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Sample records for gamma-emitting photoaffinity label

  1. delta 9-(16 alpha-/sup 125/I)iodo-19-nortestosterone: a gamma-emitting photoaffinity label for the progesterone receptor

    SciTech Connect

    Lamb, D.J.; Bullock, D.W.; Hoyte, R.M.; Hochberg, R.B.

    1988-05-01

    We have synthesized 16 alpha-iodo-4,9-estradien-17 beta-ol-3-one (delta 9-16 alpha-iodo-19-nortestosterone (delta 9-INT)) labeled with 125I (delta 9-(16 alpha-125I)INT) to provide a new gamma-emitting photoaffinity ligand for the progesterone receptor that has many advantages over the currently available (3H)R5020. We have characterized the interaction of delta 9-(16 alpha-125I)INT with the rabbit uterine progesterone receptor and have demonstrated the usefulness of this compound for studies of receptor structure. The binding of 2 nM (3H)progesterone to receptor in rabbit uterine cytosol was specifically competed for by 19-nortestosterone, 16 alpha-iodo-19-nortestosterone, and delta 9-INT. Scatchard analysis demonstrated that delta 9-(16 alpha-125I)INT and (3H)progesterone estimated the same number of binding sites in rabbit uterine cytosol, with a Kd for delta 9-(16 alpha-125I)INT of about 2.7 nM. The binding of delta 9-(16 alpha-125I)INT was inhibited by both progesterone and R5020, whereas testosterone, estradiol, and 5 alpha-dihydrotestosterone were ineffective. In cytosol, delta 9-(16 alpha-125I)INT covalently labeled the same mol wt receptor forms as (3H)R5020. Although the efficiency of cross-linking was similar for (3H)R5020 (3%) and delta 9-(16 alpha-125I)INT (4%), the radioactivity was 10-fold greater due to the higher specific activity of delta 9-(16 alpha-125I)INT and the lack of sample quench. The use of delta 9-(16 alpha-125I)INT greatly increases the sensitivity and efficiency of the photoaffinity labeling technique; it will provide a valuable tool for further studies of the progesterone receptor, allowing the detection of receptor in dilute cytosol after gel electrophoresis under denaturing conditions.

  2. Photoaffinity-labeled Cytokinins

    PubMed Central

    Theiler, Jane B.; Leonard, Nelson J.; Schmitz, Ruth Y.; Skoog, Folke

    1976-01-01

    Two new azidopurine derivatives, 2-azido-N6-(Δ2-isopentenyl)adenine and 2-azido-N6-benzyladenine, have been synthesized as potential photoaffinity labels for probing cytokinin-binding sites. The preparation and the biological activity of these compounds are described. PMID:16659772

  3. Photoaffinity labeling in target- and binding-site identification

    PubMed Central

    Smith, Ewan; Collins, Ian

    2015-01-01

    Photoaffinity labeling (PAL) using a chemical probe to covalently bind its target in response to activation by light has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites. Methods to identify the specific target proteins of hit molecules from phenotypic screens are highly valuable in early drug discovery. In this review, we summarize the principles of PAL including probe design and experimental techniques for in vitro and live cell investigations. We emphasize the need to optimize and validate probes and highlight examples of the successful application of PAL across multiple disease areas. PMID:25686004

  4. Photoaffinity labelling of high affinity dopamine binding proteins

    SciTech Connect

    Ross, G.M.; McCarry, B.E.; Mishra, R.K.

    1986-03-01

    A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-)N'-4-azidobenzamidol)-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using (/sup 3/H)-SCH 23390 (a D/sub 1/ specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked (/sup 3/H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of (/sup 3/H)-SCH 23390 binding. Compounds which compete for D/sub 1/ receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D/sub 1/ (adenylate cyclase linked) dopamine receptor.

  5. Agonist photoaffinity label for the. beta. -adrenergic receptor

    SciTech Connect

    Resek, J.F.; Ruoho, A.E.

