with tolerance to boron stress in barley

USDA-ARS?s Scientific Manuscript database

Boron (B) is an essential micronutrient required for plant growth and development. Recently, two major B-uptake proteins, BOR1 and NIP5;1 have been identified and partially characterized. BOR1 is a high-affinity B transporter involved in xylem loading in roots, and NIP5;1 acts is a major boric-acid ...

Arsenic-phosphorus interactions in the soil-plant-microbe system: Dynamics of uptake, suppression and toxicity to plants.

PubMed

Anawar, Hossain M; Rengel, Zed; Damon, Paul; Tibbett, Mark

2018-02-01

High arsenic (As) concentrations in the soil, water and plant systems can pose a direct health risk to humans and ecosystems. Phosphate (Pi) ions strongly influence As availability in soil, its uptake and toxicity to plants. Better understanding of As(V)-Pi interactions in soils and plants will facilitate a potential remediation strategy for As contaminated soils, reducing As uptake by crop plants and toxicity to human populations via manipulation of soil Pi content. However, the As(V)-Pi interactions in soil-plant systems are complex, leading to contradictory findings among different studies. Therefore, this review investigates the role of soil type, soil properties, minerals, Pi levels in soil and plant, Pi transporters, mycorrhizal association and microbial activities on As-Pi interactions in soils and hydroponics, and uptake by plants, elucidate the key mechanisms, identify key knowledge gaps and recommend new research directions. Although Pi suppresses As uptake by plants in hydroponic systems, in soils it could either increase or decrease As availability and toxicity to plants depending on the soil types, properties and charge characteristics. In soil, As(V) availability is typically increased by the addition of Pi. At the root surface, the Pi transport system has high affinity for Pi over As(V). However, Pi concentration in plant influences the As transport from roots to shoots. Mycorrhizal association may reduce As uptake via a physiological shift to the mycorrhizal uptake pathway, which has a greater affinity for Pi over As(V) than the root epidermal uptake pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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

2015-12-01

Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta. © 2015 American Society of Plant Biologists. All Rights Reserved.

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

PubMed Central

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

Molecular and functional characterization of choline transporter in the human trophoblastic cell line JEG-3 cells.

PubMed

Yara, M; Iwao, B; Hara, N; Yamanaka, T; Uchino, H; Inazu, M

2015-06-01

Choline is essential for the synthesis of the major membrane phospholipid phosphatidylcholine (PC), the methyl donor betaine and the neurotransmitter acetylcholine (ACh), which is involved in several vital biological functions that play key roles in fetal development. In this study, we examined the molecular and functional characteristics of choline uptake in the human trophoblastic cell line JEG-3. We examined [(3)H]choline uptake in the human trophoblastic cell line JEG-3. The expression of CTL1 and CTL2 was evaluated by quantitative real-time PCR, western blotting and immunocytochemistry. We demonstrated that JEG-3 cells take up [(3)H] choline by a saturable process that is mediated by a Na(+)-independent and pH-dependent transport system. The cells have two different [(3)H] choline transport systems, high- and low-affinity, with Km values of 28.4 ± 5.0 μM and 210.6 ± 55.1 μM, respectively. Cationic compounds and hemicholinium-3 (HC-3) inhibited choline uptake. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in JEG-3 cells and were localized to the plasma membrane. The present results suggest that choline is mainly transported via a high-affinity choline transport system (CTL1) and a low-affinity choline transport system (CTL2) in human trophoblastic JEG-3 cells. These transporters play an important role in the growth of the fetus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity.

PubMed

Chanda, Nripen; Kattumuri, Vijaya; Shukla, Ravi; Zambre, Ajit; Katti, Kavita; Upendran, Anandhi; Kulkarni, Rajesh R; Kan, Para; Fent, Genevieve M; Casteel, Stan W; Smith, C Jeffrey; Boote, Evan; Robertson, J David; Cutler, Cathy; Lever, John R; Katti, Kattesh V; Kannan, Raghuraman

2010-05-11

Development of cancer receptor-specific gold nanoparticles will allow efficient targeting/optimum retention of engineered gold nanoparticles within tumors and thus provide synergistic advantages in oncology as it relates to molecular imaging and therapy. Bombesin (BBN) peptides have demonstrated high affinity toward gastrin-releasing peptide (GRP) receptors in vivo that are overexpressed in prostate, breast, and small-cell lung carcinoma. We have synthesized a library of GRP receptor-avid nanoplatforms by conjugating gold nanoparticles (AuNPs) with BBN peptides. Cellular interactions and binding affinities (IC(50)) of AuNP-BBN conjugates toward GRP receptors on human prostate cancer cells have been investigated in detail. In vivo studies using AuNP-BBN and its radiolabeled surrogate (198)AuNP-BBN, exhibiting high binding affinity (IC(50) in microgram ranges), provide unequivocal evidence that AuNP-BBN constructs are GRP-receptor-specific showing accumulation with high selectivity in GRP-receptor-rich pancreatic acne in normal mice and also in tumors in prostate-tumor-bearing, severe combined immunodeficient mice. The i.p. mode of delivery has been found to be efficient as AuNP-BBN conjugates showed reduced RES organ uptake with concomitant increase in uptake at tumor targets. The selective uptake of this new generation of GRP-receptor-specific AuNP-BBN peptide analogs has demonstrated realistic clinical potential in molecular imaging via x-ray computed tomography techniques as the contrast numbers in prostate tumor sites are severalfold higher as compared to the pretreatment group (Hounsfield unit = 150).

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

PubMed Central

2012-01-01

Background The expression and activity of the different Saccharomyces cerevisiae hexose uptake systems (Hxt) and the kinetics of glucose uptake are considered essential to industrial alcoholic fermentation performance. However, the dynamics of glucose uptake kinetics during the different stages of fermentation, depending on glucose and nitrogen availability, is very poorly characterized. The objective of the present work was to examine thoroughly the alterations occurring in glucose uptake kinetics during alcoholic fermentation, by the wine strain S. cerevisiae PYCC 4072, of a synthetic grape juice basal medium with either a limiting or non-limiting initial nitrogen concentration and following nitrogen supplementation of the nitrogen-depleted sluggish fermentation. Results Independently of the initial concentration of the nitrogen source, glucose transport capacity is maximal during the early stages of fermentation and presumably sustained by the low-affinity and high-capacity glucose transporter Hxt1p. During nitrogen-limited sluggish fermentation, glucose uptake capacity was reduced to approximately 20% of its initial values (Vmax = 4.9 ± 0.8 compared to 21.9 ± 1.2 μmol h-1 10-8 cells), being presumably sustained by the low-affinity glucose transporter Hxt3p (considering the calculated Km = 39.2 ± 8.6 mM). The supplementation of the sluggish fermentation broth with ammonium led to the increase of glucose transport capacity associated to the expression of different glucose uptake systems with low and high affinities for glucose (Km = 58.2 ± 9.1 and 2.7 ± 0.4 mM). A biclustering analysis carried out using microarray data, previously obtained for this yeast strain transcriptional response to equivalent fermentation conditions, indicates that the activation of the expression of genes encoding the glucose transporters Hxt2p (during the transition period to active fermentation) and Hxt3p, Hxt4p, Hxt6p and Hxt7p (during the period of active fermentation) may have a major role in the recovery of glucose uptake rate following ammonium supplementation. These results suggest a general derepression of the glucose-repressible HXT genes and are consistent with the downregulation of Mig1p and Rgt1p. Conclusions Although reduced, glucose uptake rate during nitrogen-limited fermentation is not abrogated. Following ammonium supplementation, sluggish fermentation recovery is associated to the increase of glucose uptake capacity, related to the de novo synthesis of glucose transporters with different affinity for glucose and capacity, presumably of Hxt2p, Hxt3p, Hxt4p, Hxt6p and Hxt7p. This study is a contribution to the understanding of yeast response to different stages of alcoholic fermentation at the level of glucose uptake kinetics, in particular under nitrogen limitation or replenish, which is useful knowledge to guide fermentation practices. PMID:22846176

[Relation between location of elements in periodic table and affinity for the malignant tumor (author's transl)].

PubMed

Ando, A; Hisada, K; Ando, I

1977-10-01

Affinity of many inorganic compounds for the malignant tumor was examined, using the rats which were subcutaneously transplanted with Yoshida sarcoma. And the relations between the uptake rate into the malignant tumor and in vitro binding power to the protein were investigated in these compounds. In these experiments, the bipositive ions and anions had not affinity for the tumor tissue with a few exceptions. On the other hand, Hg, Au and Bi, which have strong binding power to the protein, showed high uptake rate into the malignant tumor. As Hg++, Au+ and Bi+++ are soft acids according to classification of Lewis acids, it was thought that these elements would bind strongly to soft base (R-SH, R-S-) present in the tumor tissue. In many hard acids (according to classification of Lewis acids), the uptake rate into the tumor was shown as a function of ionic potentials (valency/ionic radii) of the metal ions. It is presumed that the chemical bond of these hard acids in the tumor tissue is ionic bond to hard base (R-COO-, R-PO3(2-), R-SO3-, R-NH2).

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

1991-01-01

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

Expression and substrate specificity of betaine/proline transporters suggest a novel choline transport mechanism in sugar beet.

PubMed

Yamada, Nana; Sakakibara, Shota; Tsutsumi, Koichi; Waditee, Rungaroon; Tanaka, Yoshito; Takabe, Teruhiro

2011-09-15

Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells. Copyright © 2011 Elsevier GmbH. All rights reserved.

Improved xylose uptake in Saccharomyces cerevisiae due to directed evolution of galactose permease Gal2 for sugar co-consumption.

PubMed

Reznicek, O; Facey, S J; de Waal, P P; Teunissen, A W R H; de Bont, J A M; Nijland, J G; Driessen, A J M; Hauer, B

2015-07-01

Saccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed. Three rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption. Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass. © 2015 The Society for Applied Microbiology.

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

PubMed Central

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

2012-01-01

Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction and in parasympathetic nerve terminals in the periphery, as well as important memory-related circuits in the brain and also takes part in several critical functions. ACh is synthesized from choline and acetyl coenzyme-A by the enzyme choline acetyltransferase (ChAT). The formation of acetylcholine in cholinergic nerve terminals requires both the transport of choline into the cells from the extracellular space, and the activity of ChAT. High affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal, and is the acutely regulated, rate-limiting step in ACh synthesis. The HACU component of choline uptake can be differentiated from non-specific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure to simultaneously measure HACU and ChAT in cultured cells by simple radionuclide-based techniques. In this procedure we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders including Alzheimer’s disease, myasthenia gravis, and cardiovascular disease. PMID:22752895

AtCHX13 Is a Plasma Membrane K+ Transporter1[C][W][OA

PubMed Central

Zhao, Jian; Cheng, Ning-Hui; Motes, Christy M.; Blancaflor, Elison B.; Moore, Miranda; Gonzales, Naomi; Padmanaban, Senthilkumar; Sze, Heven; Ward, John M.; Hirschi, Kendal D.

2008-01-01

Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (CHX), which may mediate K+ transport; however, the vast majority of these transporters remain uncharacterized. Here, we show that AtCHX13 (At2g30240) has a role in K+ acquisition. AtCHX13 suppressed the sensitivity of yeast (Saccharomyces cerevisiae) mutant cells defective in K+ uptake. Uptake experiments using 86Rb+ as a tracer for K+ demonstrated that AtCHX13 mediated high-affinity K+ uptake in yeast and in plant cells with a Km of 136 and 196 μm, respectively. Functional green fluorescent protein-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K+ deficiency conditions, whereas overexpression of AtCHX13 reduced the sensitivity to K+ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K+ uptake, although the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K+-deficient conditions. These results indicate that one role of AtCHX13 is to promote K+ uptake into plants when K+ is limiting in the environment. PMID:18676662

Engineered Knottin Peptides: A New Class of Agents for Imaging Integrin Expression in Living Subjects

PubMed Central

Kimura, Richard H; Cheng, Zhen; Gambhir, Sanjiv Sam; Cochran, Jennifer R

2009-01-01

There is a critical need for molecular imaging agents to detect cell surface integrin receptors that are present in human cancers. Previously, we used directed evolution to engineer knottin peptides that bind with low nM affinity to integrin receptors that are overexpressed on the surface of tumor cells and the tumor neovasculature. To evaluate these peptides as molecular imaging agents, we site-specifically conjugated Cy5.5 or 64Cu-DOTA to their N-termini, and used optical and positron emission tomography (PET) imaging to measure their uptake and biodistribution in U87MG glioblastoma murine xenograft models. Near-infrared fluorescence and microPET imaging both demonstrated that integrin binding affinity plays a strong role in the tumor uptake of knottin peptides. Tumor uptake at 1 h post injection for two high affinity (IC50 ∼20 nM) 64Cu-DOTA-conjugated knottin peptides was 4.47 ± 1.21 and 4.56 ± 0.64 % injected dose/gram (%ID/g), compared to a low affinity knottin peptide (IC50 ∼0.4 μM; 1.48 ± 0.53 %ID/g) and c(RGDyK) (IC50 ∼1 μM; 2.32 ± 0.55 %ID/g), a low affinity cyclic pentapeptide under clinical development. Furthermore, 64Cu-DOTA-conjugated knottin peptides generated lower levels of non-specific liver uptake (∼2 %ID/g) compared to c(RGDyK) (∼4 %ID/g) 1 h post injection. MicroPET imaging results were confirmed by in vivo biodistribution studies. 64Cu-DOTA-conjugated knottin peptides were stable in mouse serum, and in vivo metabolite analysis showed minimal degradation in the blood or tumor upon injection. Thus, engineered integrin-binding knottin peptides show great potential as clinical diagnostics for a variety of cancers. PMID:19276378

Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

NASA Astrophysics Data System (ADS)

Taft, William C.; Delorenzo, Robert J.

1984-05-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

Micromolar-affinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations.

PubMed Central

Taft, W C; DeLorenzo, R J

1984-01-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance. PMID:6328498

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

PubMed

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

2011-03-01

The physiological responses of the arsenic-hyperaccumulator, Pteris vittata, such as arsenic uptake and chemical transformation in the fern, have been investigated. However, a few questions remain regarding arsenic treatment in hydroponics. Incubation conditions such as aeration, arsenic concentration, and incubation period might affect those responses of P. vittata in hydroponics. Arsenite uptake was low under anaerobic conditions, as previously reported. However, in an arsenite uptake experiment, phosphorous (P) starvation-dependent uptake of arsenate was observed under aerobic conditions. Time course-dependent analysis of arsenite oxidation showed that arsenite was gradually oxidized to arsenate during incubation. Arsenite oxidation was not observed in any of the control conditions, such as exposure to a nutrient solution or to culture medium only, or with the use of dried root; arsenite oxidation was only observed when live root was used. This result suggests that sufficient aeration allows the rhizosphere system to oxidize arsenite and enables the fern to efficiently take up arsenite as arsenate. X-ray absorption near edge structure (XANES) analyses showed that long-duration exposure to arsenic using a hydroponic system led to the accumulation of arsenate as the dominant species in the root tips, but not in the whole roots, partly because up-regulation of arsenate uptake by P starvation of the fern was caused and retained by long-time incubation. Analysis of concentration-dependent arsenate uptake by P. vittata showed that the uptake switched from a high-affinity transport system to a low-affinity system at high arsenate concentrations, which partially explains the increased arsenate abundance in the whole root. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Uptake of dissolved inorganic and organic nitrogen by the benthic toxic dinoflagellate Ostreopsis cf. ovata.

PubMed

Jauzein, Cécile; Couet, Douglas; Blasco, Thierry; Lemée, Rodolphe

2017-05-01

Environmental factors that shape dynamics of benthic toxic blooms are largely unknown. In particular, for the toxic dinoflagellate Ostreopsis cf. ovata, the importance of the availability of nutrients and the contribution of the inorganic and organic pools to growth need to be quantified in marine coastal environments. The present study aimed at characterizing N-uptake of dissolved inorganic and organic sources by O. cf. ovata cells, using the 15 N-labelling technique. Experiments were conducted taking into account potential interactions between nutrient uptake systems as well as variations with the diel cycle. Uptake abilities of O. cf. ovata were parameterized for ammonium (NH 4 + ), nitrate (NO 3 - ) and N-urea, from the estimation of kinetic and inhibition parameters. In the range of 0 to 10μmolNL -1 , kinetic curves showed a clear preference pattern following the ranking NH 4 + >NO 3 - >N-urea, where the preferential uptake of NH 4 + relative to NO 3 - was accentuated by an inhibitory effect of NH 4 + concentration on NO 3 - uptake capabilities. Conversely, under high nutrient concentrations, the preference for NH 4 + relative to NO 3 - was largely reduced, probably because of the existence of a low-affinity high capacity inducible NO 3 - uptake system. Ability to take up nutrients in darkness could not be defined as a competitive advantage for O. cf. ovata. Species competitiveness can also be defined from nutrient uptake kinetic parameters. A strong affinity for NH 4 + was observed for O. cf. ovata cells that may partly explain the success of this toxic species during the summer season in the Bay of Villefranche-sur-mer (France). Copyright © 2017 Elsevier B.V. All rights reserved.

Temperature Dependence of Inorganic Nitrogen Uptake: Reduced Affinity for Nitrate at Suboptimal Temperatures in Both Algae and Bacteria

PubMed Central

Reay, David S.; Nedwell, David B.; Priddle, Julian; Ellis-Evans, J. Cynan

1999-01-01

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 ≈ 3, where Q10 is the proportional change with a 10°C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures. PMID:10347046

Inhibition of heme biosynthesis prevents transcription of iron uptake genes in yeast.

PubMed

Crisp, Robert J; Pollington, Annette; Galea, Charles; Jaron, Shulamit; Yamaguchi-Iwai, Yuko; Kaplan, Jerry

2003-11-14

Yeast are capable of modifying their metabolism in response to environmental changes. We investigated the activity of the oxygen-dependent high-affinity iron uptake system of Saccharomyces cerevisiae under conditions of heme depletion. We found that the absence of heme, due to a deletion in the gene that encodes delta-aminolevulinic acid synthase (HEM1), resulted in decreased transcription of genes belonging to both the iron and copper regulons, but not the zinc regulon. Decreased transcription of the iron regulon was not due to decreased expression of the iron sensitive transcriptional activator Aft1p. Expression of the constitutively active allele AFT1-1up was unable to induce transcription of the high affinity iron uptake system in heme-depleted cells. We demonstrated that under heme-depleted conditions, Aft1p-GFP was able to cycle normally between the nucleus and cytosol in response to cytosolic iron. Despite the inability to induce transcription under low iron conditions, chromatin immunoprecipitation demonstrated that Aft1p binds to the FET3 promoter in the absence of heme. Finally, we provide evidence that under heme-depleted conditions, yeast are able to regulate mitochondrial iron uptake and do not accumulate pathologic iron concentrations, as is seen when iron-sulfur cluster synthesis is disrupted.

Brain tumor initiating cells adapt to restricted nutrition through preferential glucose uptake.

PubMed

Flavahan, William A; Wu, Qiulian; Hitomi, Masahiro; Rahim, Nasiha; Kim, Youngmi; Sloan, Andrew E; Weil, Robert J; Nakano, Ichiro; Sarkaria, Jann N; Stringer, Brett W; Day, Bryan W; Li, Meizhang; Lathia, Justin D; Rich, Jeremy N; Hjelmeland, Anita B

2013-10-01

Like all cancers, brain tumors require a continuous source of energy and molecular resources for new cell production. In normal brain, glucose is an essential neuronal fuel, but the blood-brain barrier limits its delivery. We now report that nutrient restriction contributes to tumor progression by enriching for brain tumor initiating cells (BTICs) owing to preferential BTIC survival and to adaptation of non-BTICs through acquisition of BTIC features. BTICs outcompete for glucose uptake by co-opting the high affinity neuronal glucose transporter, type 3 (Glut3, SLC2A3). BTICs preferentially express Glut3, and targeting Glut3 inhibits BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in brain tumors and other cancers; thus, tumor initiating cells may extract nutrients with high affinity. As altered metabolism represents a cancer hallmark, metabolic reprogramming may maintain the tumor hierarchy and portend poor prognosis.

Brain Tumor Initiating Cells Adapt to Restricted Nutrition through Preferential Glucose Uptake

PubMed Central

Flavahan, William A.; Wu, Qiulian; Hitomi, Masahiro; Rahim, Nasiha; Kim, Youngmi; Sloan, Andrew E.; Weil, Robert J.; Nakano, Ichiro; Sarkaria, Jann N.; Stringer, Brett W.; Day, Bryan W.; Li, Meizhang; Lathia, Justin D.; Rich, Jeremy N.; Hjelmeland, Anita B.

2013-01-01

Like all cancers, brain tumors require a continuous source of energy and molecular resources for new cell production. In normal brain, glucose is an essential neuronal fuel, but the blood-brain barrier limits its delivery. We now report that nutrient restriction contributes to tumor progression by enriching for brain tumor initiating cells (BTICs) due to preferential BTIC survival and adaptation of non-BTICs through acquisition of BTIC features. BTICs outcompete for glucose uptake by co-opting the high affinity neuronal glucose transporter, type 3 (Glut3, SLC2A3). BTICs preferentially express Glut3 and targeting Glut3 inhibits BTIC growth and tumorigenic potential. Glut3, but not Glut1, correlates with poor survival in brain tumors and other cancers; thus, TICs may extract nutrients with high affinity. As altered metabolism represents a cancer hallmark, metabolic reprogramming may instruct the tumor hierarchy and portend poor prognosis. PMID:23995067

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

PubMed

García-Garayoa, E; Allemann-Tannahill, L; Bläuenstein, P; Willmann, M; Carrel-Rémy, N; Tourwé, D; Iterbeke, K; Conrath, P; Schubiger, P A

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 [99mTc] 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.

Impact of iron on silicon utilization by diatoms in the Southern Ocean: A case study of Si/N cycle decoupling in a naturally iron-enriched area

NASA Astrophysics Data System (ADS)

Mosseri, Julie; Quéguiner, Bernard; Armand, Leanne; Cornet-Barthaux, Véronique

2008-03-01

Biogenic silica stocks and fluxes were investigated in austral summer over the naturally iron-fertilized Kerguelen Plateau and in nearby high-nutrient, low-chlorophyll (HNLC) off-plateau surface waters. The Kerguelen Plateau hosted a large-diatom bloom, with high levels of biogenic silica (BSi) but relatively low silicic acid (Si(OH) 4) uptake rates (1100±600 mmol m -2 and 8±4 mmol m -2 d -1, respectively). Diatoms of the naturally iron-enriched area presented high affinities for silicic acid, allowing them in combination with a beneficial nutrient vertical supply to grow in low silicic acid waters (<2 μM). Si(OH) 4 acid uptake rates were also compared with carbon and nitrogen uptake rates. As expected for diatoms growing in favourable nutrient conditions, and from previous artificial iron-enrichment experiments, Si:C and Si:NO 3 elemental uptake ratios of the natural diatom community of the plateau were close to 0.13 and 1, respectively. In contrast, diatom communities in the HNLC waters were composed of strongly silicified (high Si:C, Si:NO 3 uptake ratios) diatoms with low affinities for Si(OH) 4. Although the Si:NO 3 uptake ratio in the surface waters of the plateau was close to 1, the apparent consumption of nitrate on a seasonal basis was much lower (˜5 μM) than the apparent consumption of silicic acid (˜15 μM). This was mainly due to diatoms growing actively on ammonium (i.e. 39-77% of the total nitrogen uptake) produced by an intense heterotrophic activity. Thus we find that while Fe fertilization does increase N uptake with respect to Si uptake, rapid recycling of N decouples nitrogen and carbon export from silica export so that the "silicate pump" remains more efficient than that of N (or P). For this reason an iron-fertilized Southern Ocean is unlikely to experience nitrate exhaustion or export silicic acid to the global ocean.

A versatile Escherichia coli strain for identification of biotin transporters and for biotin quantification

PubMed Central

Finkenwirth, Friedrich; Kirsch, Franziska; Eitinger, Thomas

2014-01-01

Biotin is an essential cofactor of carboxylase enzymes in all kingdoms of life. The vitamin is produced by many prokaryotes, certain fungi, and plants. Animals depend on biotin uptake from their diet and in humans lack of the vitamin is associated with serious disorders. Many aspects of biotin metabolism, uptake, and intracellular transport remain to be elucidated. In order to characterize the activity of novel biotin transporters by a sensitive assay, an Escherichia coli strain lacking both biotin synthesis and its endogenous high-affinity biotin importer was constructed. This strain requires artificially high biotin concentrations for growth. When only trace levels of biotin are available, it is viable only if equipped with a heterologous high-affinity biotin transporter. This feature was used to ascribe transport activity to members of the BioY protein family in previous work. Here we show that this strain together with its parent is also useful as a diagnostic tool for wide-concentration-range bioassays. PMID:24256712

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

The tau positron-emission tomography tracer AV-1451 binds with similar affinities to tau fibrils and monoamine oxidases.

PubMed

Vermeiren, Céline; Motte, Philippe; Viot, Delphine; Mairet-Coello, Georges; Courade, Jean-Philippe; Citron, Martin; Mercier, Joël; Hannestad, Jonas; Gillard, Michel

2018-02-01

Lilly/Avid's AV-1451 is one of the most advanced tau PET tracers in the clinic. Although results obtained in Alzheimer's disease patients are compelling, discrimination of tracer uptake in healthy individuals and patients with supranuclear palsy (PSP) is less clear as there is substantial overlap of signal in multiple brain regions. Moreover, accurate quantification of [ 18 F]AV-1451 uptake in Alzheimer's disease may not be possible. The aim of the present study was to characterize the in vitro binding of AV-1451 to understand and identify potential off-target binding that could explain the poor discrimination observed in PSP patients. [ 3 H]AV-1451 and AV-1451 were characterized in in vitro binding assays using recombinant and native proteins/tissues from postmortem samples of controls and Alzheimer's disease and PSP patients. [ 3 H]AV-1451 binds to multiple sites with nanomolar affinities in brain homogenates and to tau fibrils isolated from Alzheimer's disease or PSP patients. [ 3 H]AV-1451 also binds with similarly high affinities in brain homogenates devoid of tau pathology. This unexpected binding was demonstrated to be because of nanomolar affinities of [ 3 H]AV-1451 for monoamine oxidase A and B enzymes. High affinity of AV-1451 for monoamine oxidase proteins may limit its utility as a tau PET tracer in PSP and Alzheimer's disease because of high levels of monoamine oxidase expression in brain regions also affected by tau deposition, especially if monoamine oxidase levels change over time or with a treatment intervention. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3.

PubMed

Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M

2016-01-01

The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([ 11 C]MA2) and a fluorine-18 ([ 18 F]MA3) labeled analog of a highly potent N -arylamide oxadiazole CB2 agonist (EC 50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC 50 : 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for h CB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC 50 values when compared to the originally reported trifluoromethyl analog 12 . [ 11 C]MA2 and [ 18 F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [ 11 C]MA2 and [ 18 F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted.

Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3

PubMed Central

Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M.

2016-01-01

The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2) and a fluorine-18 ([18F]MA3) labeled analog of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analog 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted. PMID:27713686

Exploring the microbially-mediated soil H2 sink: A lab-based study of the physiology and related H2 consumption of isolates from the Harvard Forest

NASA Astrophysics Data System (ADS)

Rao, D.; Meredith, L. K.; Bosak, T.; Hansel, C. M.; Ono, S.; Prinn, R. G.

2012-12-01

Atmospheric hydrogen (H2) is a secondary greenhouse gas because it attenuates the removal of methane (CH4) from the atmosphere. The largest and most uncertain term in the H2 biogeochemical cycle, microbe-mediated soil uptake, is responsible for about 80% of Earth's tropospheric H2 sink. Recently, the first H2-oxidizing soil microorganisms were discovered (genus Streptomyces) whose low-threshold, high-affinity NiFe-hydrogenase functions at ambient H2 levels (approx. 530 ppb). To better understand the ecological function of this hydrogenase, we conducted a controlled laboratory study of the H2 uptake behavior in accordance with the complex life cycle development of the streptomycetes. Several strains of the genus Streptomyces containing a high-affinity NiFe- hydrogenase were isolated from soil at the Harvard Forest. The presence of this hydrogenase, detected by PCR amplification of the hydrogenase large subunit, predicted H2 uptake behavior in wild-type streptomycetes and in phylogenetically different organisms containing more distantly related versions of the gene. H2 uptake depended on the streptomyces' life cycle, reaching a maximum during spore formation. These findings reveal connections between environmental conditions, organismal life cycle, and H2 uptake. With the rise of H2-based energy sources and a potential change in the tropospheric concentration of H2, understanding the sources and sinks of this trace gas is important for the future.

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

PubMed

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

2016-01-15

Biochar is becoming an environmentally friendly material for remediation of heavy metal contaminated soils and improving food safety. A field trial over four rice seasons was conducted to investigate the use of biochar and low Cd accumulating cultivars on Cd uptake in a heavy metal contaminated soil. Wheat straw derived biochar was applied at 0, 20 and 40 t ha(-1). Two rice cultivars with differing Cd accumulation abilities were selected in each season. The results showed that both biochar and low Cd affinity cultivars significantly reduced rice grain Cd accumulation. Biochar had no significant effect the first season but thereafter consistently reduced rice grain Cd by a maximum of 61, 86 and 57% over the next three seasons. Zn accumulation in the rice grains was not decreased by biochar application, although available soil Zn was sharply reduced (35-91%). Indica conventional rice cultivars had much lower Cd, but higher Zn and lower Cd/Zn ratios in the grain than indica hybrid cultivars. Biochar was more effective for mitigating grain Cd accumulation in low Cd affinity cultivars than in high affinity cultivars. Soil pH was sustainably increased (up to nearly 1 unit) while available Cd significantly decreased by a maximum of 85% after biochar addition. The translocation of Cd from rice roots to shoots was reduced from 20 to 80% by biochar. Low uptake affinity cultivars combined with biochar reduced late rice grain Cd concentration and Cd/Zn ratios by 69-80% and 72-80%, respectively. It indicated that the management of combining biochar and low Cd affinity cultivars should be an efficient way to remediate Cd contaminated rice paddies and reduce health risk associated with consuming rice from these soils. Copyright © 2015 Elsevier B.V. All rights reserved.

Kinetics of N-Utilization By Natural Phytoplankton Assemblages During Upwelling Events At The NW Iberian Shelf

NASA Astrophysics Data System (ADS)

Brion, N.; Elskens, M.; Dehairs, F.; Baeyens, W.

2003-04-01

The concentration-dependent uptakes of nitrate, ammonium and the effect of ammo-nium on the f-ratio were surveyed in surface waters of the NW Iberian shelf during June 1997, July 1998 and September 1999. Because relationships between rates and substrate concentrations were quite variable, ranging from linear to convex shaped curves, they were fitted to rational functions. Stepwize regression analysis yielded subsequent model equations with reasonable statistical properties which allowed describing all but all a few cases. Differentiating these equations with respect to the concentration gave the slope of the tangent to the curve, i.e., the variation in rate expected for a given perturbation of the ambient substrate concentration. The initial slope value was then used as an index to gauge the "affinity" of the plankton community for the nitrogen substrate utilization. In June 1997, the situation at the Iberian shelf showed no upwelling except near Cape Finistère. Overall, the phytoplankton community displayed a high "affinity" for both nitrate and ammonium and low f-ratio values, which is indicative of a re-generated production regime. High ammonium regeneration rates supported furthermore these observations. It was also demonstrated that the new production rates is only marginally sensitive to changes of the ambient nitrate and/or ammonium concentrations. This indicates that the production regime is rather stable throughout. Only at Cape Finistère, nitrate concentrations were high reflecting the onset of an upwelling event. In this zone, the phytoplankton community, taking advantage of its high affinity for nitrate enhanced both total N-uptake rate and f-ratio. In July 1998, the situation evolved towards an extension to the south of the upwelling event starting at Cape Finistère. In this southern zone of the upwelling the phytoplankton community displayed generally a lower affinity for nitrate (but not for ammonium) than in 1997. In spite of this lower affinity, nitrate uptake rate was dominant resulting in f-ratio values greater than 0.5, a characteristic of a new production regime. The new production rate is only marginally sensitive to increases of the ambient nitrate, but is drastically inhibited by small increases of the ambient ammonium. The situation of September 1999 was very close to that observed in July 1998, with higher nitrate concentrations in the coastal northern part of the sampling area dominated by upwelling.

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

PubMed

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

2014-05-15

Potassium (K(+)) is an essential macronutrient for plants. It is taken into the plant by the transport systems present in the plasma membranes of root epidermal and cortical cells. The identity of these systems and their regulation is beginning to be understood and the systems of K(+) transport in the model species Arabidopsis thaliana remain far better characterized than in any other plant species. Roots can activate different K(+) uptake systems to adapt to their environment, important to a sessile organism that needs to cope with a highly variable environment. The mechanisms of K(+) acquisition in the model species A. thaliana are the best characterized at the molecular level so far. According to the current model, non-selective channels are probably the main pathways for K(+) uptake at high concentrations (>10mM), while at intermediate concentrations (1mM), the inward rectifying channel AKT1 dominates K(+) uptake. Under lower concentrations of external K(+) (100μM), AKT1 channels, together with the high-affinity K(+) uptake system HAK5 contribute to K(+) acquisition, and at extremely low concentrations (<10μM) the only system capable of taking up K(+) is HAK5. Depending on the species the high-affinity system has been named HAK5 or HAK1, but in all cases it fulfills the same functions. The activation of these systems as a function of the K(+) availability is achieved by different mechanisms that include phosphorylation of AKT1 or induction of HAK5 transcription. Some of the characteristics of the systems for root K(+) uptake are shared by other organisms, whilst others are specific to plants. This indicates that some crucial properties of the ancestral of K(+) transport systems have been conserved through evolution while others have diverged among different kingdoms. Copyright © 2013 Elsevier GmbH. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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.

Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of Indium-111-labeled Lactam Bridge-Cyclized α-MSH Peptides

PubMed Central

Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

2011-01-01

The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides. Methods Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. Results DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively. Conclusion The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide. PMID:21421725

Effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized alpha-MSH peptides.

PubMed

Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

2011-04-01

The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of (111)In-labeled lactam bridge-cyclized DOTA-[X]-CycMSH(hex) {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH(2); X = GGNle, GENle, or NleGE; GG = -Gly-Gly- and GE = -Gly-Glu-} peptides. Three novel peptides (DOTA-GGNle-CycMSH(hex), DOTA-GENle-CycMSH(hex), and DOTA-NleGE-CycMSH(hex)) were designed and synthesized. The melanocortin-1 (MC1) receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of (111)In-DOTA-GGNle-CycMSH(hex) and (111)In-DOTA-GENle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. DOTA-GGNle-CycMSH(hex) and DOTA-GENle-CycMSH(hex) displayed 2.1 and 11.5 nM MC1 receptor-binding affinities, whereas DOTA-NleGE-CycMSH(hex) showed 873.4 nM MC1 receptor-binding affinity. The introduction of the -GG- linker maintained high melanoma uptake while decreasing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex). The tumor uptake of (111)In-DOTA-GGNle-CycMSH(hex) was 19.05 ± 5.04 and 18.6 ± 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. (111)In-DOTA-GGNle-CycMSH(hex) exhibited 28%, 32%, and 42% less kidney uptake than (111)In-DOTA-Nle-CycMSH(hex) we reported previously, and 61%, 65%, and 68% less liver uptake than (111)In-DOTA-Nle-CycMSH(hex) at 2, 4, and 24 h after injection, respectively. The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the (111)In-labeled lactam bridge-cyclized α-melanocyte-stimulating hormone peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex), highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide.

Autoradiographic localization of /sup 3/H-paroxetine-labeled serotonin uptake sites in rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

De Souza, E.B.; Kuyatt, B.L.

1987-01-01

Paroxetine is a potent and selective inhibitor of serotonin uptake into neurons. Serotonin uptake sites have been identified, localized, and quantified in rat brain by autoradiography with 3H-paroxetine; 3H-paroxetine binding in slide-mounted sections of rat forebrain was of high affinity (KD = 10 pM) and the inhibition affinity constant (Ki) values of various drugs in competing 3H-paroxetine binding significantly correlated with their reported potencies in inhibiting synaptosomal serotonin uptake. Serotonin uptake sites labeled by 3H-paroxetine were highly concentrated in the dorsal and median raphe nuclei, central gray, superficial layer of the superior colliculus, lateral septal nucleus, paraventricular nucleus of themore » thalamus, and the islands of Calleja. High concentrations of 3H-paroxetine binding sites were found in brainstem areas containing dopamine (substantia nigra and ventral tegmental area) and norepinephrine (locus coeruleus) cell bodies. Moderate concentrations of 3H-paroxetine binding sites were present in laminae I and IV of the frontal parietal cortex, primary olfactory cortex, olfactory tubercle, regions of the basal ganglia, septum, amygdala, thalamus, hypothalamus, hippocampus, and some brainstem areas including the interpeduncular, trigeminal, and parabrachial nuclei. Lower densities of 3H-paroxetine binding sites were found in other regions of the neocortex and very low to nonsignificant levels of binding were present in white matter tracts and in the cerebellum. Lesioning of serotonin neurons with 3,4-methylenedioxyamphetamine caused large decreases in 3H-paroxetine binding. The autoradiographic distribution of 3H-paroxetine binding sites in rat brain corresponds extremely well to the distribution of serotonin terminals and cell bodies as well as with the pharmacological sites of action of serotonin.« less

Antidepressant-like activity of VN2222, a serotonin reuptake inhibitor with high affinity at 5-HT1A receptors.

PubMed

Tordera, Rosa M; Monge, Antonio; Del Río, Joaquín; Lasheras, Berta

2002-05-03

It has been suggested that drugs combining serotonin (5-hydroxytryptamine, 5-HT) transporter blockade and 5-HT1A autoreceptor antagonism could be a novel strategy for a shorter onset of action and higher therapeutic efficacy of antidepressants. The present study was aimed at characterizing the pharmacology of 1-(3-benzo[b]tiophenyl)-3-[4-(2-methoxyphenyl)-1-piperazinyl]-1-propanol (VN2222) a new synthetic compound with high affinity at both the 5-HT transporter and 5-HT1A receptors and devoid of high affinity at other receptors studied, with the only exception of alpha1-adrenoceptors. In keeping with the binding affinity at the 5-HT transporter, VN2222 inhibited 5-HT uptake in vitro both in rat cortical synaptosomes and in mesencephalic cultures and also in vivo when administered locally into the rat ventral hippocampus. After systemic administration, VN2222 exhibited an inverted U-shape effect so the inhibition of [3H]5-HT uptake ex vivo and the increase in 5-HT extracellular levels in microdialysis experiments was observed at low doses of 0.01-0.1 mg/kg whereas higher doses were ineffective. In studies related to 5-HT1A receptor function, 0.01-0.1 microM VN2222 produced a partial inhibition of forskolin-stimulated cAMP formation behaving as a weak agonist of 5-HT1A receptors. In body temperature studies, 5 mg/kg VN2222 produced a mild hypothermic effect in mice, suggesting a weak agonist activity at presynaptic 5-HT1A receptors; much lower doses (0.01-0.5 mg/kg) partially antagonized the hypothermia induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) possibly through 5-HT transporter blockade. In the learned helplessness test in rats, an animal model for antidepressants, 1-5 mg/kg VN2222 reduced significantly the number of escape failures. Consequently, VN2222 is a new compound with a dual effect on the serotonergic system, as 5-HT uptake blocker and 5-HT1A receptor partial agonist, and with a remarkable activity in an animal model of depression with high predictive validity.

Role of Secondary Transporters and Phosphotransferase Systems in Glucose Transport by Oenococcus oeni ▿

PubMed Central

Kim, Ok Bin; Richter, Hanno; Zaunmüller, Tanja; Graf, Sabrina; Unden, Gottfried

2011-01-01

Glucose uptake by the heterofermentative lactic acid bacterium Oenococcus oeni B1 was studied at the physiological and gene expression levels. Glucose- or fructose-grown bacteria catalyzed uptake of [14C]glucose over a pH range from pH 4 to 9, with maxima at pHs 5.5 and 7. Uptake occurred in two-step kinetics in a high- and low-affinity reaction. The high-affinity uptake followed Michaelis-Menten kinetics and required energization. It accumulated the radioactivity of glucose by a factor of 55 within the bacteria. A large portion (about 80%) of the uptake of glucose was inhibited by protonophores and ionophores. Uptake of the glucose at neutral pH was not sensitive to degradation of the proton potential, Δp. Expression of the genes OEOE_0819 and OEOE_1574 (here referred to as 0819 and 1574), coding for secondary transporters, was induced by glucose as identified by quantitative real-time (RT)-PCR. The genes 1574 and 0819 were able to complement growth of a Bacillus subtilis hexose transport-deficient mutant on glucose but not on fructose. The genes 1574 and 0819 therefore encode secondary transporters for glucose, and the transports are presumably Δp dependent. O. oeni codes, in addition, for a phosphotransferase transport system (PTS) (gene OEOE_0464 [0464] for the permease) with similarity to the fructose- and mannose-specific PTS of lactic acid bacteria. Quantitative RT-PCR showed induction of the gene 0464 by glucose and by fructose. The data suggest that the PTS is responsible for Δp-independent hexose transport at neutral pH and for the residual Δp-independent transport of hexoses at acidic pH. PMID:22020640

Novel alpha-MSH peptide analogs for melanoma targeting

NASA Astrophysics Data System (ADS)

Flook, Adam Michael

Skin cancer is the one of the most diagnosed cancers in the United States with increasing incidence over the past two decades. There are three major forms of skin cancer but melanoma is the deadliest. It is estimated that 76,690 new diagnoses of melanoma and 9,480 deaths will occur in 2013. Melanoma accounts for approximately 1.6% of all cancer related deaths and is the 5 th leading diagnosed cancer in the United States. The mean survival rate of patients diagnosed with metastatic melanoma is six months, with five year survival rates of less than 5%. In this project, we describe the design and characterization of novel melanoma-targeting peptide analogs for use in diagnostic imaging of both primary and metastatic melanoma lesions. Novel alpha-MSH peptide conjugates were designed to target the melanocortin-1 receptor present and over-expressed on melanoma cells. These peptides were synthesized and their in-vitro melanocortin-1 receptor binding affinities were established in murine melanoma cells. Once binding affinities were determined, the peptides were radiolabeled with 99mTc utilizing a novel direct radiolabeling technique developed in our laboratory. The peptides were purified via reverse-phase high performance liquid chromatography and in-vivo melanoma targeting and pharmacokinetic properties were determined in B16/F1 melanoma-bearing female C57BL/6 mice. Biodistribution and SPECT/CT imaging studies were performed with the promising 99m Tc-labeled peptide conjugates. All alpha-MSH peptide conjugates tested showed low nanomolar binding affinity for the melanocortin-1 receptor. All peptides were readily radiolabeld with 99mTc with greater than 95% radiochemical purity. All 99mTc-labeled peptides displayed high specific in-vivo melanoma tumor uptake while maintaining low normal organ accumulation, and were excreted through the urinary system in a timely fashion. In addition, all tested 99mTc-labeld alpha-MSH peptides demonstrated clear visualization of in-vivo tumor lesions with SPECT/CT. While all peptides exhibited high melanoma uptake, extremely high non-specific renal uptake was of concern. After synthesis of alpha-MSH peptide conjugates containing a different amino acid linker, renal uptake was drastically reduced and a lead compound had emerged, showing favorable in-vivo melanoma targeting and uptake properties with limited amounts of non-specific renal accumulation.

Evidence for the lack of spare high-affinity insulin receptors in skeletal muscle.

PubMed Central

Camps, M; Gumà, A; Viñals, F; Testar, X; Palacín, M; Zorzano, A

1992-01-01

In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full occupancy of high-affinity binding sites. Therefore our data provide evidence for a lack of spare high-affinity insulin receptors in skeletal muscle. PMID:1323279

Evidence for the lack of spare high-affinity insulin receptors in skeletal muscle.

PubMed

Camps, M; Gumà, A; Viñals, F; Testar, X; Palacín, M; Zorzano, A

1992-08-01

In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full occupancy of high-affinity binding sites. Therefore our data provide evidence for a lack of spare high-affinity insulin receptors in skeletal muscle.

Analysing the substrate multispecificity of a proton-coupled oligopeptide transporter using a dipeptide library

PubMed Central

Ito, Keisuke; Hikida, Aya; Kawai, Shun; Lan, Vu Thi Tuyet; Motoyama, Takayasu; Kitagawa, Sayuri; Yoshikawa, Yuko; Kato, Ryuji; Kawarasaki, Yasuaki

2013-01-01

Peptide uptake systems that involve members of the proton-coupled oligopeptide transporter (POT) family are conserved across all organisms. POT proteins have characteristic substrate multispecificity, with which one transporter can recognize as many as 8,400 types of di/tripeptides and certain peptide-like drugs. Here we characterize the substrate multispecificity of Ptr2p, a major peptide transporter of Saccharomyces cerevisiae, using a dipeptide library. The affinities (Ki) of di/tripeptides toward Ptr2p show a wide distribution range from 48 mM to 0.020 mM. This substrate multispecificity indicates that POT family members have an important role in the preferential uptake of vital amino acids. In addition, we successfully establish high performance ligand affinity prediction models (97% accuracy) using our comprehensive dipeptide screening data in conjunction with simple property indices for describing ligand molecules. Our results provide an important clue to the development of highly absorbable peptides and their derivatives including peptide-like drugs. PMID:24060756

Effect of Cu(II), Cd(II) and Zn(II) on Pb(II) biosorption by algae Gelidium-derived materials.

PubMed

Vilar, Vítor J P; Botelho, Cidália M S; Boaventura, Rui A R

2008-06-15

Biosorption of Pb(II), Cu(II), Cd(II) and Zn(II) from binary metal solutions onto the algae Gelidium sesquipedale, an algal industrial waste and a waste-based composite material was investigated at pH 5.3, in a batch system. Binary Pb(II)/Cu(II), Pb(II)/Cd(II) and Pb(II)/Zn(II) solutions have been tested. For the same equilibrium concentrations of both metal ions (1 mmol l(-1)), approximately 66, 85 and 86% of the total uptake capacity of the biosorbents is taken by lead ions in the systems Pb(II)/Cu(II), Pb(II)/Cd(II) and Pb(II)/Zn(II), respectively. Two-metal results were fitted to a discrete and a continuous model, showing the inhibition of the primary metal biosorption by the co-cation. The model parameters suggest that Cd(II) and Zn(II) have the same decreasing effect on the Pb(II) uptake capacity. The uptake of Pb(II) was highly sensitive to the presence of Cu(II). From the discrete model it was possible to obtain the Langmuir affinity constant for Pb(II) biosorption. The presence of the co-cations decreases the apparent affinity of Pb(II). The experimental results were successfully fitted by the continuous model, at different pH values, for each biosorbent. The following sequence for the equilibrium affinity constants was found: Pb>Cu>Cd approximately Zn.

Effects of chronic waterborne nickel exposure on growth, ion homeostasis, acid-base balance, and nickel uptake in the freshwater pulmonate snail, Lymnaea stagnalis.

PubMed

Niyogi, Som; Brix, Kevin V; Grosell, Martin

2014-05-01

The freshwater pulmonate snail, Lymnaea stagnalis, is the most sensitive aquatic organism tested to date for Ni. We undertook a series of experiments to investigate the underlying mechanism(s) for this observed hypersensitivity. Consistent with previous experiments, juvenile snail growth in a 21-day exposure was reduced by 48% relative to the control when exposed to 1.3 μg l(-1) Ni (EC20 less than the lowest concentration tested). Ca(2+) homeostasis was significantly disrupted by Ni exposure as demonstrated by reductions in net Ca(2+) uptake, and reductions in Ca(2+) concentrations in the hemolymph and soft tissues. We also observed reduced soft tissue [Mg(2+)]. Snails underwent a significant alkalosis with hemolymph pH increasing from 8.1 to 8.3 and hemolymph TCO2 increasing from 19 to 22 mM in control versus Ni-exposed snails, respectively. Unlike in previous studies with Co and Pb, snail feeding rates were found to be unaffected by Ni at the end of the exposure. Snails accumulated Ni in the soft tissue in a concentration-dependent manner, and Ni uptake experiments with (63)Ni revealed a biphasic uptake profile - a saturable high affinity component at low exposure concentrations (36-189 nM) and a linear component at the high exposure concentrations (189-1,897 nM). The high affinity transport system had an apparent Km of 89 nM Ni(2+) and Vmax of 2.4 nmol g(-1)h(-1). This equates to a logK of 7.1, significantly higher than logK's (2.6-5.2) for any other aquatic organisms evaluated to date, which will have implications for Biotic Ligand Model development. Finally, pharmacological inhibitors that block Ca(2+) uptake pathways in snails did not inhibit Ni uptake, suggesting that the uptake of Ni does not occur via Ca(2+) uptake pathways. As with Cu and Pb, the exact mechanism for the significant disruption in Ca(2+) homeostasis and reduction in juvenile snail growth remains unknown. Copyright © 2014 Elsevier B.V. All rights reserved.

Changes in gene expression, cell physiology and toxicity of the harmful cyanobacterium Microcystis aeruginosa at elevated CO2

PubMed Central

Sandrini, Giovanni; Cunsolo, Serena; Schuurmans, J. Merijn; Matthijs, Hans C. P.; Huisman, Jef

2015-01-01

Rising CO2 concentrations may have large effects on aquatic microorganisms. In this study, we investigated how elevated pCO2 affects the harmful freshwater cyanobacterium Microcystis aeruginosa. This species is capable of producing dense blooms and hepatotoxins called microcystins. Strain PCC 7806 was cultured in chemostats that were shifted from low to high pCO2 conditions. This resulted in a transition from a C-limited to a light-limited steady state, with a ~2.7-fold increase of the cyanobacterial biomass and ~2.5-fold more microcystin per cell. Cells increased their chlorophyll a and phycocyanin content, and raised their PSI/PSII ratio at high pCO2. Surprisingly, cells had a lower dry weight and contained less carbohydrates, which might be an adaptation to improve the buoyancy of Microcystis when light becomes more limiting at high pCO2. Only 234 of the 4691 genes responded to elevated pCO2. For instance, expression of the carboxysome, RuBisCO, photosystem and C metabolism genes did not change significantly, and only a few N assimilation genes were expressed differently. The lack of large-scale changes in the transcriptome could suit a buoyant species that lives in eutrophic lakes with strong CO2 fluctuations very well. However, we found major responses in inorganic carbon uptake. At low pCO2, cells were mainly dependent on bicarbonate uptake, whereas at high pCO2 gene expression of the bicarbonate uptake systems was down-regulated and cells shifted to CO2 and low-affinity bicarbonate uptake. These results show that the need for high-affinity bicarbonate uptake systems ceases at elevated CO2. Moreover, the combination of an increased cyanobacterial abundance, improved buoyancy, and higher toxin content per cell indicates that rising atmospheric CO2 levels may increase the problems associated with the harmful cyanobacterium Microcystis in eutrophic lakes. PMID:25999931

Lysosomal Trapping Is Present in Retinal Capillary Endothelial Cells: Insight into Its Influence on Cationic Drug Transport at the Inner Blood-Retinal Barrier.

PubMed

Kubo, Yoshiyuki; Seko, Narumi; Usui, Takuya; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi

2016-01-01

Lysosomal trapping was investigated in the retinal capillary endothelial cells that are responsible for the inner blood-retinal barrier (BRB) using LysoTracker(®) Red (LTR). Using confocal microscopy on TR-iBRB2 cells, an in vitro model of the inner BRB, the presence of lysosomal trapping in retinal capillary endothelial cells was suggested since TR-iBRB2 cells exhibited punctuate intracellular localization of LTR that was attenuated by NH4Cl treatment. The study confirmed that LTR uptake by retinal capillary endothelial cells took place in a time- and temperature-dependent manner, and exhibited the 1.58-fold greater uptake at pH 8.4 than that at pH 7.4 while there was no change in uptake between pH 6.4 and pH 7.4, suggesting that passive diffusion is not enough to explain LTR uptake. The inhibition study showed the possible influence of lysosomal trapping on cationic drug transport by retinal capillary endothelial cells since LTR uptake was significantly inhibited by cationic amphiphilic drugs. Inhibition profiling and the estimation of IC50 suggested the influence of lysosomal trapping on propranolol and low-affinity pyrilamine transport while lysosomal trapping had only a partial effect on verapamil, clonidine, nicotine and high-affinity pyrilamine transport in retinal capillary endothelial cells.

Down's syndrome fibroblasts exhibit enhanced inositol uptake.

PubMed Central

Fruen, B R; Lester, B R

1990-01-01

The inositol metabolism of Down's syndrome (DS, trisomy 21) skin fibroblasts was examined. We report that DS cells accumulated [3H]inositol 2-3-fold faster than did other aneuploid or diploid controls. In contrast, trisomy 21 did not affect the uptake of choline, serine or glucose. Kinetic analysis demonstrated an increased maximal velocity of high-affinity, Na(+)-dependent, inositol transport, consistent with the expression of higher numbers of transporters by DS cells. Enhanced uptake was accompanied by a proportional increase in the incorporation of radiolabelled inositol into phospholipid. We suggest that an imbalance of inositol metabolism may contribute to plasma membrane abnormalities characteristic of DS cells. Images Fig. 4. PMID:2144418

AGE-RELATED EFFECTS OF CHLORPYRIFOS ON HIGH AFFINITY CHOLINE UPTAKE IN RAT BRAIN. (R825811)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

NO3- , PO43- and SO42- deprivation reduced LKT1-mediated low-affinity K+ uptake and SKOR-mediated K+ translocation in tomato and Arabidopsis plants.

PubMed

Ródenas, Reyes; García-Legaz, Manuel Francisco; López-Gómez, Elvira; Martínez, Vicente; Rubio, Francisco; Ángeles Botella, M

2017-08-01

Regulation of essential macronutrients acquisition by plants in response to their availability is a key process for plant adaptation to changing environments. Here we show in tomato and Arabidopsis plants that when they are subjected to NO 3 - , PO 4 3 - and SO 4 2 - deprivation, low-affinity K + uptake and K + translocation to the shoot are reduced. In parallel, these nutritional deficiencies produce reductions in the messenger levels of the genes encoding the main systems for low-affinity K + uptake and K + translocation, i.e. AKT1 and SKOR in Arabidopsis and LKT1 and the tomato homolog of SKOR, SlSKOR in tomato, respectively. The results suggest that the shortage of one nutrient produces a general downregulation of the acquisition of other nutrients. In the case of K + nutrient, one of the mechanisms for such a response resides in the transcriptional repression of the genes encoding the systems for K + uptake and translocation. © 2017 Scandinavian Plant Physiology Society.

Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design.

PubMed

Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M

2016-05-05

Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared - non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

Evaluation of metal biouptake from the analysis of bulk metal depletion kinetics at various cell concentrations: theory and application.

PubMed

Rotureau, Elise; Billard, Patrick; Duval, Jérôme F L

2015-01-20

Bioavailability of trace metals is a key parameter for assessment of toxicity on living organisms. Proper evaluation of metal bioavailability requires monitoring the various interfacial processes that control metal partitioning dynamics at the biointerface, which includes metal transport from solution to cell membrane, adsorption at the biosurface, internalization, and possible excretion. In this work, a methodology is proposed to quantitatively describe the dynamics of Cd(II) uptake by Pseudomonas putida. The analysis is based on the kinetic measurement of Cd(II) depletion from bulk solution at various initial cell concentrations using electroanalytical probes. On the basis of a recent formalism on the dynamics of metal uptake by complex biointerphases, the cell concentration-dependent depletion time scales and plateau values reached by metal concentrations at long exposure times (>3 h) are successfully rationalized in terms of limiting metal uptake flux, rate of excretion, and metal affinity to internalization sites. The analysis shows the limits of approximate depletion models valid in the extremes of high and weak metal affinities. The contribution of conductive diffusion transfer of metals from the solution to the cell membrane in governing the rate of Cd(II) uptake is further discussed on the basis of estimated resistances for metal membrane transfer and extracellular mass transport.

Anion exchange pathways for Cl sup minus transport in rabbit renal microvillus membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karniski, L.P.; Aronson, P.S.

1987-09-01

The authors evaluated the mechanisms of chloride transport in microvillus membrane vesicles isolated from the rabbit renal cortex. The presence of Cl-formate exchange was confirmed. Outward gradients of oxaloacetate, HCO{sub 3}, acetate, lactate, succinate, sulfate, and p-aminohippurate (PAH) stimulated the rate of Cl uptake minimally or not at all. However, an outward gradient of oxalate stimulated Cl uptake by 70%, and an outward Cl gradient induced uphill oxalate uptake, indicting Cl-oxalate exchange. Moreover, an outward formate gradient induced uphill oxalate uptake, indicating formate-oxalate exchange. Studies of inhibitor and substrate specificity indicated the probably operation of at least two separate anionmore » exchangers in mediating Cl transport. The Cl-formate exchanger accepted Cl and formate as substrates, had little or no affinity for oxalate, was sensitive to inhibition by furosemide, and was less sensitive to inhibition by 4,4{prime}-diisothiocyanostilbene-2,2{prime}-disulfonic acid (DIDS). The Cl (formate)-oxalate exchanger also accepted Cl and formate as substrates but had high affinity for oxalate, was highly sensitive to inhibition by DIDS, and was less sensitive to inhibition by furosemide. The Cl-formate exchanger was electroneutral, whereas the Cl (formate)-oxalate exchanger was electrogenic. They conclude that at least separate anion exchangers mediating Cl transport are present on the luminal membrane of the rabbit proximal tubule cell. These exchangers may play important roles in mediating transtubular Cl and oxalate transport in this nephron segment.« less

Relationship between Increase in Astrocytic GLT-1 Glutamate Transport and Late-LTP

ERIC Educational Resources Information Center

Pita-Almenar, Juan D.; Zou, Shengwei; Colbert, Costa M.; Eskin, Arnold

2012-01-01

Na[superscript +]-dependent high-affinity glutamate transporters have important roles in the maintenance of basal levels of glutamate and clearance of glutamate during synaptic transmission. Interestingly, several studies have shown that basal glutamate transport displays plasticity. Glutamate uptake increases in hippocampal slices during early…

IN VITRO EFFECTS OF ORGANOPHOSPHORUS ANTICHOLINESTERASES AND MUSCARINIC AGONISTS ON RAT BRAIN SYNAPTOSOMAL HIGH AFFINITY CHOLINE UPTAKE. (R825811)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter.

PubMed

Bagchi, Rammyani; Salehin, Mohammad; Adeyemo, O Sarah; Salazar, Carolina; Shulaev, Vladimir; Sherrier, D Janine; Dickstein, Rebecca

2012-10-01

The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.

Functional Assessment of the Medicago truncatula NIP/LATD Protein Demonstrates That It Is a High-Affinity Nitrate Transporter1[W][OA

PubMed Central

Bagchi, Rammyani; Salehin, Mohammad; Adeyemo, O. Sarah; Salazar, Carolina; Shulaev, Vladimir; Sherrier, D. Janine; Dickstein, Rebecca

2012-01-01

The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function. PMID:22858636

Genome Data Mining and Soil Survey for the Novel Group 5 [NiFe]-Hydrogenase To Explore the Diversity and Ecological Importance of Presumptive High-Affinity H2-Oxidizing Bacteria ▿†

PubMed Central

Constant, Philippe; Chowdhury, Soumitra Paul; Hesse, Laura; Pratscher, Jennifer; Conrad, Ralf

2011-01-01

Streptomyces soil isolates exhibiting the unique ability to oxidize atmospheric H2 possess genes specifying a putative high-affinity [NiFe]-hydrogenase. This study was undertaken to explore the taxonomic diversity and the ecological importance of this novel functional group. We propose to designate the genes encoding the small and large subunits of the putative high-affinity hydrogenase hhyS and hhyL, respectively. Genome data mining revealed that the hhyL gene is unevenly distributed in the phyla Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria. The hhyL gene sequences comprised a phylogenetically distinct group, namely, the group 5 [NiFe]-hydrogenase genes. The presumptive high-affinity H2-oxidizing bacteria constituting group 5 were shown to possess a hydrogenase gene cluster, including the genes encoding auxiliary and structural components of the enzyme and four additional open reading frames (ORFs) of unknown function. A soil survey confirmed that both high-affinity H2 oxidation activity and the hhyL gene are ubiquitous. A quantitative PCR assay revealed that soil contained 106 to 108 hhyL gene copies g (dry weight)−1. Assuming one hhyL gene copy per genome, the abundance of presumptive high-affinity H2-oxidizing bacteria was higher than the maximal population size for which maintenance energy requirements would be fully supplied through the H2 oxidation activity measured in soil. Our data indicate that the abundance of the hhyL gene should not be taken as a reliable proxy for the uptake of atmospheric H2 by soil, because high-affinity H2 oxidation is a facultatively mixotrophic metabolism, and microorganisms harboring a nonfunctional group 5 [NiFe]-hydrogenase may occur. PMID:21742924

The use of one-bead one-compound combinatorial library technology to discover high-affinity αvβ3 integrin and cancer targeting RGD ligands with a build-in handle

PubMed Central

Xiao, Wenwu; Wang, Yan; Lau, Edmond Y.; Luo, Juntao; Yao, Nianhuan; Shi, Changying; Meza, Leah; Tseng, Harry; Maeda, Yoshiko; Kumaresan, Pappanaicken; Liu, Ruiwu; Lightstone, Felice C.; Takada, Yoshikazu; Lam, Kit S.

2012-01-01

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiological processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted-therapy and cancer imaging. In this report, one-bead-one-compound (OBOC) combinatorial libraries containing the RGD motif were designed and screened against K562 myeloid leukemia cells that had been transfected with human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a build-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC50 = 0.68±0.08 μM) to some of the well-known RGD “head-to-tail” cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2 to 8 fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with biotinylated-ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake. PMID:20858725

Nickel and zinc isotope fractionation in hyperaccumulating and nonaccumulating plants.

PubMed

Deng, Teng-Hao-Bo; Cloquet, Christophe; Tang, Ye-Tao; Sterckeman, Thibault; Echevarria, Guillaume; Estrade, Nicolas; Morel, Jean-Louis; Qiu, Rong-Liang

2014-10-21

Until now, there has been little data on the isotope fractionation of nickel (Ni) in higher plants and how this can be affected by plant Ni and zinc (Zn) homeostasis. A hydroponic cultivation was conducted to investigate the isotope fractionation of Ni and Zn during plant uptake and translocation processes. The nonaccumulator Thlaspi arvense, the Ni hyperaccumulator Alyssum murale and the Ni and Zn hyperaccumulator Noccaea caerulescens were grown in low (2 μM) and high (50 μM) Ni and Zn solutions. Results showed that plants were inclined to absorb light Ni isotopes, presumably due to the functioning of low-affinity transport systems across root cell membrane. The Ni isotope fractionation between plant and solution was greater in the hyperaccumulators grown in low Zn treatments (Δ(60)Ni(plant-solution) = -0.90 to -0.63‰) than that in the nonaccumulator T. arvense (Δ(60)Ni(plant-solution) = -0.21‰), thus indicating a greater permeability of the low-affinity transport system in hyperaccumulators. Light isotope enrichment of Zn was observed in most of the plants (Δ(66)Zn(plant-solution) = -0.23 to -0.10‰), but to a lesser extent than for Ni. The rapid uptake of Zn on the root surfaces caused concentration gradients, which induced ion diffusion in the rhizosphere and could result in light Zn isotope enrichment in the hyperaccumulator N. caerulescens. In high Zn treatment, Zn could compete with Ni during the uptake process, which reduced Ni concentration in plants and decreased the extent of Ni isotope fractionation (Δ(60)Ni(plant-solution) = -0.11 to -0.07‰), indicating that plants might take up Ni through a low-affinity transport system of Zn. We propose that isotope composition analysis for transition elements could become an empirical tool to study plant physiological processes.

Differing effects of transport inhibitor on glutamate uptake by nerve terminals before and after exposure of rats to artificial gravity.

NASA Astrophysics Data System (ADS)

Borisova, T.; Krisanova, N.; Himmelreich, N.

Glutamate is the major excitatory neurotransmitter in the brain. Subsequent to its release from glutamatergic neurons and activation of receptors, it is removed from extracellular space by high affinity Na^+-dependent glutamate transporters, which utilize the Na^+/K^+ electrochemical gradient as a driving force and located in nerve terminals and astrocytes. The glutamate transporters may modify the time course of synaptic events. Like glutamate itself, glutamate transporters are somehow involved in almost all aspects of normal and abnormal brain activity (e.g. cerebral ischemia, amyotrophic lateral sclerosis, Alzheimer's disease, traumatic brain injury, epilepsy and schizophrenia). The present study assessed transporter inhibitor for the ability to inhibit glutamate uptake by synaptosomes at the normal and hypergravity conditions (rats were rotated in a long-arm centrifuge at ten-G during one-hour period). DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a newly developed competitive inhibitor of the high-affinity, Na^+-dependent glutamate transporters. As a potent, non- transported inhibitor of glutamate transporters, DL-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms. We demonstrated that DL-TBOA inhibited glutamate uptake ( 100 μM glutamate, 30 sec incubation period) in dose-dependent manner as in control as in hypergravity. The effect of this transport inhibitor on glutamate uptake by control synaptosomes and synaptosomes prepared of animals exposed to hypergravity was different. IC50 values calculated on the basis of curves of non-linear regression kinetic analysis was 18±2 μM and 11±2 μM ((P≤0,05) before and after exposure to artificial gravity, respectively. Inhibition caused by 10 μM DL-TBOA was significantly increased from 38,0±3,8 % in control group to 51,0±4,1 % in animals, exposed to hypergravity (P≤0,05). Thus, DL-TBOA had complex effect on glutamate uptake process and perhaps, became more potent under testing conditions. Recently we showed that the affinity of glutamate transporters to substrate (glutamate) was unaffected under hypergravity stress. In contrast, the studies of maximal velocity of glutamate uptake reveal the significant lowering of glutamate transporter activity in response to hypergravity loading. The effects of DL-TBOA superimpose on the preexisting reduced uptake after hypergravity and result in a higher proportion of glutamate transporters being inhibited. Such knowledge will be of value designing new therapeutic strategies under different pathological conditions.

The Organization of Controller Motifs Leading to Robust Plant Iron Homeostasis

PubMed Central

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

2016-01-01

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

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.

PubMed

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.

Kinetics of caesium and potassium absorption by roots of three grass pastures and competitive effects of potassium on caesium uptake in Cynodon sp.

NASA Astrophysics Data System (ADS)

Ayub, J. Juri; Valverde, L. Rubio; Garcia-Sanchez, M. J.; Fernandez, J. A.; Velasco, R. H.

2008-08-01

Caesium uptake by plant roots has been normally associated with the uptake of potassium as the potassium transport systems present in plants have also the capacity to transport caesium. Three grass species (Eragrostis curvula, Cynodon sp and Distichlis spicata) growing in seminatural grassland of central Argentina were selected to study their capability to incorporate Cs+ (and K+) using electrophysiological techniques. Although the 137Cs soil inventory ranged between 328-730 Bq m-2 in this region, no 137Cs activity was detected in these plants. However, all the species, submitted previously to K+ starvation, showed the uptake of both Cs+ and K+ when micromolar concentrations of these cations were present in the medium. The uptake showed saturation kinetics for both cations that could be fitted to the Michelis-Menten model. KM values were smaller for K+ than for Cs+, indicating a higher affinity for the first cation. The presence of increasing K+ concentrations in the assay medium inhibited Cs+ uptake in Cynodon sp., as expected if both cations are transported by the same transport systems. This effect is due to the competition of both ions for the union sites of the high affinity potassium transporters. In field situation, where soil concentration of Cs+ is smaller than K+ concentration, is then expectable that caesium activity in plants is not detectable. Nevertheless, the studied plants would have the capacity to incorporate caesium if its availability in soil solution increases. In addition, studies of Cs/K interaction can help us to understand the variability in transfer factors.

Enhancement of GABAergic transmission by zolpidem, an imidazopyridine with preferential affinity for type I benzodiazepine receptors.

PubMed

Biggio, G; Concas, A; Corda, M G; Serra, M

1989-02-28

The effect of zolpidem, an imidazopyridine derivative with high affinity at the type I benzodiazepine recognition site, on the function of the GABAA/ionophore receptor complex was studied in vitro. Zolpidem, mimicking the action of diazepam, increased [3H]GABA binding, enhanced muscimol-stimulated 36Cl- uptake and reduced [35S]TBPS binding in rat cortical membrane preparations. Zolpidem was less effective than diazepam on the above parameters. Zolpidem induced a lower increase of [3H]GABA binding (23 vs. 35%) and muscimol-stimulated 36Cl- uptake (22 vs. 40%) and a smaller decrease of [35S]TBPS binding (47 vs. 77%) than diazepam. The finding that zolpidem enhanced the function of GABAergic synapses with an efficacy qualitatively and quantitatively different from that of diazepam suggests that this compound is a partial agonist at the benzodiazepine recognition site. Thus, our results are consistent with the view that the biochemical and pharmacological profile of a benzodiazepine recognition site ligand reflects its efficacy to enhance GABAergic transmission. Whether the preferential affinity of zolpidem at the type I site is involved in its atypical biochemical and pharmacological profile remains to be clarified.

Effects of repeated exposure to white noise on central cholinergic activity in the rat.

PubMed

Lai, H

1988-03-01

Acute (45 min) exposure to noise has been shown to decrease sodium-dependent high-affinity choline uptake activity in the frontal cortex and hippocampus of the rat. In the present experiment, the effects of repeated noise exposure on choline uptake in these two brain regions were studied. Rats were exposed to 100-dB white noise in ten 45-min sessions. Tolerance developed to the effects of noise on choline uptake. In addition, the effects were found to be classically conditionable to cues in the exposure environment. These data may have important implications in understanding the health hazard of noise exposure in both the public and occupational environments.

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

PubMed Central

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

1989-01-01

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

Histones have high affinity for the glomerular basement membrane. Relevance for immune complex formation in lupus nephritis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schmiedeke, T.M.; Stoeckl, F.W.W.; Weber, R.

1989-06-01

An effort has been made to integrate insights on charge-based interactions in immune complex glomerulonephritis with nuclear antigen involvement in lupus nephritis. Attention was focussed on the histones, a group of highly cationic nuclear constituents, which could be expected to bind to fixed anionic sites present in the glomerular basement membrane (GBM). We demonstrated that all histone subfractions, prepared according to Johns, have a high affinity for GBM and the basement membrane of peritubular capillaries. Tissue uptake of /sup 125/I-labeled histones was measured by injecting 200 micrograms of each fraction into the left kidney via the aorta and measuring organmore » uptake after 15 min. In glomeruli isolated from the left kidneys, the following quantities of histones were found: f1, 13 micrograms; f2a (f2al + f2a2), 17 micrograms; f2b, 17 micrograms; and f3, 32 micrograms. Kinetic studies of glomerular binding showed that f1 disappeared much more rapidly than f2a. The high affinity of histones (pI between 10.5 and 11.0; mol wt 10,000-22,000) for the GBM correlates well with their ability to form aggregates (mol wt greater than 100,000) for comparison lysozyme (pI 11, mol wt 14,000), which does not aggregate spontaneously bound poorly (0.4 micrograms in isolated glomeruli). The quantity of histones and lysozyme found in the isolated glomeruli paralleled their in vitro affinity for a Heparin-Sepharose column (gradient elution studies). This gel matrix contains the sulfated, highly anionic polysaccharide heparin, which is similar to the negatively charged heparan sulfate present in the GBM. Lysozyme eluted with 0.15 M NaCl, f1 with 1 M NaCl, and f2a, f2b, and f3 could not be fully desorbed even with 2 M NaCl; 6 M guanidine-HCl was necessary.« less

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

PubMed Central

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

Efficient DNA binding and nuclear uptake by distamycin derivatives conjugated to octa-arginine sequences.