    1987-05-01

    An iodinated photosensitive derivative of norepinephrine, N-(p-azido-m-iodophenethylamidoisobutyryl)norepinephrine (NAIN), has been synthesized and characterized. Carrier-free radioiodinated NAIN ((/sup 125/I)-NAIN) was used at 1-2 x 10/sup -9/ M to photoaffinity label the ..beta..-adrenergic receptor in guinea pig lung membranes. SDS-PAGE analysis of (-)-alprenolol (10/sup -5/M) protectable (/sup 125/I)-NAIN labeling showed the same molecular weight polypeptide (65 kDa) that was specifically derivatized with the antagonist photolabel, (/sup 125/I)-IABP. Specific labeling of the ..beta..-adrenergic receptor with (/sup 125/I)-NAIN was dependent on the presence of MgCl/sub 2/ and the absence of guanyl nucleotide. GTP..gamma..S (10/sup -4/ M) abolished specific receptor labeling by (/sup 125/I)-NAIN. N-ethylmaleimide (2 mm) in the presence of (/sup 125/I)-NAIN protected against the guanyl nucleotide effect. These data are consistent with photolabeling by (/sup 125/I)-NAIN while the agonist, receptor, and GTP binding protein are in a high affinity complex.

  6. Characterization of mammalian glucose transport proteins using photoaffinity labeling techniques

    SciTech Connect

    Wadzinski, B.E.

    1989-01-01

    A carrier-free radioiodinated phenylazide derivative of forskolin, 3-iodo-4-azidophenethylamido-7-O-succinyl-deacetyl-forskolin (({sup 125}I)IAPS-forskolin), has been shown to be a highly selective photoaffinity probe for the human erythrocyte glucose transported and the glucose transport proteins found in several mammalian tissues and cultured cells where the glucose transport protein is present at a low concentration. The photoincorporation of ({sup 125}I)IAPS-forskolin into these glucose transporters was blocked by D- (but not L-) glucose, cytochalasin B, and forskolin. In addition to labeling the mammalian glucose transport proteins, ({sup 125}I)IAPS-forskolin also labeled the L-arabinose transporter from E. coli. In muscle and adipose tissues, glucose transport is markedly increased in response to insulin. ({sup 125}I)IAPS-forskolin was shown to selectivity tag the glucose transporter in membranes derived from these cells. In addition, the covalent derivatization of the transport protein in subcellular fractions of the adipocyte has provided a means to study the hormonal regulation of glucose transport. ({sup 125}I)IAPS-forskolin has also been used to label the purified human erythrocyte glucose transporter. The site of insertion has therefore been localized by analysis of the radiolabeled peptides which were produced following chemical and proteolytic digestion of the labeled transport protein.

  7. Photoaffinity labeling of uncoupler binding sites on mitochondrial membrane.

    PubMed

    Kurup, C K; Sanadi, D R

    1977-02-01

    3H 2-azido-4-nitrophenol, a photoactive uncoupler, has been synthesized, and its uncoupling action on oxidative phosphorylation and its binding to the mitochondrial membrane have been studied. The uncoupler bound covalently to the mitochondrial membrane on photoirradiation was 3-4 times that bound reversibly in the absence of light. When irradiation was carried out in the presence of serum albumin, covalent binding was significantly depressed. The pattern of loss of ATP-Pi exchange activity with increasing amounts of the uncoupler suggests that serum albumin prevents the binding of the uncoupler to the functional sites as well. Polyacrylamide gel electrophoresis of photoaffinity labeled submitochondrial particles in the presence of sodium dodecyl sulfate revealed that a 9000 dalton peptide bound high levels of uncoupler. Other proteins in the molecular weight range of 20,000-40,000 and 55,000 were also labeled. Photolysis in the presence of serum albumin or ATP decreased the covalent binding of the uncoupler to all the proteins, but particularly to the 20,000 dalton component. Soluble ATPase and the mitochondrial proteolipid purified from labeled mitochondria showed the presence of label.