PubMed

Vázquez, Olalla; Blanco-Canosa, Juan B; Vázquez, M Eugenio; Martínez-Costas, Jose; Castedo, Luis; Mascareñas, José L

2008-11-24

Efficient targeting of DNA by designed molecules requires not only careful fine-tuning of their DNA-recognition properties, but also appropriate cell internalization of the compounds so that they can reach the cell nucleus in a short period of time. Previous observations in our group on the relatively high affinity displayed by conjugates between distamycin derivatives and bZIP basic regions for A-rich DNA sites, led us to investigate whether the covalent attachment of a positively charged cell-penetrating peptide to a distamycin-like tripyrrole might yield high affinity DNA binders with improved cell internalization properties. Our work has led to the discovery of synthetic tripyrrole-octa-arginine conjugates that are capable of targeting specific DNA sites that contain A-rich tracts with low nanomolar affinity; they simultaneously exhibit excellent membrane and nuclear translocation properties in living HeLa cells.

Neurochemical correlates of. gamma. -aminobutyrate (GABA) inhibition in cat visual cortex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balcar, V.J.; Dreher, B.

1990-01-01

High affinity binding of ({sup 3}H){gamma}-aminobutyric acid (GABA) to neuronal membranes from different parts of cat visual cortex was tested for sensitivity to GABA{sub A} agonists isoguvacine and THIP, GABA{sub A} antagonist SR95531 and GABA{sub B} agonist baclofen. Some of the GABA{sub A}-binding sites were found to have a very low affinity for THIP, suggesting the presence and, possibly, uneven distribution of non-synaptic GABA{sub A} receptors in cat visual cortex. There were no differences in K{sub m} and V{sub max} values of high affinity uptake of GABA and in the potency of K{sup +}-stimulated release of GABA, between primary andmore » association cortices. Consequently, the present results indicate that despite the anatomical and physiological differences between the primary and association feline visual cortices the neurochemical characteristics of GABAergic inhibition are very similar in the two regions.« less

SOLID PHASE MICROEXTRACTION SAMPLING OF FIRE DEBRIS RESIDUES IN THE PRESENCE OF RADIONUCLIDE SURROGATE METALS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duff, M; Keisha Martin, K; S Crump, S

2007-03-23

The Federal Bureau of Investigation (FBI) Laboratory currently does not have on site facilities for handling radioactive evidentiary materials and there are no established FBI methods or procedures for decontaminating highly radioactive fire debris (FD) evidence while maintaining evidentiary value. One experimental method for the isolation of FD residue from radionuclide metals involves using solid phase microextraction (SPME) fibers to remove the residues of interest. Due to their high affinity for organics, SPME fibers should have little affinity for most (radioactive) metals. The focus of this research was to develop an examination protocol that was applicable to safe work inmore » facilities where high radiation doses are shielded from the workers (as in radioactive shielded cells or ''hot cells''). We also examined the affinity of stable radionuclide surrogate metals (Co, Ir, Re, Ni, Ba, Cs, Nb, Zr and Nd) for sorption by the SPME fibers. This was done under exposure conditions that favor the uptake of FD residues under conditions that will provide little contact between the SPME and the FD material (such as charred carpet or wood that contains commonly-used accelerants). Our results from mass spectrometric analyses indicate that SPME fibers show promise for use in the room temperature head space uptake of organic FD residue (namely, diesel fuel oil, kerosene, gasoline and paint thinner) with subsequent analysis by gas chromatography (GC) with mass spectrometric (MS) detection. No inorganic forms of ignitable fluids were included in this study.« less

AF64A depletes hippocampal high-affinity choline uptake but does not alter the density of alpha-bungarotoxin binding sites or modify the effect of exogenous choline.

PubMed

Morley, B J; Garner, L L

1990-06-11

Sodium-dependent, high-affinity choline uptake (HACU) and the density of alpha-bungarotoxin (BuTX) receptor-binding sites were measured in the hippocampus following the intraventricular infusion of ethylcholine aziridinium ion (AF64A), a neurotoxin that competes with choline at high-affinity choline transport sites and may result in the degeneration of cholinergic axons. Eight days after the infusion of AF64A into the lateral ventricles (2.5 nmol/side), HACU was depleted by 60% in the hippocampus of experimental animals in comparison with controls, but the density of BuTX-binding sites was not altered. The administration of 15 mg/ml of choline chloride in the drinking water increased the density of BuTX-binding sites, as previously reported by this laboratory. The administration of AF64A did not prevent the effect of exogenous choline on the density of binding sites, nor did choline treatment alter the effect of AF64A on HACU. These data indicate that the density of BuTX-binding sites in the hippocampus is not altered following a substantial decrease in HACU and presumed degeneration of cholinergic axons. Since the effect of exogenous choline was not prevented by AF64A treatment, the data are interpreted to support the hypothesis that the increase in the density of BuTX-binding sites following dietary choline supplementation is attributable to a direct effect of choline on receptor sites.

Substrate specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa.

PubMed

Wylie, J L; Worobec, E A

1993-07-01

Specificity of the high-affinity glucose transport system of Pseudomonas aeruginosa was examined. At a concentration of [14C]glucose near the Vmax of the system, inhibition by maltose, galactose, and xylose was detected. This inhibition is similar to that detected in earlier in vivo studies and correlates with the known specificity of OprB, a glucose-specific porin of P. aeruginosa. At a level of [14C]glucose 100 times lower, only unlabelled glucose inhibited uptake to any extent. This matches the known in vitro specificity of the periplasmic glucose binding protein. These findings were used to explain the discrepancy between earlier in vivo and in vitro results reported in the literature.

Imaging of Cerebral Amyloid Angiopathy with Bivalent 99mTc-Hydroxamamide Complexes

NASA Astrophysics Data System (ADS)

Iikuni, Shimpei; Ono, Masahiro; Watanabe, Hiroyuki; Matsumura, Kenji; Yoshimura, Masashi; Kimura, Hiroyuki; Ishibashi-Ueda, Hatsue; Okamoto, Yoko; Ihara, Masafumi; Saji, Hideo

2016-05-01

Cerebral amyloid angiopathy (CAA), characterized by the deposition of amyloid aggregates in the walls of cerebral vasculature, is a major factor in intracerebral hemorrhage and vascular cognitive impairment and is also associated closely with Alzheimer’s disease (AD). We previously reported 99mTc-hydroxamamide (99mTc-Ham) complexes with a bivalent amyloid ligand showing high binding affinity for β-amyloid peptide (Aβ(1-42)) aggregates present frequently in the form in AD. In this article, we applied them to CAA-specific imaging probes, and evaluated their utility for CAA-specific imaging. In vitro inhibition assay using Aβ(1-40) aggregates deposited mainly in CAA and a brain uptake study were performed for 99mTc-Ham complexes, and all 99mTc-Ham complexes with an amyloid ligand showed binding affinity for Aβ(1-40) aggregates and very low brain uptake. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice were carried out for bivalent 99mTc-Ham complexes ([99mTc]SB2A and [99mTc]BT2B), and they displayed excellent labeling of Aβ depositions in human CAA brain sections and high affinity and selectivity to CAA in transgenic mice. These results may offer new possibilities for the development of clinically useful CAA-specific imaging probes based on the 99mTc-Ham complex.

Imaging of Cerebral Amyloid Angiopathy with Bivalent (99m)Tc-Hydroxamamide Complexes.

PubMed

Iikuni, Shimpei; Ono, Masahiro; Watanabe, Hiroyuki; Matsumura, Kenji; Yoshimura, Masashi; Kimura, Hiroyuki; Ishibashi-Ueda, Hatsue; Okamoto, Yoko; Ihara, Masafumi; Saji, Hideo

2016-05-16

Cerebral amyloid angiopathy (CAA), characterized by the deposition of amyloid aggregates in the walls of cerebral vasculature, is a major factor in intracerebral hemorrhage and vascular cognitive impairment and is also associated closely with Alzheimer's disease (AD). We previously reported (99m)Tc-hydroxamamide ((99m)Tc-Ham) complexes with a bivalent amyloid ligand showing high binding affinity for β-amyloid peptide (Aβ(1-42)) aggregates present frequently in the form in AD. In this article, we applied them to CAA-specific imaging probes, and evaluated their utility for CAA-specific imaging. In vitro inhibition assay using Aβ(1-40) aggregates deposited mainly in CAA and a brain uptake study were performed for (99m)Tc-Ham complexes, and all (99m)Tc-Ham complexes with an amyloid ligand showed binding affinity for Aβ(1-40) aggregates and very low brain uptake. In vitro autoradiography of human CAA brain sections and ex vivo autoradiography of Tg2576 mice were carried out for bivalent (99m)Tc-Ham complexes ([(99m)Tc]SB2A and [(99m)Tc]BT2B), and they displayed excellent labeling of Aβ depositions in human CAA brain sections and high affinity and selectivity to CAA in transgenic mice. These results may offer new possibilities for the development of clinically useful CAA-specific imaging probes based on the (99m)Tc-Ham complex.

COMPARATIVE EFFECTS OF PARAOXON, CHLORPYRIFOS OXON AND MUSCARINIC AGONISTS ON HIGH AFFINITY CHOLINE UPTAKE IN RAT CORTICAL OR STRIATAL SYNAPTOSOMES. (R825811)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Increased brain uptake of targeted nanoparticles by adding an acid-cleavable linkage between transferrin and the nanoparticle core.

PubMed

Clark, Andrew J; Davis, Mark E

2015-10-06

Most therapeutic agents are excluded from entering the central nervous system by the blood-brain barrier (BBB). Receptor mediated transcytosis (RMT) is a common mechanism used by proteins, including transferrin (Tf), to traverse the BBB. Here, we prepared Tf-containing, 80-nm gold nanoparticles with an acid-cleavable linkage between the Tf and the nanoparticle core to facilitate nanoparticle RMT across the BBB. These nanoparticles are designed to bind to Tf receptors (TfRs) with high avidity on the blood side of the BBB, but separate from their multidentate Tf-TfR interactions upon acidification during the transcytosis process to allow release of the nanoparticle into the brain. These targeted nanoparticles show increased ability to cross an in vitro model of the BBB and, most important, enter the brain parenchyma of mice in greater amounts in vivo after systemic administration compared with similar high-avidity nanoparticles containing noncleavable Tf. In addition, we investigated this design with nanoparticles containing high-affinity antibodies (Abs) to TfR. With the Abs, the addition of the acid-cleavable linkage provided no improvement to in vivo brain uptake for Ab-containing nanoparticles, and overall brain uptake was decreased for all Ab-containing nanoparticles compared with Tf-containing ones. These results are consistent with recent reports of high-affinity anti-TfR Abs trafficking to the lysosome within BBB endothelium. In contrast, high-avidity, Tf-containing nanoparticles with the acid-cleavable linkage avoid major endothelium retention by shedding surface Tf during their transcytosis.

Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells of Zostera marina L.

PubMed

Rubio, L; Linares-Rueda, A; García-Sánchez, M J; Fernández, J A

2005-02-01

Zostera marina L. is an angiosperm that grows in a medium in which inorganic phosphate (P(i)) and nitrate (NO(3)(-)) are present in micromolar concentrations and must be absorbed against a steep electrochemical potential gradient. The operation of a Na(+)-dependent NO(3)(-) transport was previously demonstrated in leaf cells of this plant, suggesting that other Na(+)-coupled systems could mediate the uptake of anions. To address this question, P(i) transport was studied in leaves and roots of Z. marina, as well as NO(3)(-) uptake in roots. Electrophysiological studies demonstrated that micromolar concentrations of P(i) induced depolarizations of the plasma membrane of root cells. However, this effect was not observed in leaf cells. P(i)-induced depolarizations showed Michaelis-Menten kinetics (K(m)=1.5+/-0.6 microM P(i); D(max)=7.8+/-0.8 mV), and were not observed in the absence of Na(+). However, depolarizations were restored when Na(+) was resupplied. NO(3)(-) additions also evoked depolarizations of the plasma membrane of root cells only in the presence of Na(+). Both NO(3)(-)- and P(i)-induced depolarizations were accompanied by an increase in cytoplasmic Na(+) activity, detected by Na(+)-sensitive microelectrodes. P(i) net uptake (measured in depletion experiments) was stimulated by Na(+). These results strongly suggest that P(i) uptake in roots of Z. marina is mediated by a high-affinity Na(+)-dependent transport system. Both NO(3)(-) and P(i) transport systems exploit the steep inwardly directed electrochemical potential gradient for Na(+), considering the low cytoplasmic Na(+) activity (10.7+/-3.3 mM Na(+)) and the high external Na(+) concentration (500 mM Na(+)).

Preparation and In Vitro Photodynamic Activity of Glucosylated Zinc(II) Phthalocyanines as Underlying Targeting Photosensitizers.

PubMed

Liu, Jian-Yong; Wang, Chen; Zhu, Chun-Hui; Zhang, Zhi-Hong; Xue, Jin-Ping

2017-05-19

Two novel glucosylated zinc(ІІ) phthalocyanines 7a-7b, as well as the acetyl-protected counterparts 6a-6b, have been synthesized by the Cu(I)-catalyzed 1,3-dipolar cycloaddition between the propargylated phthalocyanine and azide-substituted glucoses. All of these phthalocyanines were characterized with various spectroscopic methods and studied for their photo-physical, photo-chemical, and photo-biological properties. With glucose as the targeting unit, phthalocyanines 7a-7b exhibit a specific affinity to MCF-7 breast cancer cells over human embryonic lung fibroblast (HELF) cells, showing higher cellular uptake. Upon illumination, both photosensitizers show high cytotoxicity with IC 50 as low as 0.032 µM toward MCF-7 cells, which are attributed to their high cellular uptake and low aggregation tendency in the biological media, promoting the generation of intracellular reactive oxygen species (ROS). Confocal laser fluorescence microscopic studies have also revealed that they have high and selective affinities to the lysosomes, but not the mitochondria, of MCF-7 cells. The results show that these two glucosylated zinc(II) phthalocyanines are potential anticancer agents for targeting photodynamic therapy.

Development of a bone-targeted pH-sensitive liposomal formulation containing doxorubicin: physicochemical characterization, cytotoxicity, and biodistribution evaluation in a mouse model of bone metastasis.

PubMed

Ferreira, Diêgo Dos Santos; Faria, Samilla Dornelas; Lopes, Sávia Caldeira de Araújo; Teixeira, Cláudia Salviano; Malachias, Angelo; Magalhães-Paniago, Rogério; de Souza Filho, José Dias; Oliveira, Bruno Luis de Jesus Pinto; Guimarães, Alexander Ramos; Caravan, Peter; Ferreira, Lucas Antônio Miranda; Alves, Ricardo José; Oliveira, Mônica Cristina

2016-01-01

Despite recent advances in cancer therapy, the treatment of bone tumors remains a major challenge. A possible underlying hypothesis, limitation, and unmet need may be the inability of therapeutics to penetrate into dense bone mineral, which can lead to poor efficacy and high toxicity, due to drug uptake in healthy organs. The development of nanostructured formulations with high affinity for bone could be an interesting approach to overcome these challenges. To develop a liposomal formulation with high affinity for hydroxyapatite and the ability to release doxorubicin (DOX) in an acidic environment for future application as a tool for treatment of bone metastases. Liposomes were prepared by thin-film lipid hydration, followed by extrusion and the sulfate gradient-encapsulation method. Liposomes were characterized by average diameter, ζ-potential, encapsulation percentage, X-ray diffraction, and differential scanning calorimetry. Release studies in buffer (pH 7.4 or 5), plasma, and serum, as well as hydroxyapatite-affinity in vitro analysis were performed. Cytotoxicity was evaluated by MTT assay against the MDA-MB-231 cell line, and biodistribution was assessed in bone metastasis-bearing animals. Liposomes presented suitable diameter (~170 nm), DOX encapsulation (~2 mg/mL), controlled release, and good plasma and serum stability. The existence of interactions between DOX and the lipid bilayer was proved through differential scanning calorimetry and small-angle X-ray scattering. DOX release was faster when the pH was in the range of a tumor than at physiological pH. The bone-targeted formulation showed a strong affinity for hydroxyapatite. The encapsulation of DOX did not interfere in its intrinsic cytotoxicity against the MDA-MB-231 cell line. Biodistribution studies demonstrated high affinity of this formulation for tumors and reduction of uptake in the heart. These results suggest that bone-targeted pH-sensitive liposomes containing DOX can be an interesting strategy for selectively delivering this drug into bone-tumor sites, increasing its activity, and reducing DOX-related toxicity.

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

PubMed Central

Heyne, R I; de Vrij, W; Crielaard, W; Konings, W N

1991-01-01

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism. PMID:1670936

[Cellular uptake of TPS-L-carnitine synthesised as transporter-based renal targeting prodrug].

PubMed

Li, Li; Zhu, Di; Sun, Xun

2012-11-01

To synthesize transporter-based renal targeting prodrug TPS-L-Carnitine and to determine its cellular uptake in vitro. Triptolide (TP) was conjugated with L-carnitine using succinate as the linker to form TPS-L-Carnitine, which could be specifically recognized by OCTN2, a cationic transporter with high affinity to L-Carnitine and is highly expressed on the apical membrane of renal proximal tubule cells. Cellular uptake assays of the prodrug and its parent drug were performed on HK-2 cells, a human proximal tubule cell line, in different temperature, concentration and in the presence of competitive inhibitors. TPS-L-Carnitine was taken up into HK-2 cells in a saturable and temperature- and concentration-dependent manner. The uptake process could be inhibited by the competitive inhibitors. The uptake of TPS-L-Carnitine was significantly higher than that of TP at 37 degrees C in the same drug concentration. TPS-L-Carnitine was taken through endocytosis mediated by transporter. TPS-L-Carnitine provides a good renal targeting property and lays the foundation for further studies in vivo.

The binding of [3H]-propylbenzilylcholine mustard by longitudinal muscle strips from guinea-pig small intestine

PubMed Central

Burgen, A.S.V.; Hiley, C.R.; Young, J.M.

1974-01-01

1 The synthesis of tritium labelled propylbenzilylcholine mustard ([3H]-PrBCM; N-2′-chloroethyl-N-[2″, 3″-3H2] propyl-2-aminoethyl benzilate) is described. 2 The uptake by muscle strips was measured and shown to be considerably increased by previous immersion of the muscle in distilled water. 3 A considerable part of the uptake is inhibited selectively by atropine, but not by nicotinic antagonists. A number of muscarinic agonists also inhibit uptake and their apparent affinity constants have been determined. 4 The uptake by atropine-sensitive sites is temperature-insensitive, whereas the other sites are temperature-sensitive. Recovery is highly temperature-sensitive and there is good agreement between recovery of sensitivity to agonists and loss of radioactivity from the muscle. PMID:4150888

Diselenolane-mediated cellular uptake.

PubMed

Chuard, Nicolas; Poblador-Bahamonde, Amalia I; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi; Matile, Stefan

2018-02-21

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides.

The role of hemoglobin oxygen affinity in oxygen transport at high altitude.

PubMed

Winslow, Robert M

2007-09-30

Hemoglobin is involved in the regulation of O(2) transport in two ways: a long-term adjustment in red cell mass is mediated by erythropoietin (EPO), a response to renal oxgyenation. Short-term, rapid-response adjustments are mediated by ventilation, cardiac output, hemoglobin oxygen affinity (P50), barriers to O(2) diffusion, and the control of local microvascular tissue perfusion. The distribution of O(2) between dissolved (PO2) and hemoglobin-bound (saturation) is the familiar oxygen equilibrium curve, whose position is noted as P50. Human hemoglobin is not genetically adapted for function at high altitude. However, more specialized species native to high altitudes (guinea pig and bar-headed goose, for example) seem to have a lower P50 than their sea level counterparts, an adaptation that presumably promotes O(2) uptake from a hypoxic environment. Humans, native to very high altitude either in the Andes or Himalayan mountains, also can increase O(2) affinity, not because of a fundamental difference in hemoglobin structure or function, but because of extreme hyperventilation and alkalosis.

Effects of Atrazine, Metolachlor, Carbaryl and Chlorothalonil on Benthic Microbes and Their Nutrient Dynamics

PubMed Central

Elias, Daniel; Bernot, Melody J.

2014-01-01

Atrazine, metolachlor, carbaryl, and chlorothalonil are detected in streams throughout the U.S. at concentrations that may have adverse effects on benthic microbes. Sediment samples were exposed to these pesticides to quantify responses of ammonium, nitrate, and phosphate uptake by the benthic microbial community. Control uptake rates of sediments had net remineralization of nitrate (−1.58 NO3 µg gdm−1 h−1), and net assimilation of phosphate (1.34 PO4 µg gdm−1 h−1) and ammonium (0.03 NH4 µg gdm−1 h−1). Metolachlor decreased ammonium and phosphate uptake. Chlorothalonil decreased nitrate remineralization and phosphate uptake. Nitrate, ammonium, and phosphate uptake rates are more pronounced in the presence of these pesticides due to microbial adaptations to toxicants. Our interpretation of pesticide availability based on their water/solid affinities supports no effects for atrazine and carbaryl, decreasing nitrate remineralization, and phosphate assimilation in response to chlorothalonil. Further, decreased ammonium and phosphate uptake in response to metolachlor is likely due to affinity. Because atrazine target autotrophs, and carbaryl synaptic activity, effects on benthic microbes were not hypothesized, consistent with results. Metolachlor and chlorothalonil (non-specific modes of action) had significant effects on sediment microbial nutrient dynamics. Thus, pesticides with a higher affinity to sediments and/or broad modes of action are likely to affect sediment microbes' nutrient dynamics than pesticides dissolved in water or specific modes of action. Predicted nutrient uptake rates were calculated at mean and peak concentrations of metolachlor and chlorothalonil in freshwaters using polynomial equations generated in this experiment. We concluded that in natural ecosystems, peak chlorothalonil and metolachlor concentrations could affect phosphate and ammonium by decreasing net assimilation, and nitrate uptake rates by decreasing remineralization, relative to mean concentrations of metolachlor and chlorothalonil. Our regression equations can complement models of nitrogen and phosphorus availability in streams to predict potential changes in nutrient dynamics in response to pesticides in freshwaters. PMID:25275369

Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient

PubMed Central

Romero-Calderón, Rafael; Krantz, David E.

2005-01-01

Polyamine transport activities have been described in diverse multicellular systems, but their bioenergetic mechanisms and molecular identity remain unclear. In the present paper, we describe a high-affinity spermine/spermidine transport activity expressed in Drosophila S2 cells. Ion-replacement experiments indicate that polyamine uptake across the cell membrane is Na+-, K+-, Cl−- and Ca2+-independent, but pH-sensitive. Additional experiments using ionophores suggest that polyamine uptake may be H+-coupled. Pharmacological experiments show that polyamine uptake in S2 cells is selectively blocked by MGBG {methylglyoxal bis(guanylhydrazone) or 1,1′-[(methylethanediylidine)-dinitrilo]diguanidine} and paraquat (N,N-dimethyl-4,4′-bipyridylium), two known inhibitors of polyamine uptake in mammalian cells. In addition, inhibitors known to block the Slc22 (solute carrier 22) family of organic anion/cation transporters inhibit spermine uptake in S2 cells. These data and the genetic tools available in Drosophila will facilitate the molecular identification and further characterization of this activity. PMID:16248856

Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient.

PubMed

Romero-Calderón, Rafael; Krantz, David E

2006-01-15

Polyamine transport activities have been described in diverse multicellular systems, but their bioenergetic mechanisms and molecular identity remain unclear. In the present paper, we describe a high-affinity spermine/spermidine transport activity expressed in Drosophila S2 cells. Ion-replacement experiments indicate that polyamine uptake across the cell membrane is Na+-, K+-, Cl-- and Ca2+-independent, but pH-sensitive. Additional experiments using ionophores suggest that polyamine uptake may be H+-coupled. Pharmacological experiments show that polyamine uptake in S2 cells is selectively blocked by MGBG {methylglyoxal bis(guanylhydrazone) or 1,1'-[(methylethanediylidine)-dinitrilo]diguanidine} and paraquat (N,N-dimethyl-4,4'-bipyridylium), two known inhibitors of polyamine uptake in mammalian cells. In addition, inhibitors known to block the Slc22 (solute carrier 22) family of organic anion/cation transporters inhibit spermine uptake in S2 cells. These data and the genetic tools available in Drosophila will facilitate the molecular identification and further characterization of this activity.

Design of Internalizing PSMA-specific Glu-ureido-based Radiotherapeuticals

PubMed Central

Wüstemann, Till; Bauder-Wüst, Ulrike; Schäfer, Martin; Eder, Matthias; Benesova, Martina; Leotta, Karin; Kratochwil, Clemens; Haberkorn, Uwe; Kopka, Klaus; Mier, Walter

2016-01-01

Despite the progress in diagnosis and treatment, prostate cancer (PCa) is one of the main causes for cancer-associated deaths among men. Recently, prostate-specific membrane antigen (PSMA) binding tracers have revolutionized the molecular imaging of this disease. The translation of these tracers into therapeutic applications is challenging because of high PSMA-associated kidney uptake. While both the tumor uptake and the uptake in the kidneys are PSMA-specific, the kidneys show a more rapid clearance than tumor lesions. Consequently, the potential of endoradiotherapeutic drugs targeting PSMA is highly dependent on a sustained retention in the tumor - ideally achieved by predominant internalization of the respective tracer. Previously, we were able to show that the pharmacokinetics of the tracers containing the Glu-urea-based binding motif can be further enhanced with a specifically designed linker. Here, we evaluate an eventual influence of the chelator moiety on the pharmacokinetics, including the tumor internalization. A series of tracers modified by different chelators were synthesized using solid phase chemistry. The conjugates were radiolabeled to evaluate the influence on the receptor binding affinity, the ligand-induced internalization and the biodistribution behavior. Competitive binding and internalization assays were performed on PSMA positive LNCaP cells and the biodistribution of the most promising compound was evaluated by positron emission tomography (PET) in LNCaP-tumor-bearing mice. Interestingly, conjugation of the different chelators did not cause significant differences: all compounds showed nanomolar binding affinities with only minor differences. PET imaging of the 68Ga-labeled CHX-A''-DTPA conjugate revealed that the chelator moiety does not impair the specificity of tumor uptake when compared to the gold standard PSMA-617. However, strong differences of the internalization ratios caused by the chelator moiety were observed: differences in internalization between 15% and 65% were observed, with the CHX-A''-DTPA conjugate displaying the highest internalization ratio. A first-in-man PET/CT study proved the high tumor uptake of this 68Ga-labeled PSMA-targeting compound. These data indicate that hydrophobic entities at the chelator mediate the internalization efficacy. Based on its specific tumor uptake in combination with its very high internalization ratio, the clinical performance of the chelator-conjugated Glu-urea-based PSMA inhibitors will be further elucidated. PMID:27279903

High affinity 3H-Phe uptake by brush border membrane vesicles from whole larvae of Aedes aegypti (AaBMVw)

USDA-ARS?s Scientific Manuscript database

Brush border membrane vesicles from whole Aedes aegypti larvae (AaBBMVw) are confirmed to be valid preparations for membrane transport studies. The Abdul-Rauf and Ellar method was used to isolate AaBBMVw that were frozen, stored for several months, transported to a distant site, thawed and used to s...

Gallium-67-labeled lactam bridge-cyclized alpha-MSH peptides with enhanced melanoma uptake and reduced renal uptake.

PubMed

Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

2012-06-20

The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of (67)Ga-DOTA-GGNle-CycMSHhex {(67)Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (67)Ga-NOTA-GGNle-CycMSHhex {(67)Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and compare with (67)Ga-DOTA-GlyGlu-CycMSH {(67)Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM) in B16/F1 melanoma cells. Both (67)Ga-NOTA-GGNle-CycMSHhex and (67)Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than (67)Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, (67)Ga-NOTA-GGNle-CycMSHhex exhibited more favorable radiolabeling conditions (>85% radiolabeling yields started at 37 °C), as well as higher tumor/kidney uptake ratios than (67)Ga-DOTA-GGNle-CycMSHhex at 0.5, 2, and 24 h postinjection. High melanoma uptake coupled with low renal uptake highlighted the potential of (67)Ga-NOTA-GGNle-CycMSHhex for melanoma imaging and therapy.

Gallium-67-Labeled Lactam Bridge-Cyclized Alpha-MSH Peptides with Enhanced Melanoma Uptake and Reduced Renal Uptake

PubMed Central

Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

2012-01-01

The purpose of this study was to examine the melanoma targeting and pharmacokinetic properties of 67Ga-DOTA-GGNle-CycMSHhex {67Ga-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} and 67Ga-NOTA-GGNle-CycMSHhex {67Ga-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2} and compare with 67Ga-DOTA-GlyGlu-CycMSH {67Ga-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-dPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]} we previously reported. DOTA-GGNle-CycMSHhex and NOTA-GGNle-CycMSHhex were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and pharmacokinetic properties of 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs. 2.1 nM) in B16/F1 melanoma cells. Both 67Ga-NOTA-GGNle-CycMSHhex and 67Ga-DOTA-GGNle-CycMSHhex exhibited dramatically enhanced melanoma uptake and reduced renal uptake than 67Ga-DOTA-GlyGlu-CycMSH in B16/F1 melanoma-bearing C57 mice. Furthermore, 67Ga-NOTA-GGNle-CycMSHhexexhibited more favorable radiolabeling conditions (> 85% radiolabeling yields started at 37°C), as well as higher tumor/kidney uptake ratios than 67Ga-DOTA-GGNle-CycMSHhex at 0.5, 2 and 24 h post-injection. High melanoma uptake coupled with low renal uptake highlighted the potential of 67Ga-NOTA-GGNle-CycMSHhexfor melanoma imaging and therapy. PMID:22621181

Interaction of GABA-mimetics with the taurine transporter (TauT, Slc6a6) in hyperosmotic treated Caco-2, LLC-PK1 and rat renal SKPT cells.

PubMed

Rasmussen, Rune Nørgaard; Lagunas, Candela; Plum, Jakob; Holm, René; Nielsen, Carsten Uhd

2016-01-20

The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues. Copyright © 2015 Elsevier B.V. All rights reserved.

Cellular Uptake of Chloroquine Is Dependent on Binding to Ferriprotoporphyrin IX and Is Independent of NHE Activity in Plasmodium falciparum

PubMed Central

Bray, Patrick G.; Janneh, Omar; Raynes, Kaylene J.; Mungthin, Mathirut; Ginsburg, Hagai; Ward, Stephen A.

1999-01-01

Here we provide definitive evidence that chloroquine (CQ) uptake in Plasmodium falciparum is determined by binding to ferriprotoporphyrin IX (FPIX). Specific proteinase inhibitors that block the degradation of hemoglobin and stop the generation of FPIX also inhibit CQ uptake. Food vacuole enzymes can generate cell-free binding, using human hemoglobin as a substrate. This binding accounts for CQ uptake into intact cells and is subject to identical inhibitor specificity. Inhibition of CQ uptake by amiloride derivatives occurs because of inhibition of CQ–FPIX binding rather than inhibition of the Na+/H+ exchanger (NHE). Inhibition of parasite NHE using a sodium-free medium does not inhibit CQ uptake nor does it alter the ability of amilorides to inhibit uptake. CQ resistance is characterized by a reduced affinity of CQ–FPIX binding that is reversible by verapamil. Diverse compounds that are known to disrupt lysosomal pH can mimic the verapamil effect. These effects are seen in sodium-free medium and are not due to stimulation of the NHE. We propose that these compounds increase CQ accumulation and overcome CQ resistance by increasing the pH of lysosomes and endosomes, thereby causing an increased affinity of binding of CQ to FPIX. PMID:10209030

Taurine uptake by human retinal pigment epithelium: implications for the transport of small solutes between the choroid and the outer retina.

PubMed

Hillenkamp, Jost; Hussain, Ali A; Jackson, Timothy L; Cunningham, Joanna R; Marshall, John

2004-12-01

To characterize the Michaelis-Menten kinetics of the taurine transporter (TT) in retinal pigment epithelium (RPE) freshly isolated from human donor eyes. To identify the rate limiting compartment in the pathway of taurine delivery from the choroidal blood supply to the outer retina composed by Bruch's-choroid (BC) and the RPE in the human older age group. In human donor samples (4 melanoma-affected eyes, and 14 control eyes; age range, 62-93 years), radiochemical techniques were used to determine the RPE taurine accumulation at various exogenous concentrations. The transport capability of human RPE was obtained from a kinetic analysis of the high-affinity carrier over a substrate concentration of 1 to 60 microM taurine. Uptake of taurine into human RPE at a taurine concentration of 1 microM was independent of donor age (P > 0.05) and averaged at 2.83 +/- 0.27 (SEM) pmol/10 minutes per 6-mm trephine. Taurine transport by human RPE was mediated by a high-affinity carrier of K(m) 50 microM and V(max) of 267 pmol/10 minutes per 5-mm disc. In human donor RPE, uptake of taurine remained viable in the age range 62 to 93 years. Taurine transport rates in the RPE were lower than across the isolated BC complex, and thus the data suggest that the former compartment houses the rate-limiting step in the delivery of taurine to the outer retina.

The use of one-bead one-compound combinatorial library technology to discover high-affinity αvβ3 integrin and cancer targeting arginine-glycine-aspartic acid ligands with a built-in handle.

PubMed

Xiao, Wenwu; Wang, Yan; Lau, Edmond Y; Luo, Juntao; Yao, Nianhuan; Shi, Changying; Meza, Leah; Tseng, Harry; Maeda, Yoshiko; Kumaresan, Pappanaicken; Liu, Ruiwu; Lightstone, Felice C; Takada, Yoshikazu; Lam, Kit S

2010-10-01

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiologic processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted therapy and cancer imaging. In this report, one-bead one-compound (OBOC) combinatorial libraries containing the arginine-glycine-aspartic acid (RGD) motif were designed and screened against K562 myeloid leukemia cells that had been transfected with the human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a built-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC(50) = 0.68 ± 0.08 μmol/L) to some of the well-known RGD "head-to-tail" cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2- to 8-fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with the biotinylated ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake.

Independent Colimitation for Carbon Dioxide and Inorganic Phosphorus

PubMed Central

Spijkerman, Elly; de Castro, Francisco; Gaedke, Ursula

2011-01-01

Simultaneous limitation of plant growth by two or more nutrients is increasingly acknowledged as a common phenomenon in nature, but its cellular mechanisms are far from understood. We investigated the uptake kinetics of CO2 and phosphorus of the algae Chlamydomonas acidophila in response to growth at limiting conditions of CO2 and phosphorus. In addition, we fitted the data to four different Monod-type models: one assuming Liebigs Law of the minimum, one assuming that the affinity for the uptake of one nutrient is not influenced by the supply of the other (independent colimitation) and two where the uptake affinity for one nutrient depends on the supply of the other (dependent colimitation). In addition we asked whether the physiological response under colimitation differs from that under single nutrient limitation. We found no negative correlation between the affinities for uptake of the two nutrients, thereby rejecting a dependent colimitation. Kinetic data were supported by a better model fit assuming independent uptake of colimiting nutrients than when assuming Liebigs Law of the minimum or a dependent colimitation. Results show that cell nutrient homeostasis regulated nutrient acquisition which resulted in a trade-off in the maximum uptake rates of CO2 and phosphorus, possibly driven by space limitation on the cell membrane for porters for the different nutrients. Hence, the response to colimitation deviated from that to a single nutrient limitation. In conclusion, responses to single nutrient limitation cannot be extrapolated to situations where multiple nutrients are limiting, which calls for colimitation experiments and models to properly predict growth responses to a changing natural environment. These deviations from single nutrient limitation response under colimiting conditions and independent colimitation may also hold for other nutrients in algae and in higher plants. PMID:22145031

Synthesis and evaluation of [125I]I-TSA as a brain nicotinic acetylcholine receptor alpha7 subtype imaging agent.

PubMed

Ogawa, Mikako; Tatsumi, Ryo; Fujio, Masakazu; Katayama, Jiro; Magata, Yasuhiro

2006-04-01

Some in vitro investigations have suggested that the nicotinic acetylcholine receptor (nAChR) alpha7 subtype is implicated in Alzheimer's disease, schizophrenia and others. Recently, we developed (R)-3'-(5-bromothiophen-2-yl)spiro[1-azabicyclo[2.2.2]octane-3,5'-[1',3']oxazolidin]-2'-one (Br-TSA), which has a high affinity and selectivity for alpha7 nAChRs. Therefore we synthesized (R)-3'-(5-[125I]iodothiophen-2-yl)spiro[1-azabicyclo[2.2.2]octane-3,5'-[1',3']oxazolidin]-2'-one ([125I]I-TSA) and evaluated its potential for the in vivo detection of alpha7 nAChR in brain. In vitro binding affinity of I-TSA was measured in rat brain homogenates. Radioiodination was accomplished by a Br-I exchange reaction. Biodistribution studies were undertaken in mice by tail vein injection of [(125)I]I-TSA. In vivo receptor blocking studies were carried out by treating mice with methyllycaconitine (MLA; 5 nmol/5 mul, i.c.v.) or nonradioactive I-TSA (50 micromol/kg, i.v.). I-TSA exhibited a high affinity and selectivity for the alpha7 nAChR (K(i) for alpha7 nAChR = 0.54 nM). Initial uptake in the brain was high (4.42 %dose/g at 5 min), and the clearance of radioactivity was relatively slow in the hippocampus (alpha7 nAChR-rich region) and was rather rapid in the cerebellum (alpha7 nAChR poor region). The hippocampus to cerebellum uptake ratio was 0.9 at 5 min postinjection, but it was increased to 1.8 at 60 min postinjection. Although the effect was not statistically significant, administration of I-TSA and MLA decreased the accumulation of radioactivity in hippocampus. Despite its high affinity and selectivity, [125I]I-TSA does not appear to be a suitable tracer for in vivo alpha7 nAChR receptor imaging studies due to its high nonspecific binding. Further structural optimization is needed.

Theranostic Value of Multimers: Lessons Learned from Trimerization of Neurotensin Receptor Ligands and Other Targeting Vectors

PubMed Central

Maschauer, Simone; Einsiedel, Jürgen; Reich, Dominik; Hübner, Harald; Gmeiner, Peter; Wester, Hans-Jürgen; Prante, Olaf; Notni, Johannes

2017-01-01

Neurotensin receptor 1 (NTS1) is overexpressed on a variety of cancer entities; for example, prostate cancer, ductal pancreatic adenocarcinoma, and breast cancer. Therefore, it represents an interesting target for the diagnosis of these cancers types by positron emission tomography (PET). The metabolically-stabilized neurotensin (NT) derivative peptide Nlys8-Lys9-Pro10-Tyr11-Tle12-Leu13-OH was elongated at the N-terminus with 6-azido norleucine and coupled with the 1,4,7-triazacyclononane-1,4,7-tris[(2-carboxyethyl)methylenephosphinic acid] (TRAP) chelator TRAP(alkyne)3 in order to synthesize a NT trimer with subnanomolar affinity and high stability. The 68Ga-labeled peptide [68Ga]Ga-TRAP(NT4)3 was characterized in vitro using the NTS1-expressing human colorectal adenocarcinoma cell line HT29. It displayed fast and high internalization rates of >90%, but also fast efflux rates of 50% over 15 min. In vivo, [68Ga]Ga-TRAP(NT4)3 showed moderate HT29 tumor uptake values of 1.7 %ID/g at 60 min post-injection (p.i.), but also high uptake and retention in the kidneys and liver. A comparison of data for trimer/monomer pairs of NT ligands and other targeting vectors (peptides and peptoids targeting integrins αvβ3, α5β1, and αvβ6, the PSMA-ligand DUPA (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), and nitroimidazoles targeting hypoxia) revealed that multimers always exhibit higher target affinities and tumor uptake, but not necessarily improved tumor-to-tissue ratios. Thus, although in vitro data are not suitable for prediction of in vivo performance, multimers are potentially superior to monomers, particularly for applications where high tumor accumulation is crucial. PMID:28287433

Affinity interactions between natural pigments and human whole saliva.

PubMed

Yao, Jiang-Wu; Lin, Feng; Tao, Tao; Lin, Chang-Jian

2011-03-01

The aim of the present study was to assess the null hypothesis that there are no differences of affinity between pigments and human whole saliva (WS), and the affinity is not influenced by the functional groups of pigments, temperatures, pH values, and salt concentrations. The affinity constants of interactions between WS and theaflavin (TF)/curcumin (Cur)/cyanidin (Cy) were determined by surface plasmon resonance (SPR) and fluorescence quenching. Mass-uptake at various temperatures, pH values, and salt concentrations was also carried out. The order of affinity of the pigments binding to WS is TF>Cur>Cy. A large number of complexes and precipitations of pigments/proteins were formed through a quick, strong, and almost irreversible binding process. The mass-uptake of pigments was affected not only by the functional groups, but also by molecular weight of pigments, temperatures, pH values, and salt concentrations. The complex of pigments may easily and rapidly deposit onto the WS film, and are difficult to remove from the WS surface. However, the complex of pigments can be reduced by properly regulating the physicochemical conditions, such as temperatures, pH values, and salt concentrations. Copyright © 2010 Elsevier Ltd. All rights reserved.

Transport of EDTA into cells of the EDTA-degrading bacterial strain DSM 9103.

PubMed

Witschel, M; Egli, T; Zehnder, A J; Wehrli, E; Spycher, M

1999-04-01

In the bacterial strain DSM 9103, which is able to grow with the complexing agent EDTA as the sole source of carbon, nitrogen and energy, the transport of EDTA into whole cells was investigated. EDTA uptake was found to be dependent on speciation: free EDTA and metal-EDTA complexes with low stability constants were readily taken up, whereas those with stability constants higher than 1016 were not transported. In EDTA-grown cells, initial transport rates of CaEDTA showed substrate-saturation kinetics with a high apparent affinity for CaEDTA (affinity constant Kt= 0.39 microM). Several uncouplers had an inhibitory effect on CaEDTA transport. CaEDTA uptake was also significantly reduced in the presence of an inhibitor of ATPase and the ionophore nigericin, which dissipates the proton gradient. Valinomycin, however, which affects the electrical potential, had little effect on uptake, indicating that EDTA transport is probably driven by the proton gradient. Of various structurally related compounds tested only Ca2+-complexed diethylenetriaminepentaacetate (CaDTPA) competitively inhibited CaEDTA transport. Uptake in fumarate-grown cells was low compared to that measured in EDTA-grown bacteria. These results strongly suggest that the first step in EDTA degradation by strain DSM 9103 consists of transport by an inducible energy-dependent carrier. Uptake experiments with 45Ca2+ in the presence and absence of EDTA indicated that Ca2+ is transported together with EDTA into the cells. In addition, these transport studies and electron-dispersive X-ray analysis of electron-dense intracellular bodies present in EDTA-grown cells suggest that two mechanisms acting simultaneously allow the cells to cope with the large amounts of metal ions taken up together with EDTA. In one mechanism the metal ions are excreted, in the other they are inactivated intracellularly in polyphosphate granules.

Transport of selenium across the plasma membrane of primary hepatocytes and enterocytes of rainbow trout.

PubMed

Misra, Sougat; Kwong, Raymond W M; Niyogi, Som

2012-05-01

Transport of essential solutes across biological membranes is one of the fundamental characteristics of living cells. Although selenium is an essential micronutrient, little is known about the cellular mechanisms of chemical species-specific selenium transport in fish. We report here the kinetic and pharmacological transport characteristics of selenite and its thiol (glutathione and l-cysteine) derivatives in primary cultures of hepatocytes and isolated enterocytes of rainbow trout. Findings from the current study suggest an apparent low-affinity linear transport system for selenite in both cell types. However, we recorded high-affinity Hill kinetics (K(d)=3.61±0.28 μmol l(-1)) in enterocytes exposed to selenite in the presence of glutathione. The uptake of selenite in the presence of thiols was severalfold higher than uptake of selenite alone (at equimolar concentration) in both hepatocytes and enterocytes. Cellular accumulation of selenium was found to be energy independent. Interestingly, we observed a decrease in selenite transport with increasing pH, whereas selenite uptake increased with increasing pH in the presence glutathione in both cell types. The cellular uptake of selenite demonstrated a pronounced competitive interaction with a structurally similar compound, sulfite. The uptake of selenite as well as its thiol derivatives was found to be sensitive to the anion transport blocker DIDS, irrespective of the cell type. Inorganic mercury (Hg(2+)) elicited an inhibition of selenite transport in both cell types, but augmented the transport of reduced forms of selenite in hepatocytes. Based on the substrate choice and comparable pharmacological properties, we advocate that multiple anion transport systems are probably involved in the cellular transport of selenite in fish.

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent.

PubMed

Howlett, Robert M; Hughes, Bethan M; Hitchcock, Andrew; Kelly, David J

2012-06-01

Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K(d) value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of (63)NiCl(2). The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.

An investigation into IgE-facilitated allergen recognition and presentation by human dendritic cells

PubMed Central

2013-01-01

Background Allergen recognition by dendritic cells (DCs) is a key event in the allergic cascade leading to production of IgE antibodies. C-type lectins, such as the mannose receptor and DC-SIGN, were recently shown to play an important role in the uptake of the house dust mite glycoallergen Der p 1 by DCs. In addition to mannose receptor (MR) and DC-SIGN the high and low affinity IgE receptors, namely FcϵRI and FcϵRII (CD23), respectively, have been shown to be involved in allergen uptake and presentation by DCs. Objectives This study aims at understanding the extent to which IgE- and IgG-facilitated Der p 1 uptake by DCs influence T cell polarisation and in particular potential bias in favour of Th2. We have addressed this issue by using two chimaeric monoclonal antibodies produced in our laboratory and directed against a previously defined epitope on Der p 1, namely human IgE 2C7 and IgG1 2C7. Results Flow cytometry was used to establish the expression patterns of IgE (FcϵRI and FcϵRII) and IgG (FcγRI) receptors in relation to MR on DCs. The impact of FcϵRI, FcϵRII, FcγRI and mannose receptor mediated allergen uptake on Th1/Th2 cell differentiation was investigated using DC/T cell co-culture experiments. Myeloid DCs showed high levels of FcϵRI and FcγRI expression, but low levels of CD23 and MR, and this has therefore enabled us to assess the role of IgE and IgG-facilitated allergen presentation in T cell polarisation with minimal interference by CD23 and MR. Our data demonstrate that DCs that have taken up Der p 1 via surface IgE support a Th2 response. However, no such effect was demonstrable via surface IgG. Conclusions IgE bound to its high affinity receptor plays an important role in Der p 1 uptake and processing by peripheral blood DCs and in Th2 polarisation of T cells. PMID:24330349

An investigation into IgE-facilitated allergen recognition and presentation by human dendritic cells.

PubMed

Sharquie, Inas K; Al-Ghouleh, Abeer; Fitton, Patricia; Clark, Mike R; Armour, Kathryn L; Sewell, Herb F; Shakib, Farouk; Ghaemmaghami, Amir M

2013-12-13

Allergen recognition by dendritic cells (DCs) is a key event in the allergic cascade leading to production of IgE antibodies. C-type lectins, such as the mannose receptor and DC-SIGN, were recently shown to play an important role in the uptake of the house dust mite glycoallergen Der p 1 by DCs. In addition to mannose receptor (MR) and DC-SIGN the high and low affinity IgE receptors, namely FcεRI and FcεRII (CD23), respectively, have been shown to be involved in allergen uptake and presentation by DCs. This study aims at understanding the extent to which IgE- and IgG-facilitated Der p 1 uptake by DCs influence T cell polarisation and in particular potential bias in favour of Th2. We have addressed this issue by using two chimaeric monoclonal antibodies produced in our laboratory and directed against a previously defined epitope on Der p 1, namely human IgE 2C7 and IgG1 2C7. Flow cytometry was used to establish the expression patterns of IgE (FcεRI and FcεRII) and IgG (FcγRI) receptors in relation to MR on DCs. The impact of FcεRI, FcεRII, FcγRI and mannose receptor mediated allergen uptake on Th1/Th2 cell differentiation was investigated using DC/T cell co-culture experiments. Myeloid DCs showed high levels of FcεRI and FcγRI expression, but low levels of CD23 and MR, and this has therefore enabled us to assess the role of IgE and IgG-facilitated allergen presentation in T cell polarisation with minimal interference by CD23 and MR. Our data demonstrate that DCs that have taken up Der p 1 via surface IgE support a Th2 response. However, no such effect was demonstrable via surface IgG. IgE bound to its high affinity receptor plays an important role in Der p 1 uptake and processing by peripheral blood DCs and in Th2 polarisation of T cells.

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

PubMed Central

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

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

NASA Technical Reports Server (NTRS)

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

1999-01-01

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

Recent advances in hyaluronic acid-decorated nanocarriers for targeted cancer therapy

PubMed Central

Wickens, Jennifer M.; Alsaab, Hashem O.; Kesharwani, Prashant; Bhise, Ketki; Amin, Mohd Cairul Iqbal Mohd; Tekade, Rakesh Kumar; Gupta, Umesh; Iyer, Arun K.

2016-01-01

The cluster-determinant 44 (CD44) receptor has a high affinity for hyaluronic acid (HA) binding and is a desirable receptor for active targeting based on its overexpression in cancer cells compared with normal body cells. The nanocarrier affinity can be increased by conjugating drug-loaded carriers with HA, allowing enhanced cancer cell uptake via the HA-CD44 receptor-mediated endocytosis pathway. In this review, we discuss recent advances in HA-based nanocarriers and micelles for cancer therapy. In vitro and in vivo experiments have repeatedly indicated HA-based nanocarriers to be a target-specific drug and gene delivery platform with great promise for future applications in clinical cancer therapy. PMID:28017836

Identification of the bacteria scavenging atmospheric CO and evaluation of the impact of land-use change on their distribution and activity

NASA Astrophysics Data System (ADS)

Constant, P.; Quiza, L.; Lalonde, I.

2013-12-01

Soil bacteria scavenging carbon monoxide (CO) are responsible for the biological sink of atmospheric CO. These bacteria mitigate an important fraction of the global emissions of CO from natural and anthropogenic sources. This ubiquitous soil ecosystem service is of critical importance since CO indirectly regulates the atmospheric lifetime of methane - the second most powerful greenhouse gas. So far, only few carboxydovore bacteria were shown to oxidize atmospheric CO. The CO-dehydrogenase (CODH) is the enzyme catalyzing the CO oxidation reaction in these bacteria. The enzyme is a dimer of heterotrimers encoded by the genes coxS, coxM and coxL. CoxL is the large subunit of the CODH. Phylogenetic analyzes revealed that coxL gene sequences encompass two main clusters: BMS and OMP groups but the version conferring a high affinity for CO and the ability to scavenge atmospheric CO is unknown. The objective of this investigation was to relate the diversity of coxL gene sequences with CO soil uptake activity and soil physicochemical properties. For this purpose, we collected soil samples in three neighbouring sites encompassing different land-use types: an undisturbed deciduous forest, a maize field and a larch monoculture. We analyzed (i) coxL diversity in the three environments, using a new coxL PCR detection assay targeting both OMP and BMS groups, (ii) CO oxidation activity using a gas chromatography assay and, (iii) soil physicochemical properties. Our results demonstrate that land-use change exerts a significant impact on coxL diversity as well as CO oxidation activity, with significant loss of the potential CO soil uptake activity following the conversion of native forest to maize or larch plantation. Most of the coxL gene sequences retrieved from the soil samples were not affiliated to sequences derived from microbial genome databases, impairing a taxonomic identification of the potential CO-oxidizing bacteria detected in soil. Canonical ordination analysis allowed us to identify coxL sequences belonging to potential high affinity CO-oxidizing bacteria, in addition to recognise environmental factors influencing their distribution and CO soil uptake activity. The activity increased with total carbon and nitrogen in soil and was inversely correlated to water content, pH, potassium and phosphorus. Candidates belonging to OMP group were identified as potential high affinity CO oxidizing bacteria. These bacteria were enriched in the laboratory and tested for their CO uptake activity. Work is currently in progress to assess the abundance and the CO uptake activity of these microorganisms in soil. Taken together, these results will be implemented into molecular models aimed at predicting CO uptake activity in soil. These models will be utilized to predict the response of the biological sink of CO to global change, while determining how land management practices could protect this important ecosystem service.

N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging.

PubMed

Gourni, Eleni; Mansi, Rosalba; Jamous, Mazen; Waser, Beatrice; Smerling, Christiane; Burian, Antje; Buchegger, Franz; Reubi, Jean Claude; Maecke, Helmut R

2014-10-01

Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Prospective Design of Anti‐Transferrin Receptor Bispecific Antibodies for Optimal Delivery into the Human Brain

PubMed Central

Kanodia, JS; Gadkar, K; Bumbaca, D; Zhang, Y; Tong, RK; Luk, W; Hoyte, K; Lu, Y; Wildsmith, KR; Couch, JA; Watts, RJ; Dennis, MS; Ernst, JA; Scearce‐Levie, K; Atwal, JK; Joseph, S

2016-01-01

Anti‐transferrin receptor (TfR)‐based bispecific antibodies have shown promise for boosting antibody uptake in the brain. Nevertheless, there are limited data on the molecular properties, including affinity required for successful development of TfR‐based therapeutics. A complex nonmonotonic relationship exists between affinity of the anti‐TfR arm and brain uptake at therapeutically relevant doses. However, the quantitative nature of this relationship and its translatability to humans is heretofore unexplored. Therefore, we developed a mechanistic pharmacokinetic‐pharmacodynamic (PK‐PD) model for bispecific anti‐TfR/BACE1 antibodies that accounts for antibody‐TfR interactions at the blood‐brain barrier (BBB) as well as the pharmacodynamic (PD) effect of anti‐BACE1 arm. The calibrated model correctly predicted the optimal anti‐TfR affinity required to maximize brain exposure of therapeutic antibodies in the cynomolgus monkey and was scaled to predict the optimal affinity of anti‐TfR bispecifics in humans. Thus, this model provides a framework for testing critical translational predictions for anti‐TfR bispecific antibodies, including choice of candidate molecule for clinical development. PMID:27299941

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2.

PubMed

Song, Jie; Baker, Nicola; Rothert, Monja; Henke, Björn; Jeacock, Laura; Horn, David; Beitz, Eric

2016-02-01

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.

[Relations between location of elements in periodic table and affinity for the kidneys (author's transl)].

PubMed

Ando, A; Hisada, K; Ando, I

1977-10-01

The distribution of many inorganic compounds in rats was investigated in order to evaluate kidney affinity of inorganic compounds. In these experiments, 30%, 10-20% and 4-10% of administered dose was localized in the kidneys in 203Hg-acetate and 203 Bi-acetate, in H198AuCl4, 103PdCl2, 201TlCl, 210Pd(NO3)2 and H2(127M)TeO3, and in Na2(51)CrO4, 54MnCl2, (114m)InCl3 and 7BeCl2, respectively. Some bipositive ions and anions was hardly taken up into the kidneys. And in many hard acids according to classification of Lewis acids, the uptake rate into the kidneys was usually small. On the other hand, Hg, Au and Bi, which have strong binding power to the protein, showed high uptake rate in the kidneys. As Hg++, Au+ and Bi+++ was soft acids according to classification of Lewis acids, it was thought that these elements would bind strongly to soft base (RSH, RS-) present in the kidney.

Description of two-metal biosorption equilibria by Langmuir-type models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chong, K.H.; Volesky, B.

A biosorbent prepared from Ascophyllum nodosum seaweed biomass, FCAN2, was examined for its sorption capacity. Equilibrium batch sorption studies were performed using two-metal systems containing either (Cu+Zn), (Cu+Cd), or (Zn+Cd). In the evaluation of the two-metal sorption system performance, simple isotherm curves had to be replaced by three-dimensional sorption isotherm surfaces. In order to describe the isotherm surfaces mathematically, three Langmuir-type models were evaluated. The apparent one-parameter Langmuir constant (b) was used to quantify FCAN2 ``affinity`` for one metal in the presence of another one. The uptake of Zn decreased drastically when Cu of Cd were present. The uptake ofmore » Cd was much more sensitive to the presence of Cu than to that of Zn. The presence of Cd and Zn alter the ``affinity`` of FCAN2 for Cu the least at high Cu equilibrium concentrations. The mathematical model of the two-metal sorption system enabled quantitative estimation of one-metal (bio)sorption inhibition due to the influence of a second metal.« less

Genome mining and functional genomics for siderophore production in Aspergillus niger.

PubMed

Franken, Angelique C W; Lechner, Beatrix E; Werner, Ernst R; Haas, Hubertus; Lokman, B Christien; Ram, Arthur F J; van den Hondel, Cees A M J J; de Weert, Sandra; Punt, Peter J

2014-11-01

Iron is an essential metal for many organisms, but the biologically relevant form of iron is scarce because of rapid oxidation resulting in low solubility. Simultaneously, excessive accumulation of iron is toxic. Consequently, iron uptake is a highly controlled process. In most fungal species, siderophores play a central role in iron handling. Siderophores are small iron-specific chelators that can be secreted to scavenge environmental iron or bind intracellular iron with high affinity. A second high-affinity iron uptake mechanism is reductive iron assimilation (RIA). As shown in Aspergillus fumigatus and Aspergillus nidulans, synthesis of siderophores in Aspergilli is predominantly under control of the transcription factors SreA and HapX, which are connected by a negative transcriptional feedback loop. Abolishing this fine-tuned regulation corroborates iron homeostasis, including heme biosynthesis, which could be biotechnologically of interest, e.g. the heterologous production of heme-dependent peroxidases. Aspergillus niger genome inspection identified orthologues of several genes relevant for RIA and siderophore metabolism, as well as sreA and hapX. Interestingly, genes related to synthesis of the common fungal extracellular siderophore triacetylfusarinine C were absent. Reverse-phase high-performance liquid chromatography (HPLC) confirmed the absence of triacetylfusarinine C, and demonstrated that the major secreted siderophores of A. niger are coprogen B and ferrichrome, which is also the dominant intracellular siderophore. In A. niger wild type grown under iron-replete conditions, the expression of genes involved in coprogen biosynthesis and RIA was low in the exponential growth phase but significantly induced during ascospore germination. Deletion of sreA in A. niger resulted in elevated iron uptake and increased cellular ferrichrome accumulation. Increased sensitivity toward phleomycin and high iron concentration reflected the toxic effects of excessive iron uptake. Moreover, SreA-deficiency resulted in increased accumulation of heme intermediates, but no significant increase in heme content. Together with the upregulation of several heme biosynthesis genes, these results reveal a complex heme regulatory mechanism. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Molecular analysis of maltotriose transport and utilization by Saccharomyces cerevisiae.

PubMed

Day, Rachel E; Rogers, Peter J; Dawes, Ian W; Higgins, Vincent J

2002-11-01

Efficient fermentation of maltotriose is a desired property of Saccharomyces cerevisiae for brewing. In a standard wort, maltotriose is the second most abundant sugar, and slower uptake leads to residual maltotriose in the finished product. The limiting factor of sugar metabolism is its transport, and there are conflicting reports on whether a specific maltotriose permease exists or whether the mechanisms responsible for maltose uptake also carry out maltotriose transport. In this study, radiolabeled maltotriose was used to show that overexpression of the maltose permease gene, MAL61, in an industrial yeast strain resulted in an increase in the rate of transport of maltotriose as well as maltose. A strain derived from W303-1A and lacking any maltose or maltotriose transporter but carrying a functional maltose transport activator (MAL63) was developed. By complementing this strain with permeases encoded by MAL31, MAL61, and AGT1, it was possible to measure their specific transport kinetics by using maltotriose and maltose. All three permeases were capable of high-affinity transport of maltotriose and of allowing growth of the strain on the sugar. Maltotriose utilization from the permease encoded by AGT1 was regulated by the same genetic mechanisms as those involving the maltose transcriptional activator. Competition studies carried out with two industrial strains, one not containing any homologue of AGT1, showed that maltose uptake and maltotriose uptake were competitive and that maltose was the preferred substrate. These results indicate that the presence of residual maltotriose in beer is not due to a genetic or physiological inability of yeast cells to utilize the sugar but rather to the lower affinity for maltotriose uptake in conjunction with deteriorating conditions present at the later stages of fermentation. Here we identify molecular mechanisms regulating the uptake of maltotriose and determine the role of each of the transporter genes in the cells.

Affinity of Tau antibodies for solubilized pathological Tau species but not their immunogen or insoluble Tau aggregates predicts in vivo and ex vivo efficacy.

PubMed

Congdon, Erin E; Lin, Yan; Rajamohamedsait, Hameetha B; Shamir, Dov B; Krishnaswamy, Senthilkumar; Rajamohamedsait, Wajitha J; Rasool, Suhail; Gonzalez, Veronica; Levenga, Josien; Gu, Jiaping; Hoeffer, Charles; Sigurdsson, Einar M

2016-08-30

A few tau immunotherapies are now in clinical trials with several more likely to be initiated in the near future. A priori, it can be anticipated that an antibody which broadly recognizes various pathological tau aggregates with high affinity would have the ideal therapeutic properties. Tau antibodies 4E6 and 6B2, raised against the same epitope region but of varying specificity and affinity, were tested for acutely improving cognition and reducing tau pathology in transgenic tauopathy mice and neuronal cultures. Surprisingly, we here show that one antibody, 4E6, which has low affinity for most forms of tau acutely improved cognition and reduced soluble phospho-tau, whereas another antibody, 6B2, which has high affinity for various tau species was ineffective. Concurrently, we confirmed and clarified these efficacy differences in an ex vivo model of tauopathy. Alzheimer's paired helical filaments (PHF) were toxic to the neurons and increased tau levels in remaining neurons. Both toxicity and tau seeding were prevented by 4E6 but not by 6B2. Furthermore, 4E6 reduced PHF spreading between neurons. Interestingly, 4E6's efficacy relates to its high affinity binding to solubilized PHF, whereas the ineffective 6B2 binds mainly to aggregated PHF. Blocking 4E6's uptake into neurons prevented its protective effects if the antibody was administered after PHF had been internalized. When 4E6 and PHF were administered at the same time, the antibody was protective extracellularly. Overall, these findings indicate that high antibody affinity for solubilized PHF predicts efficacy, and that acute antibody-mediated improvement in cognition relates to clearance of soluble phospho-tau. Importantly, both intra- and extracellular clearance pathways are in play. Together, these results have major implications for understanding the pathogenesis of tauopathies and for development of immunotherapies.

Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity

PubMed Central

Jansen, Mickel L. A.; Daran-Lapujade, Pascale; de Winde, Johannes H.; Piper, Matthew D. W.; Pronk, Jack T.

2004-01-01

Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. PMID:15066785

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

PubMed Central

Bai, Erdeni; Rosell, Federico I.; Lige, Bao; Mauk, Marcia R.; Lelj-Garolla, Barbara; Moore, Geoffrey R.; Mauk, A. Grant

2006-01-01

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents. PMID:16928194

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

PubMed Central

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

2013-01-01

Purpose: To quantify the affinity of a cationic computed tomography (CT) contrast agent (CA4+) and that of an anionic contrast agent (ioxaglate) to glycosaminoglycans (GAGs) in ex vivo cartilage tissue explants and to characterize the in vivo diffusion kinetics of CA4+ and ioxaglate in a rabbit model. Materials and Methods: All in vivo procedures were approved by the institutional animal care and use committee. The affinities of ioxaglate and CA4+ to GAGs in cartilage (six bovine osteochondral plugs) were quantified by means of a modified binding assay using micro-CT after plug equilibration in serial dilutions of each agent. The contrast agents were administered intraarticularly to the knee joints of five New Zealand white rabbits to determine the in vivo diffusion kinetics and cartilage tissue imaging capabilities. Kinetics of diffusion into the femoral groove cartilage and relative contrast agent uptake into bovine plugs were characterized by means of nonlinear mixed-effects models. Diffusion time constants (τ) were compared by using a Student t test. Results: The uptake of CA4+ in cartilage was consistently over 100% of the reservoir concentration, whereas it was only 59% for ioxaglate. In vivo, the contrast material–enhanced cartilage reached a steady CT attenuation for both CA4+ and ioxaglate, with τ values of 13.8 and 6.5 minutes, respectively (P = .04). The cartilage was easily distinguishable from the surrounding tissues for CA4+ (12 mg of iodine per milliliter); comparatively, the anionic contrast agent provided less favorable imaging results, even when a higher concentration was used (80 mg of iodine per milliliter). Conclusion: The affinity of the cationic contrast agent CA4+ to GAGs enables high-quality imaging and segmentation of ex vivo bovine and rabbit cartilage, as well as in vivo rabbit cartilage. © RSNA, 2012 Supplemental material: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.12112246/-/DC1 PMID:23192774

Inhibition of the high affinity myo-inositol transport system: a common mechanism of action of antibipolar drugs?

PubMed

Lubrich, B; van Calker, D

1999-10-01

The mechanism of action of antibipolar drugs like lithium, carbamazepine, and valproate that are used in the treatment of manic-depressive illness, is unknown. Lithium is believed to act through uncompetitive inhibition of inositolmonophosphatase, which results in a depletion of neural cells of inositol and a concomitant modulation of phosphoinositol signaling. Here, we show that lithium ions, carbamazepine, and valproate, but not the tricyclic antidepressant amitriptyline, inhibit at therapeutically relevant concentrations and with a time course similar to their clinical actions the high affinity myo-inositol transport in astrocyte-like cells and downregulate the level of the respective mRNA. Inhibition of inositol uptake could thus represent an additional pathway for inositol depletion, which might be relevant in the mechanism of action of all three antibipolar drugs.

Crystallization and preliminary X-ray diffraction analysis of a high-affinity phosphate-binding protein endowed with phosphatase activity from Pseudomonas aeruginosa PAO1

PubMed Central

Djeghader, Ahmed; Gotthard, Guillaume; Suh, Andrew; Gonzalez, Daniel; Scott, Ken; Chabriere, Eric; Elias, Mikael

2013-01-01

In prokaryotes, phosphate starvation induces the expression of numerous phosphate-responsive genes, such as the pst operon including the high-affinity phosphate-binding protein (PBP or pstS) and alkaline phosphatases such as PhoA. This response increases the cellular inorganic phosphate import efficiency. Notably, some Pseudomonas species secrete, via a type-2 secretion system, a phosphate-binding protein dubbed LapA endowed with phosphatase activity. Here, the expression, purification, crystallization and X-ray data collection at 0.87 Å resolution of LapA are described. Combined with biochemical and enzymatic characterization, the structure of this intriguing phosphate-binding protein will help to elucidate the molecular origin of its phosphatase activity and to decipher its putative role in phosphate uptake. PMID:24100568

Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis.

PubMed

Pi, Hualiang; Helmann, John D

2017-11-28

Bacterial cells modulate transcription in response to changes in iron availability. The ferric uptake regulator (Fur) senses intracellular iron availability and plays a central role in maintaining iron homeostasis in Bacillus subtilis Here we utilized FrvA, a high-affinity Fe 2+ efflux transporter from Listeria monocytogenes , as an inducible genetic tool to deplete intracellular iron. We then characterized the responses of the Fur, FsrA, and PerR regulons as cells transition from iron sufficiency to deficiency. Our results indicate that the Fur regulon is derepressed in three distinct waves. First, uptake systems for elemental iron ( efeUOB ), ferric citrate ( fecCDEF ), and petrobactin ( fpbNOPQ ) are induced to prevent iron deficiency. Second, B. subtilis synthesizes its own siderophore bacillibactin ( dhbACEBF ) and turns on bacillibactin ( feuABC ) and hydroxamate siderophore ( fhuBCGD ) uptake systems to scavenge iron from the environment and flavodoxins ( ykuNOP ) to replace ferredoxins. Third, as iron levels decline further, an "iron-sparing" response ( fsrA , fbpAB , and fbpC ) is induced to block the translation of abundant iron-utilizing proteins and thereby permit the most essential iron-dependent enzymes access to the limited iron pools. ChIP experiments demonstrate that in vivo occupancy of Fur correlates with derepression of each operon, and the graded response observed here results, at least in part, from higher-affinity binding of Fur to the "late"-induced genes.

Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture.

PubMed

Nakanishi, Takeo; Fukushi, Akimasa; Sato, Masanobu; Yoshifuji, Mayuko; Gose, Tomoka; Shirasaka, Yoshiyuki; Ohe, Kazuyo; Kobayashi, Masato; Kawai, Keiichi; Tamai, Ikumi

2011-12-05

Since in vitro cell culture models often show altered apical transporter expression, they are not necessarily suitable for the analysis of renal transport processes. Therefore, we aimed here to investigate the usefulness of primary-cultured rat proximal tubular cells (PTCs) for this purpose. After isolation of renal cortical cells from rat kidneys, PTCs were enriched and the gene expression and function of apical transporters were analyzed by means of microarray, RT-PCR and uptake experiments. RT-PCR confirmed that the major apical transporters were expressed in rat PTCs. Na(+)-dependent uptake of α-methyl-d-glucopyranoside (αMG), ergothioneine and carnitine by the PTCs suggests functional expression of Sglts, Octn1 and Octn2, respectively. Inhibition of pH-dependent glycylsarcosine uptake by low concentration of cephalexin, which is a β-lactam antibiotics recognized by Pepts, indicates a predominant role of high affinity type Pept2, but not low affinity type Pept1, in the PTCs. Moreover, the permeability ratio of [(14)C]αMG (apical to basolateral/basolateral to apical) across PTCs was 4.3, suggesting that Sglt-mediated reabsorptive transport is characterized. In conclusion, our results indicate that rat PTCs in primary culture are found to be a promising in vitro model to evaluate reabsorption processes mediated at least by Sglts, Pept2, Octn1 and Octn2.

Interaction of environmental contaminants with zebrafish organic anion transporting polypeptide, Oatp1d1 (Slco1d1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Popovic, Marta; Zaja, Roko; Fent, Karl

Polyspecific transporters from the organic anion transporting polypeptide (OATP/Oatp) superfamily mediate the uptake of a wide range of compounds. In zebrafish, Oatp1d1 transports conjugated steroid hormones and cortisol. It is predominantly expressed in the liver, brain and testes. In this study we have characterized the transport of xenobiotics by the zebrafish Oatp1d1 transporter. We developed a novel assay for assessing Oatp1d1 interactors using the fluorescent probe Lucifer yellow and transient transfection in HEK293 cells. Our data showed that numerous environmental contaminants interact with zebrafish Oatp1d1. Oatp1d1 mediated the transport of diclofenac with very high affinity, followed by high affinity towardsmore » perfluorooctanesulfonic acid (PFOS), nonylphenol, gemfibrozil and 17α-ethinylestradiol; moderate affinity towards carbaryl, diazinon and caffeine; and low affinity towards metolachlor. Importantly, many environmental chemicals acted as strong inhibitors of Oatp1d1. A strong inhibition of Oatp1d1 transport activity was found by perfluorooctanoic acid (PFOA), chlorpyrifos-methyl, estrone (E1) and 17β-estradiol (E2), followed by moderate to low inhibition by diethyl phthalate, bisphenol A, 7-acetyl-1,1,3,4,4,6-hexamethyl-1,2,3,4 tetrahydronapthalene and clofibrate. In this study we identified Oatp1d1 as a first Solute Carrier (SLC) transporter involved in the transport of a wide range of xenobiotics in fish. Considering that Oatps in zebrafish have not been characterized before, our work on zebrafish Oatp1d1 offers important new insights on the understanding of uptake processes of environmental contaminants, and contributes to the better characterization of zebrafish as a model species. - Highlights: • We optimized a novel assay for determination of Oatp1d1 interactors • Oatp1d1 is the first SLC characterized fish xenobiotic transporter • PFOS, nonylphenol, diclofenac, EE2, caffeine are high affinity Oatp1d1substrates • PFOA, chlorpyrifos-methyl, E1, E2 are strong inhibitors of Oatp1d1 • PFOA and diclofenac can block Oatp1d1 binding of DHEAS, E3S and E17ß-glucuronide.« less

Frontal Decortication and Adaptive Changes in Striatal Cholinergic Neurons: Neuropharmacological and Behavioral Implications

DTIC Science & Technology

1989-11-24

ACh); choline (Oh); apomnorphine (APO); oxotremorine (OTMN); OXI, oxiracetam; SDHACU, sodium-dependent high affinity choline uptake: PC...control group, Dunnett’s test. TABLE 3- Restoration of the ACh increasing effect of oxotremorine by piracetam in DC rats. Striatal ACh content (nmoles/g...ACh content induced by oxotremorine and apomorphine useful model for studying means to restore the deficit in stria- acting at muscarine and dopamine

Tachycardia, reduced vagal capacity, and age-dependent ventricular dysfunction arising from diminished expression of the presynaptic choline transporter.

PubMed

English, Brett A; Appalsamy, Martin; Diedrich, Andre; Ruggiero, Alicia M; Lund, David; Wright, Jane; Keller, Nancy R; Louderback, Katherine M; Robertson, David; Blakely, Randy D

2010-09-01

Healthy cardiovascular function relies on a balanced and responsive integration of noradrenergic and cholinergic innervation of the heart. High-affinity choline uptake by cholinergic terminals is pivotal for efficient ACh production and release. To date, the cardiovascular impact of diminished choline transporter (CHT) expression has not been directly examined, largely due to the transporter's inaccessibility in vivo. Here, we describe findings from cardiovascular experiments using transgenic mice that bear a CHT genetic deficiency. Whereas CHT knockout (CHT(-/-)) mice exhibit early postnatal lethality, CHT heterozygous (CHT(+/-)) mice survive, grow, and reproduce normally and exhibit normal spontaneous behaviors. However, the CHT(+/-) mouse heart displays significantly reduced levels of high-affinity choline uptake accompanied by significantly reduced levels of ACh. Telemeterized recordings of cardiovascular function in these mice revealed tachycardia and hypertension at rest. After treadmill exercise, CHT(+/-) mice exhibited slower heart rate recovery, consistent with a diminished cholinergic reserve, a contention validated through direct vagal nerve stimulation. Echocardiographic and histological experiments revealed an age-dependent decrease in fractional shortening, increased left ventricular dimensions, and increased ventricular fibrosis, consistent with ventricular dysfunction. These cardiovascular phenotypes of CHT(+/-) mice encourage an evaluation of humans bearing reduced CHT expression for their resiliency in maintaining proper heart function as well as risk for cardiovascular disease.

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

PubMed

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

1996-04-01

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

Sphagnum Mosses - Masters of Efficient N-Uptake while Avoiding Intoxication

PubMed Central

Fritz, Christian; Lamers, Leon P. M.; Riaz, Muhammad; van den Berg, Leon J. L.; Elzenga, Theo J. T. M.

2014-01-01

Peat forming Sphagnum mosses are able to prevent the dominance of vascular plants under ombrotrophic conditions by efficiently scavenging atmospherically deposited nitrogen (N). N-uptake kinetics of these mosses are therefore expected to play a key role in differential N availability, plant competition, and carbon sequestration in Sphagnum peatlands. The interacting effects of rain N concentration and exposure time on moss N-uptake rates are, however, poorly understood. We investigated the effects of N-concentration (1, 5, 10, 50, 100, 500 µM), N-form (15N - ammonium or nitrate) and exposure time (0.5, 2, 72 h) on uptake kinetics for Sphagnum magellanicum from a pristine bog in Patagonia (Argentina) and from a Dutch bog exposed to decades of N-pollution. Uptake rates for ammonium were higher than for nitrate, and N-binding at adsorption sites was negligible. During the first 0.5 h, N-uptake followed saturation kinetics revealing a high affinity (Km 3.5–6.5 µM). Ammonium was taken up 8 times faster than nitrate, whereas over 72 hours this was only 2 times. Uptake rates decreased drastically with increasing exposure times, which implies that many short-term N-uptake experiments in literature may well have overestimated long-term uptake rates and ecosystem retention. Sphagnum from the polluted site (i.e. long-term N exposure) showed lower uptake rates than mosses from the pristine site, indicating an adaptive response. Sphagnum therefore appears to be highly efficient in using short N pulses (e.g. rainfall in pristine areas). This strategy has important ecological and evolutionary implications: at high N input rates, the risk of N-toxicity seems to be reduced by lower uptake rates of Sphagnum, at the expense of its long-term filter capacity and related competitive advantage over vascular plants. As shown by our conceptual model, interacting effects of N-deposition and climate change (changes in rainfall) will seriously alter the functioning of Sphagnum peatlands. PMID:24416125

Sphagnum mosses--masters of efficient N-uptake while avoiding intoxication.

PubMed

Fritz, Christian; Lamers, Leon P M; Riaz, Muhammad; van den Berg, Leon J L; Elzenga, Theo J T M

2014-01-01

Peat forming Sphagnum mosses are able to prevent the dominance of vascular plants under ombrotrophic conditions by efficiently scavenging atmospherically deposited nitrogen (N). N-uptake kinetics of these mosses are therefore expected to play a key role in differential N availability, plant competition, and carbon sequestration in Sphagnum peatlands. The interacting effects of rain N concentration and exposure time on moss N-uptake rates are, however, poorly understood. We investigated the effects of N-concentration (1, 5, 10, 50, 100, 500 µM), N-form ((15)N-ammonium or nitrate) and exposure time (0.5, 2, 72 h) on uptake kinetics for Sphagnum magellanicum from a pristine bog in Patagonia (Argentina) and from a Dutch bog exposed to decades of N-pollution. Uptake rates for ammonium were higher than for nitrate, and N-binding at adsorption sites was negligible. During the first 0.5 h, N-uptake followed saturation kinetics revealing a high affinity (Km 3.5-6.5 µM). Ammonium was taken up 8 times faster than nitrate, whereas over 72 hours this was only 2 times. Uptake rates decreased drastically with increasing exposure times, which implies that many short-term N-uptake experiments in literature may well have overestimated long-term uptake rates and ecosystem retention. Sphagnum from the polluted site (i.e. long-term N exposure) showed lower uptake rates than mosses from the pristine site, indicating an adaptive response. Sphagnum therefore appears to be highly efficient in using short N pulses (e.g. rainfall in pristine areas). This strategy has important ecological and evolutionary implications: at high N input rates, the risk of N-toxicity seems to be reduced by lower uptake rates of Sphagnum, at the expense of its long-term filter capacity and related competitive advantage over vascular plants. As shown by our conceptual model, interacting effects of N-deposition and climate change (changes in rainfall) will seriously alter the functioning of Sphagnum peatlands.

Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants.

PubMed

Kristensen, Anders S; Larsen, Mads B; Johnsen, Laust B; Wiborg, Ove

2004-03-01

The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.

Znu is the predominant zinc importer in Yersinia pestis during in vitro growth but is not essential for virulence.

PubMed

Desrosiers, Daniel C; Bearden, Scott W; Mier, Ildefonso; Abney, Jennifer; Paulley, James T; Fetherston, Jacqueline D; Salazar, Juan C; Radolf, Justin D; Perry, Robert D

2010-12-01

Little is known about Zn homeostasis in Yersinia pestis, the plague bacillus. The Znu ABC transporter is essential for zinc (Zn) uptake and virulence in a number of bacterial pathogens. Bioinformatics analysis identified ZnuABC as the only apparent high-affinity Zn uptake system in Y. pestis. Mutation of znuACB caused a growth defect in Chelex-100-treated PMH2 growth medium, which was alleviated by supplementation with submicromolar concentrations of Zn. Use of transcriptional reporters confirmed that Zur mediated Zn-dependent repression and that it can repress gene expression in response to Zn even in the absence of Znu. Virulence testing in mouse models of bubonic and pneumonic plague found only a modest increase in survival in low-dose infections by the znuACB mutant. Previous studies of cluster 9 (C9) transporters suggested that Yfe, a well-characterized C9 importer for manganese (Mn) and iron in Y. pestis, might function as a second, high-affinity Zn uptake system. Isothermal titration calorimetry revealed that YfeA, the solute-binding protein component of Yfe, binds Mn and Zn with comparably high affinities (dissociation constants of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM, respectively), although the complete Yfe transporter could not compensate for the loss of Znu in in vitro growth studies. Unexpectedly, overexpression of Yfe interfered with the znu mutant's ability to grow in low concentrations of Zn, while excess Zn interfered with the ability of Yfe to import iron at low concentrations; these results suggest that YfeA can bind Zn in the bacterial cell but that Yfe is incompetent for transport of the metal. In addition to Yfe, we have now eliminated MntH, FetMP, Efe, Feo, a substrate-binding protein, and a putative nickel transporter as the unidentified, secondary Zn transporter in Y. pestis. Unlike other bacterial pathogens, Y. pestis does not require Znu for high-level infectivity and virulence; instead, it appears to possess a novel class of transporter, which can satisfy the bacterium's Zn requirements under in vivo metal-limiting conditions. Our studies also underscore the need for bacterial cells to balance binding and transporter specificities within the periplasm in order to maintain transition metal homeostasis.

Loss of intracellular lipid binding proteins differentially impacts saturated fatty acid uptake and nuclear targeting in mouse hepatocytes

PubMed Central

Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Martin, Gregory G.; Landrock, Kerstin K.; Landrock, Danilo; Payne, H. Ross; Kier, Ann B.

2012-01-01

The liver expresses high levels of two proteins with high affinity for long-chain fatty acids (LCFAs): liver fatty acid binding protein (L-FABP) and sterol carrier protein-2 (SCP-2). Real-time confocal microscopy of cultured primary hepatocytes from gene-ablated (L-FABP, SCP-2/SCP-x, and L-FABP/SCP-2/SCP-x null) mice showed that the loss of L-FABP reduced cellular uptake of 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazo)-aminostearic acid (a fluorescent-saturated LCFA analog) by ∼50%. Importantly, nuclear targeting of the LCFA was enhanced when L-FABP was upregulated (SCP-2/SCP-x null) but was significantly reduced when L-FABP was ablated (L-FABP null), thus impacting LCFA nuclear targeting. These effects were not associated with a net decrease in expression of key membrane proteins involved in LCFA or glucose transport. Since hepatic LCFA uptake and metabolism are closely linked to glucose uptake, the effect of glucose on L-FABP-mediated LCFA uptake and nuclear targeting was examined. Increasing concentrations of glucose decreased cellular LCFA uptake and even more extensively decreased LCFA nuclear targeting. Loss of L-FABP exacerbated the decrease in LCFA nuclear targeting, while loss of SCP-2 reduced the glucose effect, resulting in enhanced LCFA nuclear targeting compared with control. Simply, ablation of L-FABP decreases LCFA uptake and even more extensively decreases its nuclear targeting. PMID:22859366

[Methoxyflurane and ethanol do not inhibit the neuronal uptake of noradrenaline (uptake 1) at the desipramine binding site].

PubMed

Kress, H G; Schömig, E

1990-07-01

We recently demonstrated that the net accumulation of 3H-norepinephrine in the rat pheochromocytoma cell line PC12 was reduced by anesthetic concentrations of n-alkanols and the volatile anesthetics halothane, enflurane, isoflurane, and methoxyflurane. In PC12 cells, as in adrenergic neurons, norepinephrine is transported across the plasma membrane by a saturable, high-affinity, carrier-mediated mechanism (uptake1), which follows Michaelis-Menten kinetics, is energy- and sodium-dependent, and is inhibited by low concentrations of cocaine and the tricyclic antidepressant desipramine. Although uptake1 is the most important process for the removal of norepinephrine from the synaptic cleft, the net accumulation of norepinephrine within the neuron also depends on other factors including its vesicular uptake and storage within the granules, its metabolism by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT), and the efflux of its more lipophilic metabolites. In our previous report we could not exclude the contribution of any of these factors to the observed inhibitory effects of volatile substances. Therefore, the aim of the present study with ethanol and methoxyflurane was: (1) to elucidate further the exact mechanism responsible for the reduction of the norepinephrine accumulation; and (2) to investigate the anesthetics' interaction with the substrate recognition site, which is identical with the desipramine binding site on the norepinephrine carrier. METHODS. For 3H-norepinephrine uptake experiments, PC12 cells were cultured on dishes (60 mm, Nunc) coated with polyornithine. Reserpine (10 microM) was added to the culture 24 h before the experiment to deplete endogenous norepinephrine. The initial carrier-mediated transport rate (60 s) was measured as previously described. 3H-desipramine equilibrium binding was determined with isolated plasma membranes prepared from PC12 cells grown in suspension culture. The carrier-mediated uptake of 3H-norepinephrine and the specific 3H-desipramine binding were defined as those inhibited by 1 microM nisoxetine. All buffers contained 10 microM pargyline and 10 microM U-0521 to inhibit MAO and COMT. Incubations were done in the presence and absence of methoxyflurane (1% and 2% vol/vol in synthetic air containing 5% CO2) or ethanol (5% vol/vol). Media had been equilibrated with methoxyflurane by bubbling (30 min) and were routinely checked by gas chromatography. RESULTS AND DISCUSSION. Methoxyflurane and ethanol inhibited uptake1. However, reduction of uptake1 was far less pronounced than that previously found for the net accumulation of norepinephrine. Even at a vaporous concentration of 2% (corresponding with an over 15-fold half-maximal inhibitory concentration for norepinephrine accumulation), methoxyflurane produced only 58% inhibition of the high-affinity uptake...

Polyamine Uptake in Carrot Cell Cultures 1

PubMed Central

Pistocchi, Rossella; Bagni, Nello; Creus, José A.

1987-01-01

Putrescine and spermidine uptake into carrot (Daucus carota L.) cells in culture was studied. The time course of uptake showed that the two polyamines were very quickly transported into the cells, reaching a maximum absorption within 1 minute. Increasing external polyamine concentrations up to 100 millimolar showed the existence of a biphasic system with different affinities at low and high polyamine concentrations. The cellular localization of absorbed polyamines was such that a greater amount of putrescine was present in the cytoplasmic soluble fraction, while spermidine was mostly present in cell walls. The absorbed polyamines were released into the medium in the presence of increasing external concentrations of the corresponding polyamine or Ca2+. The effects of Ca2+ were different for putrescine and spermidine; putrescine uptake was slightly stimulated by 10 micromolar Ca2+ and inhibited by higher concentrations, while for spermidine uptake there was an increasing stimulation in the Ca2+ concentration range between 10 micromolar and 1 millimolar. La3+ nullified the stimulatory effect of 10 micromolar Ca2+ on putrescine uptake and that of 1 millimolar Ca2+ on spermidine uptake. La3+ at 0.5 to 1 millimolar markedly inhibited the uptake of both polyamines, suggesting that it interferes with the sites of polyamine uptake. Putrescine uptake was affected to a lesser extent by metabolic inhibitors than was spermidine uptake. It is proposed that the entry of polyamines into the cells is driven by the transmembrane electrical gradient, with a possible antiport mechanism between external and internal polyamine molecule. PMID:16665446

Recent advances in hyaluronic acid-decorated nanocarriers for targeted cancer therapy.

PubMed

Wickens, Jennifer M; Alsaab, Hashem O; Kesharwani, Prashant; Bhise, Ketki; Amin, Mohd Cairul Iqbal Mohd; Tekade, Rakesh Kumar; Gupta, Umesh; Iyer, Arun K

2017-04-01

The cluster-determinant 44 (CD44) receptor has a high affinity for hyaluronic acid (HA) binding and is a desirable receptor for active targeting based on its overexpression in cancer cells compared with normal body cells. The nanocarrier affinity can be increased by conjugating drug-loaded carriers with HA, allowing enhanced cancer cell uptake via the HA-CD44 receptor-mediated endocytosis pathway. In this review, we discuss recent advances in HA-based nanocarriers and micelles for cancer therapy. In vitro and in vivo experiments have repeatedly indicated HA-based nanocarriers to be a target-specific drug and gene delivery platform with great promise for future applications in clinical cancer therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effects of high-dose fenfluramine treatment on monoamine uptake sites in rat brain: Assessment using quantitative autoradiography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Appel, N.M.; Mitchell, W.M.; Contrera, J.F.

1990-01-01

Fenfluramine is an amphetamine derivative that in humans is used primarily as an anorectic agent in the treatment of obesity. In rats, subchronic high-dose d,l-fenfluramine treatment (24 mg/kg subcutaneously, twice daily for 4 days) causes long-lasting decreases in brain serotonin (5HT), its metabolite 5-hydroxyindoleacetic acid, and high-affinity 5HT uptake sites. Moreover, this high-dose treatment regimen causes both selective long-lasting decreases in fine-caliber 5HT-immunoreactive axons and appearance of other 5HT-immunoreactive axons with morphology characteristic of degenerating axons. Determination of the potential neurotoxic effects of fenfluramine treatment using immunohistochemistry is limited from the perspectives that staining is difficult to quantify and thatmore » it relies on presence of the antigen (in this case 5HT), and the 5HT-depleting effects of fenfluramine are well known. In the present study, we used quantitative in vitro autoradiography to assess, in detail, the density and regional distribution of (3H)paroxetine-labeled 5HT and (3H)mazindol-labeled catecholamine uptake sites in response to the high-dose fenfluramine treatment described above. Because monoamine uptake sites are concentrated on monoamine-containing nerve terminals, decreases in uptake site density would provide a quantitative assessment of potential neurotoxicity resulting from this fenfluramine treatment regimen. Marked decreases in densities of (3H)paroxetine-labeled 5HT uptake sites occurred in brain regions in which fenfluramine treatment decreased the density of 5HT-like immunostaining when compared to saline-treated control rats. These included cerebral cortex, caudate putamen, hippocampus, thalamus, and medial hypothalamus.« less

Sortase Independent and Dependent Systems for Acquisition of Haem and Haemoglobin in Listeria monocytogenes

PubMed Central

Xiao, Qiaobin; Jiang, Xiaoxu; Moore, Kyle J.; Shao, Yi; Pi, Hualiang; Dubail, Iharilalao; Charbit, Alain; Newton, Salete M.; Klebba, Phillip E.

2011-01-01

Summary We studied three Fur-regulated systems of Listeria monocytogenes: the srtB region, that encodes sortase-anchored proteins and a putative ABC transporter, and the fhu and hup operons, that produce putative ABC transporters for ferric hydroxamates and haemin (Hn)/haemoglobin (Hb), respectively. Deletion of lmo2185 in the srtB region reduced listerial [59Fe]-Hn transport, and purified Lmo2185 bound [59Fe]-Hn (KD = 12 nM), leading to its designation as a Hn/Hb binding protein (hbp2). Purified Hbp2 also acted as a hemophore, capturing and supplying Hn from the environment. Nevertheless, Hbp2 only functioned in [59Fe]-Hn transport at external concentrations less than 10 nM: at higher Hn levels its uptake occurred with equivalent affinity and rate without Hbp2. Similarly, deletion of sortase A had no effect on ferric siderophore or Hn/Hb transport at any concentration, and the srtA-independence of listerial Hn/Hb uptake distinguished it from comparable systems of Staphylococcus aureus. In the cytoplasmic membrane, the Hup transporter was specific for Hn: its lipoprotein (HupD) only showed high affinity for the iron porphyrin (KD = 26 nM). Conversely, the FhuD lipoprotein encoded by the fhu operon had broad specificity: it bound both ferric siderophores and Hn, with the highest affinity for ferrioxamine B (KD = 123 nM). Deletions of Hup permease components hupD, hupG, or hupDGC reduced Hn/Hb uptake, and complementation of ΔhupC and ΔhupG by chromosomal integration of hupC+ and hupG+ alleles on pPL2 restored growth promotion by Hn/Hb. However, ΔhupDGC did not completely eliminate [59Fe]-Hn transport, implying the existence of another cytoplasmic membrane Hn transporter. The overall KM of Hn uptake by wild-type strain EGD-e was 1 nM, and it occurred at similar rates (Vmax = 23 pMol/109 cells/min) to those of ferric siderophore transporters. In the ΔhupDBGC strain uptake occurred at a 3-fold lower rate (Vmax = 7 pMol/109 cells/min). The results show that at low (< 50 nM) levels of Hn, SrtB-dependent peptidoglycan-anchored proteins (e.g., Hbp2) bind the porphyrin, and HupDGC or another transporter completes its uptake into the cytoplasm. However, at higher concentrations Hn uptake is SrtB-independent: peptidoglycan-anchored binding proteins are dispensable because HupDGC directly absorbs and internalizes Hn. Finally, ΔhupDGC increased the LD50 of L. monocytogenes 100-fold in the mouse infection model, reiterating the importance of this system in listerial virulence. PMID:21545655

Characteristics of ammonium and nitrate uptake by phytoplankton in Lake Kinneret

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berman, T.; Sherr, B.F.; Sherr, E.

1984-03-01

Seasonal and diurnal patterns of NH/sub 4//sup +/ and NO/sub 3//sup -/ uptake were determined for Lake Kinneret phytoplankton. Nanoplankton generally, but not always, had a higher uptake of NH/sub 4//sup +/ and NO/sub 3//sup -/ than did net plankton. Ammonium was always taken up preferentially and the phytoplankton had lower apparent affinity constants (K/sub s/) for this ion than for NO/sub 3//sup -/. However, during the annual dinoflagellate bloom of Peridinium (February-May), when ambient levels of NH/sub 4//sup +/ were low and those of NO/sub 3//sup -/ were high, a considerable portion of the total N flux was derivedmore » from NO/sub 3//sup -/. The finding that NO/sub 3//sup -/ was utilized by the dinoflagellates implies that control of the amounts of this nutrient, which is predominantly supplied from watershed sources, could be important in limiting eutrophication in Lake Kinneret.« less

Nonoisotopic Assay for the Presynaptic Choline Transporter Reveals Capacity for Allosteric Modulation of Choline Uptake

PubMed Central

2012-01-01

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na+-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline KM with no change in Vmax. As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling. PMID:23077721

Nonoisotopic assay for the presynaptic choline transporter reveals capacity for allosteric modulation of choline uptake.

PubMed

Ruggiero, Alicia M; Wright, Jane; Ferguson, Shawn M; Lewis, Michelle; Emerson, Katie S; Iwamoto, Hideki; Ivy, Michael T; Holmstrand, Ericka C; Ennis, Elizabeth A; Weaver, C David; Blakely, Randy D

2012-10-17

Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na(+)-dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K(M) with no change in V(max). As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling.

Mechanistic investigations into the species differences in pinometostat clearance: impact of binding to alpha-1-acid glycoprotein and permeability-limited hepatic uptake.

PubMed

Smith, Sherri A; Gagnon, Sandra; Waters, Nigel J

2017-03-01

1. The plasma clearance of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was shown to be markedly lower in human compared to the preclinical species, mouse, rat and dog. 2. This led to vertical allometry where various interspecies scaling methods were applied to the data, with fold-errors between 4 and 13. We had previously reported the elimination and metabolic pathways of EPZ-5676 were similar across species. Therefore, the aim of this work was to explore the mechanistic basis for the species difference in clearance for EPZ-5676, focusing on other aspects of disposition. 3. The protein binding of EPZ-5676 in human plasma demonstrated a non-linear relationship suggesting saturable binding at physiologically relevant concentrations. Saturation of protein binding was not observed in plasma from preclinical species. Kinetic determinations using purified serum albumin and alpha-1-acid glycoprotein (AAG) confirmed that EPZ-5676 is a high affinity ligand for AAG with a dissociation constant (K d ) of 0.24 μM. 4. Permeability limited uptake was also considered since hepatocyte CL int was much lower in human relative to preclinical species. Passive unbound CL int for EPZ-5676 was estimated using a correlation analysis of logD and data previously reported on seven drugs in sandwich cultured human hepatocytes. 5. Incorporation of AAG binding and permeability limited hepatic uptake into the well-stirred liver model gave rise to a predicted clearance for EPZ-5676 within 2-fold of the observed value of 1.4 mL min -1  kg -1 . This analysis suggests that the marked species difference in EPZ-5676 clearance is driven by high affinity binding to human AAG as well as species-specific hepatic uptake invoking the role of transporters.

Increase of chromium tolerance in Scenedesmus acutus after sulfur starvation: Chromium uptake and compartmentalization in two strains with different sensitivities to Cr(VI).

PubMed

Marieschi, M; Gorbi, G; Zanni, C; Sardella, A; Torelli, A

2015-10-01

In photosynthetic organisms sulfate constitutes the main sulfur source for the biosynthesis of GSH and its precursor Cys. Hence, sulfur availability can modulate the capacity to cope with environmental stresses, a phenomenon known as SIR/SED (Sulfur Induced Resistance or Sulfur Enhanced Defence). Since chromate may compete for sulfate transport into the cells, in this study chromium accumulation and tolerance were investigated in relation to sulfur availability in two strains of the unicellular green alga Scenedesmus acutus with different Cr-sensitivities. Paradoxically, sulfur deprivation has been demonstrated to induce a transient increase of Cr-tolerance in both strains. Sulfur deprivation is known to enhance the sulfate uptake/assimilation pathway leading to important consequences on Cr-tolerance: (i) reduced chromate uptake due to the induction of high affinity sulfate transporters (ii) higher production of cysteine and GSH which can play a role both through the formation of unsoluble complexes and their sequestration in inert compartments. To investigate the role of the above mentioned mechanisms, Cr accumulation in total cells and in different cell compartments (cell wall, membranes, soluble and miscellaneous fractions) was analyzed in both sulfur-starved and unstarved cells. Both strains mainly accumulated chromium in the soluble fraction, but the uptake was higher in the wild-type. In this type a short period of sulfur starvation before Cr(VI) treatment lowered chromium accumulation to the level observed in the unstarved Cr-tolerant strain, in which Cr uptake seems instead less influenced by S-starvation, since no significant decrease was observed. The increase in Cr-tolerance following S-starvation seems thus to rely on different mechanisms in the two strains, suggesting the induction of a mechanism constitutively active in the Cr-tolerant strain, maybe a high affinity sulfate transporter also in the wild-type. Changes observed in the cell wall and membrane fractions suggest a strong involvement of these compartments in Cr-tolerance increase following S-starvation. Copyright © 2015 Elsevier B.V. All rights reserved.

Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter.

PubMed Central

Zelcer, Noam; Saeki, Tohru; Bot, Ilse; Kuil, Annemieke; Borst, Piet

2003-01-01

Many of the transporters involved in the transport of bile acids in the enterohepatic circulation have been characterized. The basolateral bile-acid transporter of ileocytes and cholangiocytes remains an exception. It has been suggested that rat multidrug resistance protein 3 (Mrp3) fulfills this function. Here we analyse bile-salt transport by human MRP3. Membrane vesicles from insect ( Spodoptera frugiperda ) cells expressing MRP3 show time-dependent uptake of glycocholate and taurocholate. Furthermore, sulphated bile salts were high-affinity competitive inhibitors of etoposide glucuronide transport by MRP3 (IC50 approximately 10 microM). Taurochenodeoxycholate, taurocholate and glycocholate inhibited transport at higher concentrations (IC50 approximately 100, 250 and 500 microM respectively). We used mouse fibroblast-like cell lines derived from mice with disrupted Mdr1a, Mdr1b and Mrp1 genes to generate transfectants that express the murine apical Na+-dependent bile-salt transporter (Asbt) and MRP3. Uptake of glycocholate by these cells is Na+-dependent, with a K(m) and V(max) of 29+/-7 microM and 660 +/- 63 pmol/min per mg of protein respectively and is inhibited by several organic-aniontransport inhibitors. Expression of MRP3 in these cells limits the accumulation of glycocholate and increases the efflux from cells preloaded with taurocholate or glycocholate. In conclusion, we find that MRP3 transports both taurocholate and glycocholate, albeit with low affinity, in contrast with the high-affinity transport by rat Mrp3. Our results suggest that MRP3 is unlikely to be the principal basolateral bile-acid transporter of ileocytes and cholangiocytes, but that it may have a role in the removal of bile acids from the liver in cholestasis. PMID:12220224

EFFECT OF HUMIC ACID ON UPTAKE AND TRANSFER OF COPPER FROM MICROBES TO CILIATES TO COPEPODS

EPA Science Inventory

This research is part of an ongoing project designed to determine the effect of humic acid on the uptake and transfer of metals by marine organisms at the lower end of the food chain. Binding affinities for Cu, Cd, Zn, and Cr to Suwannee River humic acid were determined at variou...

Preparation and evaluation of the tumor-specific antigen-derived synthetic mucin 1 peptide: A potential candidate for the targeting of breast carcinoma.

PubMed

Okarvi, Subhani M; Al Jammaz, Ibrahim

2016-07-01

The goal of this study was to prepare a synthetic peptide derived from breast tumor associated antigen and to evaluate its potential as a breast cancer imaging agent. A mucin 1 derived peptide was synthesized by solid-phase peptide synthesis and examined for its radiochemical and metabolic stability. The tumor cell binding affinity of (99m)Tc-MUC1 peptide was investigated on MUC1-positive T47D and MCF7 breast cancer cell lines. In vivo biodistribution was studied in normal Balb/c mice and in vivo tumor targeting and imaging in MCF7 and T47D tumor-bearing nude mice. The synthesized MUC1-derived peptide displayed high radiochemical and metabolic stability. In vitro tumor cell-binding on T47D and MCF7 cell lines demonstrated high affinity of (99m)Tc-MUC1 peptide towards human breast cancer cells (binding affinities in nanomolar range). Pharmacokinetic studies performed on Balb/c mice are characterized by an efficient clearance from the blood and excretion predominantly through the urinary system. In vivo tumor uptake in nude mice with MCF7 tumor xenografts was 2.77±0.63% ID/g as early as 1h p.i. whereas in nude mice with T47D human ductal breast epithelial cancer cells, the accumulation in the tumor was found to be 2.65±0.54% ID/g at 1h p.i. Also tumor lesion was detectable in γ-camera imaging. The tumor uptake values were always higher than the blood and muscle uptake, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. A low to moderate (<5% ID/g) accumulation and retention of (99m)Tc-MUC1 was found in the major organs (i.e., lungs, stomach, liver, intestines, kidneys, etc.) in both normal and tumor-bearing mice. This study suggests that (99m)Tc-MUC1 tumor-antigen peptide may be a potential candidate for the targeted imaging of MUC1-positive human tumors and warrants further investigation. Copyright © 2016 Elsevier Inc. All rights reserved.

Targeted silver nanoparticles for ratiometric cell phenotyping

NASA Astrophysics Data System (ADS)

Willmore, Anne-Mari A.; Simón-Gracia, Lorena; Toome, Kadri; Paiste, Päärn; Kotamraju, Venkata Ramana; Mölder, Tarmo; Sugahara, Kazuki N.; Ruoslahti, Erkki; Braun, Gary B.; Teesalu, Tambet

2016-04-01

Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 +/- 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 +/- 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo.Affinity targeting is used to deliver nanoparticles to cells and tissues. For efficient targeting, it is critical to consider the expression and accessibility of the relevant receptors in the target cells. Here, we describe isotopically barcoded silver nanoparticles (AgNPs) as a tool for auditing affinity ligand receptors in cells. Tumor penetrating peptide RPARPAR (receptor: NRP-1) and tumor homing peptide GKRK (receptor: p32) were used as affinity ligands on the AgNPs. The binding and uptake of the peptide-functionalized AgNPs by cultured PPC-1 prostate cancer and M21 melanoma cells was dependent on the cell surface expression of the cognate peptide receptors. Barcoded peptide-functionalized AgNPs were synthesized from silver and palladium isotopes. The cells were incubated with a cocktail of the barcoded nanoparticles [RPARPAR (R), GKRK (K), and control], and cellular binding and internalization of each type of nanoparticle was assessed by inductively coupled plasma mass spectrometry. The results of isotopic analysis were in agreement with data obtained using optical methods. Using ratiometric measurements, we were able to classify the PPC-1 cell line as mainly NRP-1-positive, with 75 +/- 5% R-AgNP uptake, and the M21 cell line as only p32-positive, with 89 +/- 9% K-AgNP uptake. The isotopically barcoded multiplexed AgNPs are useful as an in vitro ratiometric phenotyping tool and have potential uses in functional evaluation of the expression of accessible homing peptide receptors in vivo. Electronic supplementary information (ESI) available: TEM images of isotopic AgNPs, cell antibody staining, coadministration ICP-MS data, and biotin control particle ICP-MS data. See DOI: 10.1039/C5NR07928D

Preclinical validation of 111In-girentuximab-F(ab')2 as a tracer to image hypoxia related marker CAIX expression in head and neck cancer xenografts.

PubMed

Huizing, Fokko J; Hoeben, Bianca A W; Franssen, Gerben; Lok, Jasper; Heskamp, Sandra; Oosterwijk, Egbert; Boerman, Otto C; Bussink, Johan

2017-09-01

Hypoxia is a major cause of radio- and chemoresistance. Carbonic anhydrase IX (CAIX) is an endogenous hypoxia-related marker and an important prognostic marker. Assessment of CAIX expression may allow patient selection for hypoxia or CAIX-targeted treatment. The radioactive tracer 111 In-girentuximab-F(ab') 2 targets CAIX and can be used for SPECT imaging. Aim of this study was to validate and optimize 111 In-girentuximab-F(ab') 2 for imaging of CAIX expression in head and neck tumor xenografts. Affinity and internalization kinetics of 111 In-girentuximab-F(ab') 2 were determined in vitro using CAIX-expressing SK-RC-52 cells. Tumor targeting characteristics were determined in athymic mice with six different head and neck squamous cell carcinoma (SCCNij) xenografts. Tracer uptake was measured by ex vivo radioactivity counting. Intratumoral distribution of tracer uptake was measured using autoradiography and CAIX expression was determined immunohistochemically. 26% of the tracer was internalized into the SK-RC-52 cells within 24h. The half maximal inhibitory concentration (IC 50 ) was 0.69±0.08nM. In biodistribution studies SCCNij153 tumors showed the highest tracer uptake: 4.1±0.8ID/g at 24h p.i. Immunohistochemical and autoradiographic analyses of the xenografts showed a distinct spatial correlation between localization of the tracer and CAIX expression. 111 In-girentuximab-F(ab') 2 has a high affinity for CAIX. In vivo tumor uptake correlated strongly with CAIX expression in different head and neck xenografts. These results suggest that 111 In-girentuximab-F(ab') 2 is a promising tracer for imaging of hypoxia-related CAIX expression. Copyright © 2017 Elsevier B.V. All rights reserved.

The Dionaea muscipula ammonium channel DmAMT1 provides NH₄⁺ uptake associated with Venus flytrap's prey digestion.

PubMed

Scherzer, Sönke; Krol, Elzbieta; Kreuzer, Ines; Kruse, Jörg; Karl, Franziska; von Rüden, Martin; Escalante-Perez, Maria; Müller, Thomas; Rennenberg, Heinz; Al-Rasheid, Khaled A S; Neher, Erwin; Hedrich, Rainer

2013-09-09

Ammonium transporter (AMT/MEP/Rh) superfamily members mediate ammonium uptake and retrieval. This pivotal transport system is conserved among all living organisms. For plants, nitrogen represents a macronutrient available in the soil as ammonium, nitrate, and organic nitrogen compounds. Plants living on extremely nutrient-poor soils have developed a number of adaptation mechanisms, including a carnivorous lifestyle. This study addresses the molecular nature, function, and regulation of prey-derived ammonium uptake in the Venus flytrap, Dionaea muscipula, one of the fastest active carnivores. The Dionaea muscipula ammonium transporter DmAMT1 was localized in gland complexes where its expression was upregulated upon secretion. These clusters of cells decorating the inner trap surface are engaged in (1) secretion of an acidic digestive enzyme cocktail and (2) uptake of prey-derived nutrients. Voltage clamp of Xenopus oocytes expressing DmAMT1 and membrane potential recordings with DmAMT1-expressing Dionaea glands were used to monitor and compare electrophysiological properties of DmAMT1 in vitro and in planta. DmAMT1 exhibited the hallmark biophysical properties of a NH4(+)-selective channel. At depolarized membrane potentials (Vm = 0), the Km (3.2 ± 0.3 mM) indicated a low affinity of DmAMT1 for ammonium that increased systematically with negative going voltages. Upon hyperpolarization to, e.g., -200 mV, a Km of 0.14 ± 0.015 mM documents the voltage-dependent shift of DmAMT1 into a NH4(+) transport system of high affinity. We suggest that regulation of glandular DmAMT1 and membrane potential readjustments of the endocrine cells provide for effective adaptation to varying, prey-derived ammonium sources. Copyright © 2013 Elsevier Ltd. All rights reserved.

The cytotoxicity of 3-bromopyruvate in breast cancer cells depends on extracellular pH.

PubMed

Azevedo-Silva, João; Queirós, Odília; Ribeiro, Ana; Baltazar, Fátima; Young, Ko H; Pedersen, Peter L; Preto, Ana; Casal, Margarida

2015-04-15

Although the anti-cancer properties of 3BP (3-bromopyruvate) have been described previously, its selectivity for cancer cells still needs to be explained [Ko et al. (2001) Cancer Lett. 173, 83-91]. In the present study, we characterized the kinetic parameters of radiolabelled [14C] 3BP uptake in three breast cancer cell lines that display different levels of resistance to 3BP: ZR-75-1 < MCF-7 < SK-BR-3. At pH 6.0, the affinity of cancer cells for 3BP transport correlates with their sensitivity, a pattern that does not occur at pH 7.4. In the three cell lines, the uptake of 3BP is dependent on the protonmotive force and is decreased by MCTs (monocarboxylate transporters) inhibitors. In the SK-BR-3 cell line, a sodium-dependent transport also occurs. Butyrate promotes the localization of MCT-1 at the plasma membrane and increases the level of MCT-4 expression, leading to a higher sensitivity for 3BP. In the present study, we demonstrate that this phenotype is accompanied by an increase in affinity for 3BP uptake. Our results confirm the role of MCTs, especially MCT-1, in 3BP uptake and the importance of cluster of differentiation (CD) 147 glycosylation in this process. We find that the affinity for 3BP transport is higher when the extracellular milieu is acidic. This is a typical phenotype of tumour microenvironment and explains the lack of secondary effects of 3BP already described in in vivo studies [Ko et al. (2004) Biochem. Biophys. Res. Commun. 324, 269-275].

Receptor-Mediated Melanoma Targeting with Radiolabeled α-Melanocyte-Stimulating Hormone: Relevance of the Net Charge of the Ligand.

PubMed

Bapst, Jean-Philippe; Eberle, Alex N

2017-01-01

A majority of melanotic and amelanotic melanomas overexpress melanocortin type 1 receptors (MC1Rs) for α-melanocyte-stimulating hormone. Radiolabeled linear or cyclic analogs of α-MSH have a great potential as diagnostic or therapeutic tools for the management of malignant melanoma. Compounds such as [ 111 In]DOTA-NAP-amide exhibit high affinity for the MC1R in vitro , good tumor uptake in vivo , but they may suffer from relatively high kidney uptake and retention in vivo . We have shown previously that the introduction of negative charges into radiolabeled DOTA-NAP-amide peptide analogs may enhance their excretion and reduce kidney retention. To address the question of where to place negative charges within the ligand, we have extended these studies by designing two novel peptides, Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys(DOTA)-d-Asp-d-Asp-OH (DOTA-NAP-d-Asp-d-Asp) with three negative charges at the C -terminal end (overall net charge of the molecule -2) and DOTA-Gly-Tyr(P)-Nle-Asp-His-d-Phe-Arg-Trp-NH 2 (DOTA-Phospho-MSH 2-9 ) with two negative charges in the N -terminal region (net charge -1). The former peptide showed markedly reduced receptor affinity and biological activity by >10-fold compared to DOTA-NAP-amide as reference compound, and the latter peptide displayed similar bioactivity and receptor affinity as the reference compound. The uptake by melanoma tumor tissue of [ 111 In]DOTA-Phospho-MSH 2-9 was 7.33 ± 0.47 %ID/g 4 h after injection, i.e., almost equally high as with [ 111 In]DOTA-NAP-amide. The kidney retention was 2.68 ± 0.18 %ID/g 4 h after injection and hence 44% lower than that of [ 111 In]DOTA-NAP-amide. Over an observation period from 4 to 48 h, the tumor-to-kidney ratio of [ 111 In]DOTA-Phospho-MSH 2-9 was 35% more favorable than that of the reference compound. In a comparison of DOTA-NAP-d-Asp-d-Asp, DOTA-Phospho-MSH 2-9 and DOTA-NAP-amide with five previously published analogs of DOTA-NAP-amide that altogether cover a range of peptides with an overall net charge between +2 and -2, we now demonstrate that a net charge of -1, with the extra negative charges preferably placed in the N -terminal region, has led to the lowest kidney uptake and retention. Charges of +2 or -2 markedly increased kidney uptake and retention. In conclusion, the novel DOTA-Phospho-MSH 2-9 may represent a new lead compound for negatively charged linear MC1R ligands that can be further developed into a clinically relevant melanoma targeting radiopeptide.

Receptor-Mediated Melanoma Targeting with Radiolabeled α-Melanocyte-Stimulating Hormone: Relevance of the Net Charge of the Ligand

PubMed Central

Bapst, Jean-Philippe; Eberle, Alex N.

2017-01-01

A majority of melanotic and amelanotic melanomas overexpress melanocortin type 1 receptors (MC1Rs) for α-melanocyte-stimulating hormone. Radiolabeled linear or cyclic analogs of α-MSH have a great potential as diagnostic or therapeutic tools for the management of malignant melanoma. Compounds such as [111In]DOTA-NAP-amide exhibit high affinity for the MC1R in vitro, good tumor uptake in vivo, but they may suffer from relatively high kidney uptake and retention in vivo. We have shown previously that the introduction of negative charges into radiolabeled DOTA-NAP-amide peptide analogs may enhance their excretion and reduce kidney retention. To address the question of where to place negative charges within the ligand, we have extended these studies by designing two novel peptides, Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys(DOTA)-d-Asp-d-Asp-OH (DOTA-NAP-d-Asp-d-Asp) with three negative charges at the C-terminal end (overall net charge of the molecule −2) and DOTA-Gly-Tyr(P)-Nle-Asp-His-d-Phe-Arg-Trp-NH2 (DOTA-Phospho-MSH2-9) with two negative charges in the N-terminal region (net charge −1). The former peptide showed markedly reduced receptor affinity and biological activity by >10-fold compared to DOTA-NAP-amide as reference compound, and the latter peptide displayed similar bioactivity and receptor affinity as the reference compound. The uptake by melanoma tumor tissue of [111In]DOTA-Phospho-MSH2-9 was 7.33 ± 0.47 %ID/g 4 h after injection, i.e., almost equally high as with [111In]DOTA-NAP-amide. The kidney retention was 2.68 ± 0.18 %ID/g 4 h after injection and hence 44% lower than that of [111In]DOTA-NAP-amide. Over an observation period from 4 to 48 h, the tumor-to-kidney ratio of [111In]DOTA-Phospho-MSH2-9 was 35% more favorable than that of the reference compound. In a comparison of DOTA-NAP-d-Asp-d-Asp, DOTA-Phospho-MSH2-9 and DOTA-NAP-amide with five previously published analogs of DOTA-NAP-amide that altogether cover a range of peptides with an overall net charge between +2 and −2, we now demonstrate that a net charge of −1, with the extra negative charges preferably placed in the N-terminal region, has led to the lowest kidney uptake and retention. Charges of +2 or −2 markedly increased kidney uptake and retention. In conclusion, the novel DOTA-Phospho-MSH2-9 may represent a new lead compound for negatively charged linear MC1R ligands that can be further developed into a clinically relevant melanoma targeting radiopeptide. PMID:28491052

Interaction of the new monofunctional anticancer agent Phenanthriplatin with transporters for organic cations

NASA Astrophysics Data System (ADS)

Hucke, Anna; Park, Ga Young; Bauer, Oliver B.; Beyer, Georg; Köppen, Christina; Zeeh, Dorothea; Wehe, Christoph A.; Sperling, Michael; Schröter, Rita; Kantauskaitè, Marta; Hagos, Yohannes; Karst, Uwe; Lippard, Stephen J.; Ciarimboli, Giuliano

2018-05-01

Cancer treatment with platinum compounds is an important achievement of modern chemotherapy. However, despite the beneficial effects, the clinical impact of these agents is hampered by the development of drug resistance as well as dose-limiting side effects. The efficacy but also side effects of platinum complexes can be mediated by uptake through plasma membrane transporters. In the kidneys, plasma membrane transporters are involved in their secretion into the urine. Renal secretion is accomplished by uptake from the blood into the proximal tubules cells, followed by excretion into the urine. The uptake process is mediated mainly by organic cation transporters (OCT), which are expressed in the basolateral domain of the plasma membrane facing the blood. The excretion of platinum into the urine is mediated by exchange with protons via multidrug and toxin extrusion proteins (MATE) expressed in the apical domain of plasma membrane. Recently, the monofunctional, cationic platinum agent phenanthriplatin, which is able to escape common cellular resistance mechanisms, has been synthesized and investigated. In the present study, the interaction of phenanthriplatin with transporters for organic cations has been evaluated. Phenanthriplatin is a high affinity substrate for OCT2, but has a lower apparent affinity for MATEs. The presence of these transporters increased cytotoxicity of phenanthriplatin. Therefore, phenanthriplatin may be especially effective in the treatment of cancers that express OCTs, such as colon cancer cells. However, the interaction of phenanthriplatin with OCTs suggests that its use as chemotherapeutic agent may be complicated by OCT-mediated toxicity. Unlike cisplatin, phenanthriplatin interacts with high specificity with hMATE1 and hMATE2K in addition to hOCT2. This interaction may facilitate its efflux from the cells and thereby decrease overall efficacy and/or toxicity.

Differences in serotonin transporter binding affinity in patients with major depressive disorder and night eating syndrome.

PubMed

Lundgren, J D; Amsterdam, J; Newberg, A; Allison, K C; Wintering, N; Stunkard, A J

2009-03-01

We examined serotonin transporter (SERT) binding affinity using single photon emission computed tomography (SPECT) in patients with major depressive disorder (MDD) and night eating syndrome (NES). There are similarities between MDD and NES in affective symptoms, appetite disturbance, nighttime awakenings, and, particularly, response to selective serotonin reuptake inhibitors (SSRIs). Six non-depressed patients with NES and seven patients with MDD underwent SPECT brain imaging with 123I-ADAM, a radiopharmaceutical agent selective for SERT sites. Uptake ratios of 123I-ADAM SERT binding were obtained for the midbrain, basal ganglia, and temporal lobe regions compared to the cerebellum reference region. Patients with NES had significantly greater SERT uptake ratios (effect size range 0.64-0.84) in the midbrain, right temporal lobe, and left temporal lobe regions than those with MDD whom we had previously studied. Pathophysiological differences in SERT uptake between patients with NES and MDD suggest these are distinct clinical syndromes.

The Penicillium chrysogenum transporter PcAraT enables high-affinity, glucose-insensitive l-arabinose transport in Saccharomyces cerevisiae.

PubMed

Bracher, Jasmine M; Verhoeven, Maarten D; Wisselink, H Wouter; Crimi, Barbara; Nijland, Jeroen G; Driessen, Arnold J M; Klaassen, Paul; van Maris, Antonius J A; Daran, Jean-Marc G; Pronk, Jack T

2018-01-01

l-Arabinose occurs at economically relevant levels in lignocellulosic hydrolysates. Its low-affinity uptake via the Saccharomyces cerevisiae Gal2 galactose transporter is inhibited by d-glucose. Especially at low concentrations of l-arabinose, uptake is an important rate-controlling step in the complete conversion of these feedstocks by engineered pentose-metabolizing S. cerevisiae strains. Chemostat-based transcriptome analysis yielded 16 putative sugar transporter genes in the filamentous fungus Penicillium chrysogenum whose transcript levels were at least threefold higher in l-arabinose-limited cultures than in d-glucose-limited and ethanol-limited cultures. Of five genes, that encoded putative transport proteins and showed an over 30-fold higher transcript level in l-arabinose-grown cultures compared to d-glucose-grown cultures, only one (Pc20g01790) restored growth on l-arabinose upon expression in an engineered l-arabinose-fermenting S. cerevisiae strain in which the endogenous l-arabinose transporter, GAL2 , had been deleted. Sugar transport assays indicated that this fungal transporter, designated as Pc AraT, is a high-affinity ( K m  = 0.13 mM), high-specificity l-arabinose-proton symporter that does not transport d-xylose or d-glucose. An l-arabinose-metabolizing S. cerevisiae strain in which GAL2 was replaced by PcaraT showed 450-fold lower residual substrate concentrations in l-arabinose-limited chemostat cultures than a congenic strain in which l-arabinose import depended on Gal2 (4.2 × 10 -3 and 1.8 g L -1 , respectively). Inhibition of l-arabinose transport by the most abundant sugars in hydrolysates, d-glucose and d-xylose was far less pronounced than observed with Gal2. Expression of Pc AraT in a hexose-phosphorylation-deficient, l-arabinose-metabolizing S. cerevisiae strain enabled growth in media supplemented with both 20 g L -1 l-arabinose and 20 g L -1 d-glucose, which completely inhibited growth of a congenic strain in the same condition that depended on l-arabinose transport via Gal2. Its high affinity and specificity for l-arabinose, combined with limited sensitivity to inhibition by d-glucose and d-xylose, make Pc AraT a valuable transporter for application in metabolic engineering strategies aimed at engineering S. cerevisiae strains for efficient conversion of lignocellulosic hydrolysates.

Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1.

PubMed

Belenky, Peter A; Moga, Tiberiu G; Brenner, Charles

2008-03-28

NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.

Diffusion Limitations in Root Uptake of Cadmium and Zinc, But Not Nickel, and Resulting Bias in the Michaelis Constant1[W][OA

PubMed Central

Degryse, Fien; Shahbazi, Afsaneh; Verheyen, Liesbeth; Smolders, Erik

2012-01-01

It has long been recognized that diffusive boundary layers affect the determination of active transport parameters, but this has been largely overlooked in plant physiological research. We studied the short-term uptake of cadmium (Cd), zinc (Zn), and nickel (Ni) by spinach (Spinacia oleracea) and tomato (Lycopersicon esculentum) in solutions with or without metal complexes. At same free ion concentration, the presence of complexes, which enhance the diffusion flux, increased the uptake of Cd and Zn, whereas Ni uptake was unaffected. Competition effects of protons on Cd and Zn uptake were observed only at a very large degree of buffering, while competition of magnesium ions on Ni uptake was observed even in unbuffered solutions. These results strongly suggest that uptake of Cd and Zn is limited by diffusion of the free ion to the roots, except at very high degree of solution buffering, whereas Ni uptake is generally internalization limited. All results could be well described by a model that combined a diffusion equation with a competitive Michaelis-Menten equation. Direct uptake of the complex was estimated to be a major contribution only at millimolar concentrations of the complex or at very large ratios of complex to free ion concentration. The true Km for uptake of Cd2+ and Zn2+ was estimated at <5 nm, three orders of magnitude smaller than the Km measured in unbuffered solutions. Published Michaelis constants for plant uptake of Cd and Zn likely strongly overestimate physiological ones and should not be interpreted as an indicator of transporter affinity. PMID:22864584

Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

PubMed Central

Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

2015-01-01

AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants. PMID:25915023

Diselenolane-mediated cellular uptake† †Electronic supplementary information (ESI) available: Detailed procedures and results for all reported experiments. See DOI: 10.1039/c7sc05151d

PubMed Central

Chuard, Nicolas; Poblador-Bahamonde, Amalia I.; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi

2018-01-01

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides. PMID:29675232

Tachycardia, reduced vagal capacity, and age-dependent ventricular dysfunction arising from diminished expression of the presynaptic choline transporter

PubMed Central

English, Brett A.; Appalsamy, Martin; Diedrich, Andre; Ruggiero, Alicia M.; Lund, David; Wright, Jane; Keller, Nancy R.; Louderback, Katherine M.; Robertson, David

2010-01-01

Healthy cardiovascular function relies on a balanced and responsive integration of noradrenergic and cholinergic innervation of the heart. High-affinity choline uptake by cholinergic terminals is pivotal for efficient ACh production and release. To date, the cardiovascular impact of diminished choline transporter (CHT) expression has not been directly examined, largely due to the transporter's inaccessibility in vivo. Here, we describe findings from cardiovascular experiments using transgenic mice that bear a CHT genetic deficiency. Whereas CHT knockout (CHT−/−) mice exhibit early postnatal lethality, CHT heterozygous (CHT+/−) mice survive, grow, and reproduce normally and exhibit normal spontaneous behaviors. However, the CHT+/− mouse heart displays significantly reduced levels of high-affinity choline uptake accompanied by significantly reduced levels of ACh. Telemeterized recordings of cardiovascular function in these mice revealed tachycardia and hypertension at rest. After treadmill exercise, CHT+/− mice exhibited slower heart rate recovery, consistent with a diminished cholinergic reserve, a contention validated through direct vagal nerve stimulation. Echocardiographic and histological experiments revealed an age-dependent decrease in fractional shortening, increased left ventricular dimensions, and increased ventricular fibrosis, consistent with ventricular dysfunction. These cardiovascular phenotypes of CHT+/− mice encourage an evaluation of humans bearing reduced CHT expression for their resiliency in maintaining proper heart function as well as risk for cardiovascular disease. PMID:20601463

Hemicholinium-3 sensitive choline transport in human T lymphocytes: Evidence for use as a proxy for brain choline transporter (CHT) capacity.

PubMed

Koshy Cherian, Ajeesh; Parikh, Vinay; Wu, Qi; Mao-Draayer, Yang; Wang, Qin; Blakely, Randy D; Sarter, Martin

2017-09-01

The synaptic uptake of choline via the high-affinity, hemicholinium-3-dependent choline transporter (CHT) strongly influences the capacity of cholinergic neurons to sustain acetylcholine (ACh) synthesis and release. To advance research on the impact of CHT capacity in humans, we established the presence of the neuronal CHT protein in human T lymphocytes. Next, we demonstrated CHT-mediated choline transport in human T cells. To address the validity of T cell-based choline uptake as a proxy for brain CHT capacity, we isolated T cells from the spleen, and synaptosomes from cortex and striatum, of wild type and CHT-overexpressing mice (CHT-OXP). Choline uptake capacity in T cells from CHT-OXP mice was two-fold higher than in wild type mice, mirroring the impact of CHT over-expression on synaptosomal CHT-mediated choline uptake. Monitoring T lymphocyte CHT protein and activity may be useful for estimating human CNS cholinergic capacity and for testing hypotheses concerning the contribution of CHT and, more generally, ACh signaling in cognition, neuroinflammation and disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

Successful Coupling of a Bis-Amidoxime Uranophile with a Hydrophilic Backbone for Selective Uranium Sequestration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Piechowicz, Marek; Abney, Carter W.; Thacker, Nathan C.

The amidoxime group (-RNH2NOH) has long been used to extract uranium from seawater on account of its high affinity toward uranium. The development of tunable sorbent materials for uranium sequestration remains a research priority as well as a significant challenge. Herein, we report the design, synthesis, and uranium sorption properties of bis-amidoxime-functionalized polymeric materials (BAP 1–3). Bifunctional amidoxime monomers were copolymerized with an acrylamide cross-linker to obtain bis-amidoxime incorporation as high as 2 mmol g–1 after five synthetic steps. The resulting sorbents were able to uptake nearly 600 mg of uranium per gram of polymer after 37 days of contactmore » with a seawater simulant containing 8 ppm uranium. Moreover, the polymeric materials exhibited low vanadium uptake with a maximum capacity of 128 mg of vanadium per gram of polymer. This computationally predicted and experimentally realized selectivity of uranium over vanadium, nearly 5 to 1 w/w, is one of the highest reported to date and represents an advancement in the rational design of sorbent materials with high uptake capacity and selectivity.« less

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

PubMed

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.

Quantitative cumulative biodistribution of antibodies in mice: effect of modulating binding affinity to the neonatal Fc receptor.

PubMed

Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

2014-01-01

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.

Increased xylose affinity of Hxt2 through gene shuffling of hexose transporters in Saccharomyces cerevisiae.

PubMed

Nijland, J G; Shin, H Y; de Waal, P P; Klaassen, P; Driessen, A J M

2018-02-01

Optimizing D-xylose transport in Saccharomyces cerevisiae is essential for efficient bioethanol production from cellulosic materials. We have used a gene shuffling approach of hexose (Hxt) transporters in order to increase the affinity for D-xylose. Various libraries were transformed to a hexose transporter deletion strain, and shuffled genes were selected via growth on low concentrations of D-xylose. This screening yielded two homologous fusion proteins (fusions 9,4 and 9,6), both consisting of the major central part of Hxt2 and various smaller parts of other Hxt proteins. Both chimeric proteins showed the same increase in D-xylose affinity (8·1 ± 3·0 mmol l -1 ) compared with Hxt2 (23·7 ± 2·1 mmol l -1 ). The increased D-xylose affinity could be related to the C terminus, more specifically to a cysteine to proline mutation at position 505 in Hxt2. The Hxt2 C505P mutation increased the affinity for D-xylose for Hxt2, thus providing a way to increase D-xylose transport flux at low D-xylose concentration. The gene shuffling protocol using the highly homologues hexose transporters family provides a powerful tool to enhance the D-xylose affinity of Hxt transporters in S. cerevisiae, thus providing a means to increase the D-xylose uptake flux at low D-xylose concentrations. © 2017 The Society for Applied Microbiology.

PET/CT With 68Ga-DOTA-TATE for Diagnosis of Neuroendocrine: Differentiation in Patients With Castrate-Resistant Prostate Cancer.

PubMed

Gofrit, Ofer Nathan; Frank, Stephen; Meirovitz, Amichay; Nechushtan, Hovav; Orevi, Marina

2017-01-01

Castrate-resistant prostate cancer (CRPC) often shows histological evidence of neuroendocrine differentiation (NED). To evaluate the extent of NED in patients with CRPC, we used PET/CT with Ga-[DOTA-Tyr]-octreotate (Ga-DOTA-TATE), a somatostatin analog that binds somatostatin receptor 2 with high affinity. This radiotracer is used in imaging of neuroendocrine tumors. Twelve patients (mean age, 65 [SD, 12] years) with CRPC were studied. Their mean prostate-specific antigen level at scanning was 85.6 (SD, 144.6) ng/mL. PET/CT images were obtained after the injection of 120 to 200 MBq of Ga-DOTA-TATE. All participants had at least 1 blastic metastasis demonstrating uptake of Ga-DOTA-TATE (mean SUVmax of 5.3 [SD, 2.3]). In 6 patients, moderately high to high uptakes (SUVmax, >5) were seen. Patients with multiple bone metastases had a significantly higher SUVmax compared with patients with few metastases (mean of 5.8 vs 3.8, P = 0.05). In 4 patients, lytic bone lesions or lymph node metastases also showed uptake of the tracer (mean SUVmax of 7.2 [SD, 3.2]). Uptake of the radiotracer was also observed in bones showing normal architecture in CT, suggesting that NED cells appear early during metastases development. Uptake of Ga-DOTA-TATE is a common finding in metastases of CRPC patients, suggesting that NED is frequent in these patients. In half of the patients, widespread uptake of Ga-DOTA-TATE was observed. This suggests that the possibility of treating selected CRCP patients with anti-neuroendocrine tumor therapies should be explored and that Ga-DOTA-TATE scanning could have a role in predicting the efficacy of these treatments.

Membrane transport mechanisms of choline in human intestinal epithelial LS180 cells.

PubMed

Horie, Asuka; Ishida, Kazuya; Watanabe, Yuri; Shibata, Kaito; Hashimoto, Yukiya

2014-12-01

The aim of the present study was to investigate the membrane transport mechanisms of choline using human intestinal epithelial LS180 cells. The mRNA of choline transporter-like proteins (CTLs) was expressed significantly in LS180 cells, and the rank order was CTL1 > CTL4 > CTL3 > CTL2 > CTL5. In contrast, the mRNA expression of other choline transporters, organic cation transporter (OCT) 1, OCT2 and high-affinity choline transporter 1 (CHT1), was considerably lower in LS180 cells. Five mm unlabelled choline, hemicolinium-3 and guanidine, but not tetraethylammonium, inhibited the cellular uptake of 100 µm choline in LS180 cells. The uptake of choline into LS180 cells was virtually Na(+)-independent. The uptake of choline was significantly decreased by acidification of the extracellular pH; however, it was not increased by alkalization of the extracellular pH. In addition, both acidification and alkalization of intracellular pH decreased the uptake of choline, indicating that the choline uptake in LS180 cells is not stimulated by the outward H(+) gradient. On the other hand, the uptake of choline was decreased by membrane depolarization along with increasing extracellular K(+) concentration. In addition, the Na(+)-independent uptake of choline was saturable, and the Km value was estimated to be 108 µm. These findings suggest that the uptake of choline into LS180 cells is membrane potential-dependent, but not outward H(+) gradient-dependent. Copyright © 2014 John Wiley & Sons, Ltd.

Heparin affinity purification of extracellular vesicles

PubMed Central

Balaj, Leonora; Atai, Nadia A.; Chen, Weilin; Mu, Dakai; Tannous, Bakhos A.; Breakefield, Xandra O.; Skog, Johan; Maguire, Casey A.

2015-01-01

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity. PMID:25988257

Reversibly extracellular pH controlled cellular uptake and photothermal therapy by PEGylated mixed-charge gold nanostars.

PubMed

Wang, Shouju; Teng, Zhaogang; Huang, Peng; Liu, Dingbin; Liu, Ying; Tian, Ying; Sun, Jing; Li, Yanjun; Ju, Huangxian; Chen, Xiaoyuan; Lu, Guangming

2015-04-17

Shielding nanoparticles from nonspecific interactions with normal cells/tissues before they reach and after they leave tumors is crucial for the selective delivery of NPs into tumor cells. By utilizing the reversible protonation of weak electrolytic groups to pH changes, long-chain amine/carboxyl-terminated polyethylene glycol (PEG) decorated gold nanostars (GNSs) are designed, exhibiting reversible, significant, and sensitive response in cell affinity and therapeutic efficacy to the extracellular pH (pHe) gradient between normal tissues and tumors. This smart nanosystem shows good dispersity and unimpaired photothermal efficacy in complex bioenvironment at pH 6.4 and 7.4 even when their surface charge is neutral. One PEGylated mixed-charge GNSs with certain surface composition, GNS-N/C 4, exhibits high cell affinity and therapeutic efficacy at pH 6.4, and low affinity and almost "zero" damage to cells at pH 7.4. Remarkably, this significant and sensitive response in cell affinity and therapeutic efficacy is reversible as local pH alternated. In vivo, GNS-N/C 4 shows higher accumulation in tumors and improved photothermal therapeutic efficacy than pH-insensitive GNSs. This newly developed smart nanosystem, whose cell affinity reversibly transforms in response to pHe gradient with unimpaired biostability, provides a novel effective means of tumor-selective therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of cysteine and humic acids on bioavailability of Ag from Ag nanoparticles to a freshwater snail

USGS Publications Warehouse

Luoma, Samuel N.; Tasha Stoiber,; Croteau, Marie-Noele; Isabelle Romer,; Ruth Merrifeild,; Lead, Jamie

2016-01-01

Metal-based engineered nanoparticles (NPs) will undergo transformations that will affect their bioavailability, toxicity and ecological risk when released to the environment, including interactions with dissolved organic material. The purpose of this paper is to determine how interactions with two different types of organic material affect the bioavailability of silver nanoparticles (AgNPs). Silver uptake rates by the pond snail Lymnaea stagnalis were determined after exposure to 25 nmol l-1 of Ag as PVP AgNPs, PEG AgNPs or AgNO3, in the presence of either Suwannee River humic acid or cysteine, a high-affinity thiol-rich organic ligand. Total uptake rate of Ag from the two NPs was either increased or not strongly affected in the presence of 1 – 10 mg 1-1 humic acid. Humic substances contain relatively few strong ligands for Ag explaining their limited effects on Ag uptake rate. In contrast, Ag uptake rate was substantially reduced by cysteine. Three components of uptake from the AgNPs were quantified in the presence of cysteine using a biodynamic modeling approach: uptake of dissolved Ag released by the AgNPs, uptake of a polymer or large (>3kD) Ag-cysteine complex and uptake of the nanoparticle itself. Addition of 1:1 Ag:cysteine reduced concentrations of dissolved Ag, which contributed to, but did not fully explain the reductions in uptake. A bioavailable Ag-cysteine complex (> 3kD) appeared to be the dominant avenue of uptake from both PVP AgNPs and PEG AgNPs in the presence of cysteine. Quantifying the different avenues of uptake sets the stage for studies to assess toxicity unique to NPs.

RAPID NITRATE UPTAKE RATES AND LARGE SHORT-TERM STORAGE CAPACITIES MAY EXPLAIN WHY OPPORTUNISTIC GREEN MACROALGAE DOMINATE SHALLOW EUTROPHIC ESTUARIES1.

PubMed

Kennison, Rachel L; Kamer, Krista; Fong, Peggy

2011-06-01

We quantified the effects of initial macroalgal tissue nitrogen (N) status (depleted and enriched) and varying pulses of nitrate (NO 3 - ) concentration on uptake and storage of nitrogen in Ulva intestinalis L. and Ulva expansa (Setch.) Setch. et N. L. Gardner using mesocosms modeling shallow coastal estuaries in Mediterranean climates. Uptake of NO 3 - (μmol · g dry weight [dwt] -1  · h -1 ) was measured as loss from the water after 1, 2, 4, 8, 12, and 24 h and storage as total tissue nitrogen (% dwt) and nitrate (ppm). Both species of algae exhibited a high affinity for NO 3 - across all N pulses and initial tissue contents. There was greater NO 3 - removal from the water for depleted than enriched algae across all time intervals. In the low-N-pulse treatment, U. intestinalis and U. expansa removed all measurable NO 3 - within 8 and 12 h, respectively, and in the medium and high treatments, removal was high and then decreased over time. Maximum mean uptake rates of nitrate were greater for U. expansa (∼300 μmol · g dwt -1  · h -1 ) than U. intestinalis (∼100 μmol · g dwt -1  · h -1 ); however, uptake rates were highly variable over time. Overall, U. expansa uptake rates were double those of U. intestinalis. Maximum tissue NO 3 - for U. expansa was >1,000 ppm, five times that of U. intestinalis, suggesting that U. expansa has a greater storage capacity in this cellular pool. These results showed that opportunistic green algae with differing tissue nutrient histories were able to efficiently remove nitrate from the water across a wide range of N pulses; thus, both are highly adapted to proliferate in estuarine environments with pulsed nutrient supplies. © 2011 Phycological Society of America.

Novel channel-mediated choline transport in cholinergic neurons of the mouse retina.

PubMed

Ishii, Toshiyuki; Homma, Kohei; Mano, Asuka; Akagi, Takumi; Shigematsu, Yasuhide; Shimoda, Yukio; Inoue, Hiroyoshi; Kakinuma, Yoshihiko; Kaneda, Makoto

2017-10-01

Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X 2 purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X 2 purinoceptors acquire permeability to large cations, such as N -methyl-d-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, α,β-methylene ATP did not produce a cation current, whereas ATPγS and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl-2',4'-sulfonic acid. Accordingly, P2X 2 purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X 2 purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina. NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X 2 purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X 2 purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo. Copyright © 2017 the American Physiological Society.

Cesium Uptake by Rice Roots Largely Depends Upon a Single Gene, HAK1, Which Encodes a Potassium Transporter.

PubMed

Rai, Hiroki; Yokoyama, Saki; Satoh-Nagasawa, Namiko; Furukawa, Jun; Nomi, Takiko; Ito, Yasuka; Fujimura, Shigeto; Takahashi, Hidekazu; Suzuki, Ryuichiro; Yousra, ELMannai; Goto, Akitoshi; Fuji, Shinichi; Nakamura, Shin-Ichi; Shinano, Takuro; Nagasawa, Nobuhiro; Wabiko, Hiroetsu; Hattori, Hiroyuki

2017-09-01

Incidents at the Fukushima and Chernobyl nuclear power stations have resulted in widespread environmental contamination by radioactive nuclides. Among them, 137cesium has a 30 year half-life, and its persistence in soil raises serious food security issues. It is therefore important to prevent plants, especially crop plants, from absorbing radiocesium. In Arabidopsis thaliana, cesium ions are transported into root cells by several different potassium transporters such as high-affinity K+ transporter 5 (AtHAK5). Therefore, the cesium uptake pathway is thought to be highly redundant, making it difficult to develop plants with low cesium uptake. Here, we isolated rice mutants with low cesium uptake and reveal that the Oryza sativa potassium transporter OsHAK1, which is expressed on the surfaces of roots, is the main route of cesium influx into rice plants, especially in low potassium conditions. During hydroponic cultivation with low to normal potassium concentrations (0-206 µM: the normal potassium level in soil), cesium influx in OsHAK1-knockout lines was no greater than one-eighth that in the wild type. In field experiments, knockout lines of O. sativa HAK1 (OsHAK1) showed dramatically reduced cesium concentrations in grains and shoots, but their potassium uptake was not greatly affected and their grain yields were similar to that of the wild type. Our results demonstrate that, in rice roots, potassium transport systems other than OsHAK1 make little or no contribution to cesium uptake. These results show that low cesium uptake rice lines can be developed for cultivation in radiocesium-contaminated areas. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Neurochemical binding profiles of novel indole and benzofuran MDMA analogues.

PubMed

Shimshoni, Jakob A; Winkler, Ilan; Golan, Ezekiel; Nutt, David

2017-01-01

3,4-Methylenedioxy-N-methylamphetamine (MDMA) has been shown to be effective in the treatment of post-traumatic stress disorder (PTSD) in numerous clinical trials. In the present study, we have characterized the neurochemical binding profiles of three MDMA-benzofuran analogues (1-(benzofuran-5-yl)-propan-2-amine, 5-APB; 1-(benzofuran-6-yl)-N-methylpropan-2-amine, 6-MAPB; 1-(benzofuran-5-yl)-N-methylpropan-2-amine, 5-MAPB) and one MDMA-indole analogue (1-(1H-indol-5-yl)-2-methylamino-propan-1-ol, 5-IT). These compounds were screened as potential second-generation anti-PTSD drugs, against a battery of human and non-human receptors, transporters, and enzymes, and their potencies as 5-HT 2 receptor agonist and monoamine uptake inhibitors determined. All MDMA analogues displayed high binding affinities for 5-HT 2a,b,c and NE α2 receptors, as well as significant 5-HT, DA, and NE uptake inhibition. 5-APB revealed significant agonist activity at the 5-HT 2a,b,c receptors, while 6-MAPB, 5-MAPB, and 5-IT exhibited significant agonist activity at the 5-HT 2c receptor. There was a lack of correlation between the results of functional uptake and the monoamine transporter binding assay. MDMA analogues emerged as potent and selective monoamine oxidase A inhibitors. Based on 6-MAPB favorable pharmacological profile, it was further subjected to IC 50 determination for monoamine transporters. Overall, all MDMA analogues displayed higher monoamine receptor/transporter binding affinities and agonist activity at the 5-HT 2a,c receptors as compared to MDMA.

Trypanosoma brucei eflornithine transporter AAT6 is a low-affinity low-selective transporter for neutral amino acids.

PubMed

Mathieu, Christoph; González Salgado, Amaia; Wirdnam, Corina; Meier, Stefan; Grotemeyer, Marianne Suter; Inbar, Ehud; Mäser, Pascal; Zilberstein, Dan; Sigel, Erwin; Bütikofer, Peter; Rentsch, Doris

2014-10-01

Amino acid transporters are crucial for parasite survival since the cellular metabolism of parasitic protozoa depends on the up-take of exogenous amino acids. Amino acid transporters are also of high pharmacological relevance because they may mediate uptake of toxic amino acid analogues. In the present study we show that the eflornithine transporter AAT6 from Trypanosoma brucei (TbAAT6) mediates growth on neutral amino acids when expressed in Saccharomyces cerevisiae mutants. The transport was electrogenic and further analysed in Xenopus laevis oocytes. Neutral amino acids, proline analogues, eflornithine and acivicin induced inward currents. For proline, glycine and tryptophan the apparent affinities and maximal transport rates increased with more negative membrane potentials. Proline-induced currents were dependent on pH, but not on sodium. Although proline represents the primary energy source of T. brucei in the tsetse fly, down-regulation of TbAAT6-expression by RNAi showed that in culture TbAAT6 is not essential for growth of procyclic form trypanosomes in the presence of glucose or proline as energy source. TbAAT6-RNAi lines of both bloodstream and procyclic form trypanosomes showed reduced susceptibility to eflornithine, whereas the sensitivity to acivicin remained unchanged, indicating that acivicin enters the cell by more than one transporter.