  8. Human platelet vasopressin receptor identification by direct ultraviolet photoaffinity labeling

    SciTech Connect

    Thibonnier, M.

    1987-08-15

    Tritiated vasopressin ((/sup 3/H)AVP) was directly crosslinked to its human platelet receptor by using an ultraviolet irradiation procedure. After preincubation with (/sup 3/H)AVP, the hydrodynamic parameters of the hormone-receptor complexes solubilized with 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate were derived from Sephacryl S-300 superfine gel filtration and from sucrose density gradient ultracentrifugation experiments. The following values were obtained: Stoke's radius = 5.48 +/- 0.1 nm, apparent sedimentation coefficient = 5.55 +/- 0.1 S, and calculated molecular weight = 132,000. On sodium dodecyl sulfate-8% polyacrylamide slab gel electrophoresis under reducing conditions, (/sup 3/H)AVP preferentially and specifically labeled a 125,000-dalton protein. The labeling of this protein was suppressed by addition of excess cold vasopressin, whereas angiotensin II did not inhibit incorporation of tritiated vasopressin in this protein. These results suggest that direct UV-photoaffinity labelling with (/sup 3/H)AVP is a suitable tool for the purification of the human platelet vasopressin receptor.

  9. 2-Aryl-5-carboxytetrazole as a New Photoaffinity Label for Drug Target Identification

    PubMed Central

    2016-01-01

    Photoaffinity labels are powerful tools for dissecting ligand–protein interactions, and they have a broad utility in medicinal chemistry and drug discovery. Traditional photoaffinity labels work through nonspecific C–H/X–H bond insertion reactions with the protein of interest by the highly reactive photogenerated intermediate. Herein, we report a new photoaffinity label, 2-aryl-5-carboxytetrazole (ACT), that interacts with the target protein via a unique mechanism in which the photogenerated carboxynitrile imine reacts with a proximal nucleophile near the target active site. In two distinct case studies, we demonstrate that the attachment of ACT to a ligand does not significantly alter the binding affinity and specificity of the parent drug. Compared with diazirine and benzophenone, two commonly used photoaffinity labels, in two case studies ACT showed higher photo-cross-linking yields toward their protein targets in vitro based on mass spectrometry analysis. In the in situ target identification studies, ACT successfully captured the desired targets with an efficiency comparable to the diazirine. We expect that further development of this class of photoaffinity labels will lead to a broad range of applications across target identification, and validation and elucidation of the binding site in drug discovery. PMID:27740749

  10. Photoaffinity labeling of the progesterone receptor from human endometrial carcinoma

    SciTech Connect

    Clarke, C.L.; Satyaswaroop, P.G.

    1985-11-01

    A nude mouse model for the growth of human endometrial carcinoma and hormonal modulation of the progesterone receptor (PR) was established previously. This study describes the effect of 17 beta-estradiol and tamoxifen (TAM) on growth rate and PR concentration in a hormonally responsive human endometrial tumor (EnCa 101) grown in this experimental system and presents the first characterization of human endometrial carcinoma PR. EnCa 101 was transplanted subcutaneously into ovariectomized, BALB/c, nu/nu athymic mice and grown under 17 beta-estradiol-stimulated, TAM-stimulated, and control conditions. Both 17 beta-estradiol and TAM increased the growth rate of EnCa 101 in nude mice, and a parallel increase in the cytosol PR concentration was observed. PR was partially purified by phosphocellulose and DEAE cellulose chromatography, and the DEAE eluate was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and photoaffinity labeling with (17 alpha-methyl-TH)promegestone ((TH)R5020). Two PR-negative tumors (EnCa K and EnCa V) were also examined in parallel. Photolabeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of EnCa 101 grown in the presence of 17 beta-estradiol or TAM revealed incorporation of (3H)R5020 into proteins of molecular weight approximately 116,000 and 85,000. Labeled proteins of molecular weight 66,000, 45,000, and 35,000 were also observed. No incorporation of (TH)R5020 was observed in EnCa 101 grown in the absence of estrogen, nor was any observed in EnCa K or EnCa V.