2-Phenylbenzothiazole conjugated with cyclopentadienyl tricarbonyl [CpM(CO)3] (M = Re, (99m)Tc) complexes as potential imaging probes for β-amyloid plaques.

PubMed

Jia, Jianhua; Cui, Mengchao; Dai, Jiapei; Liu, Boli

2015-04-14

Technetium-99m-labeled cyclopentadienyl tricarbonyl complexes conjugated with the 2-phenylbenzothiazole binding motif were synthesized. The rhenium surrogates , , and were demonstrated to have moderate to high affinities for Aβ1-42 aggregates with Ki values of 142, 76, 64 and 24 nM, respectively. During the fluorescence staining of brain sections of transgenic mice and patients with Alzheimer's disease, these rhenium complexes demonstrated perfect and intense labeling of Aβ plaques. Moreover, in in vitro autoradiography, (99m)Tc-labeled complexes clearly detected β-amyloid plaques on sections of brain tissue from transgenic mice, which confirmed the sufficient affinity of these tracers for Aβ plaques. However, these compounds did not show desirable properties in vivo, especially showing poor brain uptake (below 0.5% ID g(-1)), which will hinder the further development of these tracers as brain imaging agents. Nonetheless, it is encouraging that these (99m)Tc-labeled complexes designed by a conjugate approach displayed sufficient affinities for Aβ plaques.

ImmunoPET imaging of tissue factor expression in pancreatic cancer with 89Zr-Df-ALT-836.

PubMed

Hernandez, Reinier; England, Christopher G; Yang, Yunan; Valdovinos, Hector F; Liu, Bai; Wong, Hing C; Barnhart, Todd E; Cai, Weibo

2017-10-28

Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies, including pancreatic cancer. Additionally, high TF expression was shown to strongly correlate with poor prognoses and decreased survival in pancreatic cancer patients. Herein, we exploited the potential targeting of TF for positron emission tomography (PET) imaging of pancreatic cancer. The TF-targeted tracer was developed through radiolabeling of the anti-human TF monoclonal antibody (ALT-836) with 89 Zr. The tracer was characterized by fluorescence microscopy and flow cytometry assays in BXPC-3 and PANC-1 cells, two pancreatic cancer cell lines with high and low TF expression levels, respectively. Non-invasive PET scans were acquired in tumor-bearing mice injected with 89 Zr-Df-ALT-836. Additionally, ex vivo biodistribution, blocking, and histological studies were performed to establish the affinity and specificity of 89 Zr-Df-ALT-836 for TF in vivo. 89 Zr-labeling of Df-ALT-836 was achieved in high yield and good specific activity. Flow cytometry and microscopy studies revealed no detectable difference in TF-binding affinity between ALT-836 and Df-ALT-836 in vitro. Longitudinal PET scans unveiled a lasting and prominent 89 Zr-Df-ALT-836 uptake in BXPC-3 tumors (peak at 31.5±6.0%ID/g at 48h post-injection; n=3), which was significantly abrogated (2.3±0.5%ID/g at 48h post-injection; n=3) when mice were pre-injected with a blocking dose (50mg/kg) of unlabeled ALT-836. Ex vivo biodistribution data confirmed the accuracy of the PET results, and histological analysis correlated high tumor uptake with in situ TF expression. Taken together, these results attest to the excellent affinity and TF-specificity of 89 Zr-Df-ALT-836. With elevated, persistent, and specific accumulation in TF-positive BXPC-3 tumors, PET imaging using 89 Zr-Df-ALT-836 promises to open new avenues for improving future diagnosis, stratification, and treatment response assessment in pancreatic cancer patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Hijacking membrane transporters for arsenic phytoextraction

PubMed Central

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

Use of an uncertainty analysis for genome-scale models as a prediction tool for microbial growth processes in subsurface environments.

PubMed

Klier, Christine

2012-03-06

The integration of genome-scale, constraint-based models of microbial cell function into simulations of contaminant transport and fate in complex groundwater systems is a promising approach to help characterize the metabolic activities of microorganisms in natural environments. In constraint-based modeling, the specific uptake flux rates of external metabolites are usually determined by Michaelis-Menten kinetic theory. However, extensive data sets based on experimentally measured values are not always available. In this study, a genome-scale model of Pseudomonas putida was used to study the key issue of uncertainty arising from the parametrization of the influx of two growth-limiting substrates: oxygen and toluene. The results showed that simulated growth rates are highly sensitive to substrate affinity constants and that uncertainties in specific substrate uptake rates have a significant influence on the variability of simulated microbial growth. Michaelis-Menten kinetic theory does not, therefore, seem to be appropriate for descriptions of substrate uptake processes in the genome-scale model of P. putida. Microbial growth rates of P. putida in subsurface environments can only be accurately predicted if the processes of complex substrate transport and microbial uptake regulation are sufficiently understood in natural environments and if data-driven uptake flux constraints can be applied.

A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis

PubMed Central

Goos, Sarah; Kämper, Jörg; Sauer, Norbert

2010-01-01

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. PMID:20161717

Localisation of the high-affinity choline transporter-1 in the rat skeletal motor unit.

PubMed

Lips, Katrin S; Pfeil, Uwe; Haberberger, Rainer V; Kummer, Wolfgang

2002-03-01

The rate-limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline via a high-affinity transporter. We have generated antisera against the recently identified transporter CHT1 to investigate its distribution in rat motor neurons and skeletal muscle and have used these antisera in combination with (1) antisera against the vesicular acetylcholine transporter (VAChT) to identify cholinergic synapses and (2) Alexa-488-labelled alpha-bungarotoxin to identify motor endplates. In the motor unit, immunohistochemistry and RT-PCR have demonstrated that CHT1 is restricted to motoneurons and absent from the non-neuronal ACh-synthesizing elements, e.g. skeletal muscle fibres. In addition, CHT1 is also present in parasympathetic neurons of the tongue, as evidenced by immunohistochemistry and RT-PCR. CHT1 immunoreativity is principally found at all segments (perikaryon, dendrites, axon) of the motoneuron but is enriched at neuro-neuronal and neuro-muscular synapses. This preferential localisation matches well with its anticipated pivotal role in synaptic transmitter recycling and synthesis.

Synthesis and Biological Evaluation of Cyclic [99mTc]-HYNIC-CGPRPPC as a Fibrin-Binding Peptide for Molecular Imaging of Thrombosis and Its Comparison with [99mTc]-HYNIC-GPRPP.

PubMed

Rezaeianpour, Sedigheh; Bozorgi, Atefeh Hajiagha; Moghimi, Abolghasem; Almasi, Ameneh; Balalaie, Saeed; Ramezanpour, Sorour; Nasoohi, Sanaz; Mazidi, Seyed Mohammad; Geramifar, Parham; Bitarafan-Rajabi, Ahmad; Shahhosseini, Soraya

2017-04-01

Many patients worldwide suffer from cardiovascular diseases for which an underlying factor is thrombosis. Devising a molecular imaging technique for early detection of thrombosis in a clinical setting is highly recommended. Because fibrin is a major constituent of clots and is present in all types of thrombi but absent in circulation, it is a highly specific and sensitive target for molecular imaging of thrombi. It is assumed that cyclization of peptides will improve the receptor binding affinity and stability of the peptide. In the present study, we have developed linear and cyclic fibrin-binding peptides for thrombus imaging and compared their biological properties. Linear HYNIC-GPRPP and cyclic HYNIC-CGPRPPC peptides were synthesized using a standard Fmoc strategy and radiolabeled with Tc-99m. The stability of the radiolabeled peptides in human plasma and their affinity for fibrin and blood clots were determined. Blood clearance and biodistribution were evaluated in rats and mice, respectively. The peptide with the highest affinity was injected to a live rabbit femoral thrombosis model, and scintigraphic images were obtained. In vitro studies show that peptides are stable in human plasma and have a high affinity for human fibrin. They also demonstrated fast blood clearance in rats and high thrombus uptake in the Balb/c mice femoral thrombosis model. Femoral thrombosis was visualized 30 min postinjection of cyclic peptide in a live rabbit model using single photon emission computed tomography (SPECT)/X-ray computed tomography. The results indicate that the cyclic peptide is a promising agent for molecular imaging of fibrin using SPECT.

High thermal sensitivity of blood enhances oxygen delivery in the high-flying bar-headed goose.

PubMed

Meir, Jessica U; Milsom, William K

2013-06-15

The bar-headed goose (Anser indicus) crosses the Himalaya twice a year at altitudes where oxygen (O2) levels are less than half those at sea level and temperatures are below -20°C. Although it has been known for over three decades that the major hemoglobin (Hb) component of bar-headed geese has an increased affinity for O2, enhancing O2 uptake, the effects of temperature and interactions between temperature and pH on bar-headed goose Hb-O2 affinity have not previously been determined. An increase in breathing of the hypoxic and extremely cold air experienced by a bar-headed goose at altitude (due to the enhanced hypoxic ventilatory response in this species) could result in both reduced temperature and reduced levels of CO2 at the blood-gas interface in the lungs, enhancing O2 loading. In addition, given the strenuous nature of flapping flight, particularly in thin air, blood leaving the exercising muscle should be warm and acidotic, facilitating O2 unloading. To explore the possibility that features of blood biochemistry in this species could further enhance O2 delivery, we determined the P50 (the partial pressure of O2 at which Hb is 50% saturated) of whole blood from bar-headed geese under conditions of varying temperature and [CO2]. We found that blood-O2 affinity was highly temperature sensitive in bar-headed geese compared with other birds and mammals. Based on our analysis, temperature and pH effects acting on blood-O2 affinity (cold alkalotic lungs and warm acidotic muscle) could increase O2 delivery by twofold during sustained flapping flight at high altitudes compared with what would be delivered by blood at constant temperature and pH.

The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean1[W][OA

PubMed Central

Qin, Lu; Zhao, Jing; Tian, Jiang; Chen, Liyu; Sun, Zhaoan; Guo, Yongxiang; Lu, Xing; Gu, Mian; Xu, Guohua; Liao, Hong

2012-01-01

Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N2 fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N2 fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro 33P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance. PMID:22740613

Cu-64-labeled lactam bridge-cyclized α-MSH peptides for PET imaging of melanoma.

PubMed

Guo, Haixun; Miao, Yubin

2012-08-06

The purpose of this study was to examine and compare the melanoma targeting and imaging properties of (64)Cu-NOTA-GGNle-CycMSH(hex) {(64)Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and (64)Cu-DOTA-GGNle-CycMSH(hex) {(64)Cu-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-GGNle-CycMSH(hex)}. Two lactam bridge-cyclized peptides, NOTA-GGNle-CycMSH(hex) and DOTA-GGNle-CycMSH(hex), were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSH(hex) was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSH(hex). The melanoma targeting and imaging properties of (64)Cu-NOTA-GGNle-CycMSH(hex) and (64)Cu-DOTA-GGNle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSH(hex) and DOTA-GGNle-CycMSH(hex) displayed comparable MC1 receptor binding affinities (1.6 vs 2.1 nM). The substitution of DOTA with NOTA dramatically increased the melanoma uptake and decreased the renal and liver uptake of (64)Cu-NOTA-GGNle-CycMSH(hex). The tumor uptake of (64)Cu-NOTA-GGNle-CycMSH(hex) was between 12.39 ± 1.61 and 12.71 ± 2.68% ID/g at 0.5, 2, and 4 h postinjection. The accumulation of (64)Cu-NOTA-GGNle-CycMSH(hex) activity in normal organs was lower than 1.02% ID/g except for the kidneys 2, 4, and 24 h postinjection. The tumor/liver uptake ratios of (64)Cu-NOTA-GGNle-CycMSHhex were 17.96, 16.95, and 8.02, whereas the tumor/kidney uptake ratios of (64)Cu-NOTA-GGNle-CycMSH(hex) were 2.52, 3.60, and 5.74 at 2, 4, and 24 h postinjection, respectively. Greater than 91% of the injected radioactivity cleared through the urinary system by 2 h postinjection. The substitution of DOTA with NOTA resulted in a dramatic increase in melanoma uptake and decrease in renal and liver uptake of (64)Cu-NOTA-GGNle-CycMSH(hex) as compared to (64)Cu-DOTA-GGNle-CycMSH(hex). High melanoma uptake coupled with low accumulation in nontarget organs suggested (64)Cu-NOTA-GGNle-CycMSH(hex) as a lead radiolabeled peptide for melanoma imaging and therapy.

Cu-64-Labeled Lactam Bridge-Cyclized α-MSH Peptides for PET Imaging of Melanoma

PubMed Central

Guo, Haixun; Miao, Yubin

2012-01-01

The purpose of this study was to examine and compare the melanoma targeting and imaging properties of 64Cu-NOTA-GGNle-CycMSHhex {64Cu-1,4,7-triazacyclononane-1,4,7-triacetic acid-Gly-Gly-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2} and 64Cu-DOTA-GGNle-CycMSHhex {64Cu-1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid-GGNle-CycMSHhex}. Two lactam bridge-cyclized peptides, NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex, were synthesized using fluorenylmethyloxy carbonyl (Fmoc) chemistry. The melanocortin-1 (MC1) receptor binding affinity of NOTA-GGNle-CycMSHhex was determined in B16/F1 melanoma cells and compared with DOTA-GGNle-CycMSHhex. The melanoma targeting and imaging properties of 64Cu-NOTA-GGNle-CycMSHhex and 64Cu-DOTA-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. NOTA-GGNle-CycMSHhex and DOTA-GGNle-CycMSHhex displayed comparable MC1 receptor binding affinities (1.6 vs. 2.1 nM). The substitution of DOTA with NOTA dramatically increased the melanoma uptake and decreased the renal and liver uptake of 64Cu-NOTA-GGNle-CycMSHhex. The tumor uptake of 64Cu-NOTA-GGNle-CycMSHhex was between 12.39 ± 1.61 and 12.71 ± 2.68 % ID/g at 0.5, 2 and 4 h post-injection. The accumulation of 64Cu-NOTA-GGNle-CycMSHhex activity in normal organs was lower than 1.02 % ID/g except for the kidneys 2, 4 and 24 h post-injection. The tumor/liver uptake ratios of 64Cu-NOTA-GGNle-CycMSHhex were 17.96, 16.95 and 8.02, whereas the tumor/kidney uptake ratios of 64Cu-NOTA-GGNle-CycMSHhex were 2.52, 3.60 and 5.74 at 2, 4 and 24 h post-injection, respectively. Greater than 91% of the injected radioactivity cleared through the urinary system by 2 h post-injection. The substitution of DOTA with NOTA resulted in a dramatic increase in melanoma uptake and decrease in renal and liver uptake of 64Cu-NOTA-GGNle-CycMSHhex compared to 64Cu-DOTA-GGNle-CycMSHhex. High melanoma uptake coupled with low accumulation in non-target organs suggested 64Cu-NOTA-GGNle-CycMSHhex as a lead radiolabeled peptide for melanoma imaging and therapy. PMID:22780870

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds

PubMed Central

Chen, Chiliang; Malek, Adel A.; Wargo, Matthew J.; Hogan, Deborah A.; Beattie, Gwyn A.

2017-01-01

Summary We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (Km, 2.6 μM) and, although it also binds betaine (Km, 24.2 μM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (Km, 24 μM) and the betaine-specific SBP BetX (Km, 0.6 μM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs. PMID:19919675

The ATP-binding cassette transporter Cbc (choline/betaine/carnitine) recruits multiple substrate-binding proteins with strong specificity for distinct quaternary ammonium compounds.

PubMed

Chen, Chiliang; Malek, Adel A; Wargo, Matthew J; Hogan, Deborah A; Beattie, Gwyn A

2010-01-01

We identified a choline, betaine and carnitine transporter, designated Cbc, from Pseudomonas syringae and Pseudomonas aeruginosa that is unusual among members of the ATP-binding cassette (ABC) transporter family in its use of multiple periplasmic substrate-binding proteins (SBPs) that are highly specific for their substrates. The SBP encoded by the cbcXWV operon, CbcX, binds choline with a high affinity (K(m), 2.6 microM) and, although it also binds betaine (K(m), 24.2 microM), CbcXWV-mediated betaine uptake did not occur in the presence of choline. The CbcX orthologue ChoX from Sinorhizobium meliloti was similar to CbcX in these binding properties. The core transporter CbcWV also interacts with the carnitine-specific SBP CaiX (K(m), 24 microM) and the betaine-specific SBP BetX (K(m), 0.6 microM). Unlike most ABC transporter loci, caiX, betX and cbcXWV are separated in the genome. CaiX-mediated carnitine uptake was reduced by CbcX and BetX only when they were bound by their individual ligands, providing the first in vivo evidence for a higher affinity for ligand-bound than ligand-free SBPs by an ABC transporter. These studies demonstrate not only that the Cbc transporter serves as a useful model for exploring ABC transporter component interactions, but also that the orphan SBP genes common to bacterial genomes can encode functional SBPs.

Synthesis, iron(III) complexation properties, molecular dynamics simulations and P. aeruginosa siderophore-like activity of two pyoverdine analogs.

PubMed

Antonietti, Viviane; Boudesocque, Stéphanie; Dupont, Laurent; Farvacques, Natacha; Cézard, Christine; Da Nascimento, Sophie; Raimbert, Jean-François; Socrier, Larissa; Robin, Thierry-Johann; Morandat, Sandrine; El Kirat, Karim; Mullié, Catherine; Sonnet, Pascal

2017-09-08

P. aeruginosa ranks among the top five organisms causing nosocomial infections. Among the many novel strategies for developing new therapeutics against infection, targeting iron uptake mechanism seems promising as P. aeruginosa needs iron for its growth and survival. To scavenge iron, the bacterium produces siderophores possessing a very high affinity towards Fe(III) ions such as pyoverdines. In this work, we decided to study two pyoverdine analogs, aPvd2 and aPvd3, structurally close to the endogen pyoverdine. The pFe constants calculated with the values of formation showed a high affinity of aPvd3 towards Fe(III). Molecular dynamics calculations demonstrated that aPvd3-Fe forms with Fe(III) stable 1:1 complexes in water, whereas aPvd2 does not. Only aPvd3 is able to increase the bacterial growth and represents thus an alternative to pyoverdine for iron acquisition by the bacterium. The aPvd2-3 interaction studies with a lipid membrane indicated that they were unable to interact and to cross the plasma membrane of bacteria by passive diffusion. Consequently, the penetration of aPvd3 is ruled by a transport membrane protein. These results showed that aPvd3 may be used to inhibit pyoverdine uptake or to promote the accumulation and release of antibiotics into the cell following a Trojan horse strategy. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Nitric Oxide Modulates Sodium Vitamin C Transporter 2 (SVCT-2) Protein Expression via Protein Kinase G (PKG) and Nuclear Factor-κB (NF-κB)*

PubMed Central

Portugal, Camila Cabral; da Encarnação, Thaísa Godinho; Socodato, Renato; Moreira, Sarah Rodrigues; Brudzewsky, Dan; Ambrósio, António Francisco; Paes-de-Carvalho, Roberto

2012-01-01

Ascorbate is an important antioxidant, which also displays important functions in neuronal tissues, including the retina. The retina is responsible for the initial steps of visual processing, which is further refined in cerebral high-order centers. The retina is also a prototypical model for studying physiologic aspects of cells that comprise the nervous system. Of major importance also is the cellular messenger nitric oxide (NO). Previous studies have demonstrated the significance of NO for both survival and proliferation of cultured embryonic retinal cells. Cultured retinal cells express a high-affinity ascorbate transporter, and the release of ascorbate is delicately regulated by ionotropic glutamate receptors. Therefore, we proposed whether there is interplay between the ascorbate transport system and NO signaling pathway in retinal cells. Here we show compelling evidence that ascorbate uptake is tightly controlled by NO and its downstream signaling pathway in culture. NO also modulates the expression of SVCT-2, an effect mediated by cGMP and PKG. Kinetic studies suggest that NO increases the transport capacity for ascorbate, but not the affinity of SVCT-2 for its substrate. Interestingly, NO utilizes the NF-κB pathway, in a PKG-dependent manner, to modulate both SVCT-2 expression and ascorbate uptake. These results demonstrate that NO exerts a fine-tuned control of the availability of ascorbate to cultured retinal cells and strongly reinforces ascorbate as an important bioactive molecule in neuronal tissues. PMID:22041898

Nitric oxide modulates sodium vitamin C transporter 2 (SVCT-2) protein expression via protein kinase G (PKG) and nuclear factor-κB (NF-κB).

PubMed

Portugal, Camila Cabral; da Encarnação, Thaísa Godinho; Socodato, Renato; Moreira, Sarah Rodrigues; Brudzewsky, Dan; Ambrósio, António Francisco; Paes-de-Carvalho, Roberto

2012-02-03

Ascorbate is an important antioxidant, which also displays important functions in neuronal tissues, including the retina. The retina is responsible for the initial steps of visual processing, which is further refined in cerebral high-order centers. The retina is also a prototypical model for studying physiologic aspects of cells that comprise the nervous system. Of major importance also is the cellular messenger nitric oxide (NO). Previous studies have demonstrated the significance of NO for both survival and proliferation of cultured embryonic retinal cells. Cultured retinal cells express a high-affinity ascorbate transporter, and the release of ascorbate is delicately regulated by ionotropic glutamate receptors. Therefore, we proposed whether there is interplay between the ascorbate transport system and NO signaling pathway in retinal cells. Here we show compelling evidence that ascorbate uptake is tightly controlled by NO and its downstream signaling pathway in culture. NO also modulates the expression of SVCT-2, an effect mediated by cGMP and PKG. Kinetic studies suggest that NO increases the transport capacity for ascorbate, but not the affinity of SVCT-2 for its substrate. Interestingly, NO utilizes the NF-κB pathway, in a PKG-dependent manner, to modulate both SVCT-2 expression and ascorbate uptake. These results demonstrate that NO exerts a fine-tuned control of the availability of ascorbate to cultured retinal cells and strongly reinforces ascorbate as an important bioactive molecule in neuronal tissues.

The Raf-like Kinase ILK1 and the High Affinity K+ Transporter HAK5 Are Required for Innate Immunity and Abiotic Stress Response1[OPEN

PubMed Central

Brauer, Elizabeth K.; Ahsan, Nagib; Kato, Naohiro; Coluccio, Alison E.; Thelen, Jay J.

2016-01-01

Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca2+ uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H+)/K+ symporter that mediates a high-affinity uptake during K+ deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K+ efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K+ loss. Taken together, our results position ILK1 as a link between plant defense pathways and K+ homeostasis. PMID:27208244

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent.

PubMed

Kim, Eun-Mi; Joung, Min-Hee; Lee, Chang-Moon; Jeong, Hwan-Jeong; Lim, Seok Tae; Sohn, Myung-Hee; Kim, Dong Wook

2010-07-15

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the 'click-to-chelate' protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12, [(99m)Tc(CO)(3)]13, and [(99m)Tc(CO)(3)]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)(3)]12, [Re(CO)(3)]13, and [Re(CO)(3)]14, were shown to have high binding affinities (0.13 microM, 0.06 microM, and 0.16 microM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12-14) to c-Met receptor positive U87MG cells. 2010 Elsevier Ltd. All rights reserved.

In ovo uptake, metabolism, and tissue-specific distribution of chiral PCBs and PBDEs in developing chicken embryos

PubMed Central

Li, Zong-Rui; Luo, Xiao-Jun; Huang, Li-Qian; Mai, Bi-Xian

2016-01-01

Fertilized chicken eggs were injected with environmental doses of 4 chiral polychlorinated biphenyls (PCBs) and 8 polybrominated biphenyl ethers (PBDEs) to investigate their uptake, metabolism in the embryo, and distribution in the neonate chicken. PCB95 uptake was the most efficient (80%) whereas BDE209 was the least (56%). Embryos metabolized approximately 52% of the PCBs absorbed. Though some degree of metabolism in the first 18 days, most of the PCBs and PBDEs was metabolized in the last three days, when BDE85, 99, 153, and 209 decrease by 11–37%. Enantioselective metabolism of the (+) enantiomers of PCB95, 149, and 132 and the (−) enantiomer of PCB91 was observed. The enantioselective reactivity was higher with the two penta-PCBs than the two tetra-PCBs. Liver, exhibited high affinity for high lipophilic chemicals, enrich all chemicals that was deflected in other tissues except for some special chemicals in a given tissues. Lipid composition, time of organ formation, and metabolism contribute to the distribution of chemicals in the neonate chicken. The result of this study will improve our understanding on the fate and potential adverse effects of PCBs and PBDEs in the neonate chicken. PMID:27819361

Covalent Organic Frameworks as a Decorating Platform for Utilization and Affinity Enhancement of Chelating Sites for Radionuclide Sequestration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Qi; Aguila, Briana; Earl, Lyndsey D.

The potential consequences of nuclear events and the complexity of nuclear waste management motivate the development of selective solid-phase sorbents to provide enhanced protection. In this paper, it is shown that 2D covalent organic frameworks (COFs) with unique structures possess all the traits to be well suited as a platform for the deployment of highly efficient sorbents such that they exhibit remarkable performance, as demonstrated by uranium capture. The chelating groups laced on the open 1D channels exhibit exceptional accessibility, allowing significantly higher utilization efficiency. In addition, the 2D extended polygons packed closely in an eclipsed fashion bring chelating groupsmore » in adjacent layers parallel to each other, which may facilitate their cooperation, thereby leading to high affinity toward specific ions. As a result, the amidoxime-functionalized COFs far outperform their corresponding amorphous analogs in terms of adsorption capacities, kinetics, and affinities. Specifically, COF-TpAb-AO is able to reduce various uranium contaminated water samples from 1 ppm to less than 0.1 ppb within several minutes, well below the drinking water limit (30 ppb), as well as mine uranium from spiked seawater with an exceptionally high uptake capacity of 127 mg g -1. Finally, these results delineate important synthetic advances toward the implementation of COFs in environmental remediation.« less

Covalent Organic Frameworks as a Decorating Platform for Utilization and Affinity Enhancement of Chelating Sites for Radionuclide Sequestration

DOE PAGES

Sun, Qi; Aguila, Briana; Earl, Lyndsey D.; ...

2018-03-27

The potential consequences of nuclear events and the complexity of nuclear waste management motivate the development of selective solid-phase sorbents to provide enhanced protection. In this paper, it is shown that 2D covalent organic frameworks (COFs) with unique structures possess all the traits to be well suited as a platform for the deployment of highly efficient sorbents such that they exhibit remarkable performance, as demonstrated by uranium capture. The chelating groups laced on the open 1D channels exhibit exceptional accessibility, allowing significantly higher utilization efficiency. In addition, the 2D extended polygons packed closely in an eclipsed fashion bring chelating groupsmore » in adjacent layers parallel to each other, which may facilitate their cooperation, thereby leading to high affinity toward specific ions. As a result, the amidoxime-functionalized COFs far outperform their corresponding amorphous analogs in terms of adsorption capacities, kinetics, and affinities. Specifically, COF-TpAb-AO is able to reduce various uranium contaminated water samples from 1 ppm to less than 0.1 ppb within several minutes, well below the drinking water limit (30 ppb), as well as mine uranium from spiked seawater with an exceptionally high uptake capacity of 127 mg g -1. Finally, these results delineate important synthetic advances toward the implementation of COFs in environmental remediation.« less

Spatial location of neutralizing and non-neutralizing B cell epitopes on domain 1 of ricin toxin's binding subunit.

PubMed

Rong, Yinghui; Van Slyke, Greta; Vance, David J; Westfall, Jennifer; Ehrbar, Dylan; Mantis, Nicholas J

2017-01-01

Ricin toxin's binding subunit (RTB) is a galactose-/N-acetylgalactosamine (Gal/GalNac)-specific lectin that mediates uptake and intracellular trafficking of ricin within mammalian cells. Structurally, RTB consists of two globular domains, each divided into three homologous sub-domains (α, β, γ). In this report, we describe five new murine IgG monoclonal antibodies (mAbs) against RTB: MH3, 8A1, 8B3, LF1, and LC5. The mAbs have similar binding affinities (KD) for ricin holotoxin, but displayed a wide range of in vitro toxin-neutralizing activities. Competition ELISAs indicate that the two most potent toxin-neutralizing mAbs (MH3, 8A1), as well as one of the moderate toxin-neutralizing mAbs (LF1), recognize distinct epitopes near the low affinity Gal recognition domain in RTB subdomain 1α. Evaluated in a mouse model of systemic ricin challenge, all five mAbs afforded some benefit against intoxication, but only MH3 was protective. However, neither MH3 nor 24B11, another well-characterized mAb against RTB subdomain 1α, could passively protect mice against a mucosal (intranasal) ricin challenge. This is in contrast to SylH3, a previously characterized mAb directed against an epitope near RTB's high affinity Gal/GalNac recognition element in sub-domain 2γ, which protected animals against systemic and mucosal ricin exposure. SylH3 was significantly more effective than MH3 and 24B11 at blocking ricin attachment to host cell receptors, suggesting that mucosal immunity to ricin is best imparted by antibodies that target RTB's high affinity Gal/GalNac recognition element in subdomain 2γ, not the low affinity Gal recognition domain in subdomain 1α.

Synthesis and biological characterization of (3R,4R)-4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol and its stereoisomers for monoamine transporters

PubMed Central

Kharkar, Prashant S.; Batman, Angela M.; Zhen, Juan; Beardsley, Patrick M.; Reith, Maarten E. A.

2012-01-01

In this report we describe synthesis and biological evaluation of a series of asymmetric 4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol based dihydroxy compounds where the hydroxy groups are located both on the piperidine ring and also on the N-phenylethyl side chain exo-cyclically. In vitro uptake inhibition data indicates high affinity of these molecules for the dopamine transporter (DAT) in addition to their moderate to high affinity for the norepinephrine transporter (NET). Interestingly, compounds 9b and 9d exhibited affinities for all three monoamine transporters with highest potency at DAT and NET and moderate potency at the serotonin transporter (SERT) (Ki 2.29, 78.4 and 155 nM for 9b and 1.55, 14.1 and 259 nM for 9d, respectively). Selected compounds, 9a, 9d and 9d’ were tested for their locomotor activity effects in mice, and for their ability to occasion the cocaine discriminative stimulus in rats. These test compounds generally exhibited a much longer duration of action than cocaine for elevating locomotor activity, and dose-dependently completely generalized the cocaine discriminative stimulus. PMID:19449323

Biotinylated probes of artemisinin with labeling affinity toward Trypanosoma brucei brucei target proteins.

PubMed

Konziase, Benetode

2015-08-01

We studied the target proteins of artemisinin in Trypanosoma brucei brucei using the affinity-labeling method. We designed and synthesized four biotinylated probes of artemisinin for use as molecular tools. Their in vitro trypanocidal activities (data not shown) proved that they mimicked the biological action of artemisinin. We assessed the chemical stability for all of the probes in the parasite culture medium and lysate using reversed-phase high-performance liquid chromatography (HPLC). After 3-h incubations, the probes remained undecomposed in a range of 40 to 65% in the parasite culture medium, whereas approximately 80% of the probes remained stable in the parasite lysate. Using liquid chromatography mass spectrometry (LC-MS), we demonstrated that, with respect to all of the probes, uptakes into the parasite ranging from 81 to 96% occurred after 30-min incubations. In a competitive binding assay between artemisinin and the four biotinylated probes, we searched for the trypanosomal target protein of artemisinin. Consequently, we observed that only the diazirine-free probe 5 could provide the desired result with high affinity-labeling efficiency. Using the horseradish peroxidase-tagged streptavidin-biotin method, we showed that artemisinin could specifically bind to candidate target proteins of approximately 60, 40, and 39 kDa. Copyright © 2015 Elsevier Inc. All rights reserved.

Small-animal PET of tumor damage induced by photothermal ablation with 64Cu-bis-DOTA-hypericin.

PubMed

Song, Shaoli; Xiong, Chiyi; Zhou, Min; Lu, Wei; Huang, Qian; Ku, Geng; Zhao, Jun; Flores, Leo G; Ni, Yicheng; Li, Chun

2011-05-01

The purpose of this study was to investigate the potential application of small-molecular-weight (64)Cu-labeled bis-DOTA-hypericin in the noninvasive assessment of response to photothermal ablation therapy. Bis-DOTA-hypericin was labeled with (64)Cu with high efficiency (>95% without purification). Nine mice bearing subcutaneous human mammary BT474 tumors were used. Five mice were injected intratumorally with semiconductor CuS nanoparticles, followed by near-infrared laser irradiation 24 h later (12 W/cm(2) for 3 min), and 4 mice were not treated (control group). All mice were intravenously injected with (64)Cu-bis-DOTA-hypericin (24 h after laser treatment in treated mice). Small-animal PET images were acquired at 2, 6, and 24 h after radiotracer injection. All mice were killed immediately after the imaging session for biodistribution and histology study. In vitro cell uptake and surface plasmon resonance studies were performed to validate the small-animal PET results. (64)Cu-bis-DOTA-hypericin uptake was significantly higher in the treatment group than in the control group. The percentage injected dose per gram of tissue in the treated and control groups was 1.72 ± 0.43 and 0.76 ± 0.19, respectively (P = 0.017), at 24 h after injection. Autoradiography and histology results were consistent with selective uptake of the radiotracer in the necrotic zone of the tumor induced by photothermal ablation therapy. In vitro results showed that treated BT474 cells had a higher uptake of (64)Cu-bis-DOTA-hypericin than nontreated cells. Surface plasmon resonance study showed that bis-DOTA-hypericin had higher binding affinity to phosphatidylserine and phosphatidylethanolamine than to phosphatidylcholine. (64)Cu-bis-DOTA-hypericin has a potential to image thermal therapy-induced tumor cell damage. The affinity of (64)Cu-bis-DOTA-hypericin for injured tissues may be attributed to the breakdown of the cell membrane and exposure of phosphatidylserine or phosphatidylethanolamine to the radiotracer, which binds selectively to these phospholipids.

Plant Nitrogen Acquisition Under Low Availability: Regulation of Uptake and Root Architecture

PubMed Central

Kiba, Takatoshi; Krapp, Anne

2016-01-01

Nitrogen availability is a major factor determining plant growth and productivity. Plants acquire nitrogen nutrients from the soil through their roots mostly in the form of ammonium and nitrate. Since these nutrients are scarce in natural soils, plants have evolved adaptive responses to cope with the environment. One of the most important responses is the regulation of nitrogen acquisition efficiency. This review provides an update on the molecular determinants of two major drivers of the nitrogen acquisition efficiency: (i) uptake activity (e.g. high-affinity nitrogen transporters) and (ii) root architecture (e.g. low-nitrogen-availability-specific regulators of primary and lateral root growth). Major emphasis is laid on the regulation of these determinants by nitrogen supply at the transcriptional and post-transcriptional levels, which enables plants to optimize nitrogen acquisition efficiency under low nitrogen availability. PMID:27025887

Uptake of gentamicin by separated, viable renal tubules from rabbits.

PubMed

Barza, M; Murray, T; Hamburger, R J

1980-04-01

The proximal renal tubules have a marked affinity for gentamicin; they also are the major site of nephrotoxicity caused by this drug. The uptake of radiolabeled gentamicin in separated, viable renal tubules prepared by enzymatic digestion of rabbit kidneys was studied. The preparations showed rapid initial uptake of gentamicin followed by continued slower uptake. Accumulation was not affected by pH, but was significantly inhibited by ouabain, dinitrophenol, anoxia, and hypothermia in the absence of evident cellular damage. At gentamicin concentrations of greater than 50 microgram/ml in the medium, there was competition for drug uptake. Gentamicin efflux in tubules that were taken from a medium containing antibiotic and placed into antibiotic-free fluid was slow and incomplete. From these data it appears that gentamicin uptake by separated renal tubules occurs by a process that requires metabolic energy; thereafter, the drug resides in a poorly exchangeable cellular pool.

Monocarboxylate and alpha-ketoglutarate carriers from bovine heart mitochondria. Purification by affinity chromatography on immobilized 2-cyano-4-hydroxycinnamate.

PubMed

Bolli, R; Nałecz, K A; Azzi, A

1989-10-25

2-Cyano-4-hydroxycinnamate was covalently linked, through a diazo bond, to Sepharose 4B, which had been elongated with a hydrophobic spacer. A Triton X-100 extract from bovine heart mitochondria was pre-purified by hydroxylapatite chromatography and passed through the 2-cyano-4-hydroxycinnamate affinity resin in the presence of 0.7% deoxycholate. At pH 6 and in the presence of 0.2 M sodium chloride, a single polypeptide with an Mr of 34,000 was eluted. Subsequently, at pH 8 and in the presence of 2-cyano-4-hydroxycinnamate, another single protein with an Mr of 31,500 was released. Both proteins were reconstituted into phospholipid vesicles and their transport activities were measured. High, delta pH-dependent, 2-cyanocinnamate-sensitive pyruvate uptake was measured in vesicles containing only the 34-kDa protein. alpha-Ketobutyrate and other alpha-ketomonocarboxylic acids were competitive inhibitors of the pyruvate uptake, whereas di- and tricarboxylates had only small effects. alpha-Ketoglutarate-alpha-ketoglutarate exchange could only be measured in vesicles containing the 31.5-kDa protein. The molecular weight of this protein and its functional properties were similar to those of the alpha-ketoglutarate carrier isolated by a different method (Bisaccia, Indiveri, C., and Palmieri, F. (1985) Biochim. Biophys. Acta 810, 362-369). 2-Cyano-4-hydroxycinnamate inhibited the alpha-ketoglutarate exchange in a noncompetitive manner with an apparent Ki of 0.7 mM. It is concluded that by the described affinity chromatography procedure, two mitochondrial carriers transporting alpha-ketoacids, i.e. the monocarboxylate and the alpha-ketoglutarate carrier, could be purified in a functionally active state.

FigA, a putative homolog of low-affinity calcium system member Fig1 in Saccharomyces cerevisiae, is involved in growth and asexual and sexual development in Aspergillus nidulans.

PubMed

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

2014-02-01

Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca(2+) rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roy, Caroline; Gagné, Valérie; Fernandes, Maria J.G.

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} 1.1more » 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 trapping. • Human peripheral blood leukocytes capture and concentrate quinacrine. • Polymorphonuclear leukocytes do so with higher apparent affinity. • Polymorphonuclear are also more competent than lymphocytes for pinocytosis.« less

The effect of CO2 availability on the growth, iron oxidation and CO2-fixation rates of pure cultures of Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans.

PubMed

Bryan, C G; Davis-Belmar, C S; van Wyk, N; Fraser, M K; Dew, D; Rautenbach, G F; Harrison, S T L

2012-07-01

Understanding how bioleaching systems respond to the availability of CO(2) is essential to developing operating conditions that select for optimum microbial performance. Therefore, the effect of inlet gas and associated dissolved CO(2) concentration on the growth, iron oxidation and CO(2) -fixation rates of pure cultures of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum was investigated in a batch stirred tank system. The minimum inlet CO(2) concentrations required to promote the growth of At. ferrooxidans and L. ferriphilum were 25 and 70 ppm, respectively, and corresponded to dissolved CO(2) concentrations of 0.71 and 1.57 µM (at 30°C and 37°C, respectively). An actively growing culture of L. ferriphilum was able to maintain growth at inlet CO(2) concentrations less than 30 ppm (0.31-0.45 µM in solution). The highest total new cell production and maximum specific growth rates from the stationary phase inocula were observed with CO(2) inlet concentrations less than that of air. In contrast, the amount of CO(2) fixed per new cell produced increased with increasing inlet CO(2) concentrations above 100 ppm. Where inlet gas CO(2) concentrations were increased above that of air the additional CO(2) was consumed by the organisms but did not lead to increased cell production or significantly increase performance in terms of iron oxidation. It is proposed that At. ferrooxidans has two CO(2) uptake mechanisms, a high affinity system operating at low available CO(2) concentrations, which is subject to substrate inhibition and a low affinity system operating at higher available CO(2) concentrations. L. ferriphilum has a single uptake system characterised by a moderate CO(2) affinity. At. ferrooxidans performed better than L. ferriphilum at lower CO(2) availabilities, and was less affected by CO(2) starvation. Finally, the results demonstrate the limitations of using CO(2) uptake or ferrous iron oxidation data as indirect measures of cell growth and performance across varying physiological conditions. Copyright © 2012 Wiley Periodicals, Inc.

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

NASA Astrophysics Data System (ADS)

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. Stackhouse, B. T. et al. J. Geophys. Res. Biogeosciences 120, 1764-1784 (2015). Lau, M. C. Y. et al. ISME J. 9, 1880-1891 (2015).

High-affinity nitrate/nitrite transporters NrtA and NrtB of Aspergillus nidulans exhibit high specificity and different inhibitor sensitivity

PubMed Central

Akhtar, Naureen; Karabika, Eugenia; Kinghorn, James R.; Glass, Anthony D.M.; Unkles, Shiela E.

2015-01-01

The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters. PMID:25855763

Effect of arginine methylation on the RNA recognition and cellular uptake of Tat-derived peptides.

PubMed

Li, Jhe-Hao; Chiu, Wen-Chieh; Yao, Yun-Chiao; Cheng, Richard P

2015-05-01

Arginine (Arg) methylation is a common post-translational modification that regulates gene expression and viral infection. The HIV-1 Tat protein is an essential regulatory protein for HIV proliferation, and is methylated in the cell. The basic region (residues 47-57) of the Tat protein contains six Arg residues, and is responsible for two biological functions: RNA recognition and cellular uptake. In this study, we explore the effect of three different methylation states at each Arg residue in Tat-derived peptides on the two biological functions. The Tat-derived peptides were synthesized by solid phase peptide synthesis. TAR RNA binding of the peptides was assessed by electrophoresis mobility shift assays. The cellular uptake of the peptides into Jurkat cells was determined by flow cytometry. Our results showed that RNA recognition was affected by both methylation state and position. In particular, asymmetric dimethylation at position 53 decreased TAR RNA binding affinity significantly, but unexpectedly less so upon asymmetric dimethylation at position 52. The RNA binding affinity even slightly increased upon methylation at some of the flanking Arg residues. Upon Arg methylation, the cellular uptake of Tat-derived peptides mostly decreased. Interestingly, cellular uptake of Tat-derived peptides with a single asymmetrically dimethylated Arg residue was similar to the native all Arg peptide (at 120 μM). Based on our results, TAR RNA binding apparently required both guanidinium terminal NH groups on Arg53, whereas cellular uptake apparently required guanidinium terminal NH₂ groups instead. These results should provide insight into how nature uses arginine methylation to regulate different biological functions, and should be useful for the development of functional molecules with methylated arginines. Copyright © 2015. Published by Elsevier Ltd.

18F-Fluorosulfate for PET Imaging of the Sodium-Iodide Symporter: Synthesis and Biologic Evaluation In Vitro and In Vivo.

PubMed

Khoshnevisan, Alex; Chuamsaamarkkee, Krisanat; Boudjemeline, Mehdi; Jackson, Alex; Smith, Gareth E; Gee, Antony D; Fruhwirth, Gilbert O; Blower, Philip J

2017-01-01

Anion transport by the human sodium-iodide symporter (hNIS) is an established target for molecular imaging and radionuclide therapy. Current radiotracers for PET of hNIS expression are limited to 124 I - and 18 F-BF 4 - We sought new 18 F-labeled hNIS substrates offering higher specific activity, higher affinity, and simpler radiochemical synthesis than 18 F-BF 4 - METHODS: The ability of a range of anions, some containing fluorine, to block 99m TcO 4 - uptake in hNIS-expressing cells was measured. SO 3 F - emerged as a promising candidate. 18 F-SO 3 F - was synthesized by reaction of 18 F - with SO 3 -pyridine complex in MeCN and purified using alumina and quaternary methyl ammonium solid-phase extraction cartridges. Chemical and radiochemical purity and serum stability were determined by radiochromatography. Radiotracer uptake and efflux in hNIS-transduced HCT116-C19 cells and the hNIS-negative parent cell line were evaluated in vitro in the presence and absence of a known competitive inhibitor (NaClO 4 ). PET/CT imaging and ex vivo biodistribution measurement were conducted on BALB/c mice, with and without NaClO 4 inhibition. Fluorosulfate was identified as a potent inhibitor of 99m TcO 4 - uptake via hNIS in vitro (half-maximal inhibitory concentration, 0.55-0.56 μM (in comparison with 0.29-4.5 μM for BF 4 - , 0.07 μM for TcO 4 - , and 2.7-4.7 μM for I - ). Radiolabeling to produce 18 F-SO 3 F - was simple and afforded high radiochemical purity suitable for biologic evaluation (radiochemical purity > 95%, decay-corrected radiochemical yield = 31.6%, specific activity ≥ 48.5 GBq/μmol). Specific, blockable hNIS-mediated uptake in HCT116-C19 cells was observed in vitro, and PET/CT imaging of normal mice showed uptake in thyroid, salivary glands (percentage injected dose/g at 30 min, 563 ± 140 and 32 ± 9, respectively), and stomach (percentage injected dose/g at 90 min, 68 ± 21). Fluorosulfate is a high-affinity hNIS substrate. 18 F-SO 3 F - is easily synthesized in high yield and very high specific activity and is a promising candidate for preclinical and clinical PET imaging of hNIS expression and thyroid-related disease; it is the first example of in vivo PET imaging with a tracer containing an S- 18 F bond. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Functional analysis RaZIP1 transporter of the ZIP family from the ectomycorrhizal Zn-accumulating Russula atropurpurea.

PubMed

Leonhardt, Tereza; Sácký, Jan; Kotrba, Pavel

2018-04-01

A search of R. atropurpurea transcriptome for sequences encoding the transporters of the Zrt-, Irt-like Protein (ZIP) family, which are in eukaryotes integral to Zn supply into cytoplasm, allowed the identification of RaZIP1 cDNA with a predicted product belonging to ZIP I subfamily; it was subjected to functional studies in mutant Saccharomyces cerevisiae strains. The expression of RaZIP1, but not RaZIP1 H208A or RaZIP1 H232A mutants lacking conserved-among-ZIPs transmembrane histidyls, complemented Zn uptake deficiency in zrt1Δzrt2Δ yeasts. RaZIP1 substantially increased cellular Zn uptake in this strain and added to Zn sensitivity in zrc1Δcot1Δ mutant. The Fe uptake deficiency in ftr1Δ strain was not rescued and Mn uptake was insufficient for toxicity in Mn-sensitive pmr1Δ yeasts. By contrast, RaZIP1 increased Cd sensitivity in yap1Δ strain and conferred Cd transport activity in yeasts, albeit with substantially lower efficiency compared to Zn transport. In metal uptake assays, the accumulation of Zn in zrt1Δzrt2Δ strain remained unaffected by Cd, Fe, and Mn present in 20-fold molar excess over Zn. Immunofluorescence microscopy detected functional hemagglutinin-tagged HA::RaZIP1 on the yeast cell protoplast periphery. Altogether, these data indicate that RaZIP1 is a high-affinity plasma membrane transporter specialized in Zn uptake, and improve the understanding of the cellular and molecular biology of Zn in R. atropurpurea that is known for its ability to accumulate remarkably high concentrations of Zn.

Molecular and functional characterization of riboflavin specific transport system in rat brain capillary endothelial cells.

PubMed

Patel, Mitesh; Vadlapatla, Ramya Krishna; Pal, Dhananjay; Mitra, Ashim K

2012-08-15

Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of a riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with K(m) and V(max) values of 19 ± 3 μM and 0.235 ± 0.012 pmol/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca(++)/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-riboflavin. Apical and baso-lateral uptake of [3H]-riboflavin clearly indicates that a riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to a riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. The blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration. Copyright © 2012 Elsevier B.V. All rights reserved.

Functional characterization of the organic cation transporters (OCTs) in human airway pulmonary epithelial cells.

PubMed

Ingoglia, Filippo; Visigalli, Rossana; Rotoli, Bianca Maria; Barilli, Amelia; Riccardi, Benedetta; Puccini, Paola; Dall'Asta, Valeria

2015-07-01

Organic cation transporters (OCT1-3) mediate the transport of organic cations including inhaled drugs across the cell membrane, although their role in lung epithelium hasn't been well understood yet. We address here the expression and functional activity of OCT1-3 in human airway epithelial cells A549, Calu-3 and NCl-H441. Kinetic and inhibition analyses, employing [(3)H]1-methyl-4-phenylpyridinium (MPP+) as substrate, and the compounds quinidine, prostaglandine E2 (PGE2) and corticosterone as preferential inhibitors of OCT1, OCT2, and OCT3, respectively, have been performed. A549 cells present a robust MPP+ uptake mediated by one high-affinity component (Km~50μM) which is identifiable with OCT3. Corticosterone, indeed, completely inhibits MPP+ transport, while quinidine and PGE2 are inactive and SLC22A3/OCT3 silencing with siRNA markedly lowers MPP+ uptake. Conversely, Calu-3 exhibits both a high (Km<20μM) and a low affinity (Km>0.6mM) transport components, referable to OCT3 and OCT1, respectively, as demonstrated by the inhibition analysis performed at proper substrate concentrations and confirmed by the use of specific siRNA. These transporters are active also when cells are grown under air-liquid interface (ALI) conditions. Only a very modest saturable MPP+ uptake is measurable in NCl-H441 cells and the inhibitory effect of quinidine points to OCT1 as the subtype functionally involved in this model. Finally, the characterization of MPP+ transport in human bronchial BEAS-2B cells suggests that OCT1 and OCT3 are operative. These findings could help to identify in vitro models to be employed for studies concerning the specific involvement of each transporter in drug transportation. Copyright © 2015 Elsevier B.V. All rights reserved.

The role of siderophores in metal homeostasis of members of the genus Burkholderia.

PubMed

Mathew, Anugraha; Jenul, Christian; Carlier, Aurelien L; Eberl, Leo

2016-02-01

Although members of the genus Burkholderia can utilize a high-affinity iron uptake system to sustain growth under iron-limiting conditions, many strains also produce siderophores, suggesting that they may serve alternative functions. Here we demonstrate that the two Burkholderia siderophores pyochelin and ornibactin can protect the cells from metal toxicity and thus play an alternative role in metal homeostasis. We also demonstrate that metals such as copper and zinc induce the production of ornibactin. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

No-carrier-added (18F)-N-methylspiroperidol

DOEpatents

Shiue, Chyng-Yann; Fowler, Joanna S.; Wolf, Alfred P.

1993-01-01

There is disclosed a radioligand labeled with a positron emitting radionuclide suitable for dynamic study in living humans with positron emission transaxial tomography. [.sup.18 F]-N-methylspiroperidol, exhibiting extremely high affinity for the dopamine receptors, provides enhanced uptake and retention in the brain concomitant with reduced radiation burden. These characteristics all combine to provide [.sup.18 F]-N-methylspiroperidol as a radioligand superior to known radioligands for mapping dopamine receptors in normal and disease states in the living brain. Additionally, a new synthetic procedure for this material is disclosed.

No-carrier-added (18F)-N-methylspiroperidol

DOEpatents

Shiue, Chyng-Yann; Fowler, Joanna S.; Wolf, Alfred P.

1993-07-06

There is disclosed a radioligand labeled with a positron emitting radionuclide suitable for dynamic study in living humans with positron emission transaxial tomography. [.sup.18 F]-N-methylspiroperidol, exhibiting extremely high affinity for the dopamine receptors, provides enhanced uptake and retention in the brain concomitant with reduced radiation burden. These characteristics all combine to provide [.sup.18 F]-N-methylspiroperidol as a radioligand superior to known radioligands for mapping dopamine receptors in normal and disease states in the living brain. Additionally, a new synthetic procedure for this material is disclosed.

Non-Invasive Assessment of Liver Function

PubMed Central

Helmke, Steve; Colmenero, Jordi; Everson, Gregory T.

2015-01-01

Purpose of review It is our opinion that there is an unmet need in Hepatology for a minimally- or noninvasive test of liver function and physiology. Quantitative liver function tests (QLFTs) define the severity and prognosis of liver disease by measuring the clearance of substrates whose uptake or metabolism is dependent upon liver perfusion or hepatocyte function. Substrates with high affinity hepatic transporters exhibit high “first-pass” hepatic extraction and their clearance measures hepatic perfusion. In contrast, substrates metabolized by the liver have low first-pass extraction and their clearance measures specific drug metabolizing pathways. Recent Findings We highlight one QLFT, the dual cholate test, and introduce the concept of a disease severity index (DSI) linked to clinical outcome that quantifies the simultaneous processes of hepatocyte uptake, clearance from the systemic circulation, clearance from the portal circulation, and portal-systemic shunting. Summary It is our opinion that dual cholate is a relevant test for defining disease severity, monitoring the natural course of disease progression, and quantifying the response to therapy. PMID:25714706

Ecto-F1-ATPase: A moonlighting protein complex and an unexpected apoA-I receptor

PubMed Central

Vantourout, Pierre; Radojkovic, Claudia; Lichtenstein, Laeticia; Pons, Véronique; Champagne, Eric; Martinez, Laurent O

2010-01-01

Mitochondrial ATP synthase has been recently detected at the surface of different cell types, where it is a high affinity receptor for apoA-I, the major protein component in high density lipoproteins (HDL). Cell surface ATP synthase (namely ecto-F1-ATPase) expression is related to different biological effects, such as regulation of HDL uptake by hepatocytes, endothelial cell proliferation or antitumor activity of Vγ9/Vδ2 T lymphocytes. This paper reviews the recently discovered functions and regulations of ecto-F1-ATPase. Particularly, the role of the F1-ATPase pathway(s) in HDL-cholesterol uptake and apoA-I-mediated endothelial protection suggests its potential importance in reverse cholesterol transport and its regulation might represent a potential therapeutic target for HDL-related therapy for cardiovascular diseases. Therefore, it is timely for us to better understand how this ecto-enzyme and downstream pathways are regulated and to develop pharmacologic interventions. PMID:21157968

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

PubMed

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 specific irreversible inhibitors of the polyamine transporter, which should present advantages over competitive antagonists for an efficient blockade of polyamine transport in tumor cells.

Effect of Global Regulators RpoS and Cyclic-AMP/CRP on the Catabolome and Transcriptome of Escherichia coli K12 during Carbon- and Energy-Limited Growth

PubMed Central

Egli, Thomas

2015-01-01

For heterotrophic microbes, limited availability of carbon and energy sources is one of the major nutritional factors restricting the rate of growth in most ecosystems. Physiological adaptation to this hunger state requires metabolic versatility which usually involves expression of a wide range of different catabolic pathways and of high-affinity carbon transporters; together, this allows for simultaneous utilization of mixtures of carbonaceous compounds at low concentrations. In Escherichia coli the stationary phase sigma factor RpoS and the signal molecule cAMP are the major players in the regulation of transcription under such conditions; however, their interaction is still not fully understood. Therefore, during growth of E. coli in carbon-limited chemostat culture at different dilution rates, the transcriptomes, expression of periplasmic proteins and catabolomes of strains lacking one of these global regulators, either rpoS or adenylate cyclase (cya), were compared to those of the wild-type strain. The inability to synthesize cAMP exerted a strong negative influence on the expression of alternative carbon source uptake and degradation systems. In contrast, absence of RpoS increased the transcription of genes belonging to high-affinity uptake systems and central metabolism, presumably due to reduced competition of σD with σS. Phenotypical analysis confirmed this observation: The ability to respire alternative carbon substrates and to express periplasmic high-affinity binding proteins was eliminated in cya and crp mutants, while these properties were not affected in the rpoS mutant. As expected, transcription of numerous stress defence genes was negatively affected by the rpoS knock-out mutation. Interestingly, several genes of the RpoS stress response regulon were also down-regulated in the cAMP-negative strain indicating a coordinated global regulation. The results demonstrate that cAMP is crucial for catabolic flexibility during slow, carbon-limited growth, whereas RpoS is primarily involved in the regulation of stress response systems necessary for the survival of this bacterium under hunger conditions. PMID:26204448

Switching the mode of sucrose utilization by Saccharomyces cerevisiae

PubMed Central

Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

2008-01-01

Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae. PMID:18304329

Switching the mode of sucrose utilization by Saccharomyces cerevisiae.

PubMed

Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

2008-02-27

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose uptake by the yeast cells, avoiding overflow metabolism, with the concomitant reduction in ethanol production. The use of this modified yeast strain in simpler batch culture mode can be a viable option to more complicated traditional sucrose-limited fed-batch cultures for biomass-directed processes of S. cerevisiae.

In vivo gene transfer to dopamine neurons of rat substantia nigra via the high-affinity neurotensin receptor.

PubMed Central

Alvarez-Maya, I.; Navarro-Quiroga, I.; Meraz-Ríos, M. A.; Aceves, J.; Martinez-Fong, D.

2001-01-01

BACKGROUND: Recently, we synthesized a nonviral gene vector capable of transfecting cell lines taking advantage of neurotensin (NT) internalization. The vector is NT cross-linked with poly-L-lysine, to which a plasmid DNA was bound to form a complex (NT-polyplex). Nigral dopamine neurons are able to internalize NT, thus representing a target for gene transfer via NT-polyplex. This hypothesis was tested here using reporter genes encoding green fluorescent protein or chloramphenicol acetyl transferase. MATERIALS AND METHODS: NT-polyplex was injected into the substantia nigra. Double immunofluorescence labeling was used to reveal the cell type involved in the propidium iodide-labeled polyplex internalization and reporter gene expression. RESULTS: Polyplex internalization was observed within dopamine neurons but not within glial cells, and was prevented by both hypertonic sucrose solution and SR-48692, a selective nonpeptide antagonist of NT receptors. Reporter gene expression was observed in dopamine neurons from 48 hr up to 15 days after NT-polyplex injection, and was prevented by SR-48692. However, no expression was seen when the NT-polyplex was injected into the ansiform lobule of the cerebellum, which contains low- but not high-affinity NT receptors. Neither internalization nor expression was observed in cultured glial cells, despite the NT-polyplex binding to those cells that was prevented by levocabastine, a low-affinity NT receptor antagonist. CONCLUSIONS: These results suggest that high-affinity NT receptors mediate the uptake of NT-polyplex with the subsequent reporter gene expression in vivo. NT polyfection may be used to transfer genes of physiologic interest to nigrostriatal dopamine neurons, and to produce transgenic animal models of dopamine-related diseases. PMID:11471555

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

PubMed Central

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

The Vesicular Monoamine Transporter-2: An Important Pharmacological Target for the Discovery of Novel Therapeutics to Treat Methamphetamine Abuse

PubMed Central

Nickell, Justin R.; Siripurapu, Kiran B.; Vartak, Ashish; Crooks, Peter A.; Dwoskin, Linda P.

2014-01-01

Methamphetamine abuse escalates, but no approved therapeutics are available to treat addicted individuals. Methamphetamine increases extracellular dopamine in reward-relevant pathways by interacting at vesicular monoamine transporter-2 (VMAT2) to inhibit dopamine uptake and promote dopamine release from synaptic vesicles, increasing cytosolic dopamine available for reverse transport by the dopamine transporter (DAT). VMAT2 is the target of our iterative drug discovery efforts to identify pharmacotherapeutics for methamphetamine addiction. Lobeline, the major alkaloid in Lobelia inflata, potently inhibited VMAT2, methamphetamine-evoked striatal dopamine release, and methamphetamine self-administration in rats but exhibited high affinity for nicotinic acetylcholine receptors (nAChRs). Defunctionalized, unsaturated lobeline analog, meso-transdiene (MTD), exhibited lobeline-like in vitro pharmacology, lacked nAChR affinity, but exhibited high affinity for DAT, suggesting potential abuse liability. The 2,4-dicholorophenyl MTD analog, UKMH-106, exhibited selectivity for VMAT2 over DAT, inhibited methamphetamine-evoked dopamine release, but required a difficult synthetic approach. Lobelane, a saturated, defunctionalized lobeline analog, inhibited the neurochemical and behavioral effects of methamphetamine; tolerance developed to the lobelane-induced decrease in methamphetamine self-administration. Improved drug-likeness was afforded by the incorporation of a chiral N-1,2-dihydroxypropyl moiety into lobelane to afford GZ-793A, which inhibited the neurochemical and behavioral effects of methamphetamine, without tolerance. From a series of 2,5-disubstituted pyrrolidine analogs, AV-2-192 emerged as a lead, exhibiting high affinity for VMAT2 and inhibiting methamphetamine-evoked dopamine release. Current results support the hypothesis that potent, selective VMAT2 inhibitors provide the requisite preclinical behavioral profile for evaluation as pharmacotherapeutics for methamphetamine abuse and emphasize selectivity for VMAT2 relative to DAT as a criterion for reducing abuse liability of the therapeutic. PMID:24484975

99mTc-labelled HYNIC-minigastrin with reduced kidney uptake for targeting of CCK-2 receptor-positive tumours.

PubMed

von Guggenberg, E; Dietrich, H; Skvortsova, I; Gabriel, M; Virgolini, I J; Decristoforo, C

2007-08-01

Different attempts have been made to develop a suitable radioligand for targeting CCK-2 receptors in vivo, for staging of medullary thyroid carcinoma (MTC) and other receptor-expressing tumours. After initial successful clinical studies with [DTPA(0),D: Glu(1)]minigastrin (DTPA-MG0) radiolabelled with (111)In and (90)Y, our group developed a (99m)Tc-labelled radioligand, based on HYNIC-MG0. A major drawback observed with these derivatives is their high uptake by the kidneys. In this study we describe the preclinical evaluation of the optimised shortened peptide analogue, [HYNIC(0),D: Glu(1),desGlu(2-6)]minigastrin (HYNIC-MG11). (99m)Tc labelling of HYNIC-MG11 was performed using tricine and EDDA as coligands. Stability experiments were carried out by reversed phase HPLC analysis in PBS, PBS/cysteine and plasma as well as rat liver and kidney homogenates. Receptor binding and cell uptake experiments were performed using AR4-2J rat pancreatic tumour cells. Animal biodistribution was studied in AR4-2J tumour-bearing nude mice. Radiolabelling was performed at high specific activities and radiochemical purity was >90%. (99m)Tc-EDDA-HYNIC-MG11 showed high affinity for the CCK-2 receptor and cell internalisation comparable to that of (99m)Tc-EDDA-HYNIC-MG0. Despite high stability in solution, a low metabolic stability in rat tissue homogenates was found. In a nude mouse tumour model, very low unspecific retention in most organs, rapid renal excretion with reduced renal retention and high tumour uptake were observed. (99m)Tc-EDDA-HYNIC-MG11 shows advantages over (99m)Tc-EDDA-HYNIC-MG0 in terms of lower kidney retention with unchanged uptake in tumours and CCK-2 receptor-positive tissue. However, the lower metabolic stability and impurities formed in the labelling process still leave room for further improvement.

Bilateral symmetrical adrenal hypermetabolism on FDG PET/CT due to Cushing syndrome in well differentiated neuroendocrine carcinoma.

PubMed

Aktas, G E; Soyluoglu Demir, S; Sarikaya, A

2016-01-01

The (18)F-FDG PET/CT scan has been suggested for whole-body imaging to identify ectopic adrenocorticotrophic hormone secreting tumours, but there are some challenges involved. The case of a patient is presented, who was admitted with the pre-diagnosis of ectopic ACTH syndrome. On the CT, a nodular lesion was detected in the medial segment of the right lung. The FDG uptake of the lesion seemed to be increased visually, but was not pathological quantitatively (SUVmax: 1.8) on the PET/CT. There was also diffuse increased uptake (SUVmax: 14.2) in the enlarged adrenal glands. The lesion was reported as a possible malignant lesion with low FDG affinity, such as a low grade neuroendocrine tumour, while the diffuse enlarged adrenal glands with high uptake were interpreted as diffusely hyperplasic, due to Cushing's syndrome. The patient was treated with a surgical wedge resection. The histopathological diagnosis confirmed that the tumour was a grade 1 well-differentiated neuroendocrine carcinoma. Copyright © 2015 Elsevier España, S.L.U. and SEMNIM. All rights reserved.

Assessment of interference in biosorption of a heavy metal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Figueira, M.M.; Volesky, B.; Ciminelli, V.S.T.

1997-05-20

Biosorption of heavy metals by various biological materials has been studied extensively in the last decade due to its potential particularly in wastewater treatment. The presence of a large number of metals in industrial metal-bearing solutions makes it necessary to investigate their effect on the final metal uptake by individual biosorbent materials. Nonliving biomass of Sargassum, a brown marine alga, is capable of binding more than 10% of its dry weight in toxic cadmium ions. Although ubiquitous iron interferes with Cd uptake, only approximately 4.5% of it is sequestered (biomass dry weight). Biosorption of both metals at pH 4.5 couldmore » be described by Langmuir-type isotherms with b, the affinity-related coefficient (Cd: b = 0.015; Fe: b = 0.027). The interference of Fe with Cd uptake, and vice versa, was assessed by deriving three-dimensional equilibrium two-metal sorption isotherm surfaces, smoothed and cut to reveal the inhibition effect of Fe on biosorption of Cd: at the equilibrium concentration Cf[Cd] = 1.5 mM, the presence of Fe at 1.5 mM equilibrium concentration suppressed the Cd uptake to only 76% of the original value. For 50% Cd uptake reduction, a very high equilibrium Fe presence of 4.5 mM was required. The Cd presence affected the uptake of Fe very strongly. To obtain equal values of uptake for each metal in the biosorbent, the ratio of equilibrium concentrations of 0.42 Cd to 1 Fe is necessary in the liquid phase.« less

Prochlorococcus can use the Pro1404 transporter to take up glucose at nanomolar concentrations in the Atlantic Ocean

PubMed Central

Muñoz-Marín, María del Carmen; Luque, Ignacio; Zubkov, Mikhail V.; Hill, Polly G.; Diez, Jesús; García-Fernández, José Manuel

2013-01-01

Prochlorococcus is responsible for a significant part of CO2 fixation in the ocean. Although it was long considered an autotrophic cyanobacterium, the uptake of organic compounds has been reported, assuming they were sources of limited biogenic elements. We have shown in laboratory experiments that Prochlorococcus can take up glucose. However, the mechanisms of glucose uptake and its occurrence in the ocean have not been shown. Here, we report that the gene Pro1404 confers capability for glucose uptake in Prochlorococcus marinus SS120. We used a cyanobacterium unable to take up glucose to engineer strains that express the Pro1404 gene. These recombinant strains were capable of specific glucose uptake over a wide range of glucose concentrations, showing multiphasic transport kinetics. The Ks constant of the high affinity phase was in the nanomolar range, consistent with the average concentration of glucose in the ocean. Furthermore, we were able to observe glucose uptake by Prochlorococcus in the central Atlantic Ocean, where glucose concentrations were 0.5–2.7 nM. Our results suggest that Prochlorococcus are primary producers capable of tuning their metabolism to energetically benefit from environmental conditions, taking up not only organic compounds with key limiting elements in the ocean, but also molecules devoid of such elements, like glucose. PMID:23569224

Uptake and release of polar compounds in SDB-RPS Empore disks; implications for their use as passive samplers.

PubMed

Shaw, Melanie; Eaglesham, Geoff; Mueller, Jochen F

2009-03-01

Demand for sensitive monitoring tools to detect trace levels of pollutants in aquatic environments has led to investigation of sorbents to complement the suite of passive sampling phases currently in use. Styrenedivinylbenzene-reverse phase sulfonated (SDB-RPS) sorbents have a high affinity for polar organic compounds such as herbicides. However, the applicability of the performance reference compound (PRC) concept as an in situ calibration method for passive samplers that use this or similar sampling phases has yet to be validated. In this study, laboratory based calibration experiments were conducted to compare the uptake kinetics of several key pesticides with the release of three pre-loaded PRCs in Chemcatchers using SDB-RPS Empore disks deployed with a membrane and without (naked). For compounds with log K(OW) values ranging from 1.8 to 4.0, uptake into samplers with a membrane and without was linear over 30d and 10d, respectively. While uptake was linear and reproducible, PRC loss was not linear, meaning that the dissipation rates of these PRCs cannot be used to estimate field exposure conditions on uptake rates. An alternative in situ calibration technique using PRC loaded polydimethylsiloxane (PDMS) disks deployed alongside the Empore disk samplers as a surrogate calibration phase has been tested in the current study and shows promise for future applications.

Preparation and Biological Evaluation of 64Cu Labeled Tyr3-Octreotate Using a Phosphonic Acid-Based Cross-Bridged Macrocyclic Chelator

PubMed Central

Guo, Yunjun; Ferdani, Riccardo; Anderson, Carolyn J.

2014-01-01

Somatostatin receptors (SSTr) are overexpressed in a wide range of neuroendocrine tumors, making them excellent targets for nuclear imaging and therapy, and radiolabeled somatostatin analogues have been investigated for positron emission tomography imaging and radionuclide therapy of SSTr-positive tumors, especially of the subtype-2 (SSTr2). The aim of this study was to develop a somatostatin analogue, Tyr3-octreotate (Y3-TATE), conjugated to a novel cross-bridged macrocyclic chelator, 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P). Unlike traditional cross-bridged macrocycles, such as 4, 11 - bis (carboxymethyl) - 1, 4, 8, 11 -etraazabicyclo[6.6.2]hexadecane (CB-TE2A), CB-TE1A1P-Y3-TATE was radiolabeled with 64Cu in high purity and high specific activity using mild conditions. Saturation binding assays revealed that 64Cu-CB-TE1A1P-Y3-TATE had comparable binding affinity but bound to more binding sites in AR42J rat pancreatic tumor cell membranes than 64Cu-CB-TE2A-Y3-TATE. Both radiopharmaceuticals showed comparable uptake in SSTr2 positive tissues in AR42J tumor-bearing rats. 64Cu-CB-TE1A1PY3- TATE demonstrated improved blood clearance compared to 64Cu-CB-TE2A-Y3-TATE, as the tumor/blood ratios of 64Cu-CB-TE1A1P-Y3-TATE were shown to be significantly higher than those of 64Cu-CB-TE2A-Y3-TATE at 4 and 24 h postinjection. 64Cu-CB-TE1A1P-Y3-TATE, in spite of a relatively high kidney uptake, accumulated less in nontarget organs such as liver, lung, and bone. Small animal PET/CT imaging of 64Cu-CB-TE1A1P-Y3-TATE in AR42J tumor bearing rats validated significant uptake and good contrast in the tumor. This study suggests that CB-TE1A1P is a promising bifunctional chelator for 64Cu-labeled for Y3-TATE, owing to high binding affinity and target tissue uptake, the ability to radiolabel the agent at lower temperatures, and improved tumor/nontarget organ ratios over 64Cu-CB-TE2A-Y3-TATE. PMID:22663248

The FupA/B protein uniquely facilitates transport of ferrous iron and siderophore-associated ferric iron across the outer membrane of Francisella tularensis live vaccine strain

PubMed Central

Sen, Bhaswati

2014-01-01

Francisella tularensis is a highly infectious Gram-negative pathogen that replicates intracellularly within the mammalian host. One of the factors associated with virulence of F. tularensis is the protein FupA that mediates high-affinity transport of ferrous iron across the outer membrane. Together with its paralogue FslE, a siderophore–ferric iron transporter, FupA supports survival of the pathogen in the host by providing access to the essential nutrient iron. The FupA orthologue in the attenuated live vaccine strain (LVS) is encoded by the hybrid gene fupA/B, the product of an intergenic recombination event that significantly contributes to attenuation of the strain. We used 55Fe transport assays with mutant strains complemented with the different paralogues to show that the FupA/B protein of LVS retains the capacity for high-affinity transport of ferrous iron, albeit less efficiently than FupA of virulent strain Schu S4. 55Fe transport assays using purified siderophore and siderophore-dependent growth assays on iron-limiting agar confirmed previous findings that FupA/B also contributes to siderophore-mediated ferric iron uptake. These assays further demonstrated that the LVS FslE protein is a weaker siderophore–ferric iron transporter than the orthologue from Schu S4, and may be a result of the sequence variation between the two proteins. Our results indicate that iron-uptake mechanisms in LVS differ from those in Schu S4 and that functional differences in the outer membrane iron transporters have distinct effects on growth under iron limitation. PMID:24307666

Preclinical evaluation of [99mTc/EDDA/tricine/HYNIC0, 1-Nal3, Thr8]-octreotide as a new analogue in the detection of somatostatin-receptor-positive tumors.