  11. ( sup 125 I)Iodoazidococaine, a photoaffinity label for the haloperidol-sensitive sigma receptor

    SciTech Connect

    Kahoun, J.R.; Ruoho, A.E. )

    1992-02-15

    A carrier-free radioiodinated cocaine photoaffinity label, (-)-3-({sup 125}I)iodo-4-azidococaine (({sup 125}I)IACoc), has been synthesized and used as a probe for cocaine-binding proteins. Photoaffinity labeling with 0.5 nM ({sup 125}I)IACoc resulted in selective derivatization of a 26-kDa polypeptide with the pharmacology of a sigma receptor in membranes derived from whole rat brain, rat liver, and human placenta. ({sup 125}I)IACoc labeling of the 26-kDa polypeptide was also inhibited by 10 {mu}M imipramine, amitriptyline, fluoxetine, benztropine, and tetrabenazine. The size of the ({sup 125}I)I-ACoc-labeled proteins is consistent with the size of proteins photolabeled in guinea pig brain and liver membranes by using the sigma photolabel azido-({sup 3}H)DTG. Kinetic analysis of ({sup 125}I)IACoc binding to rat liver microsomes revealed two sites with K{sub d} values of 19 and 126 pM, respectively. The presence or absence of proteolytic inhibitors during membrane preparation did not alter the size of the photolabeled sigma receptor, indicating that the 26-kDa polypeptide was not derived from a larger protein. In summary, ({sup 125}I)IACoc is a potent and highly specific photoaffinity label for the haloperidol-sensitive sigma receptor and will be useful for its biochemical and molecular characterization.

  12. Studies on the structure of the follicle-stimulating hormone receptor using photoaffinity labeling procedures

    SciTech Connect

    Smith, R.A.

    1985-01-01

    The general objective of this project was to study the structure of the follicle stimulating hormone (FSH) receptor using affinity labeling methods. A low density fraction derived from homogenates of bovine testis was found to contain high affinity and low capacity receptors specific for FSH. Electron microscopic examination of the fraction revealed structure resembling multilamellar membranous vesicles (MV). For photoaffinity labeling of the FSH receptors in MV, an azidobenzoyl-/sup 125/I-analog of human FSH was prepared (/sup 125/I-AB-hFSH) and binding of specific FSH receptors was studied. /sup 125/I-AB-hFSH binding of receptors was inhibited in a dose dependent manner by unlabeled hFSH, and binding was not prevented by structurally-related human chorionic gonadotropin (hCG). The formation of photocrosslinked protein of relative molecular mass (M/sub r/) 54,000, 64,000, 76,000, 84,000, 97,000 and 116,000 was found to be inhibited by unlabeled hFSH in a dose related manner, and to be dependent on photoactivation of the FSH derivative. The interpretation of the photoaffinity labeling experiments was that three proteins associated with the FSH receptor were photoaffinity labeled. Analysis by indirect means suggested that the three proteins were assembled to form oligomeric complexes, and based on the intensities and composition of the oligomeric species, spatial relationships of the polypeptides with respect to each other on the membrane surface were deduced. The results of photoaffinity labeling suggest the FSH receptor is composed of three subunits of M/sub r/ 38,000, 48,000, and 81,000 and exists in the membrane in part as a M/sub r/ 330,000 dimer.

  13. Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

    NASA Technical Reports Server (NTRS)

    Mednieks, M. I.; Popova, I.; Grindeland, R. E.

    1992-01-01

    Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

  14. Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats

    NASA Technical Reports Server (NTRS)

    Mednieks, M. I.; Popova, I.; Grindeland, R. E.

    1992-01-01

    Photoaffinity labeling in heart tissue of rats flown on Cosmos 2044 was used to measure the regulatory (R) subunits of adenosine monophosphate-dependent protein kinase. A significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) was observed when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was observed to be unaffected by spaceflight or any of the simulation conditions. Proteins of the S2 fraction constitute a minor (less than 10 percent) component of the total, whereas the S1 fraction contained most of the cell proteins. Changes in a relatively minor aspect of adenosine monophosphate-mediated reactions are considered to be representative of a metabolic effect.

  15. Analysis of photoaffinity-labeled aryl hydrocarbon receptor heterogeneity by two-dimensional gel electrophoresis

    SciTech Connect

    Perdew, G.H.; Hollenback, C.E. )

    1990-07-03

    The level of charge heterogeneity in the aryl hydrocarbon receptor (AhR) was examined by high-resolution denaturing two-dimensional (2D) gel electrophoresis. Hepa 1c1c7 cell cytosolic fraction was photoaffinity-labeled with 2-azido-3-({sup 125}I)-iodo-7,8-dibromodibenzo-p-dioxin and applied to isoelectric focusing (IEF) tube gels. After optimization of focusing conditions a broad peak of radioactivity was detected in the apparent pI range of 5.2-5.7. IEF tube gels were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by visualization of the radiolabeled AhR by autoradiography; three distinct isoforms were detected. The same 2D electrophoretic isoform pattern was obtained when the AhR from Hepa 1c1c7 was photoaffinity-labeled in cell culture. BP{sup r}Cl cells, a mutant line derived from Hepa 1c1c7 cells, contain an AhR that is unable to bind to DNA. Photoaffinity-labeled BP{sup r}Cl cytosolic fractions were subjected to 2D gel electrophoretic analysis resulting in essentially the same molecular weight and isoform pattern as seen in Hepa 1c1c7 cytosol. This result would suggest that if a mutation is present in the BP{sup r}Cl AhR it has not caused a significant change in its IEF pattern, although a small shift in the pI values was observed. Two-dimensional gel electrophoresis of photoaffinity-labeled cytosolic fractions from HeLa cells, the rat liver tumor cell line McA-RH777, and buffalo rat thymus revealed three isoforms, essentially the same isoform pattern as in Hepa 1c1c7 cells. This would indicate that despite the considerable molecular weight polymorphism between species the level of charge heterogeneity is high conserved.

  16. Photo-Affinity Labeling of Specific Acetylcholine-Binding Sites on Membranes

    PubMed Central

    Kiefer, Hansruedi; Lindstrom, Jon; Lennox, Edwin S.; Singer, S. J.

    1970-01-01

    Acetylcholinesterase of intact red blood cell membranes and the acetylcholine receptor at the neuromuscular junction of whole-frog sartorius muscle have been irreversibly inactivated by photo-affinity labeling with two quaternary ammonium aryl azides. The inactivation requires that the azides, at the time of their photolytic conversion to highly reactive nitrenes, are reversibly bound to the specific acetylcholine-binding sites. PMID:5275370

  17. (125I)iodoazidococaine, a photoaffinity label for the haloperidol-sensitive sigma receptor.