PubMed

Gandomkar, Mostafa; Najafi, Reza; Shafiei, Mohammad; Mazidi, Mohammad; Ebrahimi, Sayed Esmaeil Sadat

2007-08-01

Radiolabeled somatostatin analogues are important tools for the in vivo localization and targeted radionuclide therapy of somatostatin-receptor-positive tumors. The aim of this study was to evaluate a new somatostatin analogue designed for the labeling with (99m)Tc: [6-hydrazinopyridine-3-carboxylic acid (HYNIC(0)), 1-Nal(3), Thr(8)]-octreotide ([HYNIC]-NATE), using ethylenediamine-N,N'-diacetic acid (EDDA) and tricine as coligands. Synthesis was preformed on a solid phase using a standard Fmoc strategy. Labeling with (99m)Tc was performed at 100 degrees C for 10 min using SnCl(2) as a reductant. Radiochemical analysis involved ITLC and high-performance liquid chromatography methods. Peptide conjugate affinity was determined in AR4-2J cell membranes. The internalization and externalization rates were studied in sstr(2)-expressing AR4-2J cells. Biodistribution of radiopeptide was studied in rats bearing the AR4-2J tumor. Radiolabeling was performed at high specific activities, and radiochemical purity was >95%. Peptide conjugate showed high affinity binding for sstr(2). The radioligand showed a moderate and specific internalization into AR4-2J cells (14.13+/-0.61% at 4 h). In animal biodistribution studies, a receptor-specific uptake of radioactivity was observed in somatostatin-receptor-positive organs. After 4 h, uptake in the AR4-2J tumor was 1.33+/-0.23%ID/g (percentage of injected dose per gram of tissue). These data show that [(99m)Tc/EDDA/tricine/HYNIC]-NATE is a specific radioligand for the somatostatin-receptor-positive tumors and is a suitable candidate for clinical studies.

Molecular analysis of maltotriose active transport and fermentation by Saccharomyces cerevisiae reveals a determinant role for the AGT1 permease.

PubMed

Alves, Sergio L; Herberts, Ricardo A; Hollatz, Claudia; Trichez, Debora; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

2008-03-01

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known alpha-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and MAL inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high- and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K(m), 36 +/- 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.

Differential recognition of ACE inhibitors in Xenopus laevis oocytes expressing rat PEPT1 and PEPT2.

PubMed

Zhu, T; Chen, X Z; Steel, A; Hediger, M A; Smith, D E

2000-05-01

To examine the mechanism of inhibition of glycylsarcosine (GlySar) transport by quinapril and enalapril, and whether or not angiotensin converting enzyme (ACE) inhibitors are transported by PEPT2 as well as by PEPT1. Xenopus laevis oocytes were cRNA-injected with rat PEPT1 or PEPT2 and the transport kinetics of radiolabeled GlySar were studied in the absence and presence of quinapril and enalapril. The two-microelectrode voltage-clamp technique was also performed to probe the electrogenic uptake of captopril, quinapril and enalapril. Kinetic analyses demonstrated that quinapril inhibited the uptake of GlySar in a noncompetitive manner in Xenopus oocytes injected with PEPT1 or PEPT2 (Ki = 0.8 or 0.4 mM, respectively). In contrast, a competitive interaction was observed between GlySar and enalapril (Ki = 10.8 mM for PEPT1 or 4.3 mM for PEPT2). Most significantly, captopril and enalapril, but not quinapril, induced inwardly-directed currents in both PEPT1- and PEPT2-expressed oocytes. These results are unique in providing direct evidence for the substrate recognition and transport of some ACE inhibitors by the high- and low-affinity oligopeptide transporters. Our findings point to differences between PEPT1 and PEPT2 in their affinity to, rather than in their specificity for, ACE inhibitors.

Inhibiting HER3-mediated tumor cell growth with affibody molecules engineered to low picomolar affinity by position-directed error-prone PCR-like diversification.

PubMed

Malm, Magdalena; Kronqvist, Nina; Lindberg, Hanna; Gudmundsdotter, Lindvi; Bass, Tarek; Frejd, Fredrik Y; Höidén-Guthenberg, Ingmarie; Varasteh, Zohreh; Orlova, Anna; Tolmachev, Vladimir; Ståhl, Stefan; Löfblom, John

2013-01-01

The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

Toxic Compounds in Our Food: Arsenic Uptake By Rice and Potential Mitigation By Silicon

NASA Astrophysics Data System (ADS)

Seyfferth, A.; Gill, R.; Penido, E.

2014-12-01

Arsenic is a ubiquitous element in soils worldwide and has the potential to negatively impact human and ecosystem health under certain biogeochemical conditions. While arsenic is relatively immobile in most oxidized soils due to a high affinity for soil solids, arsenic becomes mobilized under reduced soil conditions due to the reductive dissolution of iron(III) oxides thereby releasing soil-bound arsenic. Since arsenic is a well-known carcinogen, this plant-soil process has the potential to negatively impact the lives of billions of rice consumers worldwide upon plant uptake and grain storage of released arsenic. Moreover, arsenic uptake by rice is excacerbated by the use of As-laden groundwater for rice irrigation. One proposed strategy to decrease arsenic uptake by rice plants is via an increase in dissolved silicon in paddy soil solution (pore-water), since silicic acid and arsenous acid share an uptake pathway. However, several soil processes that influence arsenic cycling may be affected by silicon including desorption from bulk soil, formation and mineralogy of iron(III) oxide plaque, and adsorption/desorption onto/from iron plaque; the effect of silicon on these soil processes will ultimately dictate the effectiveness of altered dissolved silicon in decreasing arsenic uptake at the root, which in turn dictates the concentration of arsenic found in grains. Furthermore, the source of silicon may impact carbon cycling and, in particular, methane emissions. Here, impacts of altered dissolved silicon on processes that affect rhizospheric biogeochemical cycling of arsenic and subsequent plant-uptake, and how it influences other biogeochemical cycles such as carbon and iron are investigated. We show that silicon can decrease arsenic uptake and grain storage under certain conditions, and that altered silicon affects the type of iron (III) oxide that comprises iron plaque.

Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake.

PubMed

Bon, Nina; Couasnay, Greig; Bourgine, Annabelle; Sourice, Sophie; Beck-Cormier, Sarah; Guicheux, Jérôme; Beck, Laurent

2018-02-09

Extracellular phosphate (P i ) can act as a signaling molecule that directly alters gene expression and cellular physiology. The ability of cells or organisms to detect changes in extracellular P i levels implies the existence of a P i -sensing mechanism that signals to the body or individual cell. However, unlike in prokaryotes, yeasts, and plants, the molecular players involved in P i sensing in mammals remain unknown. In this study, we investigated the involvement of the high-affinity, sodium-dependent P i transporters PiT1 and PiT2 in mediating P i signaling in skeletal cells. We found that deletion of PiT1 or PiT2 blunted the P i -dependent ERK1/2-mediated phosphorylation and subsequent gene up-regulation of the mineralization inhibitors matrix Gla protein and osteopontin. This result suggested that both PiTs are necessary for P i signaling. Moreover, the ERK1/2 phosphorylation could be rescued by overexpressing P i transport-deficient PiT mutants. Using cross-linking and bioluminescence resonance energy transfer approaches, we found that PiT1 and PiT2 form high-abundance homodimers and P i -regulated low-abundance heterodimers. Interestingly, in the absence of sodium-dependent P i transport activity, the PiT1-PiT2 heterodimerization was still regulated by extracellular P i levels. Of note, when two putative P i -binding residues, Ser-128 (in PiT1) and Ser-113 (in PiT2), were substituted with alanine, the PiT1-PiT2 heterodimerization was no longer regulated by extracellular P i These observations suggested that P i binding rather than P i uptake may be the key factor in mediating P i signaling through the PiT proteins. Taken together, these results demonstrate that P i -regulated PiT1-PiT2 heterodimerization mediates P i sensing independently of P i uptake. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Differences in dissolved cadmium and zinc uptake among stream insects: Mechanistic explanations

USGS Publications Warehouse

Buchwalter, D.B.; Luoma, S.N.

2005-01-01

This study examined the extent to which dissolved Cd and Zn uptake rates vary in several aquatic insect taxa commonly used as indicators of ecological health. We further attempted to explain the mechanisms underlying observed differences. By comparing dissolved Cd and Zn uptake rates in several aquatic insect species, we demonstrated that species vary widely in these processes. Dissolved uptake rates were not related to gross morphological features such as body size or gill size-features that influence water permeability and therefore have ionoregulatory importance. However, finer morphological features, specifically, the relative numbers of ionoregulatory cells (chloride cells), appeared to be related to dissolved metal uptake rates. This observation was supported by Michaelis-Menten type kinetics experiments, which showed that dissolved Cd uptake rates were driven by the numbers of Cd transporters and not by the affinities of those transporters to Cd. Calcium concentrations in exposure media similarly affected Cd and Zn uptake rates in the caddisfly Hydropsyche californica. Dissolved Cd and Zn uptake rates strongly co-varied among species, suggesting that these metals are transported by similar mechanisms.

Knockdown of the partner protein OsNAR2.1 for high-affinity nitrate transport represses lateral root formation in a nitrate-dependent manner

PubMed Central

Huang, Shuangjie; Chen, Si; Liang, Zhihao; Zhang, Chenming; Yan, Ming; Chen, Jingguang; Xu, Guohua; Fan, Xiaorong; Zhang, Yali

2015-01-01

The morphological plasticity of root systems is critical for plant survival, and understanding the mechanisms underlying root adaptation to nitrogen (N) fluctuation is critical for sustainable agriculture; however, the molecular mechanism of N-dependent root growth in rice remains unclear. This study aimed to identify the role of the complementary high-affinity NO3− transport protein OsNAR2.1 in NO3−-regulated rice root growth. Comparisons with wild-type (WT) plants showed that knockdown of OsNAR2.1 inhibited lateral root (LR) formation under low NO3− concentrations, but not under low NH4+ concentrations. 15N-labelling NO3− supplies (provided at concentrations of 0–10 mM) demonstrated that (i) defects in LR formation in mutants subjected to low external NO3− concentrations resulted from impaired NO3− uptake, and (ii) the mutants had significantly fewer LRs than the WT plants when root N contents were similar between genotypes. LR formation in osnar2.1 mutants was less sensitive to localised NO3− supply than LR formation in WT plants, suggesting that OsNAR2.1 may be involved in a NO3−-signalling pathway that controls LR formation. Knockdown of OsNAR2.1 inhibited LR formation by decreasing auxin transport from shoots to roots. Thus, OsNAR2.1 probably functions in both NO3− uptake and NO3−-signalling. PMID:26644084

Hemoglobin binding and catalytic heme extraction by IsdB near iron transporter domains.

PubMed

Bowden, Catherine F M; Verstraete, Meghan M; Eltis, Lindsay D; Murphy, Michael E P

2014-04-15

The Isd (iron-regulated surface determinant) system is a multiprotein transporter that allows bacterium Staphylococcus aureus to take up iron from hemoglobin (Hb) during human infection. In this system, IsdB is a cell wall-anchored surface protein that contains two near iron transporter (NEAT) domains, one of which binds heme. IsdB rapidly extracts heme from Hb and transfers it to IsdA for relay into the bacterial cell. Using a series of recombinant IsdB constructs that included at least one NEAT domain, we demonstrated that both domains are required to bind Hb with high affinity (KD = 0.42 ± 0.05 μM) and to extract heme from Hb. Moreover, IsdB extracted heme only from oxidized metHb, although it also bound oxyHb and the Hb-CO complex. In a reconstituted model of the biological heme relay pathway, IsdB catalyzed the transfer of heme from metHb to IsdA with a Km for metHb of 0.75 ± 0.07 μN and a kcat of 0.22 ± 0.01 s(-1). The latter is consistent with the transfer of heme from metHb to IsdB being the rate-limiting step. With both NEAT domains and the linker region present in a single contiguous polypeptide, high-affinity Hb binding was achieved, rapid heme uptake was observed, and multiple turnovers of heme extraction from metHb and transfer to IsdA were conducted, representing all known Hb-heme uptake functions of the full-length IsdB protein.

Abnormal scintigraphic evolution in AA hepatic amyloidosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lomena, F.; Rosello, R.; Pons, F.

1988-03-01

A patient with AA amyloidosis secondary to ankylosing spondylitis showed intense liver uptake of Tc-99m MDP on bone imaging. The biopsy showed hepatic amyloid deposition. A repeat bone scan with Tc-99m MDP 1 year later was negative, although the clinical signs and liver function tests of the patient had not changed. A mechanism might exist, other than the affinity of amyloid to calcium, which would explain the extraosseous uptake of pyrophosphates and diphosphonates in organs and soft tissues affected by systemic amyloidosis.

Toward a magic or imaginary bullet? Ligands for drug targeting to cancer cells: principles, hopes, and challenges

PubMed Central

Toporkiewicz, Monika; Meissner, Justyna; Matusewicz, Lucyna; Czogalla, Aleksander; Sikorski, Aleksander F

2015-01-01

There are many problems directly correlated with the systemic administration of drugs and how they reach their target site. Targeting promises to be a hopeful strategy as an improved means of drug delivery, with reduced toxicity and minimal adverse side effects. Targeting exploits the high affinity of cell-surface-targeted ligands, either directly or as carriers for a drug, for specific retention and uptake by the targeted diseased cells. One of the most important parameters which should be taken into consideration in the selection of an appropriate ligand for targeting is the binding affinity (KD). In this review we focus on the importance of binding affinities of monoclonal antibodies, antibody derivatives, peptides, aptamers, DARPins, and small targeting molecules in the process of selection of the most suitable ligand for targeting of nanoparticles. In order to provide a critical comparison between these various options, we have also assessed each technology format across a range of parameters such as molecular size, immunogenicity, costs of production, clinical profiles, and examples of the level of selectivity and toxicity of each. Wherever possible, we have also assessed how incorporating such a targeted approach compares with, or is superior to, original treatments. PMID:25733832

Synthesis and preclinical evaluation of [(11)C]PAQ as a PET imaging tracer for VEGFR-2.

PubMed

Samén, Erik; Thorell, Jan-Olov; Lu, Li; Tegnebratt, Tetyana; Holmgren, Lars; Stone-Elander, Sharon

2009-08-01

(R,S)-N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-((1-methyl-3-piperidinyl)methoxy)-4-quinazolinamine (PAQ) is a tyrosine kinase inhibitor with high affinity for the vascular endothelial growth factor receptor 2 (VEGFR-2), which plays an important role in tumour angiogenesis. The aim of this work was to develop and evaluate in mice the (11)C-labelled analogue as an in vivo tracer for VEGFR-2 expression in solid tumours. [(11)C]PAQ was synthesized by an N-methylation of desmethyl-PAQ using [(11)C]methyl iodide. The tracer's pharmacokinetic properties and its distribution in both subcutaneous and intraperitoneal tumour models were evaluated with positron emission tomography (PET). [(18)F]FDG was used as a reference tracer for tumour growth. PET results were corroborated by ex vivo and in vitro phosphor imaging and immunohistochemical analyses. In vitro assays and PET in healthy animals revealed low tracer metabolism, limited excretion over 60 min and a saturable and irreversible binding. Radiotracer uptake in subcutaneous tumour masses was low, while focal areas of high uptake (up to 8% ID/g) were observed in regions connecting the tumour to the host. Uptake was similarly high but more distributed in tumours growing within the peritoneum. The pattern of radiotracer uptake was generally different from that of the metabolic tracer [(18)F]FDG and correlated well with variations in VEGFR-2 expression determined ex vivo by immunohistochemical analysis. These results suggest that [(11)C]PAQ has potential as a noninvasive PET tracer for in vivo imaging of VEGFR-2 expression in angiogenic "hot spots".

A preliminary assessment on the use of biochar as a soil additive for reducing soil-to-plant uptake of cesium isotopes in radioactively contaminated environments

DOE PAGES

Hamilton, Terry F.; Martinelli, Roger E.; Kehl, Steven R.; ...

2015-10-19

A series of K d tracer batch experiments were conducted in this paper to assess the absorptive-desorption properties of Biochar as a potential agent to selectively sequester labile soil Cs or otherwise help reduce the uptake of Cs isotopes into plants. A parallel experiment was conducted for strontium. Fine-grained fractionated Woodlands tree Biochar was found to have a relatively high affinity for Cs ions (K d > 100) relative to coral soil (K d < 10) collected from the Marshall Islands. The Biochar material also contains an abundance of K (and Mg). Finally, these findings support a hypothesis that themore » addition of Biochar as a soil amendment may provide a simple yet effective method for reducing soil-to-plant transfer of Cs isotopes in contaminated environments.« less

Mechanism of Arsenic Adsorption Using Wheat Biomass -- a spectroscopic study

NASA Astrophysics Data System (ADS)

Calvo, Oscar; Manciu, Felicia; Maldonado, Josefina; Gardea-Torresdey, Jorge

2006-10-01

Arsenic is a trace element that is toxic to animals, humans included. Since the current Environmental Protection Agency guidelines regarding water quality standards indicate that arsenic concentrations in excess of 50 ppb are hazardous to welfare of humans, the search for new water remediation methods or improvements of previous methods have been a focus in environmental technology. Investigations of arsenic uptake have used wide range of sorbents including iron oxides and oxyhydroxides, for which it have been proved that arsenic shows high affinity. In this study, we used far-infrared spectroscopy to examine the arsenic reduction using biomaterials. pH dependence analysis by FTIR demonstrates the sorption of iron oxides and oxyhydroxides by the wheat biomass. The splitting of 350 cm-1 amorphous iron oxide vibrations is a direct proof of the arsenic uptake. In addition, there is evidence of sorption of arsenic at sulfhydryl group of cysteine existent in wheat.

Plant Nitrogen Acquisition Under Low Availability: Regulation of Uptake and Root Architecture.

PubMed

Kiba, Takatoshi; Krapp, Anne

2016-04-01

Nitrogen availability is a major factor determining plant growth and productivity. Plants acquire nitrogen nutrients from the soil through their roots mostly in the form of ammonium and nitrate. Since these nutrients are scarce in natural soils, plants have evolved adaptive responses to cope with the environment. One of the most important responses is the regulation of nitrogen acquisition efficiency. This review provides an update on the molecular determinants of two major drivers of the nitrogen acquisition efficiency: (i) uptake activity (e.g. high-affinity nitrogen transporters) and (ii) root architecture (e.g. low-nitrogen-availability-specific regulators of primary and lateral root growth). Major emphasis is laid on the regulation of these determinants by nitrogen supply at the transcriptional and post-transcriptional levels, which enables plants to optimize nitrogen acquisition efficiency under low nitrogen availability. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Evidence for rapid uptake of D-galacturonic acid in the yeast Saccharomyces cerevisiae by a channel-type transport system.

PubMed

Souffriau, Ben; den Abt, Tom; Thevelein, Johan M

2012-07-30

D-Galacturonic acid is a major component of pectins but cannot be metabolized by Saccharomyces cerevisiae. It is assumed not to be taken up. We show that yeast displays surprisingly rapid low-affinity uptake of D-galacturonic acid, strongly increasing with decreasing extracellular pH and without saturation up to 1.5 M. There was no intracellular concentration above the extracellular level and transport was reversible. Among more than 160 single and multiple deletion mutants in channels and transporters, no strain was affected in D-galacturonic acid uptake. The uptake was not inhibited by any compound tested as candidate competitive inhibitor, including D-glucuronic acid, which was also transported. The characteristics of D-galacturonic acid uptake are consistent with involvement of a channel-type system, probably encoded by multiple genes. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Radiofluorinated Rhenium Cyclized α-MSH Analogs for PET Imaging of Melanocortin Receptor 1

PubMed Central

Ren, Gang; Liu, Shuanlong; Liu, Hongguang; Miao, Zheng; Cheng, Zhen

2010-01-01

In order to accomplish in vivo molecular imaging of melanoma biomarker melanocortin 1 receptor (MC1R), several alpha-melanocyte-stimulating hormone (α-MSH) analogs have been labeled with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB) and studied as positron emission tomography (PET) probes in our recent studies. To further pursue a radiofluorinated α-MSH peptide with high clinical translation potential, we utilized 4-nitrophenyl 2-18F-fluoropropionate (18F-NFP) to radiofluorinate the transition metal rhenium cyclized α-MSH metallopeptides for PET imaging of MC1R positive malignant melanoma. Methods Metallopeptides Ac-d,Lys-ReCCMSH(Arg11) (two isomers, namely RMSH-1 and RMSH-2) were synthesized using conventional solid phase peptide synthesis chemistry and rhenium cyclization reaction. The two isomers were then conjugated with 19F-NFP or 18F-NFP. The resulting cold or radiofluorinated metallopeptides, 18/19F-FP-RMSH-1 and 18/19F-FP-RMSH-2 were further evaluated for their in vitro receptor binding affinities, in vivo biodistribution and small-animal PET imaging properties. Results The binding affinities of the 19F-FP-RMSH-1 and 19F-FP-RMSH-2) were determined to be within low nM range. In vivo studies revealed that both 18F-labeled metallopeptides possessed good tumor uptake in B16F10 murine model with high MC1R expression, while much lower uptake in A375M human melanoma xenografts. Moreover, 18F-FP-RMSH-1 displayed more favorable in vivo performance in terms of higher tumor uptake and much lower accumulation in kidney and liver, when compared to 18F-FP-RMSH-2 at 2 h post-injection (p.i.). 18F-FP-RMSH-1 also displayed lower liver and lung uptake when compared with the same peptide labeled with 18F-SFB (named as 18F-FB-RMSH-1). Small animal PET imaging of 18F-FP-RMSH-1 in mice bearing B16F10 tumors at 1 and 2 h showed good tumor imaging quality. As expected, much lower tumor uptake and poorer tumor/normal organs contrast were observed for A375M model than that of B16F10 model. 18F-FP-RMSH-1 also exhibited higher tumor uptake and better tumor retention when compared with 18F-FB-RMSH-1. Conclusion 18F-FP-RMSH-1 demonstrates significant advantages over 18F-FB-RMSH-1 and 18F-FP-RMSH-2. It is a promising PET probe for imaging MC1R positive melanoma and MC1R expression in vivo. PMID:21073170

Gracilaria lemaneiformis polysaccharide as integrin-targeting surface decorator of selenium nanoparticles to achieve enhanced anticancer efficacy.

PubMed

Jiang, Wenting; Fu, Yuanting; Yang, Fang; Yang, Yufeng; Liu, Ting; Zheng, Wenjie; Zeng, Lilan; Chen, Tianfeng

2014-08-27

The poor permeability of glioma parenchyma represents a major limit for antiglioblastoma drug delivery. Gracilaria lemaneiformis polysaccharide (GLP), which has a high binding affinity to αvβ3 integrin overexpressed in glioma cells, was employed in the present study to functionalize selenium nanoparticles (SeNPs) to achieve antiglioblastoma efficacy. GLP-SeNPs showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. In U87 glioma cell membrane, which has a high integrin expression level, GLP-SeNPs exhibited significantly higher cellular uptake than unmodified SeNPs. As expected, U87 cells exhibited a greater uptake of GLP-SeNPs than C6 cells with low integrin expression level. Furthermore, the internalization of GLP-SeNPs was inhibited by cyclo-(Arg-Gly-Asp-Phe-Lys) peptides, suggesting that cellular uptake into U87 cells and C6 cells occurred via αvβ3 integrin-mediated endocytosis. For U87 cells, the cytotoxicity of SeNPs decorated by GLP was enhanced significantly because of the induction of various apoptosis signaling pathways. Internalized GLP-SeNPs triggered intracellular reactive oxygen species downregulation. Therefore, p53, MAPKs, and AKT pathways were activated to advance cell apoptosis. These findings suggest that surface decoration of nanomaterials with GLP could be an efficient strategy for design and preparation of glioblastoma targeting nanodrugs.

Radiosynthesis and ex vivo evaluation of (R)-(-)-2-chloro-N-[1-11C-propyl]n-propylnorapomorphine.

PubMed

Palner, Mikael; McCormick, Patrick; Gillings, Nic; Begtrup, Mikael; Wilson, Alan A; Knudsen, Gitte M

2010-01-01

Several dopamine D(2) agonist radioligands have been used with positron emission tomography (PET), including [(11)C-]-(-)-MNPA, [(11)C-]-(-)-NPA and [(11)C]-(+)-PHNO. These radioligands are considered particularly powerful for detection of endogenous dopamine release, but they either provide PET brain images with limited contrast or have affinity for both D(2) and D(3) receptors. We here present the carbon-11 radiolabeling and ex vivo evaluation of 2-Cl-(-)-NPA, a novel PET-tracer candidate with high in vitro D(2)/D(3) selectivity. 2-Cl-[(11)C]-(-)-NPA and [(11)C]-(-)-NPA were synthesized by a two step N-acylation-reduction process using [(11)C]-propionyl chloride. Awake rats were injected with either tracer, via the tail vein. The rats were decapitated at various times, the brains were removed and quickly dissected, and plasma metabolites were measured. Radioligand specificity, and P-glycoprotein involvement in brain uptake, was also assessed. 2-Cl-[(11)C]-(-)-NPA and [(11)C]-(-)-NPA were produced in high specific activity and purity. 2-Cl-[(11)C]-(-)-NPA accumulated slower in the striatum than [(11)C]-(-)-NPA, reaching maximum concentrations after 30 min. The maximal striatal uptake of 2-Cl-[(11)C]-(-)-NPA (standard uptake value 0.72+/-0.24) was approximately half that of [(11)C]-(-)-NPA (standard uptake value 1.37+/-0.18). Nonspecific uptake was similar for the two compounds. 2-Cl-[(11)C]-(-)-NPA was metabolized quickly, leaving only 17% of the parent compound in the plasma after 30 min. The specific binding of 2-Cl-[(11)C]-(-)-NPA was completely blocked and inhibition of P-glycoprotein did not alter the brain uptake. Ex vivo experiments showed, despite a favorable D(2)/D(3) selectivity, that 2-Cl-[(11)C]-(-)-NPA is inferior to [(11)C]-(-)-NPA as a PET tracer in rat, because of slower brain uptake and lower specific to nonspecific binding ratio. Copyright 2010 Elsevier Inc. All rights reserved.

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

PubMed

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.

Concanavalin A conjugated biodegradable nanoparticles for oral insulin delivery

NASA Astrophysics Data System (ADS)

Hurkat, Pooja; Jain, Aviral; Jain, Ashish; Shilpi, Satish; Gulbake, Arvind; Jain, Sanjay K.

2012-11-01

Major research issues in oral protein delivery include the stabilization of protein in delivery devices which could increase its oral bioavailability. The study deals with development of oral insulin delivery system utilizing biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles and modifying its surface with Concanavalin A to increase lymphatic uptake. Surface-modified PLGA nanoparticles were characterized for conjugation efficiency of ligand, shape and surface morphology, particle size, zeta potential, polydispersity index, entrapment efficiency, and in vitro drug release. Stability of insulin in the developed formulation was confirmed by SDS-PAGE, and integrity of entrapped insulin was assessed using circular dichroism spectrum. Ex vivo study was performed on Wistar rats, which exhibited the higher intestinal uptake of Con A conjugated nanoparticles. In vivo study performed on streptozotocin-induced diabetic rats which indicate that a surface-modified nanoparticle reduces blood glucose level effectively within 4 h of its oral administration. In conclusion, the present work resulted in successful production of Con A NPs bearing insulin with sustained release profile, and better absorption and stability. The Con A NPs showed high insulin uptake, due to its relative high affinity for non-reducing carbohydrate residues i.e., fucose present on M cells and have the potential for oral insulin delivery in effective management of Type 1 diabetes condition.

Evidence of isotopic fractionation of natural uranium in cultured human cells

NASA Astrophysics Data System (ADS)

Paredes, Eduardo; Avazeri, Emilie; Malard, Véronique; Vidaud, Claude; Reiller, Pascal E.; Ortega, Richard; Nonell, Anthony; Isnard, Hélène; Chartier, Frédéric; Bresson, Carole

2016-12-01

The study of the isotopic fractionation of endogen elements and toxic heavy metals in living organisms for biomedical applications, and for metabolic and toxicological studies, is a cutting-edge research topic. This paper shows that human neuroblastoma cells incorporated small amounts of uranium (U) after exposure to 10 µM natural U, with preferential uptake of the 235U isotope with regard to 238U. Efforts were made to develop and then validate a procedure for highly accurate n(238U)/n(235U) determinations in microsamples of cells. We found that intracellular U is enriched in 235U by 0.38 ± 0.13‰ (2σ, n = 7) relative to the exposure solutions. These in vitro experiments provide clues for the identification of biological processes responsible for uranium isotopic fractionation and link them to potential U incorporation pathways into neuronal cells. Suggested incorporation processes are a kinetically controlled process, such as facilitated transmembrane diffusion, and the uptake through a high-affinity uranium transport protein involving the modification of the uranyl (UO22+) coordination sphere. These findings open perspectives on the use of isotopic fractionation of metals in cellular models, offering a probe to track uptake/transport pathways and to help decipher associated cellular metabolic processes.

Evidence of isotopic fractionation of natural uranium in cultured human cells

PubMed Central

Paredes, Eduardo; Avazeri, Emilie; Malard, Véronique; Vidaud, Claude; Reiller, Pascal E.; Ortega, Richard; Nonell, Anthony; Isnard, Hélène; Chartier, Frédéric; Bresson, Carole

2016-01-01

The study of the isotopic fractionation of endogen elements and toxic heavy metals in living organisms for biomedical applications, and for metabolic and toxicological studies, is a cutting-edge research topic. This paper shows that human neuroblastoma cells incorporated small amounts of uranium (U) after exposure to 10 µM natural U, with preferential uptake of the 235U isotope with regard to 238U. Efforts were made to develop and then validate a procedure for highly accurate n(238U)/n(235U) determinations in microsamples of cells. We found that intracellular U is enriched in 235U by 0.38 ± 0.13‰ (2σ, n = 7) relative to the exposure solutions. These in vitro experiments provide clues for the identification of biological processes responsible for uranium isotopic fractionation and link them to potential U incorporation pathways into neuronal cells. Suggested incorporation processes are a kinetically controlled process, such as facilitated transmembrane diffusion, and the uptake through a high-affinity uranium transport protein involving the modification of the uranyl (UO22+) coordination sphere. These findings open perspectives on the use of isotopic fractionation of metals in cellular models, offering a probe to track uptake/transport pathways and to help decipher associated cellular metabolic processes. PMID:27872304

Evidence of isotopic fractionation of natural uranium in cultured human cells.

PubMed

Paredes, Eduardo; Avazeri, Emilie; Malard, Véronique; Vidaud, Claude; Reiller, Pascal E; Ortega, Richard; Nonell, Anthony; Isnard, Hélène; Chartier, Frédéric; Bresson, Carole

2016-12-06

The study of the isotopic fractionation of endogen elements and toxic heavy metals in living organisms for biomedical applications, and for metabolic and toxicological studies, is a cutting-edge research topic. This paper shows that human neuroblastoma cells incorporated small amounts of uranium (U) after exposure to 10 µM natural U, with preferential uptake of the 235 U isotope with regard to 238 U. Efforts were made to develop and then validate a procedure for highly accurate n( 238 U)/n( 235 U) determinations in microsamples of cells. We found that intracellular U is enriched in 235 U by 0.38 ± 0.13‰ (2σ, n = 7) relative to the exposure solutions. These in vitro experiments provide clues for the identification of biological processes responsible for uranium isotopic fractionation and link them to potential U incorporation pathways into neuronal cells. Suggested incorporation processes are a kinetically controlled process, such as facilitated transmembrane diffusion, and the uptake through a high-affinity uranium transport protein involving the modification of the uranyl (UO 2 2+ ) coordination sphere. These findings open perspectives on the use of isotopic fractionation of metals in cellular models, offering a probe to track uptake/transport pathways and to help decipher associated cellular metabolic processes.

Multimodal molecular imaging reveals high target uptake and specificity of 111In and 68Ga labeled fibrin-binding probes for thrombus detection in rats

PubMed Central

Oliveira, Bruno L.; Blasi, Francesco; Rietz, Tyson A.; Rotile, Nicholas J.; Day, Helen; Caravan, Peter

2016-01-01

We recently showed the high target specificity and favorable imaging properties of 64Cu and Al18F positron emission tomography (PET) probes for non-invasive imaging of thrombosis. Here, our aim was to evaluate new derivatives labeled with either with 68Ga, 111In, or 99mTc as thrombus imaging agents for PET and single-photon emission computed tomography (SPECT). In this study, the feasibility and potential of these probes for thrombus imaging was assessed in detail in two animal models of arterial thrombosis. The specificity of the probes was further evaluated using a triple-isotope approach with multimodal SPECT/PET/CT imaging. Methods Radiotracers were synthesized using a known fibrin-binding peptide conjugated to NODAGA, DOTA-MA, or a diethylenetriamine ligand (DETA-PA), followed by labeling with 68Ga (FBP14, 68Ga-NODAGA), 111In (FBP15, 111In-DOTA-MA) or 99mTc (FBP16, 99mTc(CO)3-DETA-PA), respectively. PET or SPECT imaging, biodistribution, pharmacokinetics and metabolic stability were evaluated in rat models of mural and occlusive carotid artery thrombosis. In vivo target specificity was evaluated by comparing the distribution of the SPECT and PET probes with preformed 125I-labeled thrombi and with a non-binding control probe using SPECT/PET/CT imaging. Results All three radiotracers showed similar affinity to soluble fibrin fragment DD(E) (Ki = 0.53–0.83 μM). After the kidneys, the highest uptake of 68Ga-FBP14 and 111In-FBP15 was in the thrombus (1.0 ± 0.2% ID/g) with low off-target accumulation. Both radiotracers underwent fast systemic elimination (t1/2 = 8-15 min) through the kidneys, which led to highly conspicuous thrombi on PET and SPECT images. 99mTc-FBP16 displayed low target uptake and distribution consistent with aggregation and/or degradation. Triple isotope imaging experiments showed that both 68Ga-FBP14 and 111In-FBP15, but not the nonbinding derivative 64Cu-D-Cys-FBP8, detected the location of the 125I-labeled thrombus, confirming high target specificity. Conclusion 68Ga-FBP14 and 111In-FBP15 have high fibrin affinity and thrombus specificity, and represent useful PET and SPECT probes for thrombus detection. PMID:26251420

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

PubMed

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.

Action of insecticidal N-alkylamides at site 2 of the voltage-sensitive sodium channel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ottea, J.A.; Payne, G.T.; Soderlund, D.M.

1990-08-01

Nine synthetic N-alkylamides were examined as inhibitors of the specific binding of ({sup 3}H)batrachotoxinin A 20{alpha}-benzoate (({sup 3}H)BTX-B) to sodium channels and as activators of sodium uptake in mouse brain synaptoneurosomes. In the presence of scorpion (Leiurus quinquestriatus) venom, the six insecticidal analogues were active as both inhibitors of ({sup 3}H)BTX-B binding and stimulators of sodium uptake. These findings are consistent with an action of these compounds at the alkaloid activator recognition site (site 2) of the voltage-sensitive sodium channel. The three noninsecticidal N-alkylamides also inhibited ({sup 3}H)BTX-B binding but were ineffective as activators of sodium uptake. Concentration-response studies revealedmore » that some of the insecticidal amides also enhanced sodium uptake through a second, high-affinity interaction that does not involve site 2, but this secondary effect does not appear to be correlated with insecticidal activity. The activities of N-alkylamides as sodium channel activators were influenced by the length of the alkenyl chain and the location of unsaturation within the molecule. These results further define the actions of N-alkylamides on sodium channels and illustrate the significance of the multiple binding domains of the sodium channel as target sites for insect control agents.« less

Interaction of SR 33557 with skeletal muscle calcium channel blocker receptors in the baboon: characterization of its binding sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sol-Rolland, J.; Joseph, M.; Rinaldi-Carmona, M.

1991-05-01

A procedure for the isolation of primate skeletal microsomal membranes was initiated. Membranes exhibited specific enzymatic markers such as 5'-nucleotidase, Ca{sup 2}{sup +},Mg({sup 2}{sup +})-adenosine triphosphatase and an ATP-dependent calcium uptake. Baboon skeletal microsomes bound specifically with high-affinity potent Ca{sup 2}{sup +} channel blockers such as dihydropyridine, phenylalkylamine and benzothiazepine derivatives. Scatchard analysis of equilibrium binding assays with ({sup 3}H)(+)-PN 200-110, ({sup 3}H)(-)-desmethoxyverapamil (( {sup 3}H)(-)-D888) and ({sup 3}H)-d-cis-dilitiazem were consistent with a single class of binding sites for the three radioligands. The pharmacological profile of SR 33557, an original compound with calcium antagonist properties, was investigated using radioligand bindingmore » studies. SR 33557 totally inhibited the specific binding of the three main classes of Ca{sup 2}{sup +} channel effectors and interacted allosterically with them. In addition, SR 33557 bound with high affinity to a homogeneous population of binding sites in baboon skeletal muscle.« less

Hyperosmolar sodium chloride is toxic to cultured neurons and causes reduction of glucose metabolism and ATP levels, an increase in glutamate uptake, and a reduction in cytosolic calcium.

PubMed

Morland, Cecilie; Pettersen, Mi Nguyen; Hassel, Bjørnar

2016-05-01

Elevation of serum sodium, hypernatremia, which may occur during dehydration or treatment with sodium chloride, may cause brain dysfunction and damage, but toxic mechanisms are poorly understood. We found that exposure to excess NaCl, 10-100mmol/L, for 20h caused cell death in cultured cerebellar granule cells (neurons). Toxicity was due to Na(+), since substituting excess Na(+) with choline reduced cell death to control levels, whereas gluconate instead of excess Cl(-) did not. Prior to cell death from hyperosmolar NaCl, glucose consumption and lactate formation were reduced, and intracellular aspartate levels were elevated, consistent with reduced glycolysis or glucose uptake. Concomitantly, the level of ATP became reduced. Pyruvate, 10mmol/L, reduced NaCl-induced cell death. The extracellular levels of glutamate, taurine, and GABA were concentration-dependently reduced by excess NaCl; high-affinity glutamate uptake increased. High extracellular [Na(+)] caused reduction in intracellular free [Ca(2+)], but a similar effect was seen with mannitol, which was not neurotoxic. We suggest that inhibition of glucose metabolism with ensuing loss of ATP is a neurotoxic mechanism of hyperosmolar sodium, whereas increased uptake of extracellular neuroactive amino acids and reduced intracellular [Ca(2+)] may, if they occur in vivo, contribute to the cerebral dysfunction and delirium described in hypernatremia. Copyright © 2016. Published by Elsevier B.V.

Paralogous Outer Membrane Proteins Mediate Uptake of Different Forms of Iron and Synergistically Govern Virulence in Francisella tularensis tularensis*

PubMed Central

Ramakrishnan, Girija; Sen, Bhaswati; Johnson, Richard

2012-01-01

Francisella tularensis subsp. tularensis is a highly infectious bacterium causing acute disease in mammalian hosts. Mechanisms for the acquisition of iron within the iron-limiting host environment are likely to be critical for survival of this intracellular pathogen. FslE (FTT0025) and FupA (FTT0918) are paralogous proteins that are predicted to form β-barrels in the outer membrane of virulent strain Schu S4 and are unique to Francisella species. Previous studies have implicated both FupA, initially identified as a virulence factor and FslE, encoded by the siderophore biosynthetic operon, in iron acquisition. Using single and double mutants, we demonstrated that these paralogs function in concert to promote growth under iron limitation. We used a 55Fe transport assay to demonstrate that FslE is involved in siderophore-mediated ferric iron uptake, whereas FupA facilitates high affinity ferrous iron uptake. Optimal replication within J774A.1 macrophage-like cells required at least one of these uptake systems to be functional. In a mouse model of tularemia, the ΔfupA mutant was attenuated, but the ΔfslE ΔfupA mutant was significantly more attenuated, implying that the two systems of iron acquisition function synergistically to promote virulence. These studies highlight the importance of specific iron acquisition functions, particularly that of ferrous iron, for virulence of F. tularensis in the mammalian host. PMID:22661710

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

PubMed Central

Kraehling, Jan R.; Chidlow, John H.; Rajagopal, Chitra; Sugiyama, Michael G.; Fowler, Joseph W.; Lee, Monica Y.; Zhang, Xinbo; Ramírez, Cristina M.; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W.; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L.; Fernández-Hernando, Carlos; Sessa, William C.

2016-01-01

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL. PMID:27869117

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.

PubMed

Kraehling, Jan R; Chidlow, John H; Rajagopal, Chitra; Sugiyama, Michael G; Fowler, Joseph W; Lee, Monica Y; Zhang, Xinbo; Ramírez, Cristina M; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L; Fernández-Hernando, Carlos; Sessa, William C

2016-11-21

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.

Effects of amino acids on melanoma targeting and clearance properties of Tc-99m-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone peptides.

PubMed

Flook, Adam M; Yang, Jianquan; Miao, Yubin

2013-11-14

The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new (99m)Tc-labeled Arg-X-Asp-conjugated α-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg(11))CCMSH {c[Arg-Ser-Asp-DTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg(11))CCMSH, RPheD-Lys-(Arg(11))CCMSH, and RdPheD-Lys-(Arg(11))CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of (99m)Tc-RSD-Lys-(Arg(11))CCMSH, (99m)Tc-RFD-Lys-(Arg(11))CCMSH, and (99m)Tc-RfD-Lys-(Arg(11))CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The substitution of Gly with Ser, Phe, and dPhe increased the MC1 receptor binding affinities of the peptides, whereas the substitution of Gly with Nle decreased the MC1 receptor binding affinity of the peptide. (99m)Tc-RSD-Lys-(Arg(11))CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these (99m)Tc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using (99m)Tc-RSD-Lys-(Arg(11))CCMSH as an imaging probe. It is desirable to reduce the renal uptake of (99m)Tc-RSD-Lys-(Arg(11))CCMSH to facilitate its potential therapeutic application.

Quantitative cumulative biodistribution of antibodies in mice

PubMed Central

Yip, Victor; Palma, Enzo; Tesar, Devin B; Mundo, Eduardo E; Bumbaca, Daniela; Torres, Elizabeth K; Reyes, Noe A; Shen, Ben Q; Fielder, Paul J; Prabhu, Saileta; Khawli, Leslie A; Boswell, C Andrew

2014-01-01

The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn’s role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn’s role in antibody PK and catabolism at the tissue level. PMID:24572100

Effects of Amino Acids on Melanoma Targeting and Clearance Properties of Tc-99m-Labeled Arg-X-Asp-Conjugated α-Melanocyte Stimulating Hormone Peptides

PubMed Central

Flook, Adam M.; Yang, Jianquan; Miao, Yubin

2013-01-01

The purpose of this study was to examine the effects of amino acids on melanoma targeting and clearance properties of new 99mTc-labeled Arg-X-Asp-conjugated alpha-melanocyte stimulating hormone (α-MSH) peptides. RSD-Lys-(Arg11)CCMSH {c[Arg-Ser-Asp-dTyr-Asp]-Lys-Cys-Cys-Glu-His-dPhe-Arg-Trp-Cys-Arg-Pro-Val-NH2}, RNleD-Lys-(Arg11)CCMSH, RPheD-Lys-(Arg11)CCMSH and RdPheD-Lys-(Arg11)CCMSH peptides were synthesized and evaluated for their melanocortin-1 (MC1) receptor binding affinities in B16/F1 melanoma cells. The biodistribution of 99mTc-RSD-Lys-(Arg11)CCMSH, 99mTc-RFD-Lys-(Arg11)CCMSH and 99mTc-RfD-Lys-(Arg11)CCMSH were determined in B16/F1 melanoma-bearing C57 mice. The substitution of Gly with Ser, Phe and dPhe increased the MC1 receptor binding affinities of the peptides, whereas the substitution of Gly with Nle decreased the MC1 receptor binding affinity of the peptide. 99mTc-RSD-Lys-(Arg11)CCMSH exhibited the highest melanoma uptake (18.01 ± 4.22% ID/g) and the lowest kidney and liver uptake among these 99mTc-peptides. The B16/F1 melanoma lesions could be clearly visualized by SPECT/CT using 99mTc-RSD-Lys-(Arg11)CCMSH as an imaging probe. It is desirable to reduce the renal uptake of 99mTc-RSD-Lys-(Arg11)CCMSH to facilitate its potential therapeutic application. PMID:24131154

Exogenous addition of histidine reduces copper availability in the yeast Saccharomyces cerevisiae.

PubMed

Watanabe, Daisuke; Kikushima, Rie; Aitoku, Miho; Nishimura, Akira; Ohtsu, Iwao; Nasuno, Ryo; Takagi, Hiroshi

2014-07-07

The basic amino acid histidine inhibited yeast cell growth more severely than lysine and arginine. Overexpression of CTR1 , which encodes a high-affinity copper transporter on the plasma membrane, or addition of copper to the medium alleviated this cytotoxicity. However, the intracellular level of copper ions was not decreased in the presence of excess histidine. These results indicate that histidine cytotoxicity is associated with low copper availability inside cells, not with impaired copper uptake. Furthermore, histidine did not affect cell growth under limited respiration conditions, suggesting that histidine cytotoxicity is involved in deficiency of mitochondrial copper.

Computer simulation of uranyl uptake by the rough lipopolysaccharide membrane of Pseudomonas aeruginosa.

PubMed

Lins, Roberto D; Vorpagel, Erich R; Guglielmi, Matteo; Straatsma, T P

2008-01-01

Heavy metal environmental contaminants cannot be destroyed but require containment, preferably in concentrated form, in a solid or immobile form for recycling or final disposal. Microorganisms are able to take up and deposit high levels of contaminant metals, including radioactive metals such as uranium and plutonium, into their cell wall. Consequently, these microbial systems are of great interest as the basis for potential environmental bioremediation technologies. The outer membranes of Gram-negative microbes are highly nonsymmetric and exhibit a significant electrostatic potential gradient across the membrane. This gradient has a significant effect on the uptake and transport of charged and dipolar compounds. However, the effectiveness of microbial systems for environmental remediation will depend strongly on specific properties that determine the uptake of targeted contaminants by a particular cell wall. To aid in the design of microbial remediation technologies, knowledge of the factors that determine the affinity of a particular bacterial outer membrane for the most common ionic species found in contaminated soils and groundwater is of great importance. Using our previously developed model for the lipopolysaccharide (LPS) membrane of Pseudomonas aeruginosa, this work presents the potentials of mean force as the estimate of the free energy profile for uptake of sodium, calcium, chloride, uranyl ions, and a water molecule by the bacterial LPS membrane. A compatible classical parameter set for uranyl has been developed and validated. Results show that the uptake of uranyl is energetically a favorable process relative to the other ions studied. At neutral pH, this nuclide is shown to be retained on the surface of the LPS membrane through chelation with the carboxyl and hydroxyl groups located in the outer core.

Imaging Thromboembolism with Fibrin-Avid 99mTc-Peptide: Evaluation in Swine

PubMed Central

Aruva, Mohan R.; Daviau, Judy; Sharma, Shubh S.; Thakur, Mathew L.

2006-01-01

A pentapeptide, Gly-Pro-Arg-Pro-Pro, with high affinity for α-chain-fibrin was labeled with 99mTc (99mTc-TP850) and evaluated in swine to image experimental venous thromboembolism (deep vein thrombosis [DVT]) and pulmonary embolism (PE). Methods Scatchard analysis was performed to determine fibrin affinity for TP850 and the number of binding sites (receptors) per milligram of fibrin. DVT was induced in the left jugular vein and PE was induced by introducing a preformed autologous blood clot into the right atrium using a 7-French introducer sheath inserted into the right jugular vein. 99mTc-TP850 was injected at 4, 24, 48, 72, 96, or 120 h later. Animals were imaged for up to 4 h after injection, heparinized, and sacrificed. Lungs were extirpated, radiographed, and imaged, and the PE was removed. Other tissues, including blood and normal lungs, were harvested and, concomitantly, 99mTc was counted for determination of target-to-tissue ratios and the percentage injected dose per gram of tissue. Results The affinity for human fibrin was 10−9 mol/L and there were >1015 receptors per milligram of fibrin. DVT and PE were visualized for up to 4 h after injection with high DVT/blood (7.9–22.6), DVT/muscle (31.1–89.4), PE/blood (1–155), and PE/lung (0.8–245) ratios. Thereafter, the PEs fragmented spontaneously below the spatial resolution of the γ-camera and, despite the high associated radioactivity, could not be localized in vivo. The fragmented clots were detectable by scintigraphy on excised lungs and provided excellent concordance with radiograms. Conclusion 99mTc-TP850 with its modest affinity (10−9 mol/L), rapid blood clearance, and high DVT and PE uptake is a promising agent for imaging vascular thrombosis. PMID:16391200

Changes in calcium uptake rate by rat cardiac mitochondria during postnatal development.

PubMed

Bassani, R A; Fagian, M M; Bassani, J W; Vercesi, A E

1998-10-01

Ca2+ uptake, transmembrane electrical potential (Deltapsim) and oxygen consumption were measured in isolated ventricular mitochondria of rats from 3 days to 5 months of age. Estimated values of ruthenium red-sensitive, succinate-supported maximal rate of Ca2+ uptake (Vmax, expressed as nmol Ca2+/min/mg protein) were higher in neonates and gradually fell during postnatal development (from 435+/-24 at 3-6 days, to 156+/-10 in adults,P<0.001), whereas K0.5 values (approximately 10 microM were not significantly affected by age. Under similar conditions, mitochondria from adults (5 months old) and neonates (4-6 days old) showed comparable state 4 (succinate and alpha-ketoglutarate as substrates) and state 3ADP (alpha-ketoglutarate-supported) respiration rates, as well as Deltapsim values (approximately-150 mV). Respiration-independent Deltapsim and Ca2+ uptake, supported by valinomycin-induced K+ efflux were also investigated at these ages. A transient Deltapsim (approximately -30 mV) was evoked by valinomycin in both neonatal and adult mitochondria. Respiration-independent Ca2+ uptake was also transient, but its initial rate was significantly higher in neonates than in adults (49. 4+/-10.0v 28.0+/-5.7 mmol Ca2+/min/mg protein,P<0.01). These results indicate that Ca2+ uptake capacity of rat cardiac mitochondria is remarkably high just after birth and declines over the first weeks of postnatal life, without change in apparent affinity of the transporter. Increased mitochondrial Ca2+ uptake rate in neonates appears to be related to the uniporter itself, rather than to modification of the driving force of the transport. Copyright 1998 Academic Press

Further analysis of the neuropharmacological profile of 9-amino-1,2,3,4-tetrahydroacridine (THA), and alleged drug for the treatment of Alzheimer's disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Drukarch, B.; Leysen, J.E.; Stoof, J.C.

1988-01-01

In a recent study the authors have documented the acetylcholinesterase and outward K+-current inhibiting activity of 9-amino-1,2,3,4-tetrahydroacridine (THA), a drug reportedly active in the treatment of Alzheimer patients. In the present study they have investigated the effects of THA on the uptake and release of radiolabeled NA, DA and 5-HT. THA concentration-dependently inhibited the uptake of these monoamines with IC-50 values of approximately 1, 7 and 2 ..mu..M respectively. Release studies of these radiolabeled monoamines from control and reserpine pretreated tissue revealed that the THA-induced uptake inhibition does not occur at the level of the axonal membrane but at themore » level of the monoaminergic storage granules. In addition the affinity of THA for alpha-1, alpha-2 and beta-adrenoceptors, for D-2 dopamine, S-la and S-2 serotonin and for muscarinic receptors was investigated. It appeared that in concentrations up to 1 ..mu..M THA did not display any affinity towards these receptors. It is concluded from these experiments that the effects of THA on monoaminergic neurotransmission might contribute to the alleged therapeutic action of THA in Alzheimer's disease. 17 references, 3 figures, 1 table.« less

Hepatic Disposition of Gemfibrozil and Its Major Metabolite Gemfibrozil 1-O-β-Glucuronide.

PubMed

Kimoto, Emi; Li, Rui; Scialis, Renato J; Lai, Yurong; Varma, Manthena V S

2015-11-02

Gemfibrozil (GEM), which decreases serum triglycerides and low density lipoprotein, perpetrates drug-drug interactions (DDIs) with several drugs. These DDIs are primarily attributed to the inhibition of drug transporters and metabolic enzymes, particularly cytochrome P450 (CYP) 2C8 by the major circulating metabolite gemfibrozil 1-O-β-glucuronide (GG). Here, we characterized the transporter-mediated hepatic disposition of GEM and GG using sandwich-cultured human hepatocytes (SCHH) and transporter-transfect systems. Significant active uptake was noted in SCHH for the metabolite. GG, but not GEM, showed substrate affinity to organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1. In SCHH, glucuronidation was characterized affinity constants (Km) of 7.9 and 61.4 μM, and biliary excretion of GG was observed. Furthermore, GG showed active basolateral efflux from preloaded SCHH and ATP-dependent uptake into membrane vesicles overexpressing multidrug resistance-associated protein (MRP) 2, MRP3, and MRP4. A mathematical model was developed to estimate hepatic uptake and efflux kinetics of GEM and GG based on SCHH studies. Collectively, the hepatic transporters play a key role in the disposition and thus determine the local concentrations of GEM and more so for GG, which is the predominant inhibitory species against CYP2C8 and OATP1B1.

pH-sensitive interaction of HMG-CoA reductase inhibitors (statins) with organic anion transporting polypeptide 2B1.

PubMed

Varma, Manthena V; Rotter, Charles J; Chupka, Jonathan; Whalen, Kevin M; Duignan, David B; Feng, Bo; Litchfield, John; Goosen, Theunis C; El-Kattan, Ayman F

2011-08-01

The human organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) is ubiquitously expressed and may play an important role in the disposition of xenobiotics. The present study aimed to examine the role of OATP2B1 in the intestinal absorption and tissue uptake of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitors (statins). We first investigated the functional affinity of statins to the transporter as a function of extracellular pH, using OATP2B1-transfeced HEK293 cells. The results indicate that OATP2B1-mediated transport is significant for rosuvastatin, fluvastatin and atorvastatin, at neutral pH. However, OATP2B1 showed broader substrate specificity as well as enhanced transporter activity at acidic pH. Furthermore, uptake at acidic pH was diminished in the presence of proton ionophore, suggesting proton gradient as the driving force for OATP2B1 activity. Notably, passive transport rates are predominant or comparable to active transport rates for statins, except for rosuvastatin and fluvastatin. Second, we studied the effect of OATP modulators on statin uptake. At pH 6.0, OATP2B1-mediated transport of atorvastatin and cerivastatin was not inhibitable, while rosuvastatin transport was inhibited by E-3-S, rifamycin SV and cyclosporine with IC(50) values of 19.7 ± 3.3 μM, 0.53 ± 0.2 μM and 2.2 ± 0.4 μM, respectively. Rifamycin SV inhibited OATP2B1-mediated transport of E-3-S and rosuvastatin with similar IC(50) values at pH 6.0 and 7.4, suggesting that the inhibitor affinity is not pH-dependent. Finally, we noted that OATP2B1-mediated transport of E-3-S, but not rosuvastatin, is pH sensitive in intestinal epithelial (Caco-2) cells. However, uptake of E-3-S and rosuvastatin by Caco-2 cells was diminished in the presence of proton ionophore. The present results indicate that OATP2B1 may be involved in the tissue uptake of rosuvastatin and fluvastatin, while OATP2B1 may play a significant role in the intestinal absorption of several statins due to their transporter affinity at acidic pH.

Spatial location of neutralizing and non-neutralizing B cell epitopes on domain 1 of ricin toxin’s binding subunit

PubMed Central

Rong, Yinghui; Van Slyke, Greta; Vance, David J.; Westfall, Jennifer; Ehrbar, Dylan

2017-01-01

Ricin toxin’s binding subunit (RTB) is a galactose-/N-acetylgalactosamine (Gal/GalNac)-specific lectin that mediates uptake and intracellular trafficking of ricin within mammalian cells. Structurally, RTB consists of two globular domains, each divided into three homologous sub-domains (α, β, γ). In this report, we describe five new murine IgG monoclonal antibodies (mAbs) against RTB: MH3, 8A1, 8B3, LF1, and LC5. The mAbs have similar binding affinities (KD) for ricin holotoxin, but displayed a wide range of in vitro toxin-neutralizing activities. Competition ELISAs indicate that the two most potent toxin-neutralizing mAbs (MH3, 8A1), as well as one of the moderate toxin-neutralizing mAbs (LF1), recognize distinct epitopes near the low affinity Gal recognition domain in RTB subdomain 1α. Evaluated in a mouse model of systemic ricin challenge, all five mAbs afforded some benefit against intoxication, but only MH3 was protective. However, neither MH3 nor 24B11, another well-characterized mAb against RTB subdomain 1α, could passively protect mice against a mucosal (intranasal) ricin challenge. This is in contrast to SylH3, a previously characterized mAb directed against an epitope near RTB’s high affinity Gal/GalNac recognition element in sub-domain 2γ, which protected animals against systemic and mucosal ricin exposure. SylH3 was significantly more effective than MH3 and 24B11 at blocking ricin attachment to host cell receptors, suggesting that mucosal immunity to ricin is best imparted by antibodies that target RTB’s high affinity Gal/GalNac recognition element in subdomain 2γ, not the low affinity Gal recognition domain in subdomain 1α. PMID:28700745

The role of tailored biochar in increasing plant growth, and reducing bioavailability, phytotoxicity, and uptake of heavy metals in contaminated soil.

PubMed

Mohamed, Badr A; Ellis, Naoko; Kim, Chang Soo; Bi, Xiaotao

2017-11-01

Microwave-assisted catalytic pyrolysis was investigated using K 3 PO 4 and clinoptilolite to enhance biochar sorption affinity for heavy metals. The performance of resulting biochar samples was characterized through their effects on plant growth, bioavailability, phytotoxicity, and uptake of heavy metals in a sandy soil contaminated with Pb, Ni, and Co. The produced biochars have high cation-exchange capacity (CEC) and surface area, and rich in plant nutrients, which not only reduced heavy metals (Pb, Ni, and Co), bioavailability and phytotoxicity, but also increased plant growth rate by up to 145%. The effectiveness of biochar in terms of reduced phytotoxicity and plant uptake of heavy metals was further improved by mixing K 3 PO 4 and clinoptilolite with biomass through microwave pyrolysis. This may be due to the predominance of different mechanisms as 10KP/10Clino biochar has the highest micropore surface area (405 m 2 /g), high concentrations of K (206 g/kg), Ca (26.5 g/kg), Mg (6.2 g/kg) and Fe (11.9 g/kg) for ion-exchange and high phosphorus content (79.8 g/kg) for forming insoluble compounds with heavy metals. The largest wheat shoot length (143 mm) and lowest extracted amounts of Pb (107 mg/kg), Ni (2.4 mg/kg) and Co (63.9 mg/kg) were also obtained by using 10KP/10Clino biochar at 2 wt% load; while the smallest shoot length (68 mm) and highest extracted amounts of heavy metals (Pb 408 mg/kg, Ni 15 mg/kg and Co 148 mg/kg) for the samples treated with biochars were observed for soils mixed with 1 wt% 10Clino biochar. Strong negative correlations were also observed between biochar micropore surface area, CEC and the extracted amounts of heavy metals. Microwave-assisted catalytic pyrolysis of biomass has a great potential for producing biochar with high sorption affinity for heavy metals and rich nutrient contents using properly selected catalysts/additives that can increase microwave heating rate and improve biochar and bio-oil properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesis and biological characterization of (3R,4R)-4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol and its stereoisomers for activity toward monoamine transporters.

PubMed

Kharkar, Prashant S; Batman, Angela M; Zhen, Juan; Beardsley, Patrick M; Reith, Maarten E A; Dutta, Aloke K

2009-07-01

A novel series of optically active molecules based on a 4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol template were developed. Depending on stereochemistry, the compounds exhibit various degrees of affinity for three dopamine, serotonin, and norepinephrine transporters. These molecules have the potential for treating several neurological disorders such as drug abuse, depression, and attention deficit hyperactivity disorder.Herein we describe the synthesis and biological evaluation of a series of asymmetric 4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol-based dihydroxy compounds in which the hydroxy groups are located on both the piperidine ring and the N-phenylethyl side chain. In vitro uptake inhibition data of these molecules indicate high affinity for the dopamine transporter (DAT) in addition to moderate to high affinity for the norepinephrine transporter (NET). Interestingly, compounds 9 b and 9 d exhibit affinities for all three monoamine transporters, with highest potency at DAT and NET, and moderate potency at the serotonin transporter (SERT) (K(i): 2.29, 78.4, and 155 nM for 9 b and 1.55, 14.1, and 259 nM for 9 d, respectively). Selected compounds 9 a, 9 d, and 9 d' were tested for their locomotor activity effects in mice and for their ability to occasion the cocaine-discriminative stimulus in rats. These test compounds generally exhibit a much longer duration of action than cocaine for elevating locomotor activity, and completely generalize the cocaine-discriminative stimulus in a dose-dependent manner.

Complex phylogeny and gene expression patterns of members of the NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family (NPF) in wheat

PubMed Central

Buchner, Peter; Hawkesford, Malcolm J.

2014-01-01

NPF (formerly referred to as low-affinity NRT1) and ‘high-affinity’ NRT2 nitrate transporter genes are involved in nitrate uptake by the root, and transport and distribution of nitrate within the plant. The NPF gene family consists of 53 members in Arabidopsis thaliana, however only 11 of these have been functionally characterized. Although homologous genes have been identified in genomes of different plant species including some cereals, there is little information available for wheat (Triticum aestivum). Sixteen genes were identified in wheat homologous to characterized Arabidopsis low-affinity nitrate transporter NPF genes, suggesting a complex wheat NPF gene family. The regulation of wheat NFP genes by plant N-status indicated involvement of these transporters in substrate transport in relation to N-metabolism. The complex expression pattern in relation to tissue specificity, nitrate availability and senescence may be associated with the complex growth patterns of wheat depending on sink/source demands, as well as remobilization during grain filling. PMID:24913625

d-Fructose-Decorated Poly(ethylene imine) for Human Breast Cancer Cell Targeting.

PubMed

Englert, Christoph; Pröhl, Michael; Czaplewska, Justyna A; Fritzsche, Carolin; Preußger, Elisabeth; Schubert, Ulrich S; Traeger, Anja; Gottschaldt, Michael

2017-08-01

The high affinity of GLUT5 transporter for d-fructose in breast cancer cells has been discussed intensely. In this contribution, high molar mass linear poly(ethylene imine) (LPEI) is functionalized with d-fructose moieties to combine the selectivity for the GLUT5 transporter with the delivery potential of PEI for genetic material. The four-step synthesis of a thiol-group bearing d-fructose enables the decoration of a cationic polymer backbone with d-fructose via thiol-ene photoaddition. The functionalization of LPEI is confirmed by 2D NMR techniques, elemental analysis, and size exclusion chromatography. Importantly, a d-fructose decoration of 16% renders the polymers water-soluble and eliminates the cytotoxicity of PEI in noncancer L929 cells, accompanied by a reduced unspecific cellular uptake of the genetic material. In contrast, the cytotoxicity as well as the cell specific uptake is increased for triple negative MDA-MB-231 breast cancer cells. Therefore, the introduction of d-fructose shows superior potential for cell targeting, which can be assumed to be GLUT5 dependent. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In Vitro and In Vivo Evaluation of 89Zr-DS-8273a as a Theranostic for Anti-Death Receptor 5 Therapy

PubMed Central

Burvenich, Ingrid J.G.; Lee, Fook-Thean; Guo, Nancy; Gan, Hui K.; Rigopoulos, Angela; Parslow, Adam C.; O'Keefe, Graeme J.; Gong, Sylvia J.; Tochon-Danguy, Henri; Rudd, Stacey E.; Donnelly, Paul S.; Kotsuma, Masakatsu; Ohtsuka, Toshiaki; Senaldi, Giorgio; Scott, Andrew M.

2016-01-01

Background: DS-8273a, an anti-human death receptor 5 (DR5) agonistic antibody, has cytotoxic activity against human cancer cells and induces apoptosis after specific binding to DR5. DS-8273a is currently being used in clinical Phase I trials. This study evaluated the molecular imaging of DR5 expression in vivo in mouse tumor models using SPECT/CT and PET/MRI, as a tool for drug development and trial design. Methods: DS-8273a was radiolabeled with indium-111 and zirconium-89. Radiochemical purity, immunoreactivity, antigen binding affinity and serum stability were assessed in vitro. In vivo biodistribution and pharmacokinetic studies were performed, including SPECT/CT and PET/MR imaging. A dose-escalation study using a PET/MR imaging quantitative analysis was also performed to determine DR5 receptor saturability in a mouse model. Results: 111In-CHX-A″-DTPA-DS-8273a and 89Zr-Df-Bz-NCS-DS-8273a showed high immunoreactivity (100%), high serum stability, and bound to DR5 expressing cells with high affinity (Ka, 1.02-1.22 × 1010 M-1). The number of antibodies bound per cell was 32,000. In vivo biodistribution studies showed high and specific uptake of 111In-CHX-A″-DTPA-DS-8273a and 89Zr-Df-Bz-NCS-DS-8273a in DR5 expressing COLO205 xenografts, with no specific uptake in normal tissues or in DR5-negative CT26 xenografts. DR5 receptor saturation was observed in vivo by biodistribution studies and quantitative PET/MRI analysis. Conclusion: 89Zr-Df-Bz-NCS-DS-8273a is a potential novel PET imaging reagent for human bioimaging trials, and can be used for effective dose assessment and patient response evaluation in clinical trials. PMID:27924159

New Gastrin Releasing Peptide Receptor-Directed [99mTc]Demobesin 1 Mimics: Synthesis and Comparative Evaluation.

PubMed

Nock, Berthold A; Charalambidis, David; Sallegger, Werner; Waser, Beatrice; Mansi, Rosalba; Nicolas, Guillaume P; Ketani, Eleni; Nikolopoulou, Anastasia; Fani, Melpomeni; Reubi, Jean-Claude; Maina, Theodosia

2018-04-12

We have previously reported on the gastrin releasing peptide receptor (GRPR) antagonist [ 99m Tc]1, ([ 99m Tc]demobesin 1, 99m Tc-[N 4 '-diglycolate-dPhe 6 ,Leu-NHEt 13 ]BBN(6-13)). [ 99m Tc]1 has shown superior biological profile compared to analogous agonist-based 99m Tc-radioligands. We herein present a small library of [ 99m Tc]1 mimics generated after structural modifications in (a) the linker ([ 99m Tc]2, [ 99m Tc]3, [ 99m Tc]4), (b) the peptide chain ([ 99m Tc]5, [ 99m Tc]6), and (c) the C-terminus ([ 99m Tc]7 or [ 99m Tc]8). The effects of above modifications on the biological properties of analogs were studied in PC-3 cells and tumor-bearing SCID mice. All analogs showed subnanomolar affinity for the human GRPR, while most receptor-affine 4 and 8 behaved as potent GRPR antagonists in a functional internalization assay. In mice bearing PC-3 tumors, [ 99m Tc]1-[ 99m Tc]6 exhibited GRPR-specific tumor uptake, rapidly clearing from normal tissues. [ 99m Tc]4 displayed the highest tumor uptake (28.8 ± 4.1%ID/g at 1 h pi), which remained high even after 24 h pi (16.3 ± 1.8%ID/g), well surpassing that of [ 99m Tc]1 (5.4 ± 0.7%ID/g at 24 h pi).

Imaging Neurotensin Receptor in Prostate Cancer With 64Cu-Labeled Neurotensin Analogs.

PubMed

Deng, Huaifu; Wang, Hui; Zhang, He; Wang, Mengzhe; Giglio, Ben; Ma, Xiaofen; Jiang, Guihua; Yuan, Hong; Wu, Zhanhong; Li, Zibo

2017-01-01

Neurotensin receptor 1 (NTR-1) is expressed and activated in prostate cancer cells. In this study, we explore the NTR expression in normal mouse tissues and study the positron emission tomography (PET) imaging of NTR in prostate cancer models. Three 64 Cu chelators (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid [DOTA], 1,4,7-triazacyclononane-N,N',N″-triacetic acid [NOTA], or AmBaSar) were conjugated to an NT analog. Neurotensin receptor binding affinity was evaluated using cell binding assay. The imaging profile of radiolabeled probes was compared in well-established NTR + HT-29 tumor model. Stability of the probes was tested. The selected agents were further evaluated in human prostate cancer PC3 xenografts. All 3 NT conjugates retained the majority of NTR binding affinity. In HT-29 tumor, all agents demonstrated prominent tumor uptake. Although comparable stability was observed, 64 Cu-NOTA-NT and 64 Cu-AmBaSar-NT demonstrated improved tumor to background contrast compared with 64 Cu-DOTA-NT. Positron emission tomography/computed tomography imaging of the NTR expression in PC-3 xenografts showed high tumor uptake of the probes, correlating with the in vitro Western blot results. Blocking experiments further confirmed receptor specificity. Our results demonstrated that 64 Cu-labeled neurotensin analogs are promising imaging agents for NTR-positive tumors. These agents may help us identify NTR-positive lesions and predict which patients and individual tumors are likely to respond to novel interventions targeting NTR-1.

Pyrimidine Salvage in Trypanosoma brucei Bloodstream Forms and the Trypanocidal Action of Halogenated Pyrimidiness

PubMed Central

Ali, Juma A. M.; Creek, Darren J.; Burgess, Karl; Allison, Harriet C.; Field, Mark C.; Mäser, Pascal; De Koning, Harry P.

2016-01-01

African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host. However, uptake of pyrimidines in bloodstream form trypanosomes has not been investigated, making it difficult to judge the relative importance of salvage and synthesis or to design a pyrimidine-based chemotherapy. Detailed characterization of pyrimidine transport activities in bloodstream form Trypanosoma brucei brucei found that these cells express a high-affinity uracil transporter (designated TbU3) that is clearly distinct from the procyclic pyrimidine transporters. This transporter had low affinity for uridine and 2′deoxyuridine and was the sole pyrimidine transporter expressed in these cells. In addition, thymidine was taken up inefficiently through a P1-type nucleoside transporter. Of importance, the anticancer drug 5-fluorouracil was an excellent substrate for TbU3, and several 5-fluoropyrimidine analogs were investigated for uptake and trypanocidal activity; 5F-orotic acid, 5F-2′deoxyuridine displayed activity in the low micromolar range. The metabolism and mode of action of these analogs was determined using metabolomic assessments of T. brucei clonal lines adapted to high levels of these pyrimidine analogs, and of the sensitive parental strains. The analysis showed that 5-fluorouracil is incorporated into a large number of metabolites but likely exerts toxicity through incorporation into RNA. 5F-2′dUrd and 5F-2′dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase as 5F-dUMP. We present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes. PMID:23188714

Mechanistic insights into PEPT1-mediated transport of a novel antiepileptic, NP-647.