    PubMed Central

    Kahoun, J R; Ruoho, A E

    1992-01-01

    A carrier-free radioiodinated cocaine photo-affinity label, (-)-3-(125I)iodo-4-azidococaine [(125I)IACoc], has been synthesized and used as a probe for cocaine-binding proteins. Photoaffinity labeling with 0.5 nM (125I)IACoc resulted in selective derivatization of a 26-kDa polypeptide with the pharmacology of a sigma receptor in membranes derived from whole rat brain, rat liver, and human placenta. Covalent labeling of the 26-kDa polypeptide was inhibited by 1 microM haloperidol, di(2-tolyl)guanidine (DTG), 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP), dextromethorphan, and carbetapentane. Stereoselective protection of (125I)IACoc photolabeling by 3-PPP [(+)-3-PPP more potent than (-)-3-PPP] was observed. (125I)IACoc labeling of the 26-kDa polypeptide was also inhibited by 10 microM imipramine, amitriptyline, fluoxetine, benztropine, and tetrabenazine. The size of the (125I)I-ACoc-labeled proteins is consistent with the size of proteins photolabeled in guinea pig brain and liver membranes by using the sigma photolabel azido-[3H]DTG. Kinetic analysis of (125I)IACoc binding to rat liver microsomes revealed two sites with Kd values of 19 and 126 pM, respectively. The presence or absence of proteolytic inhibitors during membrane preparation did not alter the size of the photolabeled sigma receptor, indicating that the 26-kDa polypeptide was not derived from a larger protein. In summary, (125I)IACoc is a potent and highly specific photoaffinity label for the haloperidol-sensitive sigma receptor and will be useful for its biochemical and molecular characterization. Images PMID:1311097

  18. Characterization and photoaffinity labeling of the muscarinic acetylcholine receptor

    SciTech Connect

    Cremo, C.R.

    1983-01-01

    The muscarinic acetylcholine receptor, identified by tritiated L-quinuclidinyl benzilate (L-(/sup 3/H)QNB) binding, was solubilized from porcine atrial membranes using a 5:1 (w/w) ratio of digitonin and cholate. Specific binding activities of the solubilized receptor solutions usually exceeded 1.0 nmol L-(/sup 3/H)QNB sites per gram of protein, representing 75-98% total site recovery and a two- to three-fold enrichment over untreated atrial membranes. Two rapid assays for measuring the binding activities of detergent extracts were devised and compared with equilibrium dialysis. All three methods gave similar results. The equilibrium dissociation constant of the solubilized receptor for L-(/sup 3/H)QNB as determined by the three methods varied from 230 to 450 pM depending on the method and temperature. The interaction of alkyl quanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkyl guanidines with alkyl chain lengths from one to ten carbons displaced (/sup 3/H)L-quinuclidinyl bensilate ((/sup 3/H)L-QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of -1n K/sub i/ versus alkyl carbon chain number, a value of -(473 +/- 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The synthesis and properties of a radiolabeled muscarinic antagonist photoaffinity probe, (/sup 3/H) p-azidoatropine methyl iodide were reported.

  19. Derivativation of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

    SciTech Connect

    Wadzinski, B.; Shanahan, M.; Ruoho, A.

    1987-05-01

    An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin (IAPS-Fsk), has been synthesized, purified, and characterized. The K/sub i/ for inhibition of 3-0-methylglucose transport by TAPS-Fsk in human erythrocytes was found to be 0.1 uM. The carrier-free radioiodinated label has been shown to be a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes with 1-10 nM (I-125)IAPS-Fsk and analysis by SDS-PAGE showed specific derivatization of a broad band with an apparent molecular weight of 40-70 kDa. Photoincorporation using 2 nM (I-125)IAPS-Fsk was protected with D-glucose, cytochalasin B, and forskolin. No protection was observed with L-glucose. Endo-B-galactosidase digestion and trypsinization of (I-125)IAPS-Fsk labelled erythrocytes reduced the specifically radiolabelled transporter to 40 kDa and 18 kDa respectively. (I-125)-IAPS-Fsk will be used to study the structural aspects of the glucose transporter.