PubMed

Khomane, Kailas S; Nandekar, Prajwal P; Wahlang, Banrida; Bagul, Pravin; Shaikh, Naeem; Pawar, Yogesh B; Meena, Chhuttan Lal; Sangamwar, Abhay T; Jain, Rahul; Tikoo, K; Bansal, Arvind K

2012-09-04

The present study, in general, is aimed to uncover the properties of the transport mechanism or mechanisms responsible for the uptake of NP-647 into Caco-2 cells and, in particular, to understand whether it is a substrate for the intestinal oligopeptide transporter, PEPT1 (SLC15A1). NP-647 showed a carrier-mediated, saturable transport with Michaelis-Menten parameters K(m) = 1.2 mM and V(max) = 2.2 μM/min. The effect of pH, sodium ion (Na(+)), glycylsarcosine and amoxicillin (substrates of PEPT1), and sodium azide (Na(+)/K(+)-ATPase inhibitor) on the flux rate of NP-647 was determined. Molecular docking and molecular dynamics simulation studies were carried out to investigate molecular interactions of NP-647 with transporter using homology model of human PEPT1. The permeability coefficient (P(appCaco-2)) of NP-647 (32.5 × 10(-6) cm/s) was found to be four times higher than that of TRH. Results indicate that NP-647 is transported into Caco-2 cells by means of a carrier-mediated, proton-dependent mechanism that is inhibited by Gly-Sar and amoxicillin. In turn, NP-647 also inhibits the uptake of Gly-Sar into Caco-2 cells and, together, this evidence suggests that PEPT1 is involved in the process. Docking and molecular dynamics simulation studies indicate high affinity of NP-647 toward PEPT1 binding site as compared to TRH. High permeability of NP-647 over TRH is attributed to its increased hydrophobicity which increases its affinity toward PEPT1 by interacting with the hydrophobic pocket of the transporter through hydrophobic forces.

Reinforcing Doses of Intravenous Cocaine Produce Only Modest Dopamine Uptake Inhibition.

PubMed

Brodnik, Zachary D; Ferris, Mark J; Jones, Sara R; España, Rodrigo A

2017-02-15

The reinforcing efficacy of cocaine is thought to stem from inhibition of the dopamine transporter (DAT) and subsequent increases in extracellular dopamine concentrations in the brain. In humans, this hypothesis has generally been supported by positron emission tomography imaging studies where the percent of DATs occupied by cocaine is used as a measure of cocaine activity in the brain. Interpretation of these studies, however, often relies on the assumption that measures of DAT occupancy directly correspond with functional DAT blockade. In the current studies, we used in vivo and in vitro fast scan cyclic voltammetry in mice to measure dopamine uptake inhibition following varying doses of cocaine as well as two high affinity DAT inhibitors. We then compared dopamine clearance rates following these drug treatments to dopamine clearance obtained from DAT knockout mice as a proxy for complete DAT blockade. We found that administration of abused doses of cocaine resulted in approximately 2% of maximal DAT blockade. Overall, our data indicate that abused doses of cocaine produce a relatively modest degree of DA uptake inhibition, and suggest that the relationship between DAT occupancy and functional blockade of the DAT is more complex than originally posited.

Fatty acid uptake by isolated rat heart myocytes represents a carrier-mediated transport process.

PubMed Central

Stremmel, W

1988-01-01

The mechanism by which fatty acids enter cardiomyocytes is unclear. Therefore, the influx kinetics of [3H]oleate into isolated rat heart myocytes were examined. Cells were incubated at 37 degrees C with [3H]oleate bound to albumin in various molar ratios and the initial rate of uptake (V0) was determined as a function of the unbound oleate concentration in the medium. V0 was saturable with increasing oleate concentrations incubated (Km 78 nM; Vmax 1.9 nmol X min-1 per 10(6) cells) and temperature dependent with an optimum at 37 degrees C. Furthermore, binding of [3H]oleate to isolated plasma membranes of cardiomyocytes was saturable, revealing a KD of 42 nM, and was inhibited by heat denaturation or trypsin pretreatment of the membranes. From these membranes a single 40-kD protein with high affinity for a variety of long chain fatty acids was isolated. With a monospecific antibody to this membrane protein, binding as well as cellular influx of [3H]oleate was selectively inhibited. These data indicate that at least a portion of myocardial fatty acid uptake is mediated by a specific membrane protein. Images PMID:3343344

Pharmacological evaluation of [123I]-CLINDE: a radioiodinated imidazopyridine-3-acetamide for the study of peripheral benzodiazepine binding sites (PBBS).

PubMed

Mattner, Filomena; Mardon, Karine; Katsifis, Andrew

2008-04-01

The study aims to evaluate the iodinated imidazopyridine, N',N'-diethyl-6-Chloro-(4'-[(123)I]iodophenyl)imidazo[1,2-a]pyridine-3-acetamide ([(123)I]-CLINDE) as a tracer for the study of peripheral benzodiazepine binding sites (PBBS). In vitro studies were performed using membrane homogenates and sections from kidney, adrenals, and brain cortex of Sprague-Dawley (SD) rats and incubated with [(123)I]-CLINDE. For in vivo studies, the rats were injected with [(123)I]-CLINDE. In competition studies, PBBS-specific drugs PK11195 and Ro 5-4864 and the CBR specific drug Flumazenil were injected before the radiotracer. In vitro binding studies in adrenal, kidney, and cortex mitochondrial membranes indicated that [(123)I]-CLINDE binds with high affinity to PBBS, K(d) = 12.6, 0.20, and 3.84 nM, respectively. The density of binding sites was 163, 5.3, and 0.34 pmol/mg protein, respectively. In vivo biodistribution indicated high uptake in adrenals (5.4), heart (1.5), lungs (1.5), kidney (1.5) %ID/g at 6 h p.i. In the central nervous system (CNS), the olfactory bulbs displayed the highest uptake; up to six times the activity in blood. Pre-administration of unlabeled CLINDE, PK11195 and Ro 5-4864 (1 mg/kg) reduced the uptake of [(123)I]-CLINDE by 70-55% in olfactory bulbs. In the kidney and heart, a reduction of 60-80% ID/g was observed, while an increase was observed in the adrenals requiring 10 mg/kg for significant displacement. Flumazenil had no effect on uptake in peripheral organs and brain. Metabolite analysis indicated >90% of the radioactivity in the above tissues was intact [(123)I]-CLINDE. [(123)I]-CLINDE displays high and selective uptake for the PBBS and warrants further development as a probe for imaging PBBS using single photon emission computed tomography (SPECT).

Combating oil spill problem using plastic waste

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saleem, Junaid, E-mail: junaidupm@gmail.com; Ning, Chao; Barford, John

Highlights: • Up-cycling one type of pollution i.e. plastic waste and successfully using it to combat the other type of pollution i.e. oil spill. • Synthesized oil sorbent that has extremely high oil uptake of 90 g/g after prolonged dripping of 1 h. • Synthesized porous oil sorbent film which not only facilitates in oil sorption but also increases the affinity between sorbent and oil by means of adhesion. - Abstract: Thermoplastic polymers (such as polypropylene, polyethylene, polyethylene terephthalate (PET) and high density polyethylene (HDPE)) constitute 5–15% of municipal solid waste produced across the world. A huge quantity of plasticmore » waste is disposed of each year and is mostly either discarded in landfills or incinerated. On the other hand, the usage of synthetic polymers as oil sorbents, in particular, polyolefins, including polypropylene (PP), and polyethylene (PE) are the most commonly used oil sorbent materials mainly due to their low cost. However, they possess relatively low oil absorption capacities. In this work, we provide an innovative way to produce a value-added product such as oil-sorbent film with high practical oil uptake values in terms of g/g from waste HDPE bottles for rapid oil spill remedy.« less

Initial in vivo PET imaging of 5-HT1A receptors with 3-[18F]mefway

PubMed Central

Wooten, Dustin W; Hillmer, Ansel T; Murali, Dhanabalan; Barnhart, Todd E; Thio, Joanne P; Bajwa, Alisha K; Bonab, Ali A; Normandin, Marc D; Schneider, Mary L; Mukherjee, Jogeshwar; Christian, Bradley T

2014-01-01

4-trans-[18F]Mefway is a PET radiotracer with high affinity for 5-HT1A receptors. Our preliminary work indicated the positional isomer, 3-[18F]mefway, would be suitable for PET imaging of 5-HT1A receptors. We now compare the in vivo behaviour of 3-mefway with 4-mefway to evaluate 3-[18F]mefway as a potential 5-HT1A PET radiotracer. Two male rhesus macaques were given bolus injections of both 3- and 4-trans-[18F]mefway in separate experiments. 90 minute dynamic PET scans were acquired. TACs were extracted in the mesial temporal lobe (MTL) and caudal anterior cingulate gyrus (cACg). The cerebellum (CB) was used as a reference region. In vivo behavior of the radiotracers in the CB was compared based upon the ratio of normalized PET uptake for 3- and 4-trans-[18F]mefway. Specific binding was compared by examining MTL/CB and cACg/CB ratios. The subject-averaged ratio of 3-[18F]mefway to 4-trans-[18F]mefway in the cerebellum was 0.96 for 60-90 minutes. MTL/CB reached plateaus of ~2.7 and ~6 by 40 minutes and 90 minutes for 3- and 4-trans-[18F]mefway, respectively. cACg/CB reached plateaus of ~2.5 and ~6 by 40 minutes and 70 minutes for 3- and 4-trans-[18F]mefway, respectively. The short pseudoequilibration times and sufficient uptake of 3-[18F]mefway may be useful in studies requiring short scan times. Furthermore, the similar nondisplaceable clearance in the CB to 4-trans-[18F]mefway suggests the lower BPND of 3-[18F]mefway is due to a lower affinity. The lower affinity of 3-[18F]mefway may make it useful for measuring changes in endogenous 5-HT levels, however, this remains to be ascertained. PMID:25143866

Structure-Activity Relationships of Substituted Cathinones, with Transporter Binding, Uptake, and Release

PubMed Central

Wolfrum, Katherine M.; Reed, John F.; Kim, Sunyoung O.; Swanson, Tracy; Johnson, Robert A.; Janowsky, Aaron

2017-01-01

Synthetic cathinones are components of “bath salts” and have physical and psychologic side effects, including hypertension, paranoia, and hallucinations. Here, we report interactions of 20 “bath salt” components with human dopamine, serotonin, and norepinephrine transporters [human dopamine transporter (hDAT), human serotonin transporter (hSERT), and human norepinephrine transporter (hNET), respectively] heterologously expressed in human embryonic kidney 293 cells. Transporter inhibitors had nanomolar to micromolar affinities (Ki values) at radioligand binding sites, with relative affinities of hDAT>hNET>hSERT for α-pyrrolidinopropiophenone (α-PPP), α-pyrrolidinobutiophenone, α-pyrrolidinohexiophenone, 1-phenyl-2-(1-pyrrolidinyl)-1-heptanone, 3,4-methylenedioxy-α-pyrrolidinopropiophenone, 3,4-methylenedioxy-α-pyrrolidinobutiophenone, 4-methyl-α-pyrrolidinopropiophenone, α-pyrrolidinovalerophenone, 4-methoxy-α-pyrrolidinovalerophenone, α-pyrrolidinopentiothiophenone (alpha-PVT), and α-methylaminovalerophenone, and hDAT>hSERT>hNET for methylenedioxypentedrone. Increasing the α-carbon chain length increased the affinity and potency of the α-pyrrolidinophenones. Uptake inhibitors had relative potencies of hDAT>hNET>hSERT except α-PPP and α-PVT, which had highest potencies at hNET. They did not induce [3H]neurotransmitter release. Substrates can enter presynaptic neurons via transporters, and the substrates methamphetamine and 3,4-methylenedioxymethylamphetamine are neurotoxic. We determined that 3-fluoro-, 4-bromo-, 4-chloro-methcathinone, and 4-fluoroamphetamine were substrates at all three transporters; 5,6-methylenedioxy-2-aminoindane (MDAI) and 4-methylethcathinone (4-MEC) were substrates primarily at hSERT and hNET; and 3,4-methylenedioxy-N-ethylcathinone (ethylone) and 5-methoxy-methylone were substrates only at hSERT and induced [3H]neurotransmitter release. Significant correlations between potencies for inhibition of uptake and for inducing release were observed for these and additional substrates. The excellent correlation of efficacy at stimulating release versus Ki/IC50 ratios suggested thresholds of binding/uptake ratios above which compounds were likely to be substrates. Based on their potencies at hDAT, most of these compounds have potential for abuse and addiction. 4-Bromomethcathinone, 4-MEC, 5-methoxy-methylone, ethylone, and MDAI, which have higher potencies at hSERT than hDAT, may have empathogen psychoactivity. PMID:27799294

Glucocorticoid-dependent induction of interleukin-6 receptor expression in human hepatocytes facilitates interleukin-6 stimulation of amino acid transport.

PubMed

Fischer, C P; Bode, B P; Takahashi, K; Tanabe, K K; Souba, W W

1996-05-01

The authors studied the effects of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on glutamine and alanine transport in isolated human hepatocytes. They also evaluated the role of dexamethasone in modulating this response and its effects on the expression of the plasma membrane high-affinity IL-6 receptor. Animal studies indicate that cytokines are important mediators of the increased hepatic amino acid uptake that occurs during cancer and sepsis, but studies in human tissues are lacking. The control of transport by cytokines and cytokine receptor expression in the liver may provide a mechanism by which hepatocytes can modulate amino acid availability during catabolic disease states. Human hepatocytes were isolated from wedge biopsy specimens and plated in 24-well trays. Interleukin-6 and TNF-alpha, in combination with the synthetic glucocorticoid dexamethasone, were added to hepatocytes in culture, and the transport of radiolabeled glutamine and alanine was measured. Fluorescent-activated cell sorter (FACS) analysis was used to study the effects of dexamethasone on IL-6 receptor number in the well-differentiated human hepatoma HepG2. Both IL-6 and TNF-alpha exerted a small stimulatory effect on alanine and glutamine transport. Dexamethasone alone did not alter transport rates, but pretreatment of cells augmented the effects of both cytokines on carrier-mediated amino acid uptake. Dexamethasone pretreatment and a combination of IL-6 and TNF-alpha resulted in a greater than twofold increase in transport activity. Fluorescent-activated cell sorter analysis demonstrated that dexamethasone induced a threefold increase in the expression of high-affinity IL-6 receptors. Interleukin-6 and TNF-alpha work coordinately with glucocorticoids to stimulate amino acid uptake in human hepatocytes. Dexamethasone exerts a permissive effect on cytokine-mediated increases in transport by increasing IL-6 receptor expression on the cell surface. It is likely that this upregulation of IL-6 receptors "primes" human liver cells for subsequent stimulation by cytokines. The resulting increase in hepatic amino acid transport provides the liver with substrate to support key metabolic pathways during catabolic states.

Correlating single-molecule and ensemble-average measurements of peptide adsorption onto different inorganic materials.

PubMed

Kim, Seong-Oh; Jackman, Joshua A; Mochizuki, Masahito; Yoon, Bo Kyeong; Hayashi, Tomohiro; Cho, Nam-Joon

2016-06-07

The coating of solid-binding peptides (SBPs) on inorganic material surfaces holds significant potential for improved surface functionalization at nano-bio interfaces. In most related studies, the goal has been to engineer peptides with selective and high binding affinity for a target material. The role of the material substrate itself in modulating the adsorption behavior of a peptide molecule remains less explored and there are few studies that compare the interaction of one peptide with different inorganic substrates. Herein, using a combination of two experimental techniques, we investigated the adsorption of a 16 amino acid-long random coil peptide to various inorganic substrates - gold, silicon oxide, titanium oxide and aluminum oxide. Quartz crystal microbalance-dissipation (QCM-D) experiments were performed in order to measure the peptide binding affinity for inorganic solid supports at the ensemble average level, and atomic force microscopy (AFM) experiments were conducted in order to determine the adhesion force of a single peptide molecule. A positive trend was observed between the total mass uptake of attached peptide and the single-molecule adhesion force on each substrate. Peptide affinity for gold was appreciably greater than for the oxide substrates. Collectively, the results obtained in this study offer insight into the ways in which inorganic materials can differentially influence and modulate the adhesion of SBPs.

Distribution and function of the peptide transporter PEPT2 in normal and cystic fibrosis human lung.

PubMed

Groneberg, D A; Eynott, P R; Döring, F; Dinh, Q Thai; Oates, T; Barnes, P J; Chung, K F; Daniel, H; Fischer, A

2002-01-01

Aerosol administration of peptide based drugs has an important role in the treatment of various pulmonary and systemic diseases. The characterisation of pulmonary peptide transport pathways can lead to new strategies in aerosol drug treatment. Immunohistochemistry and ex vivo uptake studies were established to assess the distribution and activity of the beta-lactam transporting high affinity proton coupled peptide transporter PEPT2 in normal and cystic fibrosis human airway tissue. PEPT2 immunoreactivity in normal human airways was localised to cells of the tracheal and bronchial epithelium and the endothelium of small vessels. In peripheral lung immunoreactivity was restricted to type II pneumocytes. In sections of cystic fibrosis lung a similar pattern of distribution was obtained with signals localised to endothelial cells, airway epithelium, and type II pneumocytes. Functional ex vivo uptake studies with fresh lung specimens led to an uptake of the fluorophore conjugated dipeptide derivative D-Ala-L-Lys-AMCA into bronchial epithelial cells and type II pneumocytes. This uptake was competitively inhibited by dipeptides and cephalosporins but not ACE inhibitors, indicating a substrate specificity as described for PEPT2. These findings provide evidence for the expression and function of the peptide transporter PEPT2 in the normal and cystic fibrosis human respiratory tract and suggest that PEPT2 is likely to play a role in the transport of pulmonary peptides and peptidomimetics.

Distribution and function of the peptide transporter PEPT2 in normal and cystic fibrosis human lung

PubMed Central

Groneberg, D; Eynott, P; Doring, F; Thai, D; Oates, T; Barnes, P; Chung, K; Daniel, H; Fischer, A

2002-01-01

Background: Aerosol administration of peptide based drugs has an important role in the treatment of various pulmonary and systemic diseases. The characterisation of pulmonary peptide transport pathways can lead to new strategies in aerosol drug treatment. Methods: Immunohistochemistry and ex vivo uptake studies were established to assess the distribution and activity of the ß-lactam transporting high affinity proton coupled peptide transporter PEPT2 in normal and cystic fibrosis human airway tissue. Results: PEPT2 immunoreactivity in normal human airways was localised to cells of the tracheal and bronchial epithelium and the endothelium of small vessels. In peripheral lung immunoreactivity was restricted to type II pneumocytes. In sections of cystic fibrosis lung a similar pattern of distribution was obtained with signals localised to endothelial cells, airway epithelium, and type II pneumocytes. Functional ex vivo uptake studies with fresh lung specimens led to an uptake of the fluorophore conjugated dipeptide derivative D-Ala-L-Lys-AMCA into bronchial epithelial cells and type II pneumocytes. This uptake was competitively inhibited by dipeptides and cephalosporins but not ACE inhibitors, indicating a substrate specificity as described for PEPT2. Conclusions: These findings provide evidence for the expression and function of the peptide transporter PEPT2 in the normal and cystic fibrosis human respiratory tract and suggest that PEPT2 is likely to play a role in the transport of pulmonary peptides and peptidomimetics. PMID:11809991

Iron transport and storage in the coccolithophore: Emiliania huxleyi.

PubMed

Hartnett, Andrej; Böttger, Lars H; Matzanke, Berthold F; Carrano, Carl J

2012-11-01

Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies for iron uptake being distinguished: strategy I plants use a mechanism involving soil acidification and induction of Fe(III)-chelate reductase (ferrireductase) and Fe(II) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the coccolithophore Emiliania huxleyi. Short term radio-iron uptake studies indicate that iron is taken up by Emiliania in a time and concentration dependent manner consistent with an active transport process. Based on inhibitor studies it appears that iron is taken up directly as Fe(iii). However if a reductive step is involved the Fe(II) must not be accessible to the external environment. Upon long term exposure to (57)Fe we have been able, using a combination of Mössbauer and XAS spectroscopies, to identify a single metabolite which displays spectral features similar to the phosphorus-rich mineral core of bacterial and plant ferritins.

Absorption of polycyclic aromatic hydrocarbons by a highly absorptive polymeric medium.

PubMed

Francisco, Olga; Idowu, Ifeoluwa; Friesen, Kelsey L; McDougall, Matthew; Choi, Sara Seoin; Bolluch, Patrique; Daramola, Oluwadamilola; Johnson, Wesley; Palace, Vince; Stetefeld, Jörg; Tomy, Gregg T

2018-06-01

The efficacy of a lightly cross-linked polymeric bead to absorb polycyclic aromatic hydrocarbons (PAHs) from the surface of fresh- and salt-water in a simulated oil-spill scenario was assessed in this study. A layer of PAHs at the water surface was created by first preparing the PAHs in hexane and then carefully spiking this mixture onto the surface of water. Beads were then applied to the surface of the organic phase and the amount of hydrocarbons absorbed by the beads was examined at prescribed time intervals and at different temperatures. Absorption of PAHs into the beads was exhaustive with ∼86 ± 4% being selectively removed from the organic phase by 120 s. First order reaction rates best described the uptake kinetics and absorption rates ranged from 0.0085 (naphthalene) to 0.0325 s- 1 (dibenzo[a,h]anthracene). Absorption of PAHs into the beads was driven by molecular volume (A 3 ). Uptake rates increased markedly for PAHs with molecular volumes between 130 A 3 and 190 A 3 . Beyond this molecular volume there was no apparent change in the rate of uptake. This study shows that these polymeric beads have a high affinity for PAHs and can be used under various environmental conditions with negligible difference in absorptive efficacy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synthesis and evaluation of C-11, F-18 and I-125 small molecule radioligands for detecting oxytocin receptors

PubMed Central

Smith, Aaron L.; Freeman, Sara M.; Stehouwer, Jeffery S.; Inoue, Kiyoshi; Voll, Ronald J.; Young, Larry J.; Goodman, Mark M.

2013-01-01

Compounds 1–4 were synthesized and investigated for selectivity and potency for the oxytocin receptor (OTR) to determine their viability as radioactive ligands. Binding assays determined 1–4 to have high binding affinity for both the human and rodent OTR and also have high selectivity for the human OTR over human vasopressin V1a receptors (V1aR). Inadequate selectivity for OTR over V1aR was found for rodent receptors in all four compounds. The radioactive (C-11, F-18, and I-125) derivatives of 1–4 were synthesized and investigated for use as autoradiography and positron emission tomography (PET) ligands. Receptor autoradiography performed with [125I]1 and [125I]2 on rodent brain slices provided the first small molecule radioligand images of the OTR and V1aR. Biodistribution studies determined [125I]1 and [125I]2 were adequate for in vivo peripheral investigations, but not for central investigations due to low uptake within the brain. A biodistribution study with [18F]3 suggested brain uptake occurred slowly over time. PET imaging studies with [18F]3 and [11C]4 using a rat model provided insufficient uptake in the brain over a 90 and 45 min scan times respectively to merit further investigations in non-human primates. PMID:22425346

Development of new PTK7-targeting aptamer-fluorescent and -radiolabelled probes for evaluation as molecular imaging agents: Lymphoma and melanoma in vivo proof of concept.

PubMed

Calzada, Victoria; Moreno, María; Newton, Jessica; González, Joel; Fernández, Marcelo; Gambini, Juan Pablo; Ibarra, Manuel; Chabalgoity, Alejandro; Deutscher, Susan; Quinn, Thomas; Cabral, Pablo; Cerecetto, Hugo

2017-02-01

Aptamers are single-stranded oligonucleotides that recognize molecular targets with high affinity and specificity. Aptamer that selectively bind to the protein tyrosine kinase-7 (PTK7) receptor, overexpressed on many cancers, has been labelled as probes for molecular imaging of cancer. Two new PTK7-targeting aptamer probes were developed by coupling frameworks from the fluorescent dye AlexaFluor647 or the 6-hydrazinonicotinamide (HYNIC) chelator-labelled to 99m Tc. The derivatizations via a 5'-aminohexyl terminal linker were done at room temperature and under mild buffer conditions. Physicochemical and biological controls for both imaging agents were performed verifying the integrity of the aptamer-conjugates by HPLC. Recognition of melanoma (B16F1) and lymphoma (A20) mouse cell lines by the aptamer was studied using cell binding, flow cytometry and confocal microscopy. Finally, in vivo imaging studies in tumour-bearing mice were performed. The new probes were able to bind to melanoma and lymphoma cell lines in vitro, the in vivo imaging in tumour-bearing mice showed different uptake behaviours showing for the fluorescent conjugate good uptake by B cell lymphoma while the radiolabelled conjugate did not display tumour uptake due to its high extravascular distribution, and both showed rapid clearance properties in tumour-bearing mice. Copyright © 2016 Elsevier Ltd. All rights reserved.

Purification of organic acids by chromatography with strong anionic resins: Investigation of uptake mechanisms.

PubMed

Lemaire, Julien; Blanc, Claire-Line; Lutin, Florence; Théoleyre, Marc-André; Stambouli, Moncef; Pareau, Dominique

2016-08-05

Bio-based organic acids are promising renewable carbon sources for the chemical industry. However energy-consuming purification processes are used, like distillation or crystallization, to reach high purities required in some applications. That is why preparative chromatography was studied as an alternative separation technique. In a previous work dealing with the purification of lactic, succinic and citric acids, the Langmuir model was insufficient to explain the elution profiles obtained with a strong anionic resin. Consequently the Langmuir model was coupled with a usual ion-exchange model to take into account the retention of their conjugate bases (<2%), which are commonly neglected at low pH (<1.5). Elution simulations with both uptake mechanisms fitted very well with experimental pulse tests. Only two parameters were optimized (equilibrium constant of acid uptake and ion-exchange selectivity coefficient of conjugate base) and their value were coherent with experimental and resin suppliers' data. These results confirmed that the singular tailing and apparent delay observed with succinic and citric acids can be explained by the high affinity of succinate and citrate for resin cationic sites. The model was implemented in a preparative chromatography simulation program in order to optimize operating parameters of our pilot-scale ISMB unit (Improved Simulated Moving Bed). The comparison with experimental ISMB profiles was conclusive. Copyright © 2016 Elsevier B.V. All rights reserved.

Chalcone Based Homodimeric PET Agent, 11C-(Chal)2DEA-Me, for Beta Amyloid Imaging: Synthesis and Bioevaluation.

PubMed

Chauhan, Kanchan; Tiwari, Anjani K; Chadha, Nidhi; Kaul, Ankur; Singh, Ajai Kumar; Datta, Anupama

2018-04-02

Homodimeric chalcone based 11 C-PET radiotracer, 11 C-(Chal) 2 DEA-Me, was synthesized, and binding affinity toward beta amyloid (Aβ) was evaluated. The computational studies revealed multiple binding of the tracer at the recognition sites of Aβ fibrils. The bivalent ligand 11 C-(Chal) 2 DEA-Me displayed higher binding affinity compared to the corresponding monomer, 11 C-Chal-Me, and classical Aβ agents. The radiolabeling yield with carbon-11 was 40-55% (decay corrected) with specific activity of 65-90 GBq/μmol. A significant ( p < 0.0001) improvement in the binding affinity of 11 C-(Chal) 2 DEA-Me with synthetic Aβ42 aggregates over the monomer, 11 C-Chal-Me, demonstrates the utility of the bivalent approach. The PET imaging and biodistribution data displayed suitable brain pharmacokinetics of both ligands with higher brain uptake in the case of the bivalent ligand. Metabolite analysis of healthy ddY mouse brain homogenates exhibited high stability of the radiotracers in the brain with >93% intact tracer at 30 min post injection. Both chalcone derivatives were fluorescent in nature and demonstrated significant changes in the emission properties after binding with Aβ42. The preliminary analysis indicates high potential of 11 C-(Chal) 2 DEA-Me as in vivo Aβ42 imaging tracer and highlights the significance of the bivalent approach to achieve a higher biological response for detection of early stages of amyloidosis.

Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3*

PubMed Central

Roy, Adhiraj; Kim, Jeong-Ho

2014-01-01

Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations. PMID:24451370

(18)F-FBPA as a tumor specific tracer of L-type amino acid transporter 1 (LAT1): PET evaluation in tumor and inflammation compared to (18)F-FDG and (11)C-methionine.

PubMed

Watabe, Tadashi; Hatazawa, Jun

2015-01-01

(18)F-FDG-PET is used worldwide for oncology patients. However, we sometimes encounter false positive cases of (18)F-FDG PET, such as moderate uptake in the inflammatory lesion, because (18)F-FDG accumulates not only in the cancer cells but also in the inflammatory cells (macrophage, granulation tissue, etc). To overcome this limitation of (18)F-FDG, we started to use (4-borono-2- [(18)F]fluoro-L-phenylalanine) (18)F-FBPA, an artificial amino acid tracer which is focusing attention as a tumor specific PET tracer. Physiological accumulation of (18)F-FBPA is limited in the kidney and urinary tract in humans, which enable preferable evaluation of uptake in the abdominal organs compared to (11)C-methionine ((11)C-MET). The purpose of this study was to evaluate (18)F-FBPA as a tumor specific tracer by in vitro cellular uptake analysis focusing on the selectivity of L-type amino acid transporter 1 (LAT1), which is specifically expressed in tumor cells, and in vivo PET analysis in rat xenograft and inflammation models compared to (18)F-FDG and (11)C-methionine. Uptake inhibition and efflux experiments were performed in HEK293-LAT1 and LAT2 cells using cold BPA, cold (18)F-FBPA, and hot (18)F-FBPA to evaluate LAT affinity and transport capacity. Position emission tomography studies were performed in rat xenograft model of C6 glioma 2 weeks after the implantation (n=9, body weight=197±10.5g) and subcutaneous inflammation model 4 days after the injection of turpentine oil (n=9, body weight=197±14.4g). Uptake on static PET images were compared among (18)F-FBPA at 60-70min post injection, (18)F-FDG at 60-70min, and (11)C-MET at 20-30min in the tumors and the inflammatory lesions by maximum standardized uptake value (SUVmax). Cellular uptake analysis showed no significant difference in inhibitory effect and efflux of LAT1 between cold (18)F-FBPA and cold BPA, suggesting the same affinity and transport capacity via LAT1. Uptake of (18)F-FBPA via LAT1 was superior to LAT2 by the concentration dependent uptake analysis. Position emission tomography analysis using SUVmax showed significantly higher accumulation of (18)F-FDG in the tumor and the inflammatory lesions (7.19±2.11 and 4.66±0.63, respectively) compared to (18)F-FBPA (3.23±0.40 and 1.86±0.19, respectively) and (11)C-MET (3.39±0.43 and 1.63±0.11, respectively) (P<0.01 by Tukey test). No significant difference was observed between (18)F-FBPA and (11)C-MET. (18)F-FBPA showed high selectivity of LAT1 by in vitro cellular uptake analysis, suggesting the potential as a tumor-specific substrate. In vivo PET analysis showed significantly lower uptake of (18)F-FBPA and (11)C-MET in the inflammatory lesions compared to (18)F-FDG, suggesting comparable utility of (18)F-FBPA PET to (11)C-MET PET in differentiating between the tumor and the inflammation.

Tunable ultrasmall visible-to-extended near-infrared emitting silver sulfide quantum dots for integrin-targeted cancer imaging.

PubMed

Tang, Rui; Xue, Jianpeng; Xu, Baogang; Shen, Duanwen; Sudlow, Gail P; Achilefu, Samuel

2015-01-27

The large size of many near-infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. In this study, we developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from 500 to 1200 nm and a QD core diameter between 1.5 and 9 nm. Conjugation of a tumor-avid cyclic pentapeptide (Arg-Gly-Asp-DPhe-Lys) resulted in monodisperse, water-soluble QDs (hydrodynamic diameter < 10 nm) without loss of the peptide's high binding affinity to tumor-associated integrins (KI = 1.8 nM/peptide). Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and noncytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

Ultrasmall visible-to-near-infrared emitting silver-sulfide quantum dots for cancer detection and imaging

NASA Astrophysics Data System (ADS)

Tang, Rui; Xu, Baogang; Shen, Duanwen; Sudlow, Gail; Achilefu, Samuel

2018-02-01

The large size of many near infrared (NIR) fluorescent nanoparticles prevents rapid extravasation from blood vessels and subsequent diffusion to tumors. This confines in vivo uptake to the peritumoral space and results in high liver retention. We developed a viscosity modulated approach to synthesize ultrasmall silver sulfide quantum dots (QDs) with distinct tunable light emission from visible to near-infrared in spectrum and a QD core diameter between less than 5 nm. Further functionalization of these Ag2S QDs with different type of molecules such as targeting peptides, retains monodisperse, relatively small water soluble QDs without loss of the functionality of the peptide's high binding affinity to cancerous tumor. Fluorescence and electron microscopy showed that selective integrin-mediated internalization was observed only in cancer cells treated with the peptide-labeled QDs, demonstrating that the unlabeled hydrophilic nanoparticles exhibit characteristics of negatively charged fluorescent dye molecules, which typically do not internalize in cells. The biodistribution profiles of intravenously administered QDs in different mouse models of cancer reveal an exceptionally high tumor-to-liver uptake ratio, suggesting that the small sized QDs evaded conventional opsonization and subsequent high uptake in the liver and spleen. The seamless tunability of the QDs over a wide spectral range with only a small increase in size, as well as the ease of labeling the bright and non-cytotoxic QDs with biomolecules, provides a platform for multiplexing information, tracking the trafficking of single molecules in cells, and selectively targeting disease biomarkers in living organisms without premature QD opsonization in circulating blood.

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

PubMed

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

2014-04-01

Positron emission tomography (PET) and PET/computed tomography (PET-CT) studies with (11)C- or (18)F-labeled choline derivatives are used for PET imaging in glioblastoma patients. However, the nature of the choline transport system in glioblastoma is poorly understood. In this study, we performed a functional characterization of [methyl-(3)H]choline uptake and sought to identify the transporters that mediate choline uptake in the human glioblastoma cell lines A-172 and U-251MG. In addition, we examined the influence of anti-cancer drugs and central nervous system drugs on the transport of [methyl-(3)H]choline. High- and low-affinity choline transport systems were present in A-172 cells, U-251MG cells and astrocytes, and these were Na(+)-independent and pH-dependent. Cell viability in A-172 cells was not affected by choline deficiency. However, cell viability in U-251MG cells was significantly inhibited by choline deficiency. Both A-172 and U-251MG cells have two different choline transporters, choline transporter-like protein 1 (CTL1) and CTL2. In A-172 cells, CTL1 is predominantly expressed, whereas in U-251MG cells, CTL2 is predominantly expressed. Treatment with anti-cancer drugs such as cisplatin, etoposide and vincristine influenced [methyl-(3)H]choline uptake in U-251MG cells, but not A-172 cells. Central nervous system drugs such as imipramine, fluvoxamine, paroxetine, reboxetine, citalopram and donepezil did not affect cell viability or [methyl-(3)H]choline uptake. The data presented here suggest that CTL1 and CTL2 are functionally expressed in A-172 and U-251MG cells and are responsible for [methyl-(3)H]choline uptake that relies on a directed H(+) gradient as a driving force. Furthermore, while anti-cancer drugs altered [methyl-(3)H]choline uptake, central nervous system drugs did not affect [methyl-(3)H]choline uptake. Copyright © 2014 Elsevier Inc. All rights reserved.

Drug composition matters: the influence of carrier concentration on the radiochemical purity, hydroxyapatite affinity and in-vivo bone accumulation of the therapeutic radiopharmaceutical 188Rhenium-HEDP.

PubMed

Lange, R; de Klerk, J M H; Bloemendal, H J; Ramakers, R M; Beekman, F J; van der Westerlaken, M M L; Hendrikse, N H; Ter Heine, R

2015-05-01

(188)Rhenium-HEDP is an effective bone-targeting therapeutic radiopharmaceutical, for treatment of osteoblastic bone metastases. It is known that the presence of carrier (non-radioactive rhenium as ammonium perrhenate) in the reaction mixture during labeling is a prerequisite for adequate bone affinity, but little is known about the optimal carrier concentration. We investigated the influence of carrier concentration in the formulation on the radiochemical purity, in-vitro hydroxyapatite affinity and the in-vivo bone accumulation of (188)Rhenium-HEDP in mice. The carrier concentration influenced hydroxyapatite binding in-vitro as well as bone accumulation in-vivo. Variation in hydroxyapatite binding with various carrier concentrations seemed to be mainly driven by variation in radiochemical purity. The in-vivo bone accumulation appeared to be more complex: satisfactory radiochemical purity and hydroxyapatite affinity did not necessarily predict acceptable bio-distribution of (188)Rhenium-HEDP. For development of new bisphosphonate-based radiopharmaceuticals for clinical use, human administration should not be performed without previous animal bio-distribution experiments. Furthermore, our clinical formulation of (188)Rhenium-HEDP, containing 10 μmol carrier, showed excellent bone accumulation that was comparable to other bisphosphonate-based radiopharmaceuticals, with no apparent uptake in other organs. Radiochemical purity and in-vitro hydroxyapatite binding are not necessarily predictive of bone accumulation of (188)Rhenium-HEDP in-vivo. The formulation for (188)Rhenium-HEDP as developed by us for clinical use exhibits excellent bone uptake and variation in carrier concentration during preparation of this radiopharmaceutical should be avoided. Copyright © 2015 Elsevier Inc. All rights reserved.

Oligolysine-based saccharide clusters: synthesis and specificity.

PubMed Central

Frison, Natacha; Marceau, Philippe; Roche, Annie-Claude; Monsigny, Michel; Mayer, Roger

2002-01-01

In search of specific and highly selective sugar clusters for cell receptors, such as membrane lectins, various disaccharides were coupled to small peptide cores through an amide bond. In a first step, the reducing disaccharides, i.e. lactose and three different dimannoses, were converted into glycosyl-pyroglutamyl-beta-alanine derivatives. The free carboxylic group of these conjugates was then coupled to the alpha and epsilon amino groups of the core peptide (Lys( n )-Ala-Cys-NH2) with n =1 to 5, with complete substitution leading to homogeneous glycoclusters. The thiol group of the cysteine residue was used to tag the glycosylated oligolysines upon reaction with fluorescein iodoacetamide. The affinity of these glycoclusters towards two plant lectins was assessed by surface plasmon resonance. The selectivity of their cell uptake was investigated by flow cytometry using two types of cells: a human hepatoma cell line (HepG2 cells) expressing the plasma membrane galactose-specific lectin, and monocyte-derived dendritic cells expressing the plasma membrane mannose-specific lectin. The glycoclusters containing four or five disaccharides were shown to bind plant lectins and cell surface membrane lectins with a narrow selectivity and with a high affinity. PMID:12119048

Identification and expression analyses of two genes encoding putative low-affinity nitrate transporters from Nicotiana plumbaginifolia.

PubMed

Fraisier, V; Dorbe, M F; Daniel-Vedele, F

2001-01-01

Higher plants have both high- and low-affinity nitrate uptake systems (HATS and LATS respectively). Here we report the isolation and characterization of two genes, NpNRT1.1 and NpNRT1.2, from Nicotiana plumbaginifolia whose structural features suggest that they both belong to the NRT1 gene family, which is involved in the LATS. Amino acid sequence alignment showed that the N. plumbaginifolia proteins have greater similarity to their corresponding tomato homologues than to each other. Genomic Southern blot analysis indicates that there are probably more than two members of this family in N. plumbaginifolia. Northern blot analysis shows that NpNRT1.2 expression is restricted strictly to roots, whereas NpNRT1.1, in addition to roots, is expressed at a basal level in all other plant organs. Likewise, differential expression in response to external treatments with various N sources was observed for these two genes: NpNRT1.1 can be considered as a constitutively expressed gene whereas NpNRT1.2 expression is dependent strictly on high nitrate concentrations. Finally, over-expression of a gene involved in the HATS does not lead to any modification of LATS gene expression.

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

PubMed Central

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

2015-01-01

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

The dopamine precursor L-dihydroxyphenylalanine is transported by the amino acid transporters rBAT and LAT2 in renal cortex.

PubMed

Quiñones, Henry; Collazo, Roberto; Moe, Orson W

2004-07-01

The intrarenal autocrine-paracrine dopamine (DA) system is critical for Na(+) homeostasis. l-Dihydroxyphenylalanine (l-DOPA) uptake from the glomerular filtrate and plasma provides the substrate for DA generation by the renal proximal tubule. The transporter(s) responsible for proximal tubule l-DOPA uptake has not been characterized. Renal cortical poly-A(+) RNA injected into Xenopus laevis oocytes induced l-DOPA uptake in a time- and dose-dependent fashion with biphasic K(m)s in the millimolar and micromolar range and independent of inward Na(+), K(+), or H(+) gradients, suggesting the presence of low- and high-affinity l-DOPA carriers. Complementary RNA from two amino acid transporters yielded l-DOPA uptake significantly above water-injected controls the rBAT/b(0,+)AT dimer (rBAT) and the LAT2/4F2 dimer (LAT2). In contradistinction to renal cortical poly-A(+), l-DOPA kinetics of rBAT and LAT2 showed classic Michaelis-Menton kinetics with K(m)s in the micromolar and millimolar range, respectively. Sequence-specific antisense oligonucleotides to rBAT or LAT2 (AS) caused inhibition of rBAT and LAT2 cRNA-induced l-DOPA transport and cortical poly-A(+)-induced arginine and phenylalanine transport. However, the same ASs only partially blocked poly-A(+)-induced l-DOPA transport. In cultured kidney cells, silencing inhibitory RNA (siRNA) to rBAT significantly inhibited l-DOPA uptake. We conclude that rBAT and LAT2 can mediate apical and basolateral l-DOPA uptake into the proximal tubule, respectively. Additional l-DOPA transport mechanisms exist in the renal cortex that remain to be identified.

Ascorbic Acid Efflux from Human Brain Microvascular Pericytes: Role of Re-uptake

PubMed Central

May, James M.; Qu, Zhi-chao

2015-01-01

Microvascular pericytes take up ascorbic acid on the ascorbate transporter SVCT2. Intracellular ascorbate then protects the cells against apoptosis induced by culture at diabetic glucose concentrations. To investigate whether pericytes might also provide ascorbate to the underlying endothelial cells, we studied ascorbate efflux from human pericytes. When loaded with ascorbate to intracellular concentrations of 0.8–1.0 mM, almost two-thirds of intracellular ascorbate effluxed from the cells over 2 h. This efflux was opposed by ascorbate re-uptake from the medium, since preventing re-uptake by destroying extracellular ascorbate with ascorbate oxidase increased ascorbate loss even further. Ascorbate re-uptake occurred on the SVCT2, since its blockade by replacing medium sodium with choline, by the SVCT2 inhibitor sulfinpyrazone, or by extracellular ascorbate accelerated ascorbate loss from the cells. This was supported by finding that net efflux of radiolabeled ascorbate was increased by unlabeled extracellular ascorbate with a half-maximal effect in the range of the high affinity Km of the SVCT2. Intracellular ascorbate did not inhibit its efflux. To assess the mechanism of ascorbate efflux, known inhibitors of volume-regulated anion channels (VRACs) were tested. These potently inhibited ascorbate transport into cells on the SVCT2, but not its efflux. An exception was the anion transport inhibitor DIDS, which, despite inhibition of ascorbate uptake, also inhibited net efflux at 25–50 µM. These results suggest that ascorbate efflux from vascular pericytes occurs on a DIDS-inhibitable transporter or channel different from VRACs. Further, ascorbate efflux is opposed by re-uptake of ascorbate on the SVCT2, providing a potential regulatory mechanism. PMID:26340060

Evidence for a specific uptake and retention mechanism for 25-hydroxyvitamin D (25OHD) in skeletal muscle cells.

PubMed

Abboud, M; Puglisi, D A; Davies, B N; Rybchyn, M; Whitehead, N P; Brock, K E; Cole, L; Gordon-Thomson, C; Fraser, D R; Mason, R S

2013-09-01

Little is known about the mechanism for the prolonged residence time of 25-hydroxyvitamin D (25OHD) in blood. Several lines of evidence led us to propose that skeletal muscle could function as the site of an extravascular pool of 25OHD. In vitro studies investigated the capacity of differentiated C2 murine muscle cells to take up and release 25OHD, in comparison with other cell types and the involvement of the membrane protein megalin in these mechanisms. When C2 cells are differentiated into myotubes, the time-dependent uptake of labeled 25OHD is 2-3 times higher than in undifferentiated myoblasts or nonmuscle osteoblastic MG63 cells (P < .001). During in vitro release experiments (after 25OHD uptake), myotubes released only 32% ± 6% stored 25OHD after 4 hours, whereas this figure was 60% ± 2% for osteoblasts (P < .01). Using immunofluorescence, C2 myotubes and primary rat muscle fibers were, for the first time, shown to express megalin and cubilin, endocytotic receptors for the vitamin D binding protein (DBP), which binds nearly all 25OHD in the blood. DBP has a high affinity for actin in skeletal muscle. A time-dependent uptake of Alexafluor-488-labeled DBP into mature muscle cells was observed by confocal microscopy. Incubation of C2 myotubes (for 24 hours) with receptor-associated protein, a megalin inhibitor, led to a 40% decrease in 25OHD uptake (P < .01). These data support the proposal that 25OHD, after uptake into mature muscle cells, is held there by DBP, which has been internalized via membrane megalin and is retained by binding to actin.

Recent Advances in Chemical Modification of Peptide Nucleic Acids

PubMed Central

Rozners, Eriks

2012-01-01

Peptide nucleic acid (PNA) has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification. PMID:22991652

High Temperature Stable Separator for Lithium Batteries Based on SiO2 and Hydroxypropyl Guar Gum

PubMed Central

Carvalho, Diogo Vieira; Loeffler, Nicholas; Kim, Guk-Tae; Passerini, Stefano

2015-01-01

A novel membrane based on silicon dioxide (SiO2) and hydroxypropyl guar gum (HPG) as binder is presented and tested as a separator for lithium-ion batteries. The separator is made with renewable and low cost materials and an environmentally friendly manufacturing processing using only water as solvent. The separator offers superior wettability and high electrolyte uptake due to the optimized porosity and the good affinity of SiO2 and guar gum microstructure towards organic liquid electrolytes. Additionally, the separator shows high thermal stability and no dimensional-shrinkage at high temperatures due to the use of the ceramic filler and the thermally stable natural polymer. The electrochemical tests show the good electrochemical stability of the separator in a wide range of potential, as well as its outstanding cycle performance. PMID:26512701

Structure of the Escherichia coli phosphonate binding protein PhnD and rationally optimized phosphonate biosensors.

PubMed

Alicea, Ismael; Marvin, Jonathan S; Miklos, Aleksandr E; Ellington, Andrew D; Looger, Loren L; Schreiter, Eric R

2011-12-02

The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate (Pn) uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by ~70° between the two states. Extensive hydrogen bonding and electrostatic interactions stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into Pn uptake by bacteria and facilitated the rational design of high signal-to-noise Pn biosensors based on both coupled small-molecule dyes and autocatalytic fluorescent proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cell-penetrating peptides and antimicrobial peptides: how different are they?

PubMed Central

Henriques, Sónia Troeira; Melo, Manuel Nuno; Castanho, Miguel A. R. B.

2006-01-01

Some cationic peptides, referred to as CPPs (cell-penetrating peptides), have the ability to translocate across biological membranes in a non-disruptive way and to overcome the impermeable nature of the cell membrane. They have been successfully used for drug delivery into mammalian cells; however, there is no consensus about the mechanism of cellular uptake. Both endocytic and non-endocytic pathways are supported by experimental evidence. The observation that some AMPs (antimicrobial peptides) can enter host cells without damaging their cytoplasmic membrane, as well as kill pathogenic agents, has also attracted attention. The capacity to translocate across the cell membrane has been reported for some of these AMPs. Like CPPs, AMPs are short and cationic sequences with a high affinity for membranes. Similarities between CPPs and AMPs prompted us to question if these two classes of peptides really belong to unrelated families. In this Review, a critical comparison of the mechanisms that underlie cellular uptake is undertaken. A reflection and a new perspective about CPPs and AMPs are presented. PMID:16956326

Fungal Zinc Homeostasis - A Tug of War Between the Pathogen and Host.

PubMed

Walencik, Paulina K; Watly, Joanna; Rowinska-Zyrek, Magdalena

2016-01-01

In the last decade, drug resistant invasive mycoses have become significantly more common and new antifungal drugs and ways to specifically deliver them to the fungal cell are being looked for. One of the biggest obstacles in finding such comes from the fact that fungi share essential metabolic pathways with humans. One significant difference in the metabolism of those two cells that can be challenged when looking for possible selective therapeutics is the uptake of zinc, a nutrient crucial for the fungal survival and virulence. This work summarizes the recent advances in the biological inorganic chemistry of zinc metabolism in fungi. The regulation of zinc uptake, various types of its transmembrane transport, storage and the maintenance of intracellular zinc homeostasis is discussed in detail, with a special focus on the concept of a constant 'tug of war' over zinc between the fungus and its host, with the host trying to withhold essential Zn(II), and the fungus counteracting by producing high-affinity zinc binding molecules.

Structure of the Escherichia coli Phosphonate Binding Protein PhnD and Rationally Optimized Phosphonate Biosensors

PubMed Central

Alicea, Ismael; Marvin, Jonathan S.; Miklos, Aleksandr E.; Ellington, Andrew D.; Looger, Loren L.; Schreiter, Eric R.

2012-01-01

The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by ~70° between the two states. Extensive hydrogen bonding and electrostatic interactions stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into phosphonate uptake by bacteria and facilitated the rational design of high signal-to-noise phosphonate biosensors based both on coupled small molecule dyes and autocatalytic fluorescent proteins. PMID:22019591

Calcium release microdomains and mitochondria.

PubMed

Kohlhaas, Michael; Maack, Christoph

2013-05-01

The processes of excitation-contraction (EC) coupling consume large amounts of energy that need to be replenished by oxidative phosphorylation in the mitochondria. Since Ca(2+) activates key enzymes of the Krebs cycle in the mitochondrial matrix, it is important to understand the mechanisms and kinetics of mitochondrial Ca(2+) uptake to delineate how in cardiac myocytes, energy supply is efficiently matched to demand. In recent years, the identification of various proteins involved in mitochondrial Ca(2+) signalling and the tethering of mitochondria to the sarcoplasmic reticulum (SR) has considerably advanced the field and supported the concept of a mitochondrial Ca(2+) microdomain, in which Ca(2+) concentrations are high enough to overcome the low Ca(2+) affinity of the principal mitochondrial Ca(2+) uptake mechanism, the Ca(2+) uniporter. Furthermore, defects in EC coupling that occur in heart failure disrupt SR-mitochondrial Ca(2+) crosstalk and may cause energetic deficit and oxidative stress, both factors that are thought to be causally involved in the initiation and progression of the disease.

Structure of the Escherichia coli Phosphonate Binding Protein PhnD and Rationally Optimized Phosphonate Biosensors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alicea, Ismael; Marvin, Jonathan S.; Miklos, Aleksandr E.

2012-09-17

The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate (Pn) uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by {approx}70{sup o} between the two states. Extensive hydrogen bonding and electrostatic interactionsmore » stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into Pn uptake by bacteria and facilitated the rational design of high signal-to-noise Pn biosensors based on both coupled small-molecule dyes and autocatalytic fluorescent proteins.« less

Targeting of follicle stimulating hormone peptide-conjugated dendrimers to ovarian cancer cells

NASA Astrophysics Data System (ADS)

Modi, Dimple A.; Sunoqrot, Suhair; Bugno, Jason; Lantvit, Daniel D.; Hong, Seungpyo; Burdette, Joanna E.

2014-02-01

Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles.Ovarian cancer is the most lethal gynecological malignancy. Current treatment modalities include a combination of surgery and chemotherapy, which often lead to loss of fertility in premenopausal women and a myriad of systemic side effects. To address these issues, we have designed poly(amidoamine) (PAMAM) dendrimers to selectively target the follicle stimulating hormone receptor (FSHR), which is overexpressed by tumorigenic ovarian cancer cells but not by immature primordial follicles and other non-tumorigenic cells. Fluorescein-labeled generation 5 (G5) PAMAM dendrimers were conjugated with the binding peptide domain of FSH (FSH33) that has a high affinity to FSHR. The targeted dendrimers exhibited high receptor selectivity to FSHR-expressing OVCAR-3 cells, resulting in significant uptake and downregulation of an anti-apoptotic protein survivin, while showing minimal interactions with SKOV-3 cells that do not express FSHR. The selectivity of the FSH33-targeted dendrimers was further validated in 3D organ cultures of normal mouse ovaries. Immunostaining of the conjugates revealed their selective binding and uptake by ovarian surface epithelium (OSE) cells that express FSHR, while sparing the immature primordial follicles. In addition, an in vivo study monitoring tissue accumulation following a single intraperitoneal (i.p.) injection of the conjugates showed significantly higher accumulation of FSH33-targeted dendrimers in the ovary and oviduct compared to the non-targeted conjugates. These proof-of-concept findings highlight the potential of these FSH33-targeted dendrimers to serve as a delivery platform for anti-ovarian cancer drugs, while reducing their systemic side effects by preventing nonspecific uptake by the primordial follicles. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr05042d

Nanocellulose-Zeolite Composite Films for Odor Elimination.

PubMed

Keshavarzi, Neda; Mashayekhy Rad, Farshid; Mace, Amber; Ansari, Farhan; Akhtar, Farid; Nilsson, Ulrika; Berglund, Lars; Bergström, Lennart

2015-07-08

Free standing and strong odor-removing composite films of cellulose nanofibrils (CNF) with a high content of nanoporous zeolite adsorbents have been colloidally processed. Thermogravimetric desorption analysis (TGA) and infrared spectroscopy combined with computational simulations showed that commercially available silicalite-1 and ZSM-5 have a high affinity and uptake of volatile odors like ethanethiol and propanethiol, also in the presence of water. The simulations showed that propanethiol has a higher affinity, up to 16%, to the two zeolites compared with ethanethiol. Highly flexible and strong free-standing zeolite-CNF films with an adsorbent loading of 89 w/w% have been produced by Ca-induced gelation and vacuum filtration. The CNF-network controls the strength of the composite films and 100 μm thick zeolite-CNF films with a CNF content of less than 10 vol % displayed a tensile strength approaching 10 MPa. Headspace solid phase microextraction (SPME) coupled to gas chromatography-mass spectroscopy (GC/MS) analysis showed that the CNF-zeolite films can eliminate the volatile thiol-based odors to concentrations below the detection ability of the human olfactory system. Odor removing zeolite-cellulose nanofibril films could enable improved transport and storage of fruits and vegetables rich in odors, for example, onion and the tasty but foul-smelling South-East Asian Durian fruit.

Identification and Characterization of ML352: A Novel, Noncompetitive Inhibitor of the Presynaptic Choline Transporter

PubMed Central

2015-01-01

The high-affinity choline transporter (CHT) is the rate-limiting determinant of acetylcholine (ACh) synthesis, yet the transporter remains a largely undeveloped target for the detection and manipulation of synaptic cholinergic signaling. To expand CHT pharmacology, we pursued a high-throughput screen for novel CHT-targeted small molecules based on the electrogenic properties of transporter-mediated choline transport. In this effort, we identified five novel, structural classes of CHT-specific inhibitors. Chemical diversification and functional analysis of one of these classes identified ML352 as a high-affinity (Ki = 92 nM) and selective CHT inhibitor. At concentrations that fully antagonized CHT in transfected cells and nerve terminal preparations, ML352 exhibited no inhibition of acetylcholinesterase (AChE) or cholineacetyltransferase (ChAT) and also lacked activity at dopamine, serotonin, and norepinephrine transporters, as well as many receptors and ion channels. ML352 exhibited noncompetitive choline uptake inhibition in intact cells and synaptosomes and reduced the apparent density of hemicholinium-3 (HC-3) binding sites in membrane assays, suggesting allosteric transporter interactions. Pharmacokinetic studies revealed limited in vitro metabolism and significant CNS penetration, with features predicting rapid clearance. ML352 represents a novel, potent, and specific tool for the manipulation of CHT, providing a possible platform for the development of cholinergic imaging and therapeutic agents. PMID:25560927

Multimodal Molecular Imaging Reveals High Target Uptake and Specificity of 111In- and 68Ga-Labeled Fibrin-Binding Probes for Thrombus Detection in Rats.

PubMed

Oliveira, Bruno L; Blasi, Francesco; Rietz, Tyson A; Rotile, Nicholas J; Day, Helen; Caravan, Peter

2015-10-01

We recently showed the high target specificity and favorable imaging properties of 64Cu and Al18F PET probes for noninvasive imaging of thrombosis. Here, our aim was to evaluate new derivatives labeled with either with 68Ga, 111In, or 99mTc as thrombus imaging agents for PET and SPECT. In this study, the feasibility and potential of these probes for thrombus imaging was assessed in detail in 2 animal models of arterial thrombosis. The specificity of the probes was further evaluated using a triple-isotope approach with multimodal SPECT/PET/CT imaging. Radiotracers were synthesized using a known fibrin-binding peptide conjugated to 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid monoamide (DOTA-MA), or a diethylenetriamine ligand (DETA-propanoic acid [PA]), followed by labeling with 68Ga (FBP14, 68Ga-NODAGA), 111In (FBP15, 111In-DOTA-MA), or 99mTc (FBP16, 99mTc(CO)3-DETA-PA), respectively. PET or SPECT imaging, biodistribution, pharmacokinetics, and metabolic stability were evaluated in rat models of mural and occlusive carotid artery thrombosis. In vivo target specificity was evaluated by comparing the distribution of the SPECT and PET probes with preformed 125I-labeled thrombi and with a nonbinding control probe using SPECT/PET/CT imaging. All 3 radiotracers showed affinity similar to soluble fibrin fragment DD(E) (inhibition constant=0.53-0.83 μM). After the kidneys, the highest uptake of 68Ga-FBP14 and 111In-FBP15 was in the thrombus (1.0±0.2 percentage injected dose per gram), with low off-target accumulation. Both radiotracers underwent fast systemic elimination (half-life, 8-15 min) through the kidneys, which led to highly conspicuous thrombi on PET and SPECT images. 99mTc-FBP16 displayed low target uptake and distribution consistent with aggregation or degradation. Triple-isotope imaging experiments showed that both 68Ga-FBP14 and 111In-FBP15, but not the nonbinding derivative 64Cu-D-Cys-FBP8, detected the location of the 125I-labeled thrombus, confirming high target specificity. 68Ga-FBP14 and 111In-FBP15 have high fibrin affinity and thrombus specificity and represent useful PET and SPECT probes for thrombus detection. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes.

PubMed

Storey, Stephen M; McIntosh, Avery L; Huang, Huan; Landrock, Kerstin K; Martin, Gregory G; Landrock, Danilo; Payne, H Ross; Atshaves, Barbara P; Kier, Ann B; Schroeder, Friedhelm

2012-04-15

A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in <1 min and initial rate and maximal uptake of HDL free cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null mice and hepatocytes. Taken together, these results suggest that L-FABP, particularly in the absence of SCP-2, plays a significant role in HDL-mediated cholesterol uptake in cultured primary hepatocytes.

Influence of DOTA chelator position on biodistribution and targeting properties of (111)In-labeled synthetic anti-HER2 affibody molecules.

PubMed

Perols, Anna; Honarvar, Hadis; Strand, Joanna; Selvaraju, Ramkumar; Orlova, Anna; Karlström, Amelie Eriksson; Tolmachev, Vladimir

2012-08-15

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with (99m)Tc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z(HER2:S1) at three different positions: DOTA-A1-Z(HER2:S1) (N-terminus), DOTA-K58-Z(HER2:S1) (C-terminus), and DOTA-K50-Z(HER2:S1) (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K(D) values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z(HER2:S1), DOTA-K50-Z(HER2:S1), and DOTA-K58-Z(HER2:S1), respectively. Z(HER2:S1)-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-Z(HER2:S1) (64 °C) and DOTA-K58-Z(HER2:S1) (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with (111)In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-Z(HER2:S1)and DOTA-K58-Z(HER2:S1) showed the highest uptake. Overall, DOTA-K58-Z(HER2:S1) provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.

Iron uptake and storage in the HAB dinoflagellate Lingulodinium polyedrum.

PubMed

Yarimizu, Kyoko; Cruz-López, Ricardo; Auerbach, Hendrik; Heimann, Larissa; Schünemann, Volker; Carrano, Carl J

2017-12-01

The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies being distinguished: Strategy I involves the induction of an Fe(III)-chelate reductase (ferrireductase) along with Fe(II) or Fe(III) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine, plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the harmful algal bloom dinoflagellate Lingulodinium polyedrum. L. polyedrum is an armored dinoflagellate with a mixotrophic lifestyle and one of the most common bloom species on Southern California coast widely noted for its bioluminescent properties and as a producer of yessotoxins. Short term radio-iron uptake studies indicate that iron is taken up by L. polyedrum in a time dependent manner consistent with an active transport process. Based on inhibitor and other studies it appears that a reductive-oxidative pathway such as that found in yeast and the green alga Chlamydomonas reinhardtii is likely. Of the various iron sources tested vibrioferrin, a photoactive and relatively weak siderophore produced by potentially mutualistic Marinobacter bacterial species, was the most efficient. Other more stable and non-photoactive siderophores such as ferrioxamine E were ineffective. Several pieces of data including long term exposure to 57 Fe using Mössbauer spectroscopy suggest that L. polyedrum does not possess an iron storage system but rather presumably relies on an efficient iron uptake system, perhaps mediated by mutualistic interactions with bacteria.

ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment.

PubMed

Shi, Sixiang; Hong, Hao; Orbay, Hakan; Graves, Stephen A; Yang, Yunan; Ohman, Jakob D; Liu, Bai; Nickles, Robert J; Wong, Hing C; Cai, Weibo

2015-07-01

To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and (64)Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of (64)Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of (64)Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. (64)Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management.

Preclinical Evaluation of 18F-JNJ64349311, a Novel PET Tracer for Tau Imaging.

PubMed

Declercq, Lieven; Rombouts, Frederik; Koole, Michel; Fierens, Katleen; Mariën, Jonas; Langlois, Xavier; Andrés, José Ignacio; Schmidt, Mark; Macdonald, Gregor; Moechars, Diederik; Vanduffel, Wim; Tousseyn, Thomas; Vandenberghe, Rik; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy

2017-06-01

In this study, we have synthesized and evaluated 18 F-JNJ64349311, a tracer with high affinity for aggregated tau (inhibition constant value, 8 nM) and high (≥500×) in vitro selectivity for tau over β-amyloid, in comparison with the benchmark compound 18 F-AV1451 ( 18 F-T807) in mice, rats, and a rhesus monkey. Methods: In vitro binding characteristics were determined for Alzheimer's disease, progressive supranuclear palsy, and corticobasal degeneration patient brain tissue slices using autoradiography studies. Ex vivo biodistribution studies were performed in mice. Radiometabolites were quantified in the brain and plasma of mice and in the plasma of a rhesus monkey using high-performance liquid chromatography. Dynamic small-animal PET studies were performed in rats and a rhesus monkey to evaluate tracer pharmacokinetics in the brain. Results: Mouse biodistribution studies showed moderate initial brain uptake and rapid brain washout. Radiometabolite analyses after injection of 18 F-JNJ64349311 in mice showed the presence of a polar radiometabolite in plasma, but not in the brain. Semiquantitative autoradiography studies on postmortem tissue sections of human Alzheimer's disease brains showed highly displaceable binding to tau-rich regions. No specific binding was, however, found on human progressive supranuclear palsy and corticobasal degeneration brain slices. Small-animal PET scans of Wistar rats revealed moderate initial brain uptake (SUV, ∼1.5 at 1 min after injection) and rapid brain washout. Gradual bone uptake was, however, also observed. Blocking and displacement did not affect brain time-activity curves, suggesting no off-target specific binding of the tracer in the healthy rat brain. A small-animal PET scan of a rhesus monkey revealed moderate initial brain uptake (SUV, 1.9 at 1 min after injection) with a rapid washout. In the monkey, no bone uptake was detected during the 120-min scan. Conclusion: This biologic evaluation suggests that 18 F-JNJ64349311 is a promising tau PET tracer candidate, with a favorable pharmacokinetic profile, as compared with 18 F-AV1451. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

The effect of monoclonal antibodies to the human transferrin receptor on transferrin and iron uptake by rat and rabbit reticulocytes.

PubMed

McArdle, H J; Morgan, E H

1984-02-10

The effect of monoclonal antibodies to the human transferrin receptor on transferrin and iron uptake by rat and rabbit reticulocytes has been examined. The antibodies used were as follows: T58/1.4, B3/25.4, 42/6.3, T56/14.3.1, and 43/31. The effects were the same, irrespective of the antibody. Transferrin and iron uptake were stimulated in both rat and rabbit reticulocytes. The stimulation was not due to an increase in the number or affinity of the receptors, but rather to an increase in the rate of turnover of the receptors. Electron microscopy suggested that the antibody acted by facilitating the formation of coated pits containing the transferrin-receptor complex.

Microphysical explanation of the RH-dependent water affinity of biogenic organic aerosol and its importance for climate

DOE PAGES

Rastak, N.; Pajunoja, A.; Acosta Navarro, J. C.; ...

2017-04-28

A large fraction of atmospheric organic aerosol (OA) originates from natural emissions that are oxidized in the atmosphere to form secondary organic aerosol (SOA). Isoprene (IP) and monoterpenes (MT) are the most important precursors of SOA originating from forests. The climate impacts from OA are currently estimated through parameterizations of water uptake that drastically simplify the complexity of OA. We combine laboratory experiments, thermodynamic modeling, field observations, and climate modeling to (1) explain the molecular mechanisms behind RH-dependent SOA water-uptake with solubility and phase separation; (2) show that laboratory data on IP- and MT-SOA hygroscopicity are representative of ambient datamore » with corresponding OA source profiles; and (3) demonstrate the sensitivity of the modeled aerosol climate effect to assumed OA water affinity. We conclude that the commonly used single-parameter hygroscopicity framework can introduce significant error when quantifying the climate effects of organic aerosol. The results highlight the need for better constraints on the overall global OA mass loadings and its molecular composition, including currently underexplored anthropogenic and marine OA sources.« less

Microphysical explanation of the RH-dependent water affinity of biogenic organic aerosol and its importance for climate

NASA Astrophysics Data System (ADS)

Rastak, N.; Pajunoja, A.; Acosta Navarro, J. C.; Ma, J.; Song, M.; Partridge, D. G.; Kirkevâg, A.; Leong, Y.; Hu, W. W.; Taylor, N. F.; Lambe, A.; Cerully, K.; Bougiatioti, A.; Liu, P.; Krejci, R.; Petäjä, T.; Percival, C.; Davidovits, P.; Worsnop, D. R.; Ekman, A. M. L.; Nenes, A.; Martin, S.; Jimenez, J. L.; Collins, D. R.; Topping, D. O.; Bertram, A. K.; Zuend, A.; Virtanen, A.; Riipinen, I.

2017-05-01

A large fraction of atmospheric organic aerosol (OA) originates from natural emissions that are oxidized in the atmosphere to form secondary organic aerosol (SOA). Isoprene (IP) and monoterpenes (MT) are the most important precursors of SOA originating from forests. The climate impacts from OA are currently estimated through parameterizations of water uptake that drastically simplify the complexity of OA. We combine laboratory experiments, thermodynamic modeling, field observations, and climate modeling to (1) explain the molecular mechanisms behind RH-dependent SOA water-uptake with solubility and phase separation; (2) show that laboratory data on IP- and MT-SOA hygroscopicity are representative of ambient data with corresponding OA source profiles; and (3) demonstrate the sensitivity of the modeled aerosol climate effect to assumed OA water affinity. We conclude that the commonly used single-parameter hygroscopicity framework can introduce significant error when quantifying the climate effects of organic aerosol. The results highlight the need for better constraints on the overall global OA mass loadings and its molecular composition, including currently underexplored anthropogenic and marine OA sources.

Characterization of the Physicochemical Properties of β-Cyclodextrin-Divinyl Sulfone Polymer Carrier-Bile Acid Systems.

PubMed

Mohamed, Mohamed H; Wang, Chen; Peru, Kerry M; Headley, John V; Wilson, Lee D

2017-08-07

Herein, we report on the systematic design and characterization of cross-linked polymer carriers containing β-cyclodextrin (β-CD) and divinyl sulfone (DVS). The polymer carriers were prepared at variable feed ratios (β-CD-DVS; 1:1, 1:2, 1:3, and 1:6) and characterized using spectroscopy (IR, 1 H solution NMR, and 13 C CP-MAS solids NMR spectroscopy), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and a dye decolorization method using phenolphthalein. Uptake studies were carried out at pH 9.00 for the polymer carriers using single component bile acids (cholic acid, deoxycholic acid, glycodeoxycholic acid, and taurodeoxycholic acid). Equilibrium uptake results were evaluated by the Langmuir isotherm model where variable equilibrium parameters were related to the relative apolar character of the bile acid. The Langmuir model yields a carrier/bile acid binding affinity of ∼10 3 M -1 where the lipophilic inclusion sites of the polymer play a prominent role, while the DVS linker framework sites have a lower adsorption affinity, in accordance with the greater hydrophilic character of such sites.

Kinetics and equilibrium of solute diffusion into human hair.

PubMed

Wang, Liming; Chen, Longjian; Han, Lujia; Lian, Guoping

2012-12-01

The uptake kinetics of five molecules by hair has been measured and the effects of pH and physical chemical properties of molecules were investigated. A theoretical model is proposed to analyze the experimental data. The results indicate that the binding affinity of solute to hair, as characterized by hair-water partition coefficient, scales to the hydrophobicity of the solute and decreases dramatically as the pH increases to the dissociation constant. The effective diffusion coefficient of solute depended not only on the molecular size as most previous studies suggested, but also on the binding affinity as well as solute dissociation. It appears that the uptake of molecules by hair is due to both hydrophobic interaction and ionic charge interaction. Based on theoretical considerations of the cellular structure, composition and physical chemical properties of hair, quantitative-structure-property-relationships (QSPR) have been proposed to predict the hair-water partition coefficient (PC) and the effective diffusion coefficient (D (e)) of solute. The proposed QSPR models fit well with the experimental data. This paper could be taken as a reference for investigating the adsorption properties for polymeric materials, fibres, and biomaterials.

Microphysical explanation of the RH-dependent water affinity of biogenic organic aerosol and its importance for climate.

PubMed

Rastak, N; Pajunoja, A; Acosta Navarro, J C; Ma, J; Song, M; Partridge, D G; Kirkevåg, A; Leong, Y; Hu, W W; Taylor, N F; Lambe, A; Cerully, K; Bougiatioti, A; Liu, P; Krejci, R; Petäjä, T; Percival, C; Davidovits, P; Worsnop, D R; Ekman, A M L; Nenes, A; Martin, S; Jimenez, J L; Collins, D R; Topping, D O; Bertram, A K; Zuend, A; Virtanen, A; Riipinen, I

2017-05-28

A large fraction of atmospheric organic aerosol (OA) originates from natural emissions that are oxidized in the atmosphere to form secondary organic aerosol (SOA). Isoprene (IP) and monoterpenes (MT) are the most important precursors of SOA originating from forests. The climate impacts from OA are currently estimated through parameterizations of water uptake that drastically simplify the complexity of OA. We combine laboratory experiments, thermodynamic modeling, field observations, and climate modeling to (1) explain the molecular mechanisms behind RH-dependent SOA water-uptake with solubility and phase separation; (2) show that laboratory data on IP- and MT-SOA hygroscopicity are representative of ambient data with corresponding OA source profiles; and (3) demonstrate the sensitivity of the modeled aerosol climate effect to assumed OA water affinity. We conclude that the commonly used single-parameter hygroscopicity framework can introduce significant error when quantifying the climate effects of organic aerosol. The results highlight the need for better constraints on the overall global OA mass loadings and its molecular composition, including currently underexplored anthropogenic and marine OA sources.

Microphysical explanation of the RH‐dependent water affinity of biogenic organic aerosol and its importance for climate

PubMed Central

Rastak, N.; Pajunoja, A.; Acosta Navarro, J. C.; Ma, J.; Song, M.; Partridge, D. G.; Kirkevåg, A.; Leong, Y.; Hu, W. W.; Taylor, N. F.; Lambe, A.; Cerully, K.; Bougiatioti, A.; Liu, P.; Krejci, R.; Petäjä, T.; Percival, C.; Davidovits, P.; Worsnop, D. R.; Ekman, A. M. L.; Nenes, A.; Martin, S.; Jimenez, J. L.; Collins, D. R.; Topping, D.O.; Bertram, A. K.; Zuend, A.; Virtanen, A.

2017-01-01

Abstract A large fraction of atmospheric organic aerosol (OA) originates from natural emissions that are oxidized in the atmosphere to form secondary organic aerosol (SOA). Isoprene (IP) and monoterpenes (MT) are the most important precursors of SOA originating from forests. The climate impacts from OA are currently estimated through parameterizations of water uptake that drastically simplify the complexity of OA. We combine laboratory experiments, thermodynamic modeling, field observations, and climate modeling to (1) explain the molecular mechanisms behind RH‐dependent SOA water‐uptake with solubility and phase separation; (2) show that laboratory data on IP‐ and MT‐SOA hygroscopicity are representative of ambient data with corresponding OA source profiles; and (3) demonstrate the sensitivity of the modeled aerosol climate effect to assumed OA water affinity. We conclude that the commonly used single‐parameter hygroscopicity framework can introduce significant error when quantifying the climate effects of organic aerosol. The results highlight the need for better constraints on the overall global OA mass loadings and its molecular composition, including currently underexplored anthropogenic and marine OA sources. PMID:28781391

Microphysical explanation of the RH-dependent water affinity of biogenic organic aerosol and its importance for climate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rastak, N.; Pajunoja, A.; Acosta Navarro, J. C.

A large fraction of atmospheric organic aerosol (OA) originates from natural emissions that are oxidized in the atmosphere to form secondary organic aerosol (SOA). Isoprene (IP) and monoterpenes (MT) are the most important precursors of SOA originating from forests. The climate impacts from OA are currently estimated through parameterizations of water uptake that drastically simplify the complexity of OA. We combine laboratory experiments, thermodynamic modeling, field observations, and climate modeling to (1) explain the molecular mechanisms behind RH-dependent SOA water-uptake with solubility and phase separation; (2) show that laboratory data on IP- and MT-SOA hygroscopicity are representative of ambient datamore » with corresponding OA source profiles; and (3) demonstrate the sensitivity of the modeled aerosol climate effect to assumed OA water affinity. We conclude that the commonly used single-parameter hygroscopicity framework can introduce significant error when quantifying the climate effects of organic aerosol. The results highlight the need for better constraints on the overall global OA mass loadings and its molecular composition, including currently underexplored anthropogenic and marine OA sources.« less

Twins in spirit - episode I: comparative preclinical evaluation of [(68)Ga]DOTATATE and [(68)Ga]HA-DOTATATE.

PubMed

Schottelius, Margret; Šimeček, Jakub; Hoffmann, Frauke; Willibald, Marina; Schwaiger, Markus; Wester, Hans-Jürgen

2015-01-01

Recently, an intra-patient comparison demonstrated that the somatostatin (sst) ligand [(68)Ga]HA-DOTATATE ([(68)Ga]DOTA-3-iodo-Tyr(3)-octreotate) provides PET images comparable to or superior to those obtained with [(68)Ga]DOTATATE. To provide a comprehensive basis for nevertheless observed slight differences in tracer biodistribution and dosimetry, the characteristics of [(68)Ga]HA-DOTATATE were investigated in a detailed preclinical study. Affinities of (nat)Ga-HA-DOTATATE and (nat)Ga-DOTATATE to sst1-5 were determined using membrane preparations and [(125)I]SST-28 as radioligand. Internalization into AR42J cells was studied in dual-tracer studies with [(125)I]TOC as internal reference. Biodistribution was investigated using AR42J tumor-bearing CD1 mice, and specificity of tracer uptake was confirmed in competition studies by coinjection of 0.8 mg TOC/kg. Sst2 affinities (IC50) of [(nat)Ga]HA-DOTATATE (1.4 ± 0.8 nM, logP: -3.16) and [(nat)Ga]DOTATATE (1.2 ± 0.6 nM, logP: -3.69) were nearly identical. Both compounds displayed IC50 > 1 μM for sst1,3,4, while sst5 affinity was markedly increased for (nat)Ga-HA-DOTATATE (102 ± 65 nM vs >1 μM for (nat)Ga-DOTATATE). [(nat)Lu]HA-DOTATATE and [(nat)Lu]DOTATATE showed slightly lower, identical sst2 affinities (2.0 ± 1.6 and 2.0 ± 0.8 nM, respectively) and sst3 affinities of 93 ± 1 and 162 ± 16 nM. Internalization of [(68)Ga]HA-DOTATATE was tenfold higher than that of [(125)I]TOC but only sixfold higher for [(68)Ga]DOTATATE and [(177)Lu]HA-DOTATATE. While [(68)Ga]HA-DOTATATE and [(68)Ga]DOTATATE had shown similar target- and non-target uptake in patients, biodistribution studies in mice at 1 h post injection (n = 5) revealed slightly increased non-specific uptake of [(68)Ga]HA-DOTATATE in the blood, liver, and intestines (0.7 ± 0.3, 1.0 ± 0.2, and 4.0 ± 0.7 %iD/g vs 0.3 ± 0.1, 0.5 ± 0.1, and 2.7 ± 0.8 %iD/g for [(68)Ga]DOTATATE). However, sst-mediated accumulation of [(68)Ga]HA-DOTATATE in the pancreas, adrenals, and tumor was significantly enhanced (36.6 ± 4.3, 10.8 ± 3.2, and 33.6 ± 10.9 %iD/g vs 26.1 ± 5.0, 5.1 ± 1.4, and 24.1 ± 4.9 %iD/g, respectively). Consequently, tumor/background ratios for [(68)Ga]HA-DOTATATE in the AR42J model are comparable or slightly increased compared to [(68)Ga]DOTATATE. The present preclinical data fully confirm the general biodistribution pattern and excellent in vivo sst-targeting characteristics previously observed for [(68)Ga]HA-DOTATATE in patients. The effect of slightly enhanced lipophilicity on background accumulation and normal organ dose is compensated by the high uptake of [(68)Ga]HA-DOTATATE in tumor. Thus, [(68)Ga]HA-DOTATATE represents a fully adequate, freely available substitute for [(68)Ga]DOTATATE and, given the superb sst-targeting characteristics of [(177)Lu]HA-DOTATATE in vitro, potential applicability for sst-targeted PRRT.

L-leucine, L-methionine, and L-phenylalanine share a Na(+)/K (+)-dependent amino acid transporter in shrimp hepatopancreas.

PubMed

Duka, Ada; Ahearn, Gregory A

2013-08-01

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of (3)H-L-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. (3)H-L-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na(+)- and K(+)-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. (3)H-L-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, L-methionine, at 20 mM significantly (p < 0.0001) inhibited the 2-min uptakes of 1 mM (3)H-L-leucine in both Na(+)- and K(+)-containing incubation media. The residual (3)H-L-leucine uptake in the two media were significantly greater than zero (p < 0.001), but not significantly different from each other (p > 0.05) and may represent an L-methionine- and cation-independent transport system. (3)H-L-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [L-leucine], following the carrier-mediated Michaelis-Menten equation. In NaCl, (3)H-L-leucine influx displayed a low apparent K M (high affinity) and low apparent J max, while in KCl the transport exhibited a high apparent K M (low affinity) and high apparent J max. L-methionine or L-phenylalanine (7 and 20 mM) were competitive inhibitors of (3)H-L-leucine influxes in both NaCl and KCl media, producing a significant (p < 0.01) increase in (3)H-L-leucine influx K M, but no significant response in (3)H-L-leucine influx J max. Potassium was a competitive inhibitor of sodium co-transport with (3)H-L-leucine, significantly (p < 0.01) increasing (3)H-L-leucine influx K M in the presence of sodium, but having negligible effect on (3)H-L-leucine influx J max in the same medium. These results suggest that shrimp BBMV transport (3)H-L-leucine by a single L-methionine- and L-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na(+) or K(+) acting as co-transport drivers binding to shared activator sites.

In Vivo d-Serine Hetero-Exchange through Alanine-Serine-Cysteine (ASC) Transporters Detected by Microelectrode Biosensors

PubMed Central

2013-01-01

d-Serine, a co-agonist of N-methyl d-aspartate (NMDA) receptors, has been implicated in neurological and psychiatric disorders such as cerebral ischemia, lateral amyotrophic sclerosis, or schizophrenia. d-Serine signaling represents an important pharmacological target for treating these diseases; however, the biochemical mechanisms controlling extracellular d-serine levels in vivo are still unclear. d-Serine heteroexchange through small neutral amino acid transporters has been shown in cell cultures and brain slices and could provide a biochemical mechanism for the control of d-serine extracellular concentration in vivo. Alternatively, exocytotic d-serine release has also been proposed. In this study, the dynamics of d-serine release and clearance were explored in vivo on a second-by-second time scale using microelectrode biosensors. The rate of d-serine clearance in the rat frontal cortex after a microionophoretic injection revealed a transporter-mediated uptake mechanism. d-Serine uptake was blocked by small neutral l-amino acids, implicating alanine-serine-cysteine (ASC) transporters, in particular high affinity Asc-1 and low affinity ASCT2 transporters. Interestingly, changes in alanine, serine, or threonine levels resulted in d-serine release through ASC transporters. Asc-1, but not ASCT2, appeared to release d-serine in response to changes in amino acid concentrations. Finally, neuronal silencing by tetrodotoxin increased d-serine extracellular concentration by an ASC-transporter-dependent mechanism. Together, these results indicate that d-serine heteroexchange through ASC transporters is present in vivo and may constitute a key component in the regulation of d-serine extracellular concentration. PMID:23581544

Molecular mechanisms for generating transmembrane proton gradients

PubMed Central

Gunner, M.R.; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

2013-01-01

Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side. PMID:23507617

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin-GnRH-III bioconjugates.

PubMed

Hegedüs, Rózsa; Manea, Marilena; Orbán, Erika; Szabó, Ildikó; Kiss, Eva; Sipos, Eva; Halmos, Gábor; Mező, Gábor

2012-10-01

Here we report on the synthesis and biochemical characterization (enzymatic stability, cellular uptake, in vitro antitumor activity, membrane interaction and GnRH-receptor binding affinity) of novel short-chain fatty acid (SCFA) acylated daunorubicin-GnRH-III bioconjugates, which may serve as drug delivery systems for targeted cancer chemotherapy. Ser in position 4 of GnRH-III was replaced by Lys, followed by the acylation of its ε-amino group with various fatty acids. SCFAs are potentially chemoprotective agents by suppressing the growth of cancer cells and therefore may enhance the antitumor activity of the bioconjugates. We found that all synthesized bioconjugates had high cytostatic effect in vitro, were stable in cell culture medium for 6 h and degraded in the presence of rat liver lysosomal homogenate leading to the formation of an oxime bond-linked daunorubicin-Lys as the smallest active metabolite. In the presence of α-chymotrypsin, all compounds were digested, the degradation rate strongly depending on the type of fatty acid. The bioconjugate containing Lys(nBu) in position 4 was taken up most efficiently by the cancer cells and exerted higher in vitro cytostatic effect than the previously developed GnRH-III((4)Lys(Ac), (8)Lys(Dau = Aoa)) or the parent GnRH-III(Dau = Aoa) bioconjugate. Our results could be explained by the increased binding affinity of the newly developed compound containing Lys(nBu) to the GnRH receptors. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Development of radiohalogenated muscarinic ligands for the in vivo imaging of m-AChR by nuclear medicine techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

McPherson, D.W.; Luo, H.; Knapp, F.F. Jr.

1994-06-01

Alterations in the density of acetylcholinergic muscarinic receptors (m-AChR) have been observed in various dementias. This has spurred interest in the development of radiohalogenated ligands which can be used for the non-invasive in vivo detection of m-AChR by nuclear medicine techniques. We have developed a new ligand 1-azabicyclo[2.2.2]oct-3-yl ({alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IQNP,12) which demonstrates high affinity for the muscarinic receptor. When labeled with radioiodine it has been shown to be selective and specific for m-ACHR. Initial studies on the separation and in vivo evaluation of the various isomers of IQNP have shown that the stereochemistry of the chiral centers and the configurationmore » around the double bond play an important role in m-AChR subtype specificity. In vivo evaluation of these stereoisomers demonstrate that E-(R,R)-IQNP has a high affinity for the M{sub 1} muscarinic subtype while Z-(R,R)-IQNP demonstrate a high affinity for M{sub 1} and M{sub 2} receptor subtypes. These data demonstrate IQNP (12) has potential for use in the non-evasive in vivo detection of m-AChR by single photon emission computed tomography (SPECT). A brominated analogue, ``BrQNP,`` in which the iodine has been replaced by a bromine atom, has also been prepared and was shown to block the in vivo uptake of IQNP in the brain and heart and therefore has potential for positron emission tomographic (PET) studies of m-AChR.« less

Variations in phytodetritus derived carbon uptake of the intertidal foraminifera Ammonia tepida and Haynesina germanica

NASA Astrophysics Data System (ADS)

Wukovits, Julia; Bukenberger, Patrick; Enge, Annekatrin; Wanek, Wolfgang; Watzka, Margarete; Heinz, Petra

2016-04-01

Phytodetritus represents a major component of particulate organic carbon in intertidal mudflats. Estuaries and tidal currents yield an extensive amount of these particles that display a substantial nutrient source for littoral food webs. For benthic foraminifera, a group of marine protists, phytodetritus serves as the main food source. Foraminifera are considered to play a significant role in marine carbon turnover processes and show seasonally very high population densities in intertidal sediments. Therefore, it is important to gather explicit data about the specific carbon uptake behavior of intertidal foraminiferal species. In this study, laboratory feeding experiments were carried out to observe phytodetrital carbon uptake of foraminiferal specimen collected in the German Wadden Sea. Artificially produced phytodetritus was labelled with 13C to follow carbon ingestion into foraminiferal cytoplasm over time at different simulated conditions. The experiments were performed with monocultures under exclusion of other meiofauna. Chlorophyte detritus (Dunaliella tertiolecta) was fed to the two common species Ammonia tepida and Haynesina germanica. Ammonia tepida showed a significantly higher affinity to this food source than H. germanica. Testing the effect of temperature revealed a significant decrease of carbon ingestion with increasing temperature in H. germanica. Observations focusing on A. tepida showed a rising phytodetrital carbon content in the biomass of juvenile individuals in contrast to adult foraminifera. In general, carbon uptake reaches saturation levels a few hours after food supply. Furthermore, A. tepida benefits from constant availability of fresh food rather than from a high amount of phytodetritus derived from a single food pulse. Our investigations showed that the foraminiferal impact on intertidal processing of phytodetrital carbon sources is species specific, temperature related and depends on developmental stage and input dynamics. Additionally, the presented data reveal the quantitative level of food derived carbon gathered within foraminiferal biomass.

Human, rat and chicken small intestinal Na+-Cl−-creatine transporter: functional, molecular characterization and localization

PubMed Central

Peral, M J; García-Delgado, M; Calonge, M L; Durán, J M; De La Horra, M C; Wallimann, T; Speer, O; Ilundáin, A A

2002-01-01

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [14C]Creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na+- and Cl−-dependent, with a probable stoichiometry of 2 Na+: 1 Cl−: 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a Km for creatine of 29 μm. [14C]Creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, β-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na+- and Cl−-dependent, apical creatine uptake. PMID:12433955

The Bad Berka dose protocol: comparative results of dosimetry in peptide receptor radionuclide therapy using (177)Lu-DOTATATE, (177)Lu-DOTANOC, and (177)Lu-DOTATOC.

PubMed

Schuchardt, Christiane; Kulkarni, Harshad R; Prasad, Vikas; Zachert, Carolin; Müller, Dirk; Baum, Richard P

2013-01-01

The objective of this study is to analyze the in vivo behavior of the (177)Lu-labeled peptides DOTATATE, DOTANOC, and DOTATOC used for peptide receptor radionuclide therapy (PRRNT) of neuroendocrine tumors (NETs), by measuring organ and tumor kinetics and by performing dosimetric calculations. Two hundred fifty-three patients (group 1) with metastasized NET who underwent PRRNT were examined. Out of these, 185 patients received (177)Lu-DOTATATE, 9 were treated with (177)Lu-DOTANOC, and 59 with (177)Lu-DOTATOC. Additionally, 25 patients receiving, in consecutive PRRNT cycles, DOTATATE followed by DOTATOC (group 2) and 3 patients receiving DOTATATE and DOTANOC (group 3) were analyzed. Dosimetric calculations (according to MIRD scheme) were performed using OLINDA software. In group 1, DOTATOC exhibited the lowest and DOTANOC the highest uptake and therefore mean absorbed dose in normal organs (whole body, kidney, and spleen). In group 2, there was a significant difference between DOTATATE and DOTATOC concerning kinetics and normal organ doses. (177)Lu-DOTATOC had the lowest uptake/dose delivered to normal organs and highest tumor-to-kidney ratio. There were no significant differences between the three peptides concerning tumor kinetics and mean absorbed tumor dose. The study demonstrates a correlation between high affinity of DOTANOC in vitro and high uptake in normal organs/whole body in vivo, resulting in a higher whole-body dose. DOTATOC exhibited the lowest uptake and dose delivered to normal tissues and the best tumor-to-kidney ratio. Due to large interpatient variability, individual dosimetry should be performed for each therapy cycle.

A high-affinity [ 18F]-labeled phosphoramidate peptidomimetic PSMA-targeted inhibitor for PET imaging of prostate cancer

DOE PAGES

Ganguly, Tanushree; Dannoon, Shorouk; Hopkins, Mark R.; ...

2015-06-09

Here in this study, a structurally modified phosphoramidate scaffold, with improved prostate-specific membrane antigen (PSMA) avidity, stability and in vivo characteristics, as a PET imaging agent for prostate cancer (PCa), was prepared and evaluated. p-Fluorobenzoyl-aminohexanoate and 2-(3-hydroxypropyl)glycine were introduced into the PSMA-targeting scaffold yielding phosphoramidate 5. X-ray crystallography was performed on the PSMA/5 complex. [ 18F]5 was synthesized, and cell uptake and internalization studies were conducted in PSMA(+) LNCaP and CWR22Rv1 cells and PSMA(-) PC-3 cells. In vivo PET imaging and biodistribution studies were performed at 1 and 4 h post injection in mice bearing CWR22Rv1 tumor, with or withoutmore » blocking agent. The crystallographic data showed interaction of the p-fluorobenzoyl group with an arene-binding cleft on the PSMA surface. In vitro studies revealed elevated uptake of [ 18F]5 in PSMA(+) cells (2.2% in CWR22Rv1 and 12.1% in LNCaP) compared to PSMA(-) cells (0.08%) at 4 h. In vivo tumor uptake of 2.33% ID/g and tumor-to-blood ratio of 265:1 was observed at 4 h. In conclusion, we have successfully synthesized, radiolabeled and evaluated a new PSMA-targeted PET agent. The crystal structure of the PSMA/5 complex highlighted the interactions within the arene-binding cleft contributing to the overall complex stability. The high target uptake and rapid non-target clearance exhibited by [ 18F]5 in PSMA(+) xenografts substantiates its potential use for PET imaging of PCa.« less

Preclinical properties and human in vivo assessment of 123 I-ABC577 as a novel SPECT agent for imaging amyloid-β

PubMed Central

Okumura, Yuki; Kobayashi, Ryohei; Onishi, Takako; Shoyama, Yoshinari; Barret, Olivier; Alagille, David; Jennings, Danna; Marek, Kenneth; Seibyl, John; Tamagnan, Gilles; Tanaka, Akihiro; Shirakami, Yoshifumi

2016-01-01

Abstract Non-invasive imaging of amyloid-β in the brain, a hallmark of Alzheimer’s disease, may support earlier and more accurate diagnosis of the disease. In this study, we assessed the novel single photon emission computed tomography tracer 123 I-ABC577 as a potential imaging biomarker for amyloid-β in the brain. The radio-iodinated imidazopyridine derivative 123 I-ABC577 was designed as a candidate for a novel amyloid-β imaging agent. The binding affinity of 123 I-ABC577 for amyloid-β was evaluated by saturation binding assay and in vitro autoradiography using post-mortem Alzheimer’s disease brain tissue. Biodistribution experiments using normal rats were performed to evaluate the biokinetics of 123 I-ABC577. Furthermore, to validate 123 I-ABC577 as a biomarker for Alzheimer’s disease, we performed a clinical study to compare the brain uptake of 123 I-ABC577 in three patients with Alzheimer’s disease and three healthy control subjects. 123 I-ABC577 binding was quantified by use of the standardized uptake value ratio, which was calculated for the cortex using the cerebellum as a reference region. Standardized uptake value ratio images were visually scored as positive or negative. As a result, 123 I-ABC577 showed high binding affinity for amyloid-β and desirable pharmacokinetics in the preclinical studies. In the clinical study, 123 I-ABC577 was an effective marker for discriminating patients with Alzheimer’s disease from healthy control subjects based on visual images or the ratio of cortical-to-cerebellar binding. In patients with Alzheimer’s disease, 123 I-ABC577 demonstrated clear retention in cortical regions known to accumulate amyloid, such as the frontal cortex, temporal cortex, and posterior cingulate. In contrast, less, more diffuse, and non-specific uptake without localization to these key regions was observed in healthy controls. At 150 min after injection, the cortical standardized uptake value ratio increased by ∼60% in patients with Alzheimer’s disease relative to healthy control subjects. Both healthy control subjects and patients with Alzheimer’s disease showed minimal 123 I-ABC577 retention in the white matter. These observations indicate that 123 I-ABC577 may be a useful single photon emission computed tomography imaging maker to identify amyloid-β in the human brain. The availability of an amyloid-β tracer for single photon emission computed tomography might increase the accessibility of diagnostic imaging for Alzheimer’s disease. PMID:26490333

Quantitative in vivo receptor binding. I. Theory and application to the muscarinic cholinergic receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frey, K.A.; Ehrenkaufer, R.L.; Beaucage, S.

1985-02-01

A novel approach to in vivo receptor binding experiments is presented which allows direct quantitation of binding site densities. The method is based on an equilibrium model of tracer uptake and is designed to produce a static distribution proportional to receptor density and to minimize possible confounding influences of regional blood flow, blood-brain barrier permeability, and nonspecific binding. This technique was applied to the measurement of regional muscarinic cholinergic receptor densities in rat brain using (/sup 3/H)scopolamine. Specific in vivo binding of scopolamine demonstrated saturability, a pharmacologic profile, and regional densities which are consistent with interaction of the tracer withmore » the muscarinic receptor. Estimates of receptor density obtained with the in vivo method and in vitro measurements in homogenates were highly correlated. Furthermore, reduction in striatal muscarinic receptors following ibotenic acid lesions resulted in a significant decrease in tracer uptake in vivo, indicating that the correlation between scopolamine distribution and receptor density may be used to demonstrate pathologic conditions. We propose that the general method presented here is directly applicable to investigation of high affinity binding sites for a variety of radioligands.« less

Characterization of cross-linked cellulosic ion-exchange adsorbents: 2. Protein sorption and transport.

PubMed

Angelo, James M; Cvetkovic, Aleksandar; Gantier, Rene; Lenhoff, Abraham M

2016-03-18

Adsorption behavior in the HyperCel family of cellulosic ion-exchange materials (Pall Corporation) was characterized using methods to assess, quantitatively and qualitatively, the dynamics of protein uptake as well as static adsorption as a function of ionic strength and protein concentration using several model proteins. The three exchangers studied all presented relatively high adsorptive capacities under low ionic strength conditions, comparable to commercially available resins containing polymer functionalization aimed at increasing that particular characteristic. The strong cation- and anion-exchange moieties showed higher sensitivity to increasing salt concentrations, but protein affinity on the salt-tolerant STAR AX HyperCel exchanger remained strong at ionic strengths normally used in downstream processing to elute material fully during ion-exchange chromatography. Very high uptake rates were observed in both batch kinetics experiments and time-series confocal laser scanning microscopy, suggesting low intraparticle transport resistances relative to external film resistance, even at higher bulk protein concentrations where the opposite is typically observed. Electron microscopy imaging of protein adsorbed phases provided additional insight into particle structure that could not be resolved in previous work on the bare resins. Copyright © 2016 Elsevier B.V. All rights reserved.

Phytoremediation of organochlorine pesticides: Concept, method, and recent developments.

PubMed

Singh, Tanvi; Singh, Dileep K

2017-09-02

Rapid increase in industrialization of world economy in the past century has resulted in significantly high emission of anthropogenic chemicals in the ecosystem. The organochlorine pesticides (OCPs) are a great risk to the global environment and endanger the human health due to their affinity for dispersion, transportation over long distances, and bioaccumulation in the food chain. Phytoremediation is a promising technology that aims to make use of plants and associated bacteria for the treatment of groundwater and soil polluted by these contaminants. Processes known to be involved in phytoremediation of OCPs include phytoaccumulation, rhizoremediation, and phytotransformation. Vegetation has been accounted to considerably amplify OCP elimination from soil, in contrast to non-planted soil, attributable to both, uptake within plant tissues and high microbial degradation of OCP within the root zone. Developing transgenic plants is a promising approach to enhance phytoremediation capabilities. Recent advances in the application of phytoremediation technique for OCPs, including uptake by plants and plant-microbe association in the rhizosphere for the enhanced degradation and mineralization of these pollutants, is presented in this review. Additionally, some attempts to improve this technique using transgenesis and role of certain enzymes are also discussed.

Evaluation of the plant growth-promoting activity of Pseudomonas nitroreducens in Arabidopsis thaliana and Lactuca sativa.

PubMed

Trinh, Cao Son; Lee, Hyeri; Lee, Won Je; Lee, Seok Jin; Chung, Namhyun; Han, Juhyeong; Kim, Jongyun; Hong, Suk-Whan; Lee, Hojoung

2018-06-01

Pseudomonas nitroreducens: strain IHB B 13561 (PnIHB) enhances the growth of Arabidopsis thaliana and Lactuca sativa via the stimulation of cell development and nitrate absorption. Plant growth-promoting rhizobacteria (PGPR) enhance plant development through various mechanisms; they improve the uptake of soil resources by plants to greatly promote plant growth. Here, we used Arabidopsis thaliana seedlings and Lactuca sativa to screen the growth enhancement activities of a purified PGPR, Pseudomonas nitroreducens strain IHB B 13561 (PnIHB). When cocultivated with PnIHB, both species of plants exhibited notably improved growth, particularly in regard to biomass. Quantitative reverse transcription polymerase chain reaction analysis indicated high expression levels of the nitrate transporter genes, especially NRT2.1, which plays a major role in the high-affinity nitrate transport system in roots. Moreover, enhanced activity of the cyclin-B1 promoter was observed when wild-type 'Columbia-0' Arabidopsis seedlings were exposed to PnIHB, whereas upregulation of cyclin-B also occurred in the inoculated lettuce seedlings. Overall, these results suggest that PnIHB improves A. thaliana and L. sativa growth via specific pathways involved in the promotion of cell development and enhancement of nitrate uptake.

Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

NASA Astrophysics Data System (ADS)

Brew, Helen; Attwell, David

1987-06-01

Glutamate is taken up avidly by glial cells in the central nervous system1. Glutamate uptake may terminate the transmitter action of glutamate released from neurons1, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique2. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

Effects of calcium on hepatocyte iron uptake from transferrin, iron-pyrophosphate and iron-ascorbate.

PubMed

Nilsen, T

1991-10-16

Calcium stimulates hepatocyte iron uptake from transferrin, ferric-iron-pyrophosphate and ferrous-iron-ascorbate. Maximal stimulation of iron uptake is observed at 1-1.5 mM of extra-cellular calcium and the effect is reversible and immediate. Neither the receptor affinity for transferrin, nor the total amounts of transferrin associated with the cells or the rate of transferrin endocytosis are significantly affected by calcium. In the presence of calcium the rate of iron uptake of non-transferrin bound iron increases abruptly at approximate 17 degrees C and 27 degrees C and as assessed by Arrhenius plots, the activation energy is reduced in a calcium dependent manner at approx. 27 degrees C. At a similar temperature, i.e., between 25 degrees C and 28 degrees C, calcium increases the rates of cellular iron uptake from transferrin in a way that is not reflected in the rate of transferrin endocytosis. By the results of this study it is concluded that calcium increases iron transport across the plasma membrane by a mechanism dependent on membrane fluidity.

Deanol acetamidobenzoate inhibits the blood-brain barrier transport of choline.

PubMed

Millington, W R; McCall, A L; Wurtman, R J

1978-10-01

Competition by deanol (dimethylaminoethanol) with choline for uptake from the bloodstream into the brain was demonstrated by simultaneous intracarotid administration of carbon 14-labeled choline with deanol (plus tritiated water and indium 113m, to calculate a brain uptake index) and by measuring the brain uptake of 14C-labeled choline mixed with sera from rats pretreated with deanol (300 or 500 mg/kg 8 or 30 minutes earlier). The inhibition constant for inhibition of choline uptake by deanol (159 micrograms) was actually lower than the Michaelis constant for choline itself (442 micrograms); hence, the affinity of the carrier mechanism for deanol is at least as great as it is for choline. Deanol administration also elevated blood choline levels; thus, the effect of the drug on brain choline (and acetylcholine) levels is the result of the increase it produces in blood choline and the suppression it causes in choline uptake. These findings may explain discrepant results from laboratories seeking increases in brain acetylcholine or clinical improvement in patients with tardive dyskinesia after deanol treatment.

Solitary BioY Proteins Mediate Biotin Transport into Recombinant Escherichia coli

PubMed Central

Finkenwirth, Friedrich; Kirsch, Franziska

2013-01-01

Energy-coupling factor (ECF) transporters form a large group of vitamin uptake systems in prokaryotes. They are composed of highly diverse, substrate-specific, transmembrane proteins (S units), a ubiquitous transmembrane protein (T unit), and homo- or hetero-oligomeric ABC ATPases. Biotin transporters represent a special case of ECF-type systems. The majority of the biotin-specific S units (BioY) is known or predicted to interact with T units and ABC ATPases. About one-third of BioY proteins, however, are encoded in organisms lacking any recognizable T unit. This finding raises the question of whether these BioYs function as transporters in a solitary state, a feature ascribed to certain BioYs in the past. To address this question in living cells, an Escherichia coli K-12 derivative deficient in biotin synthesis and devoid of its endogenous high-affinity biotin transporter was constructed as a reference strain. This organism is particularly suited for this purpose because components of ECF transporters do not naturally occur in E. coli K-12. The double mutant was viable in media containing either high levels of biotin or a precursor of the downstream biosynthetic path. Importantly, it was nonviable on trace levels of biotin. Eight solitary bioY genes of proteobacterial origin were individually expressed in the reference strain. Each of the BioYs conferred biotin uptake activity on the recombinants, which was inferred from uptake assays with [3H]biotin and growth of the cells on trace levels of biotin. The results underscore that solitary BioY transports biotin across the cytoplasmic membrane. PMID:23836870

Negatively Cooperative Binding of High Density Lipoprotein to the HDL Receptor SR-BI†

PubMed Central

Nieland, Thomas J.F.; Xu, Shangzhe; Penman, Marsha; Krieger, Monty

2011-01-01

Scavenger receptor class B, type I (SR-BI) is a high-density lipoprotein (HDL) receptor, which also binds low density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a co-receptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high affinity binding sites (one site model). We have re-investigated the ligand concentration dependence of 125I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [3H]CE from [3H]CE-HDL using an expanded range of ligand concentrations (<1 µg protein/ml, lower than previously reported). Scatchard and non-linear least squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites, or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects (‘ lattice model’). Similar results were observed for LDL. Application of the ‘infinite dilution’ dissociation rate method established that the binding of 125I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport and cell signaling. PMID:21254782

Relationships between functional traits and inorganic nitrogen acquisition among eight contrasting European grass species

PubMed Central

Grassein, Fabrice; Lemauviel-Lavenant, Servane; Lavorel, Sandra; Bahn, Michael; Bardgett, Richard D.; Desclos-Theveniau, Marie; Laîné, Philippe

2015-01-01

Backgrounds and Aims Leaf functional traits have been used as a basis to categoize plants across a range of resource-use specialization, from those that conserve available resources to those that exploit them. However, the extent to which the leaf functional traits used to define the resource-use strategies are related to root traits and are good indicators of the ability of the roots to take up nitrogen (N) are poorly known. This is an important question because interspecific differences in N uptake have been proposed as one mechanism by which species’ coexistence may be determined. This study therefore investigated the relationships between functional traits and N uptake ability for grass species across a range of conservative to exploitative resource-use strategies. Methods Root uptake of NH4+ and NO3–, and leaf and root functional traits were measured for eight grass species sampled at three grassland sites across Europe, in France, Austria and the UK. Species were grown in hydroponics to determine functional traits and kinetic uptake parameters (Imax and Km) under standardized conditions. Key Results Species with high specific leaf area (SLA) and shoot N content, and low leaf and root dry matter content (LDMC and RDMC, respectively), which are traits associated with the exploitative syndrome, had higher uptake and affinity for both N forms. No trade-off was observed in uptake between the two forms of N, and all species expressed a higher preference for NH4+. Conclusions The results support the use of leaf traits, and especially SLA and LDMC, as indicators of the N uptake ability across a broad range of grass species. The difficulties associated with assessing root properties are also highlighted, as root traits were only weakly correlated with leaf traits, and only RDMC and, to a lesser extent, root N content were related to leaf traits. PMID:25471096

Glutamine, glutamate, and other possible regulators of alpha-ketoglutarate and malate uptake by synaptic terminals.

PubMed

Shank, R P; Campbell, G L

1984-04-01

The uptake of alpha-ketoglutarate and malate by rat brain synaptosomal preparations was found to be affected by a variety of substances at physiologically relevant concentrations. Glutamine altered the uptake of alpha-ketoglutarate by causing an apparent reduction in the substrate-carrier affinity and an increase in Vmax. In contrast, glutamine did not appear to affect the Vmax of malate uptake, but it did increase markedly the uptake velocity at low concentrations of malate. L-Glutamate and L-aspartate were comparatively strong inhibitors of alpha-ketoglutarate and malate uptake. N-Acetylaspartate was a weak inhibitor of alpha-ketoglutarate uptake, a finding that contrasts with our previous observation that this compound potently inhibited alpha-ketoglutarate uptake by synaptosomes obtained from the cerebellum of 8- to 14-day-old mice. Ca2+ exhibited a variable effect but usually enhanced the uptake of alpha-ketoglutarate. The addition of small amounts of postmicrosomal supernatant to the incubation medium enhanced the uptake of alpha-ketoglutarate by low-density synaptosomes. By comparison, the uptake of glutamate, glutamine, gamma-aminobutyric acid, and several other amino acids was not affected. The enhancement of alpha-ketoglutarate uptake by the supernatant was due to a heat labile substance that was retained by dialysis tubing (MW cutoff = 8,000) and Amicon filter cones (CF 25), and was precipitated by ammonium sulfate at 60% saturation. In experiments in which the metabolic conversion of [U-14C] alpha-ketoglutarate to glutamate, aspartate, glutamine, and gamma-aminobutyric acid was determined, the presence of glutamine and glutamate in the incubation medium did not affect the pattern of labelling appreciably.

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

PubMed Central

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

1992-01-01

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

Physiological characterisation of a pH- and calcium-dependent sodium uptake mechanism in the freshwater crustacean, Daphnia magna.

PubMed

Glover, Chris N; Wood, Chris M

2005-03-01

Daphnia are highly sensitive to sodium metabolism disruption caused by aquatic acidification and ionoregulatory toxicants, due to their finely balanced ion homeostasis. Nine different water chemistries of varying pH (4, 6 and 8) and calcium concentration (0, 0.5 and 1 mmol l(-1)) were used to delineate the mechanism of sodium influx in Daphnia magna. Lowering water pH severely inhibited sodium influx when calcium concentration was high, but transport kinetic analysis revealed a stimulated sodium influx capacity (J(max)) when calcium was absent. At low pH increasing water calcium levels decreased J(max) and raised K(m) (decreased sodium influx affinity), while at high pH the opposite pattern was observed (elevated J(max) and reduced K(m)). These effects on sodium influx were mirrored by changes in whole body sodium levels. Further examination of the effect of calcium on sodium influx showed a severe inhibition of sodium uptake by 100 micromol l(-1) calcium gluconate at both low (50 micromol l(-1)) and high (1000 micromol l(-1)) sodium concentrations. At high sodium concentrations, stimulated sodium influx was noted with elevated calcium levels. These results, in addition to data showing amiloride inhibition of sodium influx (K(i)=180 micromol l(-1)), suggest a mechanism of sodium influx in Daphnia magna that involves the electrogenic 2Na(+)/1H(+) exchanger.

Co-ordination of NDH and Cup proteins in CO2 uptake in cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Han, Xunling; Sun, Nan; Xu, Min; Mi, Hualing

2017-06-01

High and low affinity CO2-uptake systems containing CupA (NDH-1MS) and CupB (NDH-1MS'), respectively, have been identified in Synechocystis sp. PCC 6803, but it is yet unknown how the complexes function in CO2 uptake. In this work, we found that deletion of cupB significantly lowered the growth of cells, and deletion of both cupA and cupB seriously suppressed the growth below pH 7.0 even under 3% CO2. The rate of photosynthetic oxygen evolution was decreased slightly by deletion of cupA but significantly by deletion of cupB and more severely by deletion of both cupA and cupB, especially in response to changed pH conditions under 3% CO2. Furthermore, we found that assembly of CupB into NDH-1MS' was dependent on NdhD4 and NdhF4. NDH-1MS' was not affected in the NDH-1MS-degradation mutant and NDH-1MS was not affected in the NDH-1MS'-degradation mutants, indicating the existence of independent CO2-uptake systems under high CO2 conditions. The light-induced proton gradient across thylakoid membranes was significantly inhibited in ndhD-deletion mutants, suggesting that NdhDs functions in proton pumping. The carbonic anhydrase activity was suppressed partly in the cupA- or cupB-deletion mutant but severely in the mutant with both cupA and cupB deletion, indicating that CupA and CupB function in conversion of CO2 to HCO3-. In turn, deletion of cup genes lowered the transthylakoid membrane proton gradient and deletion of ndhDs decreased the CO2 hydration. Our results suggest that NDH-1M provides an alkaline region to activate Cup proteins involved in CO2 uptake. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Trinucleotide Insertions, Deletions, and Point Mutations in Glucose Transporters Confer K+ Uptake in Saccharomyces cerevisiae

PubMed Central

Liang, Hong; Ko, Christopher H.; Herman, Todd; Gaber, Richard F.

1998-01-01

Deletion of TRK1 and TRK2 abolishes high-affinity K+ uptake in Saccharomyces cerevisiae, resulting in the inability to grow on typical synthetic growth medium unless it is supplemented with very high concentrations of potassium. Selection for spontaneous suppressors that restored growth of trk1Δ trk2Δ cells on K+-limiting medium led to the isolation of cells with unusual gain-of-function mutations in the glucose transporter genes HXT1 and HXT3 and the glucose/galactose transporter gene GAL2. 86Rb uptake assays demonstrated that the suppressor mutations conferred increased uptake of the ion. In addition to K+, the mutant hexose transporters also conferred permeation of other cations, including Na+. Because the selection strategy required such gain of function, mutations that disrupted transporter maturation or localization to the plasma membrane were avoided. Thus, the importance of specific sites in glucose transport could be independently assessed by testing for the ability of the mutant transporter to restore glucose-dependent growth to cells containing null alleles of all of the known functional glucose transporter genes. Twelve sites, most of which are conserved among eukaryotic hexose transporters, were revealed to be essential for glucose transport. Four of these have previously been shown to be essential for glucose transport by animal or plant transporters. Eight represented sites not previously known to be crucial for glucose uptake. Each suppressor mutant harbored a single mutation that altered an amino acid(s) within or immediately adjacent to a putative transmembrane domain of the transporter. Seven of 38 independent suppressor mutations consisted of in-frame insertions or deletions. The nature of the insertions and deletions revealed a striking DNA template dependency: each insertion generated a trinucleotide repeat, and each deletion involved the removal of a repeated nucleotide sequence. PMID:9447989

The organic anion transport polypeptide 1d1 (Oatp1d1) mediates hepatocellular uptake of phalloidin and microcystin into skate liver.

PubMed

Meier-Abt, F; Hammann-Hänni, A; Stieger, B; Ballatori, N; Boyer, J L

2007-02-01

Organic anion transporting polypeptides (rodent Oatp; human OATP) mediate cellular uptake of numerous organic compounds including xenobiotic toxins into mammalian hepatocytes. In the little skate Leucoraja erinacea a liver-specific Oatp (Oatp1d1, also called sOatp) has been identified and suggested to represent an evolutionarily ancient precursor of the mammalian liver OATP1B1 (human), Oatp1b2 (rat), and OATP1B3 (human). The present study tested whether Oatp1d1 shares functional transport activity of the xenobiotic oligopeptide toxins phalloidin and microcystin with the mammalian liver Oatps/OATPs. The phalloidin analogue [(3)H]-demethylphalloin was taken up into skate hepatocytes with high affinity (Km approximately 0.4 microM), and uptake could be inhibited by phalloidin and a variety of typical Oatp/OATP substrates such as bromosulfophthalein, bile salts, estrone-3-sulfate, cyclosporine A and high concentrations of microcystin-LR (Ki approximately 150 microM). When expressed in Xenopus laevis oocytes Oatp1d1 increased uptake of demethylphalloin (Km approximately 2.2 microM) and microcystin-LR (Km approximately 27 microM) 2- to 3-fold over water-injected oocytes, whereas the alternative skate liver organic anion transporter, the dimeric Ostalpha/beta, exhibited no phalloidin and only minor microcystin-LR transport. Also, the closest mammalian Oatp1d1 orthologue, the human brain and testis OATP1C1, did not show any phalloidin transport activity. These results demonstrate that the evolutionarily ancient Oatp1d1 is able to mediate uptake of cyclic oligopeptide toxins into skate liver. The findings support the notion that Oatp1d1 is a precursor of the liver-specific mammalian Oatps/OATPs and that its transport properties are closely associated with certain forms of toxic liver injury such as for example protein phosphatase inhibition by the water-borne toxin microcystin.

Safety and biodistribution of 111In-amatuximab in patients with mesothelin expressing cancers using Single Photon Emission Computed Tomography-Computed Tomography (SPECT-CT) imaging

PubMed Central

Adler, Stephen; Mena, Esther; Kurdziel, Karen; Maltzman, Julia; Wallin, Bruce; Hoffman, Kimberly; Pastan, Ira; Paik, Chang Hum; Choyke, Peter; Hassan, Raffit

2015-01-01

Amatuximab is a chimeric high-affinity monoclonal IgG1/k antibody targeting mesothelin that is being developed for treatment of mesothelin-expressing cancers. Considering the ongoing clinical development of amatuximab in these cancers, our objective was to characterize the biodistribution, and dosimetry of 111Indium (111In) radiolabelled amatuximab in mesothelin-expressing cancers. Between October 2011 and February 2013, six patients including four with malignant mesothelioma and two with pancreatic adenocarcinoma underwent Single Photon Emission Computed Tomography-Computed Tomography (SPECT/CT) imaging following administration of 111In amatuximab. SPECT/CT images were obtained at 2–4 hours, 24–48 hours and 96–168 hours after radiotracer injection. In all patients, tumor to background ratios (TBR) consistently met or exceeded an uptake of 1.2 (range 1.2–62.0) which is considered the minimum TBR that can be visualized. TBRs were higher in tumors of patients with mesothelioma than pancreatic adenocarcinoma. 111In-amatuximab uptake was noted in both primary tumors and metastatic sites. The radiotracer dose was generally well-tolerated and demonstrated physiologic uptake in the heart, liver, kidneys and spleen. This is the first study to show tumor localization of an anti-mesothelin antibody in humans. Our results show that 111In-amatuximab was well tolerated with a favorable dosimetry profile. It localizes to mesothelin expressing cancers with a higher uptake in mesothelioma than pancreatic cancer. PMID:25756664

Sodium-dependent nitrate transport at the plasma membrane of leaf cells of the marine higher plant Zostera marina L.

PubMed

García-Sánchez, M J; Jaime, M P; Ramos, A; Sanders, D; Fernández, J A

2000-03-01

NO(3)(-) is present at micromolar concentrations in seawater and must be absorbed by marine plants against a steep electrochemical potential difference across the plasma membrane. We studied NO(3)(-) transport in the marine angiosperm Zostera marina L. to address the question of how NO(3)(-) uptake is energized. Electrophysiological studies demonstrated that micromolar concentrations of NO(3)(-) induced depolarizations of the plasma membrane of leaf cells. Depolarizations showed saturation kinetics (K(m) = 2.31 +/- 0.78 microM NO(3)(-)) and were enhanced in alkaline conditions. The addition of NO(3)(-) did not affect the membrane potential in the absence of Na(+), but depolarizations were restored when Na(+) was resupplied. NO(3)(-)-induced depolarizations at increasing Na(+) concentrations showed saturation kinetics (K(m) = 0.72 +/- 0.18 mM Na(+)). Monensin, an ionophore that dissipates the Na(+) electrochemical potential, inhibited NO(3)(-)-evoked depolarizations by 85%, and NO(3)(-) uptake (measured by depletion from the external medium) was stimulated by Na(+) ions and by light. Our results strongly suggest that NO(3)(-) uptake in Z. marina is mediated by a high-affinity Na(+)-symport system, which is described here (for the first time to our knowledge) in an angiosperm. Coupling the uptake of NO(3)(-) to that of Na(+) enables the steep inwardly-directed electrochemical potential for Na(+) to drive net accumulation of NO(3)(-) within leaf cells.

Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae.

PubMed

Elbing, Karin; Larsson, Christer; Bill, Roslyn M; Albers, Eva; Snoep, Jacky L; Boles, Eckhard; Hohmann, Stefan; Gustafsson, Lena

2004-09-01

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.

Role of Hexose Transport in Control of Glycolytic Flux in Saccharomyces cerevisiae

PubMed Central

Elbing, Karin; Larsson, Christer; Bill, Roslyn M.; Albers, Eva; Snoep, Jacky L.; Boles, Eckhard; Hohmann, Stefan; Gustafsson, Lena

2004-01-01

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which has distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations. PMID:15345416

The effect of feeding high corn oil on fatty-acid-binding-protein isolated from rat liver.

PubMed

Catalá, A

1987-12-01

Fatty-acid-binding-protein isolated from liver of rats receiving normal or high fat diet was studied by three different methods. The effect of high fat diet on the thermal stability of the protein was determined employing differential scanning calorimetry. Fatty acids have a stabilizing effect on the thermal stability of the protein. In order to determine the relative binding affinity of native and delipidated protein a Sephadex G-50 assay was employed using [1-14C] oleate as ligand. The delipidated protein exhibited greater binding of oleate than did the native material. Increases in the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes in vitro were also observed when protein obtained from both sources were delipidated. The results suggest that high corn oil diet would modify the properties of fatty-acid-binding-protein in the uptake and cytosolic transport of long-chain fatty acids.

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

PubMed

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.

The biochemical characterization of two phosphate transport systems in Phytomonas serpens.

PubMed

Vieira-Bernardo, Rodrigo; Gomes-Vieira, André Luiz; Carvalho-Kelly, Luiz Fernando; Russo-Abrahão, Thais; Meyer-Fernandes, José Roberto

2017-02-01

Inorganic phosphate (P i ) is an essential nutrient for all organisms because it is required for a variety of biochemical processes, such as signal transduction and the synthesis of phosphate-containing biomolecules. Assays of 32 P i uptake performed in the absence or in the presence of Na + indicated the existence of a Na + -dependent and a Na + -independent P i transporter in Phytomonas serpens. Phylogenetic analysis of two hypothetical protein sequences of Phytomonas (EM1) showed similarities to the high-affinity P i transporters of Saccharomyces cerevisiae: Pho84, a Na + -independent P i transporter, and Pho89, a Na + -dependent P i transporter. Plasma membrane depolarization by FCCP, an H + ionophore, strongly decreased P i uptake via both Na + -independent and Na + -dependent carriers, indicating that a membrane potential is essential for P i influx. In addition, the furosemide-sensitive Na + -pump activity in the cells grown in low P i conditions was found to be higher than the activity detected in the plasma membrane of cells cultivated at high P i concentration, suggesting that the up-regulation of the Na + -ATPase pump could be related to the increase of P i uptake by the Pho89p Na + :P i symporter. Here we characterize for the first time two inorganic phosphate transporters powered by Na + and H + gradients and activated by low P i availability in the phytopathogen P. serpens. Copyright © 2016 Elsevier Inc. All rights reserved.

Bacteriostatic action of streptomycin on ribosomally resistant mutants (rpsL) of Salmonella typhimurium.

PubMed Central

Fernández, R O; Antón, D N

1987-01-01

Incubation of streptomycin-resistant (rpsL) mutants of Salmonella typhimurium in alkaline nutrient medium containing streptomycin brought about an inhibition of cell growth that was readily reversed by removing the antibiotic or neutralizing the medium. Growth inhibition was maximal at pH 8.2 and a streptomycin concentration of 800 micrograms/ml. A similar amount of dihydrostreptomycin had a negligible effect, and 10-times-higher concentrations of this antibiotic were required to reproduce the streptomycin action. Addition of streptomycin (400 micrograms/ml) to rpsL cells in alkaline (pH 8.2) nutrient medium caused inhibition of protein and DNA synthesis and also, but to a lower degree, of RNA synthesis. This effect on macromolecular synthesis was not due to ATP deprivation, since ATP content rose after addition of the antibiotic. At pH 8.2, the rate of entrance of streptomycin increased fourfold with respect to the rate at pH 7.0, leading to a large accumulation of streptomycin into rpsL cells. Uptake of the antibiotic was halted by addition of KCN or chloramphenicol. Equal uptake was obtained with 800 micrograms of dihydrostreptomycin or 400 micrograms of streptomycin per ml, yet the former did not affect cell growth at that concentration. It is concluded that high pH stimulates streptomycin and dihydrostreptomycin uptake by rpsL strains but only streptomycin accumulation causes growth inhibition in cells lacking the high-affinity ribosomal site. PMID:2449121

Interaction of Monobenzamidine-Linked Trypanocides with the Trypanosoma brucei P2 Aminopurine Transporter

PubMed Central

Stewart, Mhairi L.; Boussard, Cyrille; Brun, Reto; Gilbert, Ian H.; Barrett, Michael P.

2005-01-01

Single benzamidine group-carrying compounds were shown to interact with the Trypanosoma brucei P2 aminopurine transporter. Replacement of the amidine with a guanidine group decreased affinity. Trypanocidal activity was evident, but compounds were equally toxic against trypanosomes lacking the P2 transporter, which indicates additional uptake routes for monobenzamidine-derived compounds. PMID:16304196

Effect of hot water extracted hardwood and softwood chips on particleboard properties

Treesearch

Manuel Raul Pelaez-Samaniego; Vikram Yadama; Tsai Garcia-Perez; Eini Lowell; Thomas Amidon

2014-01-01

The affinity of particleboard (PB) to water is one of the main limitations for using PB in moisture-rich environments. PB dimensional stability and durability can be improved by reducing the available hydroxyl groups in wood through hemicellulose removal, for example, by hot water extraction (HWE), which increases wood resistance to moisture uptake. The resulting...

44Sc-DOTA-BN[2-14]NH2 in comparison to 68Ga-DOTA-BN[2-14]NH2 in pre-clinical investigation. Is 44Sc a potential radionuclide for PET?

PubMed

Koumarianou, E; Loktionova, N S; Fellner, M; Roesch, F; Thews, O; Pawlak, D; Archimandritis, S C; Mikolajczak, R

2012-12-01

In the present study we demonstrate the in vitro and in vivo comparison of the (44)Sc and (68)Ga labeled DOTA-BN[2-14]NH(2). (44)Sc is a positron emitter with a half life of 3.92 h. Hence it could be used for PET imaging with ligands requiring longer observation time than in the case of (68)Ga. The binding affinity of (nat)Sc-DOTA-BN[2-14]NH(2) and (nat)Ga-DOTA-BN[2-14]NH(2) to GRP receptors was studied in competition to [(125)I-Tyr(4)]-Bombesin in the human prostate cancer cell line PC-3. A preliminary biodistribution in normal rats was performed, while first microPET images were assessed in male Copenhagen rats bearing the androgen-independent Dunning R-3327-AT-1 prostate cancer tumor. The affinity to GRP receptors in the PC-3 cell line was higher for (nat)Ga-DOTA-BN[2-14]NH(2) (IC(50)(nM)=0.85 ± 0.06) than that of (nat)Sc-DOTA-BN[2-14]NH(2) (IC(50) (nM)=6.49 ± 0.13). The internalization rate of (68)Ga labeled DOTA-BN[2-14]NH(2) was slower than that of (44)Sc, but their final internalization percents were comparable. (68)Ga-DOTA-BN[2-14]NH(2) was externalized faster than (44)Sc-DOTA-BN[2-14]NH(2). The biodistribution of (44)Sc-DOTA-BN[2-14]NH(2) and (68)Ga-DOTA-BN[2-14]NH(2) in normal rats revealed a higher uptake in target organs and tissues of the first one while both excreted mainly through urinary tract. In microPET images both tracers were accumulated in the tumor with similar uptake patterns. Despite the differences in the receptor affinity both the (68)Ga- and the (44)Sc-labeled DOTA-BN[2-14]NH(2) tracers showed comparable distribution and similar time constants of uptake and elimination. Moreover no differences in tumor accumulation (neither in the overall uptake nor in the dynamics) were observed from the microPet imaging. From that perspective the use of either (44)Sc or (68)Ga for detecting tumors with GRP receptors is equivalent. Copyright © 2012 Elsevier Ltd. All rights reserved.

Lectins as endocytic ligands: an assessment of lectin binding and uptake to rabbit conjunctival epithelial cells.

PubMed

Qaddoumi, Mohamed; Lee, Vincent H L

2004-07-01

To investigate the binding and uptake pattern of three plant lectins in rabbit conjunctival epithelial cells (RCECs) with respect to their potential for enhancing cellular macromolecular uptake. Three fluorescein-labeled plant lectins (Lycoperison esculentum, TL; Solanum tuberosum, STL; and Ulex europaeus 1, UEA-1) were screened with respect to time-, concentration-, and temperature-dependent binding and uptake. Chitin (30 mg/ml) and L-alpha-fucose (10 mM) were used as inhibitory sugars to correct for nonspecific binding of TL or STL and UEA-1, respectively. Confocal microscopy was used to confirm internalization of STL. The binding and uptake of all three lectins in RCECs was time-dependent (reaching a plateau at 1-2 h period) and saturable at 1-h period. The rank order of affinity constants (km) was STL>TL>UEA-1 with values of 0.39>0.48>4.81 microM, respectively. However, maximal, specific binding/uptake potential was in the order UEA-1>STL>TL with values of 53.7, 52.3, and 15.0 nM/mg of cell protein, respectively. Lectins showed temperature dependence in their uptake, with STL exhibiting the highest endocytic capacity. Internalized STL was visualized by confocal microscopy to be localized to the cell membrane and cytoplasm. Based on favorable binding and uptake characteristics, potato lectin appears to be a useful candidate for further investigation as an ocular drug delivery system.

No-carrier-added [.sup.18 F]-N-fluoroalkylspiroperidols

DOEpatents

Shiue, Chyng-Yann; Wolf, Alfred P.; Bai, Lan-Qin; Teng, Ren-Tui

1989-01-01

There is disclosed radioligands labeled with the position emitting radionuclide [.sup.18 F] suitable for dynamic study in living humans with position emission transaxial tomography. These new [.sup.18 F]-N-fluoroalkylspiroperidols, wherein the alkyl group contains from 2-6 carbon atoms, exhibit extremely high affinity for the dopamine receptors and provide enhanced uptake and retention in the brain concomitant with reduced radiation burden. These characteristics all combine to make these new radioligands useful for mapping dopamine receptors in normal and disease states in the living brain. Additionally, a new synthetic procedure for these radioligands as well as a new procedure for preparing the radiolabeled alkyl halide alkylating reagents are also disclosed.

Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2.

PubMed

Barrett, M P; Tetaud, E; Seyfang, A; Bringaud, F; Baltz, T

1995-12-15

The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.

Gas Phase Sensing of Alcohols by Metal Organic Framework–Polymer Composite Materials

PubMed Central

2017-01-01

Affinity layers play a crucial role in chemical sensors for the selective and sensitive detection of analytes. Here, we report the use of composite affinity layers containing Metal Organic Frameworks (MOFs) in a polymeric matrix for sensing purposes. Nanoparticles of NH2-MIL-53(Al) were dispersed in a Matrimid polymer matrix with different weight ratios (0–100 wt %) and drop-casted on planar capacitive transducer devices. These coated devices were electrically analyzed using impedance spectroscopy and investigated for their sensing properties toward the detection of a series of alcohols and water in the gas phase. The measurements indicated a reversible and reproducible response in all devices. Sensor devices containing 40 wt % NH2-MIL-53(Al) in Matrimid showed a maximum response for methanol and water. The sensor response time slowed down with increasing MOF concentration until 40 wt %. The half time of saturation response (τ0.5) increased by ∼1.75 times for the 40 wt % composition compared to devices coated with Matrimid only. This is attributed to polymer rigidification near the MOF/polymer interface. Higher MOF loadings (≥50 wt %) resulted in brittle coatings with a response similar to the 100 wt % MOF coating. Cross-sensitivity studies showed the ability to kinetically distinguish between the different alcohols with a faster response for methanol and water compared to ethanol and 2-propanol. The observed higher affinity of the pure Matrimid polymer toward methanol compared to water allows also for a higher uptake of methanol in the composite matrices. Also, as indicated by the sensing studies with a mixture of water and methanol, the methanol uptake is independent of the presence of water up to 6000 ppm of water. The NH2-MIL-53(Al) MOFs dispersed in the Matrimid matrix show a sensitive and reversible capacitive response, even in the presence of water. By tuning the precise compositions, the affinity kinetics and overall affinity can be tuned, showing the promise of this type of chemical sensors. PMID:28440621

Design and Evaluation of New Tc-99m-Labeled Lactam Bridge-Cyclized Alpha-MSH Peptides for Melanoma Imaging

PubMed Central

Guo, Haixun; Gallazzi, Fabio; Miao, Yubin

2013-01-01

The purpose of this study was to examine the melanoma targeting and imaging properties of new 99mTc-labeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (α-MSH) peptides using bifunctional chelating agents. MAG3-GGNle-CycMSHhex, AcCG3-GGNle-CycMSHhex and HYNIC-GGNle-CycMSHhex peptides were synthesized and their melanocortin-1 (MC1) receptor binding affinities were determined in B16/F1 melanoma cells. The biodistribution of 99mTc-MAG3-GGNle-CycMSHhex, 99mTc-AcCG3-GGNle-CycMSHhex, 99mTc(CO)3-HYNIC-GGNle-CycMSHhex and 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice at 2 h post-injection to select a lead peptide for further evaluation. The melanoma targeting and imaging properties of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex were further examined because of its high melanoma uptake and fast urinary clearance. The IC50 values of MAG3-GGNle-CycMSHhex, AcCG3-GGNle-CycMSHhex and HYNIC-GGNle-CycMSHhex were 1.0 ± 0.05, 1.2 ± 0.19 and 0.6 ± 0.04 nM in B16/F1 melanoma cells, respectively. Among these four 99mTc-peptides, 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex exhibited the highest melanoma uptake (14.14 ± 4.90% ID/g) and fastest urinary clearance (91.26 ± 1.96% ID) at 2 h post-injection. 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex showed high tumor to normal organ uptake ratios except for the kidneys. The tumor/kidney uptake ratios of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex were 2.50 and 3.55 at 4 and 24 h post-injection. The melanoma lesions were clearly visualized by SPECT/CT using 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex as an imaging probe at 2 h post-injection. Overall, high melanoma uptake coupled with fast urinary clearance of 99mTc(EDDA)-HYNIC-GGNle-CycMSHhex highlighted its potential for metastatic melanoma detection in the future. PMID:23418722

Arsenate transport by sodium/phosphate cotransporter type IIb

DOE Office of Scientific and Technical Information (OSTI.GOV)

Villa-Bellosta, Ricardo, E-mail: rvilla@unizar.e; Sorribas, Victor, E-mail: sorribas@unizar.e

2010-08-15

Arsenic is a metalloid that causes the dysfunction of critical enzymes, oxidative stress, and malignancies. In recent years several transporters of As{sup III} have been identified, including aquaglyceroporins (AQP) and multidrug resistance proteins (MRP). As{sup V} transport, however, has not been sufficiently studied because it has been assumed that arsenate is taken up by mammalian cells through inorganic phosphate (Pi) transporters. In this paper we have analyzed the role of Pi transporters in the uptake of arsenate by directly using {sup 73}As{sup V} as a radiotracer in phosphate transporter-expressing Xenopus laevis oocytes. The affinities of Pi transporters for H{sub 3}AsO{submore » 4} were lower than the affinities for Pi. NaPiIIa, NaPiIIc, Pit1, and Pit2 showed a K{sub m} for arsenate that was > 1 mM (i.e., at least ten times lower than the affinities for Pi). The NaPiIIb isoform showed the highest affinity for As{sup V} in mouse (57 {mu}M), rat (51 {mu}M), and human (9.7 {mu}M), which are very similar to the affinities for Pi. Therefore, NaPiIIb can have a prominent role in the toxicokinetics of arsenic following oral exposure to freshwater or food contaminated with As{sup V}.« less

Monoamine transporter and receptor interaction profiles of a new series of designer cathinones.

PubMed

Simmler, L D; Rickli, A; Hoener, M C; Liechti, M E

2014-04-01

Psychoactive β-keto amphetamines (cathinones) are sold as "bath salts" or "legal highs" and recreationally abused. We characterized the pharmacology of a new series of cathinones, including methedrone, 4-methylethcathinone (4-MEC), 3-fluoromethcathinone (3-FMC), pentylone, ethcathinone, buphedrone, pentedrone, and N,N-dimethylcathinone. We investigated norepinephrine (NE), dopamine (DA), and serotonin (5-HT) uptake inhibition using human embryonic kidney 293 (HEK 293) cells that express the respective human monoamine transporter, the drug-induced efflux of NE, DA, and 5-HT from monoamine-preloaded cells, and binding affinity to monoamine transporters and receptors. All of the cathinones were potent NE uptake inhibitors but differed in their DA vs. 5-HT transporter inhibition profiles and monoamine release effects. Methedrone was a more potent 5-HT than DA transporter inhibitor and released NE and 5-HT similar to para-methoxymethamphetamine (PMMA), para-methoxyamphetamine (PMA), 4-methylthioamphetamine (4-MTA), and 3,4-methylenedioxymethamphetamine (MDMA). 4-MEC and pentylone equipotently inhibited all of the monoamine transporters and released 5-HT. Ethcathinone and 3-FMC inhibited NE and DA uptake and released NE, and 3-FMC also released DA similar to N-ethylamphetamine and methamphetamine. Pentedrone and N,N-dimethylcathinone were non-releasing NE and DA uptake inhibitors as previously shown for pyrovalerone cathinones. Buphedrone preferentially inhibited NE and DA uptake and also released NE. None of the cathinones bound to rodent trace amine-associated receptor 1, in contrast to the non-β-keto-amphetamines. None of the cathinones exhibited relevant binding to other monoamine receptors. In summary, we found considerable differences in the monoamine transporter interaction profiles among different cathinones and compared with related amphetamines. Copyright © 2013 Elsevier Ltd. All rights reserved.

Synthesis and evaluation of novel radioiodinated nicotinamides for malignant melanoma.

PubMed

Liu, Xiang; Pham, Tien Q; Berghofer, Paula; Chapman, Janette; Greguric, Ivan; Mitchell, Peter; Mattner, Filomena; Loc'h, Christian; Katsifis, Andrew

2008-10-01

A series of iodonicotinamides based on the melanin-binding iodobenzamide compound N-2-diethylaminoethyl-4-iodobenzamide was prepared and evaluated for the potential imaging and staging of disseminated metastatic melanoma. [(123)I]Iodonicotinamides were prepared by iododestannylation reactions using no-carrier-added iodine-123 and evaluated in vivo by biodistribution and competition studies and by single photon emission computed tomography (SPECT) imaging in black and albino nude mice bearing B16F0 murine melanotic and A375 human amelanotic melanoma tumours, respectively. The iodonicotinamides displayed low-affinity binding for sigma(1)-sigma(2) receptors (K(i)>300 nM). In biodistribution studies in mice, N-(2-(diethylamino)ethyl)-5-[(123)I]iodonicotinamide ([(123)I]1) exhibited the fastest and highest uptake of the nicotinamide series in the B16F0 tumour at 1 h ( approximately 8% ID/g), decreasing slowly over time. No uptake was observed in the A375 tumour. Clearance from the animals by urinary excretion was more rapid for N-alkyl-nicotinamides than for piperazinyl derivatives. At 1 h postinjection, the urinary excretion was 66% ID for [(123)I]1, while the gastrointestinal tract amounted to 17% ID. Haloperidol was unable to reduce the uptake of [(123)I]1 in pigmented mice, indicating that this uptake was likely due to an interaction with melanin. SPECT imaging of [(123)I]1 in black mice bearing the B16F0 melanoma indicated that the radioactivity was predominately located in the tumour and eyes. No specific localisation was observed in nude mice bearing A375 amelanotic tumours. These findings suggest that [(123)I]1, which displays high tumour uptake with rapid clearance from the body, could be a promising imaging agent for the detection of melanotic tumours.

Small substrate transport and mechanism of a molybdate ATP binding cassette transporter in a lipid environment.

PubMed

Rice, Austin J; Harrison, Alistair; Alvarez, Frances J D; Davidson, Amy L; Pinkett, Heather W

2014-05-23

Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Centrifuge-induced hypergravity: [ 3H]GABA and L-[ 14C]glutamate uptake, exocytosis and efflux mediated by high-affinity, sodium-dependent transporters

NASA Astrophysics Data System (ADS)

Borisova, T. A.; Himmelreich, N. H.

The effects of centrifuge-induced hypergravity on the presynaptic events have been investigated in order to provide further insight into regulation of glutamate and GABA neurotransmission and correlation between excitatory and inhibitory responses under artificial gravity conditions. Exposure of animals to hypergravity (centrifugation of rats at 10 G for 1 h) has been found to cause changes in the synaptic processes of brain, in particular neurotransmitter release and uptake in rat brain synaptosomes. Hypergravity loading resulted in more than two-fold enhancement of GABA transporter activity ( Vmax increased from 1.4 ± 0.3 nmol/min/mg of protein in the control group to 3.3 ± 0.59 nmol/min/mg of protein for the animals exposed to hypergravity ( P ⩽ 0.05)). The maximal velocity of L-[ 14C]glutamate uptake decreased from 12.5 ± 3.2 to 5.6 ± 0.9 nmol/min/mg of protein under artificial gravity conditions. Depolarization-evoked exocytotic release of the neurotransmitters has also changed in response to hypergravity. It increased for GABA (7.2 ± 0.54% and 11.74 ± 1.2% of total accumulated label for control and hypergravity, respectively ( P ⩽ 0.05)), but reduced for glutamate (14.4 ± 0.7% and 6.2 ± 1.9%, for control and hypergravity, respectively). Thus, comparative analysis of the neurotransmitter uptake and release has demonstrated that short-term centrifuge-induced 10 G hypergravity loading intensified inhibitory and attenuated excitatory processes in nerve terminals. The activation or reduction of neurotransmitter uptake appeared to be coupled with similarly directed alterations of the neurotransmitter release.

A novel Tc-99m and fluorescence-labeled arginine-arginine-leucine-containing peptide as a multimodal tumor imaging agent in a murine tumor model.

PubMed

Kim, Myoung Hyoun; Kim, Seul-Gi; Kim, Dae-Weung

2018-06-15

We developed a Tc-99m and TAMRA-labeled peptide, Tc-99m arginine-arginine-leucine (RRL) peptide (TAMRA-GHEG-ECG-RRL), to target tumor cells and evaluated the diagnostic performance of Tc-99m TAMRA-GHEG-ECG-RRL as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-RRL was synthesized using Fmoc solid-phase peptide synthesis. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with PC-3 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-RRL complexes were prepared in high yield (>96%). The K d of Tc-99m TAMRA-GHEG-ECG-RRL determined by saturation binding was 41.7 ± 7.8 nM. Confocal microscopy images of PC-3 cells incubated with TAMRA-GHEG-ECG-RRL showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of RRL. Specific uptake of Tc-99m TAMRA-GHEG-ECG-RRL was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In conclusion, in vivo and in vitro studies revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tc-99m TAMRA-GHEG-ECG-RRL has potential as a dual-modality tumor imaging agent. Copyright © 2018 John Wiley & Sons, Ltd.

Synthesis and evaluation of Tc-99m and fluorescence-labeled elastin-derived peptide, VAPG for multimodal tumor imaging in murine tumor model.

PubMed

Kim, Myoung Hyoun; Kim, Chang Guhn; Kim, Seul-Gi; Kim, Dae-Weung

2017-12-01

We developed a Tc-99m and fluorescence-labeled peptide, Tc-99m TAMRA-GHEG-ECG-VAPG to target tumor cells and evaluated the diagnostic performance as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-VAPG was synthesized by using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-VAPG with Tc-99m was done by using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with SW620 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry by using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-VAPG complexes were prepared in high yield (>96%). The K d of Tc-99m TAMRA-GHEG-ECG-VAPG determined by saturation binding was 16.8 ± 3.6 nM. Confocal microscopy images of SW620 cells incubated with TAMRA-GHEG-ECG-VAPG showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-VAPG in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of VAPG. Specific uptake of Tc-99m TAMRA-GHEG-ECG-VAPG was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In vivo and in vitro studies revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-VAPG in tumor cells. Tc-99m TAMRA-GHEG-ECG-VAPG has potential as a dual-modality tumor imaging agent. Copyright © 2017 John Wiley & Sons, Ltd.

Synthesis and evaluation of Tc-99m-labeled RRL-containing peptide as a non-invasive tumor imaging agent in a mouse fibrosarcoma model.

PubMed

Kim, Dae-Weung; Kim, Woo Hyoung; Kim, Myoung Hyoun; Kim, Chang Guhn

2015-11-01

Arginine-arginine-leucine (RRL) is considered a tumor endothelial cell-specific binding sequence. RRL-containing peptide targeting tumor vessels is an excellent candidate for tumor imaging. In this study, we developed RRL-containing hexapeptides and evaluated their feasibility as a tumor imaging agent in a HT-1080 fibrosarcoma-bearing murine model. The hexapeptide, glutamic acid-cysteine-glycine (ECG)-RRL was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling efficiency was evaluated using instant thin-layer chromatography. Uptake of Tc-99m ECG-RRL within HT-1080 cells was evaluated in vitro by confocal microscopy and cellular binding affinity was calculated. Gamma images were acquired In HT-1080 fibrosarcoma tumor-bearing mice, and the tumor-to-muscle uptake ratio was calculated. The inflammatory-to-normal muscle uptake ratio was also calculated in an inflammation mouse model. A biodistribution study was performed to calculate %ID/g. A high yield of Tc-99m ECG-RRL complexes was prepared after Tc-99m radiolabeling. Binding of Tc-99m ECG-RRL to tumor cells had was confirmed by in vitro studies. Gamma camera imaging in the murine model showed that Tc-99m ECG-RRL accumulated substantially in the subcutaneously engrafted tumor and that tumoral uptake was blocked by co-injecting excess RRL. Moreover, Tc-99m ECG-RRL accumulated minimally in inflammatory lesions. We successfully developed Tc-99m ECG-RRL as a new tumor imaging candidate. Specific tumoral uptake of Tc-99m ECG-RRL was evaluated both in vitro and in vivo, and it was determined to be a good tumor imaging candidate. Additionally, Tc-99m ECG-RRL effectively distinguished between cancerous tissue and inflammatory lesions.

Hepatic uptake and biliary excretion of manganese in the little skate, Leucoraja erinacea.

PubMed

Madejczyk, Michael S; Boyer, James L; Ballatori, Nazzareno

2009-05-01

The liver is a major organ involved in regulating whole body manganese (Mn) homeostasis; however, the mechanisms of Mn transport across the hepatocyte basolateral and canalicular membranes remain poorly defined. To gain insight into these transport steps, the present study measured hepatic uptake and biliary excretion of Mn in an evolutionarily primitive marine vertebrate, the elasmobranch Leucoraja erinacea, the little skate. Mn was rapidly removed from the recirculating perfusate of isolated perfused skate livers in a dose-dependent fashion; however, only a small fraction was released into bile (<2% in 6 h). Mn was also rapidly taken up by freshly isolated skate hepatocytes in culture. Mn uptake was inhibited by a variety of divalent metals, but not by cesium. Analysis of the concentration-dependence of Mn uptake revealed of two components, with apparent K(m) values 1.1+/-0.1 microM and 112+/-29 microM. The K(m) value for the high-affinity component was similar to the measured skate blood Mn concentration, 1.9+/-0.5 microM. Mn uptake was reduced by nearly half when bicarbonate was removed from the culture medium, but was unaffected by a change in pH from 6.5 to 8.5, or by substitution of Na with Li or K. Mn efflux from the hepatocytes was also rapid, and was inhibited when cells were treated with 0.5 mM 2,4-dinitrophenol to deplete ATP levels. These data indicate that skate liver has efficient mechanisms for removing Mn from the sinusoidal circulation, whereas overall biliary excretion is low and appears to be mediated in part by an ATP-sensitive mechanism.

Hepatic uptake and biliary excretion of manganese in the little skate, Leucoraja erinacea

PubMed Central

Madejczyk, Michael S.; Boyer, James L.; Ballatori, Nazzareno

2008-01-01

The liver is a major organ involved in regulating whole body manganese (Mn) homeostasis; however, the mechanisms of Mn transport across the hepatocyte basolateral and canalicular membranes remain poorly defined. To gain insight into these transport steps, the present study measured hepatic uptake and biliary excretion of Mn in an evolutionarily primitive marine vertebrate, the elasmobranch Leucoraja erinacea, the little skate. Mn was rapidly removed from the recirculating perfusate of isolated perfused skate livers in a dose-dependent fashion; however, only a small fraction was released into bile (<2% in 7h). Mn was also rapidly taken up by freshly isolated skate hepatocytes in culture. Mn uptake was inhibited by a variety of divalent metals, but not by cesium. Analysis of the concentration-dependence of Mn uptake revealed of two components, with apparent Km values 1.1 ± 0.1 μM and 112 ± 29 μM. The Km value for the high-affinity component was similar to the measured skate blood Mn concentration, 1.9 ± 0.5 μM. Mn uptake was reduced by nearly half when bicarbonate was removed from the culture medium, but was unaffected by a change in pH from 6.5 to 8.5, or by substitution of Na with Li or K. Mn efflux from the hepatocytes was also rapid, and was inhibited when cells were treated with 0.5 mM 2,4-dinitrophenol to deplete ATP levels. These data indicate that skate liver has efficient mechanisms for removing Mn from the sinusoidal circulation, whereas overall biliary excretion is low and appears to be mediated in part by an ATP-sensitive mechanism. PMID:19141331

Noninvasive positron emission tomography imaging of cell death using a novel small-molecule probe, (18)F labeled bis(zinc(II)-dipicolylamine) complex.

PubMed

Wang, Hongliang; Tang, Xiaolan; Tang, Ganghua; Huang, Tingting; Liang, Xiang; Hu, Kongzhen; Deng, Huaifu; Yi, Chang; Shi, Xinchong; Wu, Kening

2013-08-01

The synthetic bis(zinc(II)-dipicolylamine) (DPAZn2) coordination complexes are known to have a high specific and selective affinity to target the exposed phosphatidylserine (PS) on the surface of dead and dying cells. An (18)F-labeled DPAZn2 complex (4-(18)F-Fluoro-benzoyl-bis(zinc(II)-dipicolylamine), (18)F-FB-DPAZn2) as positron emission tomography (PET) tracer was developed and evaluated for in vivo imaging of tumor treated with a chemical agent. The in vitro cell stain studies revealed that fluorescent DPAZn2 complexes (Dansyl-DPAZn2) stained the same cells (apoptotic and necrotic cells) as fluorescein isothiocyanate (FITC) labeled Annexin V (FITC-Annexin V). The radiosynthesis of (18)F-FB-DPAZn2 was achieved through the amidation the precursor bis(2,2'-dipicolylamine) derivative (DPA2) with the prosthetic group N-succinimidyl-4-[(18)F]-fluorobenzoate ((18)F-SFB) and chelation with zinc nitrate. In the biodistribution study, the fast clearance of (18)F-FB-DPAZn2 from blood and kidney was observed and high uptake in liver and intestine within 90 min postinjection was also found. For the PET imaging, significantly higher tumor uptake of (18)F-FB-DPAZn2 was observed in the adriamycin (ADM)-treated Hepa1-6 hepatocellular carcinoma-bearing mice than that in the untreated tumor-model mice, while a slightly decreased tumor uptake of (18)F-FDG was found in the ADM-treated tumor-bearing mice. The results indicate that (18)F-FB-DPAZn2 has the similar capability of apoptosis detection as FITC-Annexin V and seems to be a potential PET tracer for noninvasive evaluation and monitoring of anti-tumor chemotherapy. The high uptake of (18)F-FB-DPAZn2 in the abdomen needs to optimize the structure for improving its pharmacokinetics characteristics in the future work.

Reexamining the role of choline transporter-like (Ctlp) proteins in choline transport.

PubMed

Zufferey, Rachel; Santiago, Teresa C; Brachet, Valerie; Ben Mamoun, Choukri

2004-02-01

In Saccharomyces cerevisiae, choline enters the cell via a single high-affinity transporter, Hnmlp. hnm1delta cells lacking HNM1 gene are viable. However, they are unable to transport choline suggesting that no additional active choline transporters are present in this organism. A complementation study of a choline auxotrophic mutant, ctrl-ise (hnm1-ise), using a cDNA library from Torpedo marmorata electric lobe identified a membrane protein named Torpedo marmorata choline transporter-like, tCtl1p. tCtllp was proposed to mediate a high-affinity choline transport (O'Regan et al., 1999, Proc. Natl. Acad. Sci.). Homologs of tCtl1p have been identified in other organisms, including yeast (Pns1p, YOR161c) and are postulated to function as choline transporters. Here we provide several lines of evidence indicating that Ctlp proteins are not involved in choline transport. Loss of PNS1 has no effect on choline transport and overexpression of either PNS1 or tCTL1 does not restore choline uptake activity of choline transport-defective mutants. The data presented here call into question the role of proteins of the CTL family in choline transport and suggest that the mechanism by which tCTL1 complements hnm1-ise mutant is independent of its ability to transport choline.

Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

PubMed Central

Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

2013-01-01

Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (K D values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

Epigallocatechin-3-gallate (EGCG)-stabilized selenium nanoparticles coated with Tet-1 peptide to reduce amyloid-β aggregation and cytotoxicity.

PubMed

Zhang, Jingnan; Zhou, Xianbo; Yu, Qianqian; Yang, Licong; Sun, Dongdong; Zhou, Yanhui; Liu, Jie

2014-06-11

Alzheimer's disease (AD), the most common neurodegenerative disease, is caused by an accumulation of amyloid-β (Aβ) plaque deposits in the brains. Evidence is increasingly showing that epigallocatechin-3-gallate (EGCG) can partly protect cells from Aβ-mediated neurotoxicity by inhibiting Aβ aggregation. In order to better understand the process of Aβ aggregation and amyloid fibril disaggregation and reduce the cytotoxicity of EGCG at high doses, we attached EGCG onto the surface of selenium nanoparticles (EGCG@Se). Given the low delivery efficiency of EGCG@Se to the targeted cells and the involvement of selenoprotein in antioxidation and neuroprotection, which are the key factors for preventing the onset and progression of AD, we synthesized EGCG-stabilized selenium nanoparticles coated with Tet-1 peptide (Tet-1-EGCG@Se, a synthetic selenoprotein analogue), considering the affinity of Tet-1 peptide to neurons. We revealed that Tet-1-EGCG@Se can effectively inhibit Aβ fibrillation and disaggregate preformed Aβ fibrils into nontoxic aggregates. In addition, we found that both EGCG@Se and Tet-1-EGCG@Se can label Aβ fibrils with a high affinity, and Tet-1 peptides can significantly enhance the cellular uptake of Tet-1-EGCG@Se in PC12 cells rather than in NIH/3T3 cells.

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

USDA-ARS?s Scientific Manuscript database

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

In vitro evaluation of (99m)Tc-EDDA/tricine-HYNIC-Q-Litorin in gastrin-releasing peptide receptor positive tumor cell lines.

PubMed

Yurt Lambrecht, Fatma; Durkan, Kübra; Ozgür, Aykut; Gündüz, Cumhur; Avcı, Cığır Biray; Susluer, Sunde Yılmaz

2013-05-01

Bombesin and its derivatives exhibit a high affinity for gastrin-releasing peptide receptor (GRPr), which is over-expressed in a variety of human cancers (prostate, pancreatic, lung, etc.). The aim of this study was to investigate the in vitro potential of the hydrazinonicotinamide (HYNIC)-Q-Litorin. (99m)Tc labeling was performed by using different co-ligands: tricine and ethylenediamine diacetic acid (EDDA). The radiochemical stability of radiolabeled peptide conjugates was checked at room temperature and in cysteine solution up to 24 h. The in vitro cell uptake of (99m)Tc-EDDA-HYNIC-Q-Litorin and (99m)Tc-tricine-HYNIC-Q-Litorin were evaluated on pancreatic tumor and control cell lines. Optimum specific activity and incubation time were determined for all the cell lines. The results showed that the cell uptake of the radiolabeled peptide conjugates in tumor cell lines were higher than in the control cell line. The findings of this study indicated the need for further development of in vivo study as a radiopharmaceutical for pancreatic tumor imaging.

Crystallization and preliminary X-ray diffraction analysis of the phosphate-binding protein PhoX from Xanthomonas citri

PubMed Central

Pegos, Vanessa R.; Medrano, Francisco Javier; Balan, Andrea

2014-01-01

Xanthomonas axonopodis pv. citri (X. citri) is an important bacterium that causes citrus canker disease in plants in Brazil and around the world, leading to significant economic losses. Determination of the physiology and mechanisms of pathogenesis of this bacterium is an important step in the development of strategies for its containment. Phosphate is an essential ion in all microrganisms owing its importance during the synthesis of macromolecules and in gene and protein regulation. Interestingly, X. citri has been identified to present two periplasmic binding proteins that have not been further characterized: PstS, from an ATP-binding cassette for high-affinity uptake and transport of phosphate, and PhoX, which is encoded by an operon that also contains a putative porin for the transport of phosphate. Here, the expression, purification and crystallization of the phosphate-binding protein PhoX and X-ray data collection at 3.0 Å resolution are described. Biochemical, biophysical and structural data for this protein will be helpful in the elucidation of its function in phosphate uptake and the physiology of the bacterium. PMID:25484207

Diversity and Evolutionary History of Iron Metabolism Genes in Diatoms

PubMed Central

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

2015-01-01

Ferroproteins arose early in Earth’s history, prior to the emergence of oxygenic photosynthesis and the subsequent reduction of bioavailable iron. Today, iron availability limits primary productivity in about 30% of the world’s oceans. Diatoms, responsible for nearly half of oceanic primary production, have evolved molecular strategies for coping with variable iron concentrations. Our understanding of the evolutionary breadth of these strategies has been restricted by the limited number of species for which molecular sequence data is available. To uncover the diversity of strategies marine diatoms employ to meet cellular iron demands, we analyzed 367 newly released marine microbial eukaryotic transcriptomes, which include 47 diatom species. We focused on genes encoding proteins previously identified as having a role in iron management: iron uptake (high-affinity ferric reductase, multi-copper oxidase, and Fe(III) permease); iron storage (ferritin); iron-induced protein substitutions (flavodoxin/ferredoxin, and plastocyanin/cytochrome c6) and defense against reactive oxygen species (superoxide dismutases). Homologs encoding the high-affinity iron uptake system components were detected across the four diatom Classes suggesting an ancient origin for this pathway. Ferritin transcripts were also detected in all Classes, revealing a more widespread utilization of ferritin throughout diatoms than previously recognized. Flavodoxin and plastocyanin transcripts indicate possible alternative redox metal strategies. Predicted localization signals for ferredoxin identify multiple examples of gene transfer from the plastid to the nuclear genome. Transcripts encoding four superoxide dismutase metalloforms were detected, including a putative nickel-coordinating isozyme. Taken together, our results suggest that the majority of iron metabolism genes in diatoms appear to be vertically inherited with functional diversity achieved via possible neofunctionalization of paralogs. This refined view of iron use strategies in diatoms elucidates the history of these adaptations, and provides potential molecular markers for determining the iron nutritional status of different diatom species in environmental samples. PMID:26052941

Hypoxia modifies nuclear calcium uptake pathways in the cerebral cortex of the guinea-pig fetus.

PubMed

Zanelli, S A; Spandou, E; Mishra, O P; Delivoria-Papadopoulos, M

2005-01-01

Nuclear Ca2+ signals are thought to play a critical role in the initiation and progression of programmed cell death. The present study tests the hypothesis that hypoxia alters nuclear Ca2+ transport pathways and leads to an increase in nuclear Ca(2+)-influx in cerebral cortical neuronal nuclei. To test this hypothesis the effect of tissue hypoxia on high affinity Ca(2+)-ATPase activity and the binding characteristics of inositol 1,4,5-triphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) receptors were studied in neuronal nuclei from the cerebral cortex of guinea-pig fetuses. Results show increased high-affinity Ca(2+)-ATPase activity (nmol/mg protein/h) in the hypoxic group 969.7+/-79 as compared with 602.4+/-90.9 in the normoxic group, P<0.05. The number of IP3 receptors (Bmax, fmol/mg protein) increased from 61+/-21 in the normoxic group to 164+/-49 in the hypoxic group, P<0.05. K(d) values did not change following hypoxia. In contrast, IP4 receptor Bmax (fmol/mg protein) and K(d) (nM) values increased from 360+/-32 in the normoxic group to 626+/-136 in the hypoxic group (P<0.001) and, from 26+/-1 in the normoxic group to 61+/-9 in the hypoxic group (P<0.001), respectively. 45Ca(2+)-influx (pmol/mg protein) significantly increased from 6.3+/-1.9 in the normoxic group to 10.9+/-1.1 the hypoxic group (P<0.001). The data show that hypoxia modifies nuclear Ca2+ transport pathways and results in increased nuclear Ca(2+)-influx. We speculate that hypoxia increases nuclear Ca2+ uptake from the cytoplasm to the nucleoplasm, resulting in increased transcription of proapoptotic genes and subsequent activation of programmed cell death pathways.

Amino-Terminal β-Amyloid Antibody Blocks β-Amyloid-Mediated Inhibition of the High-Affinity Choline Transporter CHT.

PubMed

Cuddy, Leah K; Seah, Claudia; Pasternak, Stephen H; Rylett, R Jane

2017-01-01

Alzheimer's disease (AD) is a common age-related neurodegenerative disorder that is characterized by progressive cognitive decline. The deficits in cognition and attentional processing that are observed clinically in AD are linked to impaired function of cholinergic neurons that release the neurotransmitter acetylcholine (ACh). The high-affinity choline transporter (CHT) is present at the presynaptic cholinergic nerve terminal and is responsible for the reuptake of choline produced by hydrolysis of ACh following its release. Disruption of CHT function leads to decreased choline uptake and ACh synthesis, leading to impaired cholinergic neurotransmission. We report here that cell-derived β-amyloid peptides (Aβ) decrease choline uptake activity and cell surface CHT protein levels in SH-SY5Y neural cells. Moreover, we make the novel observation that the amount of CHT protein localizing to early endosomes and lysosomes is decreased significantly in cells that have been treated with cell culture medium that contains Aβ peptides released from neural cells. The Aβ-mediated loss of CHT proteins from lysosomes is prevented by blocking lysosomal degradation of CHT with the lysosome inhibitor bafilomycin A1 (BafA 1 ). BafA 1 also attenuated the Aβ-mediated decrease in CHT cell surface expression. Interestingly, however, lysosome inhibition did not block the effect of Aβ on CHT activity. Importantly, neutralizing Aβ using an anti-Aβ antibody directed at the N-terminal amino acids 1-16 of Aβ, but not by an antibody directed at the mid-region amino acids 22-35 of Aβ, attenuates the effect of Aβ on CHT activity and trafficking. This indicates that a specific N-terminal Aβ epitope, or specific conformation of soluble Aβ, may impair CHT activity. Therefore, Aβ immunotherapy may be a more effective therapeutic strategy for slowing the progression of cognitive decline in AD than therapies designed to promote CHT cell surface levels.

Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

PubMed

Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

2016-01-01

Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer.

Evaluation of [11C]TAZA for amyloid β plaque imaging in postmortem human Alzheimer's disease brain region and whole body distribution in rodent PET/CT.

PubMed

Pan, Min-Liang; Mukherjee, Meenakshi T; Patel, Himika H; Patel, Bhavin; Constantinescu, Cristian C; Mirbolooki, M Reza; Liang, Christopher; Mukherjee, Jogeshwar

2016-04-01

Alzheimer's disease (AD) is a neurodegenerative disease characterized by Aβ plaques in the brain. The aim of this study was to evaluate the effectiveness of a novel radiotracer, 4-[(11) C]methylamino-4'-N,N-dimethylaminoazobenzene ([(11)C]TAZA), for binding to Aβ plaques in postmortem human brain (AD and normal control (NC)). Radiosyntheses of [(11)C]TAZA, related [(11)C]Dalene ((11)C-methylamino-4'-dimethylaminostyrylbenzene), and reference [(11)C]PIB were carried out using [(11)C]methyltriflate prepared from [(11) C]CO(2) and purified using HPLC. In vitro binding affinities were carried out in human AD brain homogenate with Aβ plaques labeled with [(3) H]PIB. In vitro autoradiography studies with the three radiotracers were performed on hippocampus of AD and NC brains. PET/CT studies were carried out in normal rats to study brain and whole body distribution. The three radiotracers were produced in high radiochemical yields (>40%) and had specific activities >37 GBq/μmol. TAZA had an affinity, K(i) = 0.84 nM and was five times more potent than PIB. [(11)C]TAZA bound specifically to Aβ plaques present in AD brains with gray matter to white matter ratios >20. [(11)C]TAZA was displaced by PIB (>90%), suggesting similar binding site for [(11)C]TAZA and [(11)C]PIB. [(11)C]TAZA exhibited slow kinetics of uptake in the rat brain and whole body images showed uptake in interscapular brown adipose tissue (IBAT). Binding in brain and IBAT were affected by preinjection of atomoxetine, a norepinephrine transporter blocker. [(11)C]TAZA exhibited high binding to Aβ plaques in human AD hippocampus. Rat brain kinetics was slow and peripheral binding to IBAT needs to be further evaluated. © 2016 Wiley Periodicals, Inc.

Development of a novel cyclic RGD peptide for multiple targeting approaches of liposomes to tumor region.

PubMed

Amin, Mohamadreza; Mansourian, Mercedeh; Koning, Gerben A; Badiee, Ali; Jaafari, Mahmoud Reza; Ten Hagen, Timo L M

2015-12-28

Liposomes containing cytotoxic agents and targeted with Arg-Gly-Asp based peptides have frequently been used against αvβ3 integrin on tumor neovasculature. However, like many other ligand modified liposomes these preparations suffered from enhanced uptake by the reticulo endothelial system (RES) and off-targeted interaction with integrin receptors vastly expressed in normal organs causing poor biodistribution and toxic effects. Here we mainly focus on development of a RGD-modified liposomal delivery system to enhance both targeting selectivity and tumor uptake. First, sterically stabilized liposomal doxorubicin (SSLD) prepared and decorated with cRGDfK and RGDyC peptides differ in their physical properties. Stability assessments as well as in vitro and in vivo studies revealed that increasing the peptide hydrophobicity promotes the therapeutic efficacy of RGD-SSLD in a C-26 tumor model due to decreased recognition by RES and opsonization and limited off-targeted interactions. Then a novel N-methylated RGD peptide was designed and its capability in targeting integrin presenting cells was comprehensively assessed both in vitro and in vivo. RGDf[N-methyl]C promotes the liposome internalization by HUVEC via integrin mediated endocytosis. Intravital microscopy in window chamber bearing mice illustrated the capability of RGDf[N-methyl]C-liposomes in targeting both tumor vasculature and tumor cells in murine B16F0 and human BLM tumor models. Quantitative biodistribution in mice bearing B16F0 tumor revealed its high affinity to tumor with no considerable affinity to normal organs. Treatment by high dose of RGDf[N-methyl]C-SSLD was found more effective than non-targeted SSLD and no toxic side effect was observed. In conclusion, the RGDf[N-methyl]C-liposome was found promising in targeting tumor vasculature as well as other cells inside the tumor. Copyright © 2015 Elsevier B.V. All rights reserved.

Physiological and molecular genetic evaluation of the dechlorination agent, pyridine-2,6-bis(monothiocarboxylic acid) (PDTC) as a secondary siderophore of Pseudomonas.

PubMed

Lewis, Thomas A; Leach, Lynne; Morales, Sergio; Austin, Paula R; Hartwell, Hadley J; Kaplan, Benjamin; Forker, Cynthia; Meyer, Jean-Marie

2004-02-01

The bacterial metabolite and transition metal chelator pyridine-2,6-dithiocarboxylic acid (PDTC), promotes a novel and effective means of dechlorination of the toxic and carcinogenic pollutant, carbon tetrachloride. Pyridine-2,6-dithiocarboxylic acid has been presumed to act as a siderophore in the Pseudomonas strains known to produce it. To explore further the physiological function of PDTC production, we have examined its regulation, the phenotype of PDTC-negative (pdt) mutants, and envelope proteins associated with PDTC in P. putida strain DSM 3601. Aspects of the regulation of PDTC production and outer membrane protein composition were consistent with siderophore function. Pyridine-2,6-dithiocarboxylic acid production was coordinated with production of the well-characterized siderophore pyoverdine; exogenously added pyoverdine led to decreased PDTC production, and added PDTC led to decreased pyoverdine production. Positive regulation of a chromosomal pdtI-xylE transcriptional fusion, and of a 66 kDa outer membrane protein (IROMP), was seen in response to exogenous PDTC. Tests with transition metal chelators indicated that PDTC could provide a benefit under conditions of metal limitation; the loss of PDTC biosynthetic capacity caused by a pdtI transposon insertion resulted in increased sensitivity to 1,10-phenanthroline, a chelator that has high affinity for a range of divalent transition metals (e.g. Fe(2+), Cu(2+), Zn(2+)). Exogenously added PDTC could also suppress a phenotype of pyoverdine-negative (Pvd-) mutants, that of sensitivity to EDDHA, a chelator with higher affinity and specificity for Fe(3+). Measurement of 59Fe incorporation showed uptake from 59Fe:PDTC by DSM 3601 grown in low-iron medium, but not by cells grown in high iron medium, or by the pdtI mutant, which did not show expression of the 66 kDa envelope protein. These data verified a siderophore function for PDTC, and have implicated it in the uptake of transition metals in addition to iron.

The Pharmacological Profile of a Novel Highly Potent Bisphosphonate, OX14 (1-Fluoro-2-(Imidazo-[1,2-α]Pyridin-3-yl)-Ethyl-Bisphosphonate).

PubMed

Lawson, Michelle A; Ebetino, Frank H; Mazur, Adam; Chantry, Andrew D; Paton-Hough, Julia; Evans, Holly R; Lath, Darren; Tsoumpra, Maria K; Lundy, Mark W; Dobson, Roy Lm; Quijano, Michael; Kwaasi, Aaron A; Dunford, James E; Duan, Xuchen; Triffitt, James T; Jeans, Gwyn; Russell, R Graham G

2017-09-01

Bisphosphonates are widely used in the treatment of clinical disorders characterized by increased bone resorption, including osteoporosis, Paget's disease, and the skeletal complications of malignancy. The antiresorptive potency of the nitrogen-containing bisphosphonates on bone in vivo is now recognized to depend upon two key properties, namely mineral binding affinity and inhibitory activity on farnesyl pyrophosphate synthase (FPPS), and these properties vary independently of each other in individual bisphosphonates. The better understanding of structure activity relationships among the bisphosphonates has enabled us to design a series of novel bisphosphonates with a range of mineral binding properties and antiresorptive potencies. Among these is a highly potent bisphosphonate, 1-fluoro-2-(imidazo-[1,2 alpha]pyridin-3-yl)-ethyl-bisphosphonate, also known as OX14, which is a strong inhibitor of FPPS, but has lower binding affinity for bone mineral than most of the commonly studied bisphosphonates. The aim of this work was to characterize OX14 pharmacologically in relation to several of the bisphosphonates currently used clinically. When OX14 was compared to zoledronate (ZOL), risedronate (RIS), and minodronate (MIN), it was as potent at inhibiting FPPS in vitro but had significantly lower binding affinity to hydroxyapatite (HAP) columns than ALN, ZOL, RIS, and MIN. When injected i.v. into growing Sprague Dawley rats, OX14 was excreted into the urine to a greater extent than the other bisphosphonates, indicating reduced short-term skeletal uptake and retention. In studies in both Sprague Dawley rats and C57BL/6J mice, OX14 inhibited bone resorption, with an antiresorptive potency equivalent to or greater than the comparator bisphosphonates. In the JJN3-NSG murine model of myeloma-induced bone disease, OX14 significantly prevented the formation of osteolytic lesions (p < 0.05). In summary, OX14 is a new, highly potent bisphosphonate with lower bone binding affinity than other clinically relevant bisphosphonates. This renders OX14 an interesting potential candidate for further development for its potential skeletal and nonskeletal benefits. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

Target-specific copper hybrid T7 phage particles.

PubMed

Dasa, Siva Sai Krishna; Jin, Qiaoling; Chen, Chin-Tu; Chen, Liaohai

2012-12-18

Target-specific nanoparticles have attracted significant attention recently, and have greatly impacted life and physical sciences as new agents for imaging, diagnosis, and therapy, as well as building blocks for the assembly of novel complex materials. While most of these particles are synthesized by chemical conjugation of an affinity reagent to polymer or inorganic nanoparticles, we are promoting the use of phage particles as a carrier to host organic or inorganic functional components, as well as to display the affinity reagent on the phage surface, taking advantage of the fact that some phages host well-established vectors for protein expression. An affinity reagent can be structured in a desired geometry on the surface of phage particles, and more importantly, the number of the affinity reagent molecules per phage particle can be precisely controlled. We previously have reported the use of the T7 phage capsid as a template for synthesizing target-specific metal nanoparticles. In this study herein, we reported the synthesis of nanoparticles using an intact T7 phage as a scaffold from which to extend 415 copies of a peptide that contains a hexahistidine (6His) motif for capture of copper ions and staging the conversion of copper ions to copper metal, and a cyclic Arginine-Glycine-Aspartic Acid (RGD4C) motif for targeting integrin and cancer cells. We demonstrated that the recombinant phage could load copper ions under low bulk copper concentrations without interfering with its target specificity. Further reduction of copper ions to copper metal rendered a very stable copper hybrid T7 phage, which prevents the detachment of copper from phage particles and maintains the phage structural integrity even under harsh conditions. Cancer cells (MCF-7) can selectively uptake copper hybrid T7 phage particles through ligand-mediated transmembrane transportation, whereas normal control cells (MCF-12F) uptake 1000-fold less. We further demonstrated that copper hybrid T7 phage could be endocytosed by cancer cells in culture.

Competitive Al3+ Inhibition of Net Mg2+ Uptake by Intact Lolium multiflorum Roots 1

PubMed Central

Rengel, Zdenko; Robinson, Donald L.

1989-01-01

Aluminum impairs uptake of Mg2+, but the mechanisms of this inhibition are not understood. The depletion technique was used to monitor net Mg2+ uptake from nutrient solution by intact, 23-day-old plants of ryegrass (Lolium multiflorum Lam., cv Gulf and Wilo). Activities of Mg2+ and monomeric Al species in nutrient solution were calculated and used as the basis for expressing the results. The kinetics of net Mg2+ absorption was resolved into (a) a transpiration-dependent uptake component, (b) a metabolically mediated, discontinuous saturable component that is Al3+ sensitive and p-chloromercuribenzene sulfonic acid (PCMBS) resistant, and (c) a linear, carbonyl cyanide m-chlorophenylhydrazone resistant, Al3+ sensitive component that might be a type of facilitated diffusion. Lowering the pH from 6.0 to 4.2 exerted a noncompetitive inhibition of net Mg2+ uptake, while aluminum at 6.6 micromolar Al3+ activity exerted competitive inhibition of net Mg2+ uptake at pH 4.2. The Al3+-induced effect was obvious after 30 minutes. Cultivar-specific ability to retain a higher affinity for Mg2+ by postulated transport proteins in the presence of Al3+ might be one of the mechanisms of differential Al tolerance among ryegrass cultivars. PMID:16667193

Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

PubMed

Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

2015-06-01

Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

Antidepressant Binding Site in a Bacterial Homologue of Neurotransmitter Transporters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh,S.; Yamashita, A.; Gouaux, E.

Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibitionmore » exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 {angstrom} above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.« less

Uptake of L-cystine via an ABC transporter contributes defense of oxidative stress in the L-cystine export-dependent manner in Escherichia coli.

PubMed

Ohtsu, Iwao; Kawano, Yusuke; Suzuki, Marina; Morigasaki, Susumu; Saiki, Kyohei; Yamazaki, Shunsuke; Nonaka, Gen; Takagi, Hiroshi

2015-01-01

Intracellular thiols like L-cystine and L-cystine play a critical role in the regulation of cellular processes. Here we show that Escherichia coli has two L-cystine transporters, the symporter YdjN and the ATP-binding cassette importer FliY-YecSC. These proteins import L-cystine, an oxidized product of L-cystine from the periplasm to the cytoplasm. The symporter YdjN, which is expected to be a new member of the L-cystine regulon, is a low affinity L-cystine transporter (Km = 1.1 μM) that is mainly involved in L-cystine uptake from outside as a nutrient. E. coli has only two L-cystine importers because ΔydjNΔyecS mutant cells are not capable of growing in the minimal medium containing L-cystine as a sole sulfur source. Another protein YecSC is the FliY-dependent L-cystine transporter that functions cooperatively with the L-cystine transporter YdeD, which exports L-cystine as reducing equivalents from the cytoplasm to the periplasm, to prevent E. coli cells from oxidative stress. The exported L-cystine can reduce the periplasmic hydrogen peroxide to water, and then generated L-cystine is imported back into the cytoplasm via the ATP-binding cassette transporter YecSC with a high affinity to L-cystine (Km = 110 nM) in a manner dependent on FliY, the periplasmic L-cystine-binding protein. The double disruption of ydeD and fliY increased cellular levels of lipid peroxides. From these findings, we propose that the hydrogen peroxide-inducible L-cystine/L-cystine shuttle system plays a role of detoxification of hydrogen peroxide before lipid peroxidation occurs, and then might specific prevent damage to membrane lipids.

Biodistribution and catabolism of (18)F-labeled neurotensin(8-13) analogs.

PubMed

Bergmann, Ralf; Scheunemann, Matthias; Heichert, Christoph; Mäding, Peter; Wittrisch, Holm; Kretzschmar, Marion; Rodig, Heike; Tourwé, Dirk; Iterbeke, Koen; Chavatte, Kris; Zips, Daniel; Reubi, Jean Claude; Johannsen, Bernd

2002-01-01

4-([(18)F]fluoro)benzoyl-neurotensin(8-13) ((18)FB-Arg(8)-Arg(9)-Pro(10)-Tyr(11)- Ile(12)-Leu(13)-OH, 1) and two analogs stabilized in one and two positions ((18)FB-Arg(8)psi(CH(2)NH)Arg(9)-Pro(10)-Tyr(11)- Ile(12)-Leu(13)-OH, 2, (18)FB-Arg(8)psi(CH(2)NH)Arg(9)-Pro(10)-Tyr(11)-Tle(12)-Leu(13)-OH, 3) were synthesized in a radiochemical yield of 25-36% and a specific activity of 5-15 GBq/mmol. The peptides were evaluated in vitro and in vivo for their potential to image tumors overexpressing neurotensin receptor 1 (NTR1) by positron emission tomography (PET). All analogs exhibited in vitro binding affinity in the low nanomolar range to NTR1-expressing human tumors, measured by quantitative receptor autoradiography, HT-29 and WiDr cells, and to sections of tumors derived from these cell lines in mice. The radiotracers were internalized in the cells in vitro, and the fluorinated peptides were able to mobilize intracellular Ca(2+) of WiDr cells. In in vivo studies in rats and in mice bearing HT-29 cell tumors, only a moderate uptake of the radioligands into the studied tumors was observed, presumed to be due to degradation in vivo and fast elimination by the kidneys. In comparison with the other analogs, the specific tumor uptake expressed as tumor-to-muscle relation was highest for the radioligand 3. The blood clearance of 3 was reduced by co-injection of peptidase inhibitors. The catabolic pathways of the radiofluorinated peptides were elucidated. The results suggest that the high binding affinity to NTR1 and the stabilization against proteolytic degradation are not yet sufficient for tumor imaging by PET.

Radioiodination and preclinical evaluation of 4-benzyl-1-(3-[125I]-iodobenzylsulfonyl)piperidine as a breast tumor imaging tracer in mouse.

PubMed

Sadeghzadeh, Masoud; Alirezapour, Behrouz; Charkhlooie, Ghorban Ali; Baghery, Maryam Keshavarz; Khorouti, Amir

2017-05-01

4-Benzyl-1-(3-iodobenzylsulfonyl)piperidine, 4-B-IBSP, has shown high-binding affinity to both sigma (σ) receptors in our previous work. In current study, radiolabeling and preclinical evaluation of 4-benzyl-1-(3-[ 125 I]-iodobenzylsulfonyl)piperidine, 4-B-[ 125 I]IBSP, in human ductal breast carcinoma (T47D) cells and in breast adenocarcinoma-bearing BALB/c mice are described. Radioiodination of this new σ ligand was performed by a palladium-catalyzed stannylation approach followed by oxidative iododestannylation reaction using Iodo-Gen. Competition-binding assays for binding of 4-B-[ 125 I]IBSP to guinea pig brain membranes and to T47D cells were performed with known σ ligands. The selectivity and binding characteristics (B max and K d ) were analyzed. In vitro stability and in vivo blood metabolism studies were also evaluated. Moreover, biodistribution studies were performed in normal and into the tumor-bearing mice at interval time points post-injection (p.i.). Both in vitro and in vivo blockade experiments were done in the presence of the σ receptors blocking agents. Radioiodinated ligand was obtained in high yield and high specific activity. The σ inhibition constants (K i , nM) for 4-(3-iodobenzyl)-1-(benzylsulfonyl)piperazine (4-IBBSPz), (+)-pentazocine, haloperidol, DTG, and 4-B-IBSP were 1.37 ± 0.19, 3.90 ± 0.77, 2.69 ± 0.33, 30.62 ± 2.01, and 0.61 ± 0.05, respectively. 4-B-[ 125 I]IBSP bound to σ receptor sites preferably to very high-affinity binding sites on T47D cells. The radioligand showed acceptable in vitro and in vivo stabilities in the blood pool. However, in vivo biodistribution studies in normal Swiss albino mice revealed fast clearance of 4-B-[ 125 I]IBSP from blood and the other normal organs. Biodistribution experiments of 4-B-[ 125 I]IBSP in breast adenocarcinoma tumor-bearing BALB/c mice showed a relatively high tumor uptake at 30 min p.i. (4.13 ± 0.95) that reaches to 1.57 ± 0.24 even after 240 min p.i. A pre-injection of 4-B-IBSP and haloperidol with 4-B-[ 125 I]IBSP resulted in 36-57% decrease in activity in the tumor, liver, and brain at 60 min p.i. The high affinity of 4-B-[ 125 I]IBSP to σ receptor-binding sites, its relatively high uptake, and preferential retention in the tumor as well as an increasing trend observed in the tumor to blood and in the tumor to muscle ratios suggests that an iodine-123 labeled counterpart, 4-B-[ 123 I]IBSP, would be a promising σ radioligand for pursuing further studies to assess its potential for breast tumors imaging with SPECT.

Understanding the in vivo uptake kinetics of a phosphatidylethanolamine-binding agent 99mTc-Duramycin

PubMed Central

Audi, Said; Li, Zhixin; Capacete, Joseph; Liu, Yu; Fang, Wei; Shu, Laura G.; Zhao, Ming

2013-01-01

Introduction 99mTc-Duramycin is a peptide-based molecular probe that binds specifically to phosphatidylethanolamine (PE). The goal was to characterize the kinetics of molecular interactions between 99mTc-Duramycin and the target tissue. Methods High level of accessible PE is induced in cardiac tissues by myocardial ischemia (30 min) and reperfusion (120 min) in Sprague Dawley rats. Target binding and biodistribution of 99mTc-duramycin was captured using SPECT/CT. To quantify the binding kinetics, the presence of radioactivity in ischemic versus normal cardiac tissues was measured by gamma counting at 3, 10, 20, 60 and 180 min after injection. A partially inactivated form of 99mTc-Duramycin was analyzed in the same fashion. A compartment model was developed to quantify the uptake kinetics of 99mTc-Duramycin in normal and ischemic myocardial tissue. Results 99mTc-duramycin binds avidly to the damaged tissue with a high target-to-background radio. Compartment modeling shows that accessibility of binding sites in myocardial tissue to 99mTc-Duramycin is not a limiting factor and the rate constant of target binding in the target tissue is at 2.2 ml/nmol/min/g. The number of available binding sites for 99mTc-Duramycin in ischemic myocardium was estimated at 0.14 nmol/g. Covalent modification of D15 resulted in a 9 fold reduction in binding affinity. Conclusion 99mTc-Duramycin accumulates avidly in target tissues in a PE-dependent fashion. Model results reflect an efficient uptake mechanism, consistent with the low molecular weight of the radiopharmaceutical and the relatively high density of available binding sites. These data help better define the imaging utilities of 99mTc-Duramycin as a novel PE-binding agent. PMID:22534031

Anorectic activities of serotonin uptake inhibitors: correlation with their potencies at inhibiting serotonin uptake in vivo and /sup 3/H-mazindol binding in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angel, I.; Taranger, M.A.; Claustre, Y.

1988-01-01

The mechanism of anorectic action of several serotonin uptake inhibitors was investigated by comparing their anorectic potencies with several biochemical and pharmacological properties and in reference to the novel compound SL 81.0385. The anorectic effect of the potent serotonin uptake inhibitor SL 81.0385 was potentiated by pretreatment with 5-hydroxytryptophan and blocked by the serotonin receptor antagonist metergoline. A good correlation was obtained between the ED/sub 50/ values of anorectic action and the ED/sub 50/ values of serotonin uptake inhibition in vivo (but not in vitro) for several specific serotonin uptake inhibitors. Most of the drugs tested displaced (/sup 3/H)-mazindol frommore » its binding to the anorectic recognition site in the hypothalamus, except the pro-drug zimelidine which was inactive. Excluding zimelidine, a good correlation was obtained between the affinities of these drugs for (/sup 3/H)-mazindol binding and their anorectic action indicating that their anorectic activity may be associated with an effect mediated through this site. Taken together these results suggest that the anorectic action of serotonin uptake inhibitors is directly associated to their ability to inhibit serotonin uptake and thus increasing the synaptic levels of serotonin. The interactions of these drugs with the anorectic recognition site labelled with (/sup 3/H)-mazindol is discussed in connection with the serotonergic regulation of carbohydrate intake.« less

Radioiodinated Exendin-4 Is Superior to the Radiometal-Labelled Glucagon-Like Peptide-1 Receptor Probes Overcoming Their High Kidney Uptake

PubMed Central

Läppchen, Tilman; Tönnesmann, Roswitha; Eersels, Jos; Meyer, Philipp T.; Maecke, Helmut R.; Rylova, Svetlana N.

2017-01-01

GLP-1 receptors are ideal targets for preoperative imaging of benign insulinoma and for quantifying the beta cell mass. The existing clinical tracers targeting GLP-1R are all agonists with low specific activity and very high kidney uptake. In order to solve those issues we evaluated GLP-1R agonist Ex-4 and antagonist Ex(9–39) radioiodinated at Tyr40 side by side with [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4 (68Ga-Ex-4) used in the clinic. The Kd, Bmax, internalization and binding kinetics of [Nle14,125I-Tyr40-NH2]Ex-4 and [Nle14,125I-Tyr40-NH2]Ex(9–39) were studied in vitro using Ins-1E cells. Biodistribution and imaging studies were performed in nude mice bearing Ins-1E xenografts. In vitro evaluation demonstrated high affinity binding of the [Nle14,125I-Tyr40-NH2]Ex-4 agonist to the Ins-1E cells with fast internalization kinetics reaching a plateau after 30 min. The antagonist [Nle14,125I-Tyr40-NH2]Ex(9–39) did not internalize and had a 4–fold higher Kd value compared to the agonist. In contrast to [Nle14,125I-Tyr40-NH2]Ex(9–39), which showed low and transient tumor uptake, [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated excellent in vivo binding properties with tumor uptake identical to that of 68Ga-Ex-4, but substantially lower kidney uptake resulting in a tumor-to-kidney ratio of 9.7 at 1 h compared to 0.3 with 68Ga-Ex-4. Accumulation of activity in thyroid and stomach for both peptides, which was effectively blocked by irenat, confirms that in vivo deiodination is the mechanism behind the low kidney retention of iodinated peptides. The 124I congener of [Nle14,125I-Tyr40-NH2]Ex-4 demonstrated a similar favourable biodistribution profile in the PET imaging studies in contrast to the typical biodistribution pattern of [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4. Our results demonstrate that iodinated Ex-4 is a very promising tracer for imaging of benign insulinomas. It solves the problem of high kidney uptake of the radiometal-labelled tracers by improving the tumor-to-kidney ratio measured for [Nle14,Lys40(Ahx-DOTA-68Ga)NH2]Ex-4 by 32 fold. PMID:28103285

Experience with indium-111 and yttrium-90-labeled somatostatin analogs.

PubMed

Virgolini, I; Traub, T; Novotny, C; Leimer, M; Füger, B; Li, S R; Patri, P; Pangerl, T; Angelberger, P; Raderer, M; Burggasser, G; Andreae, F; Kurtaran, A; Dudczak, R

2002-01-01

The high level expression of somatostatin receptors (SSTR) on various tumor cells has provided the molecular basis for successful use of radiolabeled octreotide / lanreotide analogs as tumor tracers in nuclear medicine. Other (nontumoral) potential indications for SSTR scintigraphy are based on an increased lymphocyte binding at sites of inflammatory or immunologic diseases such as thyroid-associated ophthalmology. The vast majority of human tumors seem to over-express the one or the other of five distinct hSSTR subtype receptors. Whereas neuroendocrine tumors frequently overexpress hSSTR2, intestinal adenocarcinomas seem to overexpress more often hSSTR3 or hSSTR4, or both of these hSSTR. In contrast to In-DTPA-DPhe(1)-octreotide (OctreoScan(R)) which binds to hSSTR2 and 5 with high affinity (Kd 0.1-5 nM), to hSSTR3 with moderate affinity (K(d) 10-100 nM) and does not bind to hSSTR1 and hSSTR4, (111)In / (90)Y-DOTA-lanreotide was found to bind to hSSTR2, 3, 4, and 5 with high affinity, and to hSSTR1 with lower affinity (K(d) 200 nM). Based on its unique hSSTR binding profile, (111)In-DOTA-lanreotide was suggested to be a potential radioligand for tumor diagnosis, and (90)Y-DOTA-lanreotide suitable for receptor-mediated radionuclide therapy. As opposed to (111)In-DTPA-DPhe(1)-octreotide and (111)In-DOTA-DPhe(1)-Tyr(3)-octreotide, discrepancies in the scintigraphic results were seen in about one third of (neuroendocrine) tumor patients concerning both the tumor uptake as well as detection of tumor lesions. On a molecular level, these discrepancies seem to be based on a "higherrdquuo; high-affinity binding of (111)In-DOTA-DPhe(1)-Tyr(3)-octreotide to hSSTR2 (K(d) 0.1-1 nM). Other somatostatin analogs with divergent affinity to the five known hSSTR subtype receptors have also found their way into the clinics, such as (99m)Tc-depreotide (NeoSpect(R); NeoTect(R)). Most of the imaging results are reported for neuroendocrine tumors (octreotide analogs) or nonsmall cell lung cancer ((99m)Tc-depreotide), indicating high diagnostic cabability of this type of receptor tracers. Consequently to their use as receptor imaging agents, hSSTR recognizing radioligands have also been implemented for experimental receptor-targeted radionuclide therapy. Beneficial results were reported for high-dose treatment with (111)In-DTPA-DPhe(1)-octreotide, based on the emission of Auger electrons. The Phase IIa study "MAURITIUS" (Multicenter Analysis of a Universal Receptor Imaging and Treatment Initiative, a eUropean Study) showed in progressive cancer patients (therapy entry criteria) with a calculated tumor dose > 10 Gy / GBq (90)Y-DOTA-lanreotide, the proof-of-principle for treating tumor patients with peptide receptor imaging agents. In the "MAURITIUS" study, cummulative treatment doses up to 200 mCi (90)Y-DOTA-lanreotide were given as short-term infusion. Overall treatment results in 70 patients indicated stable tumor disease in 35% of patients and regressive tumor disease in 10% of tumor patients with different tumor entities expressing hSSTR. No acute or chronic severe hematological toxicity, change in renal or liver function parameters due to (90)Y-DOTA-lanreotide treatment, were reported. (90)Y-DOTA-DPhe(1)-Tyr(3)-octreotide may show a higher tumor uptake in neuroendocrine tumor lesions and may therefore be superior for treatment in patients with neuroendocrine tumors. However, there is only limited excess to long-term and survival data at present. Potential indications for (90Y-DOTA-lanreotide are radioiodine-negative thyroid cancer, hepatocellular cancer and lung cancer. Besides newer approaches and recent developments of 188)Re-labeled radioligands, no clinical results on the treatment response are yet available. In conclusion, several radioligands have been implemented on the basis of peptide receptor recognition throughout the last decade. A plentitude of preclinical data and clinical studies confirm their potential use in diagnosis as well as "proof-of-principle" for therapy of cancer patients. However, an optimal radiopeptide formulatioents. However, an optimal radiopeptide formulation does not yet exist for receptor-targeted radionuclide therapy. Ongoing developments may result in peptides more suitable for this kind of receptor-targeted radionuclide therapy.

Effect of aging on intestinal absorption of aromatic amino acids in vitro in the rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Navab, F.; Winter, C.G.

Whole-thickness everted jejunal rings were used to measure uptake of L-tyrosine (L-Tyr), L-phenylalanine (L-Phe), and L-tryptophan (L-Trp) in 6-, 12-, and 24-mo-old rats. The rate of uptake of all tree amino acids (1 mM) was significantly reduced after 20 min of incubation in 24-mo-old compared with 6-mo-old rats. Results of influx (2 min) of 0.5-40.0 mL L-Phe and L-Trp suggested an increased affinity but decreased capacity for the transporter with age; these differences were significant for L-Trp. Respective values for apparent K{sub t} and V{sub max} are given.

Pharmacokinetics of differently designed immunoliposome formulations in rats with or without hepatic colon cancer metastases.

PubMed

Koning, G A; Morselt, H W; Gorter, A; Allen, T M; Zalipsky, S; Kamps, J A; Scherphof, G L

2001-09-01

Compare pharmacokinetics of tumor-directed immunoliposomes in healthy and tumor-bearing rats (hepatic colon cancer metastases). A tumor cell-specific monoclonal antibody was attached to polyethyleneglycol-stabilized liposomes, either in a random orientation via a lipid anchor (MPB-PEG-liposomes) or uniformly oriented at the distal end of the PEG chains (Hz-PEG-liposomes). Pharmacokinetics and tissue distribution were determined using [3H]cholesteryloleylether or bilayer-anchored 5-fluoro[3H]deoxyuridine-dipalmitate ([3H]FUdR-dP) as a marker. In healthy animals clearance of PEG-(immuno)liposomes was almost log-linear and only slightly affected by antibody attachment; in tumor-bearing animals all liposomes displayed biphasic clearance. In normal and tumor animals blood elimination increased with increasing antibody density; particularly for the Hz-PEG-liposomes, and was accompanied by increased hepatic uptake, probably due to increased numbers of macrophages induced by tumor growth. The presence of antibodies on the liposomes enhanced tumor accumulation: uptake per gram tumor tissue (2-4% of dose) was similar to that of liver. Remarkably, this applied to tumor-specific and irrelevant antibody. Increased immunoliposome uptake by trypsin-treated Kupffer cells implicated involvement of high-affinity Fc-receptors on activated macrophages. Tumor growth and immunoliposome characteristics (antibody density and orientation) determine immunoliposome pharmacokinetics. Although with a long-circulating immunoliposome formulation, efficiently retaining the prodrug FUdR-dP, we achieved enhanced uptake by hepatic metastases, this was probably not mediated by specific interaction with the tumor cells, but rather by tumor-associated macrophages.

Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide

PubMed Central

Bisogno, Tiziana; Hanuš, Lumír; De Petrocellis, Luciano; Tchilibon, Susanna; Ponde, Datta E; Brandi, Ines; Moriello, Aniello Schiano; Davis, John B; Mechoulam, Raphael; Di Marzo, Vincenzo

2001-01-01

(−)-Cannabidiol (CBD) is a non-psychotropic component of Cannabis with possible therapeutic use as an anti-inflammatory drug. Little is known on the possible molecular targets of this compound. We investigated whether CBD and some of its derivatives interact with vanilloid receptor type 1 (VR1), the receptor for capsaicin, or with proteins that inactivate the endogenous cannabinoid, anandamide (AEA).CBD and its enantiomer, (+)-CBD, together with seven analogues, obtained by exchanging the C-7 methyl group of CBD with a hydroxy-methyl or a carboxyl function and/or the C-5′ pentyl group with a di-methyl-heptyl (DMH) group, were tested on: (a) VR1-mediated increase in cytosolic Ca2+ concentrations in cells over-expressing human VR1; (b) [14C]-AEA uptake by RBL-2H3 cells, which is facilitated by a selective membrane transporter; and (c) [14C]-AEA hydrolysis by rat brain membranes, which is catalysed by the fatty acid amide hydrolase.Both CBD and (+)-CBD, but not the other analogues, stimulated VR1 with EC50=3.2 – 3.5 μM, and with a maximal effect similar in efficacy to that of capsaicin, i.e. 67 – 70% of the effect obtained with ionomycin (4 μM). CBD (10 μM) desensitized VR1 to the action of capsaicin. The effects of maximal doses of the two compounds were not additive.(+)-5′-DMH-CBD and (+)-7-hydroxy-5′-DMH-CBD inhibited [14C]-AEA uptake (IC50=10.0 and 7.0 μM); the (−)-enantiomers were slightly less active (IC50=14.0 and 12.5 μM). CBD and (+)-CBD were also active (IC50=22.0 and 17.0 μM).CBD (IC50=27.5 μM), (+)-CBD (IC50=63.5 μM) and (−)-7-hydroxy-CBD (IC50=34 μM), but not the other analogues (IC50>100 μM), weakly inhibited [14C]-AEA hydrolysis.Only the (+)-isomers exhibited high affinity for CB1 and/or CB2 cannabinoid receptors.These findings suggest that VR1 receptors, or increased levels of endogenous AEA, might mediate some of the pharmacological effects of CBD and its analogues. In view of the facile high yield synthesis, and the weak affinity for CB1 and CB2 receptors, (−)-5′-DMH-CBD represents a valuable candidate for further investigation as inhibitor of AEA uptake and a possible new therapeutic agent. PMID:11606325

Up-regulation of angiotensin II receptors by in vitro differentiation of murine N1E-115 neuroblastoma cells.

PubMed

Reagan, L P; Ye, X H; Mir, R; DePalo, L R; Fluharty, S J

1990-12-01

In vitro differentiation of murine neuroblastoma N1E-115 cells induced by low serum (0.5%) and dimethyl sulfoxide (1.5%) increased the uptake of 45Ca2+ as well as basal and forskolin-stimulated adenylate cyclase activity. Associated with these biochemical indices of differentiation was an increase in the density of binding sites for the angiotensin II (Ang II) receptor agonist 125I-[Sar1]-Ang II and the antagonist 125I-[Sar1,Ile8]-Ang II (125I-SARILE). This up-regulation was apparent within 24 hr and was maximal at 72 hr. Other manipulations that independently increased intracellular cAMP or Ca2+ levels produced a qualitatively similar up-regulation of Ang II receptors. In vitro differentiation did not diminish the specificity of these receptors for Ang-II related peptides. Sarcosine-substituted Ang II receptor antagonists such as [Sar1,Gly8]-Ang II, [Sar1,Thr8]-Ang II, or SARILE itself competed for 125I-SARILE in a monophasic fashion, whereas the competition displayed by the agonists Ang II, angiotensin III, and Crinia-Ang II for 125I-SARILE-labeled sites was biphasic, consisting of distinct high and low affinity components. Moreover, in vitro differentiation predominantly increased the density of high affinity sites for angiotensin III and Crinia-Ang II, but the lower affinity site for Ang II, and in all three cases the majority of this increased binding was insensitive to guanine nucleotides. Collectively, these results demonstrate that the expression of Ang II receptors on neuron-like cells is regulated by the biochemical events accompanying differentiation and suggest that the biphasic nature of the binding of some angiotensin agonists may be indicative of multiple receptor subtypes.

Genes responding to water deficit in apple (Malus × domestica Borkh.) roots.

PubMed

Bassett, Carole Leavel; Baldo, Angela M; Moore, Jacob T; Jenkins, Ryan M; Soffe, Doug S; Wisniewski, Michael E; Norelli, John L; Farrell, Robert E

2014-07-08

Individual plants adapt to their immediate environment using a combination of biochemical, morphological and life cycle strategies. Because woody plants are long-lived perennials, they cannot rely on annual life cycle strategies alone to survive abiotic stresses. In this study we used suppression subtractive hybridization to identify genes both up- and down-regulated in roots during water deficit treatment and recovery. In addition we followed the expression of select genes in the roots, leaves, bark and xylem of 'Royal Gala' apple subjected to a simulated drought and subsequent recovery. In agreement with studies from both herbaceous and woody plants, a number of common drought-responsive genes were identified, as well as a few not previously reported. Three genes were selected for more in depth analysis: a high affinity nitrate transporter (MdNRT2.4), a mitochondrial outer membrane translocase (MdTOM7.1), and a gene encoding an NPR1 homolog (MpNPR1-2). Quantitative expression of these genes in apple roots, bark and leaves was consistent with their roles in nutrition and defense. Additional genes from apple roots responding to drought were identified using suppression subtraction hybridization compared to a previous EST analysis from the same organ. Genes up- and down-regulated during drought recovery in roots were also identified. Elevated levels of a high affinity nitrate transporter were found in roots suggesting that nitrogen uptake shifted from low affinity transport due to the predicted reduction in nitrate concentration in drought-treated roots. Suppression of a NPR1 gene in leaves of drought-treated apple trees may explain in part the increased disease susceptibility of trees subjected to dehydrative conditions.

Effect of magnesium complexation by fluoroquinolones on their antibacterial properties.

PubMed Central

Lecomte, S; Baron, M H; Chenon, M T; Coupry, C; Moreau, N J

1994-01-01

By using infrared and 19F nuclear magnetic resonance spectroscopies, we localized the binding site and measured the affinity of magnesium for six fluoroquinolones. It was proven that magnesium is situated between the ketone and the carboxylate groups. We determined the binding constants for the 1:1 Mg(2+)-drug complex in solution. Sparfloxacin and pefloxacin, with affinity constants (Ka) of (10.1 +/- 0.6) x 10(2) M-1 and (21 +/- 1) x 10(2) M-1, respectively, were the least and the most bound, respectively. The trend of the affinities of the assayed fluoroquinolones for magnesium was correlated with their antimicrobial activities against four bacteria and with their accumulation by these bacteria. The reference strain, Escherichia coli KL16, and two resistant mutants, NalA (gyrase mutation) and NalB (uptake defect), plus Staphylococcus aureus 209P were used. It appeared that, in every case, an impairment of accumulation is responsible for the increase in the MICs observed upon the addition of magnesium. Images PMID:7695267

Effects of linker modification on tumor-to-kidney contrast of 68Ga-labeled PSMA-targeted imaging probes.

PubMed

Kuo, Hsiou-Ting; Pan, Jinhe; Zhang, Zhengxing; Lau, Joseph; Merkens, Helen; Zhang, Chengcheng; Colpo, Nadine; Lin, Kuo-Shyan; Benard, Francois

2018-06-19

68Ga-PSMA-11 is currently the most popular prostate-specific membrane antigen (PSMA) radioligand used in the clinic to detect prostate cancer and metastases. However, the high uptake of 68Ga-PSMA-11 in kidneys can create halo-artifacts resulting in lower detection sensitivity for lesions adjacent to the kidneys. In this study, we developed two 68Ga-labeled PSMA-targeted tracers, 68Ga-HTK01166 and 68Ga-HTK01167, based on 68Ga-PSMA-617 with the goal of improving tumor-to-kidney ratio compared to 68Ga-PSMA-11. The 2-naphthylalanine (2-Nal) in PSMA-617 was replaced with 2-indanylglycine (Igl) or 3,3-diphenylalanine (Dip) to synthesize HTK01166 and HTK01167, respectively. Binding affinities (Ki) of Ga-PSMA-11, Ga-PSMA-617, Ga-HTK01166 and Ga-HTK01167 to PSMA were 3.13 ± 0.40, 1.23 ± 0.08, 5.74 ± 2.48 and 25.7 ± 9.84 nM, respectively, as determined by in vitro competition binding assays. 68Ga labeling was performed in HEPES buffer with microwave heating, and 68Ga-labeled PSMA-11, PSMA-617, HTK01166 and HTK01167 were obtained in 46 - 69% average decay-corrected radiochemical yield with >99% radiochemical purity and 62.9 - 152 GBq/μmol average specific activity. PET imaging and biodistribution studies were performed in mice bearing PSMA-expressing LNCap prostate cancer xenografts. All tracers enabled clear visualization of tumors in PET images with excellent tumor-to-background contrast. The uptake values (%ID/g) for tumor and kidneys at 1 h post-injection were 8.91 ± 0.86 and 204 ± 70.6 for 68Ga-PSMA-11, 16.7 ± 2.30 and 29.2 ± 5.14 for 68Ga-PSMA-617, 14.1 ± 4.40 and 147 ± 59.6 for 68Ga-HTK01166, and 7.79 ± 1.65 and 4.30 ± 1.80 for 68Ga-HTK01167. The tumor-to-kidney ratios for 68Ga-labeled PSMA-11, PSMA-617, HTK01166 and HTK01167 were 0.05 ± 0.02, 0.63 ± 0.10, 0.10 ± 0.02 and 1.98 ± 0.63, respectively. Compared with 68Ga-PSMA-617, 68Ga-HTK01166 showed comparable tumor uptake, and almost 5-fold higher kidney uptake; whereas 68Ga-HTK01167 exhibited lower tumor and kidney uptake. Compared with 68Ga-PSMA-11, 68Ga-HTK01167 had similar tumor uptake and tumor-to-blood contrast ratio (23.8 ± 6.71 vs 20.4 ± 4.98) but higher tumor-to-background contrast ratios for other background organs especially for kidneys. Our data indicate that substitution of 2-Nal in PSMA-617 with other lipophilic amino acid can modulate PSMA binding affinity and their pharmacokinetics in vivo.

CHX14 is a plasma membrane K-efflux transporter that regulates K(+) redistribution in Arabidopsis thaliana.

PubMed

Zhao, Jian; Li, Penghui; Motes, Christy M; Park, Sunghun; Hirschi, Kendal D

2015-11-01

Potassium (K(+) ) is essential for plant growth and development, yet the molecular identity of many K(+) transporters remains elusive. Here we characterized cation/H(+) exchanger (CHX) 14 as a plasma membrane K(+) transporter. CHX14 expression was induced by elevated K(+) and histochemical analysis of CHX14 promoter::GUS transgenic plants indicated that CHX14 was expressed in xylem parenchyma of root and shoot vascular tissues of seedlings. CHX14 knockout (chx14) and CHX14 overexpression seedlings displayed different growth phenotypes during K(+) stress as compared with wild-type seedlings. Roots of mutant seedlings displayed higher K(+) uptake rates than wild-type roots. CHX14 expression in yeast cells deficient in K(+) uptake renders the mutant cells more sensitive to deficiencies of K(+) in the medium. CHX14 mediates K(+) efflux in yeast cells loaded with high K(+) . Uptake experiments using (86) Rb(+) as a tracer for K(+) with both yeast and plant mutants demonstrated that CHX14 expression in yeast and in planta mediated low-affinity K(+) efflux. Functional green fluorescent protein (GFP)-tagged versions of CHX14 were localized to both the yeast and plant plasma membranes. Taken together, we suggest that CHX14 is a plasma membrane K(+) efflux transporter involved in K(+) homeostasis and K(+) recirculation. © 2015 John Wiley & Sons Ltd.

Functional expression of SGLTs in rat brain.

PubMed

Yu, Amy S; Hirayama, Bruce A; Timbol, Gerald; Liu, Jie; Basarah, Ernest; Kepe, Vladimir; Satyamurthy, Nagichettiar; Huang, Sung-Cheng; Wright, Ernest M; Barrio, Jorge R

2010-12-01

This work provides evidence of previously unrecognized uptake of glucose via sodium-coupled glucose transporters (SGLTs) in specific regions of the brain. The current understanding of functional glucose utilization in brain is largely based on studies using positron emission tomography (PET) with the glucose tracer 2-deoxy-2-[F-18]fluoro-D-glucose (2-FDG). However, 2-FDG is only a good substrate for facilitated-glucose transporters (GLUTs), not for SGLTs. Thus, glucose accumulation measured by 2-FDG omits the role of SGLTs. We designed and synthesized two high-affinity tracers: one, α-methyl-4-[F-18]fluoro-4-deoxy-D-glucopyranoside (Me-4FDG), is a highly specific SGLT substrate and not transported by GLUTs; the other one, 4-[F-18]fluoro-4-deoxy-D-glucose (4-FDG), is transported by both SGLTs and GLUTs and will pass through the blood brain barrier (BBB). In vitro Me-4FDG autoradiography was used to map the distribution of uptake by functional SGLTs in brain slices with a comparable result from in vitro 4-FDG autoradiography. Immunohistochemical assays showed that uptake was consistent with the distribution of SGLT protein. Ex vivo 4-FDG autoradiography showed that SGLTs in these areas are functionally active in the normal in vivo brain. The results establish that SGLTs are a normal part of the physiology of specific areas of the brain, including hippocampus, amygdala, hypothalamus, and cerebral cortices. 4-FDG PET imaging also established that this BBB-permeable SGLT tracer now offers a functional imaging approach in humans to assess regulation of SGLT activity in health and disease.

Glucose Elevates NITRATE TRANSPORTER2.1 Protein Levels and Nitrate Transport Activity Independently of Its HEXOKINASE1-Mediated Stimulation of NITRATE TRANSPORTER2.1 Expression1[W][OPEN

PubMed Central

de Jong, Femke; Thodey, Kate; Lejay, Laurence V.; Bevan, Michael W.

2014-01-01

Mineral nutrient uptake and assimilation is closely coordinated with the production of photosynthate to supply nutrients for growth. In Arabidopsis (Arabidopsis thaliana), nitrate uptake from the soil is mediated by genes encoding high- and low-affinity transporters that are transcriptionally regulated by both nitrate and photosynthate availability. In this study, we have studied the interactions of nitrate and glucose (Glc) on gene expression, nitrate transport, and growth using glucose-insensitive2-1 (gin2-1), which is defective in sugar responses. We confirm and extend previous work by showing that HEXOKINASE1-mediated oxidative pentose phosphate pathway (OPPP) metabolism is required for Glc-mediated NITRATE TRANSPORTER2.1 (NRT2.1) expression. Treatment with pyruvate and shikimate, two products derived from intermediates of the OPPP that are destined for amino acid production, restores wild-type levels of NRT2.1 expression, suggesting that metabolites derived from OPPP metabolism can, together with Glc, directly stimulate high levels of NRT2.1 expression. Nitrate-mediated NRT2.1 expression is not influenced by gin2-1, showing that Glc does not influence NRT2.1 expression through nitrate-mediated mechanisms. We also show that Glc stimulates NRT2.1 protein levels and transport activity independently of its HEXOKINASE1-mediated stimulation of NRT2.1 expression, demonstrating another possible posttranscriptional mechanism influencing nitrate uptake. In gin2-1 plants, nitrate-responsive biomass growth was strongly reduced, showing that the supply of OPPP metabolites is essential for assimilating nitrate for growth. PMID:24272701

Underexpression of the Na+-dependent neutral amino acid transporter ASCT2 in the spontaneously hypertensive rat kidney.

PubMed

Pinho, Maria João; Pinto, Vanda; Serrão, Maria Paula; Jose, Pedro A; Soares-da-Silva, Patrício

2007-07-01

This study examined the inward transport of l-[(14)C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[(14)C]alanine uptake was highly dependent on extracellular Na(+). Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na(+)-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na(+)-dependent l-alanine transport under V(max) conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K(m) values. Differences in magnitude of Na(+)-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na(+)-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.

Matching the decay half-life with the biological half-life: ImmunoPET imaging with 44 Sc-labeled Cetuximab Fab fragment

DOE PAGES

Chakravarty, Rubel; Goel, Shreya; Valdovinos, Hector F.; ...

2014-11-11

Scandium-44 (t 1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with 44Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-( para-isothiocyanato-phenyl)propyl]- trans-(S,S)-cyclohexane-1,2-diamine- N,N,N',N'',N''-pentaacetic acid (CHX-A"-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastomamore » (U87MG) tumor-bearing mice was investigated after 44Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ~12% ID/g at 4 h postinjection) of 44Sc–CHX-A"-DTPA–Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. As a result, this successful strategy for immunoPET imaging of EGFR expression using 44Sc–CHX-''-DTPA–Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.« less

Matching the Decay Half-Life with the Biological Half-Life: ImmunoPET Imaging with 44Sc-Labeled Cetuximab Fab Fragment

PubMed Central

2015-01-01

Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with 44Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N′,N″,N″-pentaacetic acid (CHX-A″-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after 44Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ∼12% ID/g at 4 h postinjection) of 44Sc–CHX-A″-DTPA–Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. This successful strategy for immunoPET imaging of EGFR expression using 44Sc–CHX-A″-DTPA–Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments. PMID:25389697

Characterization of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini.

PubMed

Talkad, V D; Patto, R J; Metz, D C; Turner, R J; Fortune, K P; Bhat, S T; Gardner, J D

1994-10-20

By measuring binding of [125I]CCK-8 and [3H]L-364,718 to rat pancreatic acini we demonstrated directly that the pancreatic CCK receptor can exist in three different affinity states with respect to CCK--high affinity, low affinity and very low affinity. Binding of [125I]CCK-8 reflects interaction of the tracer with the high and low affinity states, whereas binding of [3H]L-364,718 reflects interaction of the tracer with the low and very low affinity states. Treating acini with carbachol abolished the high affinity state of the CCK receptor and converted approximately 25% of the low affinity receptors to the very low affinity state. Carbachol treatment was particularly useful in establishing the values of Kd for the high and low affinity states for different CCK receptor agonists and antagonists. Of the various CCK receptor agonists tested, CCK-8 had the highest affinity for the high affinity state (Kd approximately 1 nM), whereas CCK-JMV-180 had the highest affinity for the low (Kd 7 nM) and very low affinity (Kd 200 nM) states. Gastrin and de(SO4)CCK-8 had affinities for the high and low affinity states of the receptor that were 100- to 400-fold less than those of CCK-8 but had affinities for the very low affinity state that were only 3- to 10-fold less than that of CCK-8. CCK receptor antagonists showed several patterns in interacting with the different states of the CCK receptor. L-364,718 had the same affinity for each state of the CCK receptor. CR1409 and Bt2cGMP each had similar affinities for the high and low affinity states and lower affinity for the very low affinity state. L-365,260 and CCK-JMV-179 had the highest affinity for the low affinity state and lower affinities for the high and very low affinity states. Different CCK receptor agonists caused the same maximal stimulation of amylase secretion but showed different degrees of amplification in terms of the relationship between their abilities to stimulate amylase secretion and their abilities to occupy the low affinity state of the CCK receptor. When amplification was expressed quantitatively as the value of Kd for the low affinity state divided by the corresponding EC50 for stimulating amylase secretion the values were CCK-8 (1000), de(SO)CCK-8 (1500), gastrin (100) and CCK-JMV-180 (Menozzi, D., Vinayek, R., Jensen, R.T. and Gardner, J.D. (1991) J. Biol. Chem. 266, 10385-1091).(ABSTRACT TRUNCATED AT 400 WORDS)

Selection and Characterization of Single Stranded DNA Aptamers for the Hormone Abscisic Acid

PubMed Central

Gonzalez, Victor M.; Millo, Enrico; Sturla, Laura; Vigliarolo, Tiziana; Bagnasco, Luca; Guida, Lucrezia; D'Arrigo, Cristina; De Flora, Antonio; Salis, Annalisa; Martin, Elena M.; Bellotti, Marta; Zocchi, Elena

2013-01-01

The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98±0.14 μM and 0.80±0.07 μM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays. PMID:23971905

Self-Assembly of Amphiphilic Dendrimers: The Role of Generation and Alkyl Chain Length in siRNA Interaction

PubMed Central

Márquez-Miranda, Valeria; Araya-Durán, Ingrid; Camarada, María Belén; Comer, Jeffrey; Valencia-Gallegos, Jesús A.; González-Nilo, Fernando Danilo

2016-01-01

An ideal nucleic-acid transfection system should combine the physical and chemical characteristics of cationic lipids and linear polymers to decrease cytotoxicity and uptake limitations. Previous research described new types of carriers termed amphiphilic dendrimers (ADs), which are based on polyamidoamine dendrimers (PAMAM). These ADs display the cell membrane affinity advantage of lipids and preserve the high affinity for DNA possessed by cationic dendrimers. These lipid/dendrimer hybrids consist of a low-generation, hydrophilic dendron (G2, G1, or G0) bonded to a hydrophobic tail. The G2-18C AD was reported to be an efficient siRNA vector with significant gene silencing. However, shorter tail ADs (G2-15C and G2-13C) and lower generation (G0 and G1) dendrimers failed as transfection carriers. To date, the self-assembly phenomenon of this class of amphiphilic dendrimers has not been molecularly explored using molecular simulation methods. To gain insight into these systems, the present study used coarse-grained molecular dynamics simulations to describe how ADs are able to self-assemble into an aggregate, and, specifically, how tail length and generation play a key role in this event. Finally, explanations are given for the better efficiency of G2/18-C as gene carrier in terms of binding of siRNA. This knowledge could be relevant for the design of novel, safer ADs with well-optimized affinity for siRNA. PMID:27377641

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamilton, Terry F.; Martinelli, Roger E.; Kehl, Steven R.

A series of K d tracer batch experiments were conducted in this paper to assess the absorptive-desorption properties of Biochar as a potential agent to selectively sequester labile soil Cs or otherwise help reduce the uptake of Cs isotopes into plants. A parallel experiment was conducted for strontium. Fine-grained fractionated Woodlands tree Biochar was found to have a relatively high affinity for Cs ions (K d > 100) relative to coral soil (K d < 10) collected from the Marshall Islands. The Biochar material also contains an abundance of K (and Mg). Finally, these findings support a hypothesis that themore » addition of Biochar as a soil amendment may provide a simple yet effective method for reducing soil-to-plant transfer of Cs isotopes in contaminated environments.« less