  20. Synthesis of azidotubulin: a photoaffinity label for tubulin-binding proteins.

    PubMed

    Balczon, R D; Brinkley, B R

    1989-10-17

    A photoaffinity label for the identification of tubulin-binding proteins was synthesized from phosphocellulose-purified bovine brain tubulin and (N-hydroxysuccinimidyl)-4-azidosalicylic acid. The azidotubulin derivative retained the ability to undergo temperature-dependent microtubule assembly and disassembly. When incubated with purified tau protein, the azidotubulin and tau formed cross-linked complexes upon photoactivation. When 125I-labeled azidotubulin was used to photoaffinity label tubulin-binding proteins within the kinetochore of isolated mammalian chromosomes, a 130-kDa band was identified on autoradiographs of SDS-polyacrylamide gels of the 125I-labeled azidotubulin/chromosome preparations. The 130-kDa complex was isolated by antitubulin affinity chromatography and analyzed by immunoblotting using both antitubulin and kinetochore-specific sera obtained from human patients with the autoimmune disease scleroderma CREST. The immunoblots demonstrated that the 130-kDa band that was observed on autoradiographs was a complex of a subunit of the tubulin dimer and an 80-kDa CREST-specific kinetochore protein. The binding of azidotubulin to the 80-kDa kinetochore protein was significantly decreased when chromosomes were treated with a mixture of 9 parts underivatized tubulin to 1 part azidotubulin prior to photolysis. The formation of the 130-kDa azidotubulin/kinetochore protein complex was not inhibited by pretreating the chromosomes with CREST serum prior to incubation with azidotubulin. Azidotubulin should be a useful probe for the identification and characterization of tubulin-binding proteins.

  1. Synthesis and characterization of photoaffinity labelling reagents towards the Hsp90 C-terminal domain.

    PubMed

    Simon, Binto; Huang, Xuexia; Ju, Huangxian; Sun, Guoxuan; Yang, Min

    2017-02-21

    Glucosyl-novobiocin-based diazirine photoaffinity labelling reagents (PALs) were designed and synthesized to probe the Hsp90 C-terminal domain unknown binding pocket and the structure-activity relationship. Five PALs were successfully synthesized from novobiocin in six consecutive steps employing phase transfer catalytic glycosylation. Reactions were monitored and guided by analytical LC/MS which led to different strategies of adding either a PAL precursor or a sugar moiety first. The structures and bonding linkages of these compounds were characterised by various 2D-NMR spectroscopy and MS techniques. Synthetic techniques provide powerful probes for unknown protein binding pockets.

  2. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

    1989-01-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  3. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

    1989-01-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  4. Identification of rat brain opioid (enkephalin) receptor by photoaffinity labeling

    SciTech Connect

    Yeung, C.W.

    1986-01-01

    A photoreactive, radioactive enkephalin derivative was prepared and purified by high performance liquid chromatography. Rat brain and spinal cord plasma membranes were incubated with this radioiodinated photoprobe and were subsequently photolysed. Autoradiography of the sodium dodecyl sulfate gel electrophoresis of the solubilized and reduced membranes showed that a protein having an apparent molecular weight of 46,000 daltons was specifically labeled, suggesting that this protein may be the opioid (enkephalin) receptor.

  5. Base-Mediated One-Pot Synthesis of Aliphatic Diazirines for Photoaffinity Labeling.

    PubMed

    Wang, Lei; Tachrim, Zetryana Puteri; Kurokawa, Natsumi; Ohashi, Fumina; Sakihama, Yasuko; Hashidoko, Yasuyuki; Hashimoto, Makoto

    2017-08-22

    Aliphatic diazirines have been widely used as prominent photophores for photoaffinity labeling owing to their relatively small size which can reduce the steric effect on the natural interaction between ligands and proteins. Based on our continuous efforts to develop efficient methods for the synthesis of aliphatic diazirines, we present here a comprehensive study about base-mediated one-pot synthesis of aliphatic diazirines. It was found that potassium hydroxide (KOH) can also promote the construction of aliphatic diazirine with good efficiency. Importantly, KOH is cheaper, highly available, and easily handled and stored compared with the previously used base, potassium tert-butoxide (t-BuOK). Gram-scale study showed that it owned great advantages in being used for the large-scale production of aliphatic diazirines. This protocol is highly neat and the desired products can be easily isolated and purified. As the first comprehensive study of the base-mediated one-pot synthesis of aliphatic diazirines, this work provided good insight into the preparation and utilization of diazirine-based photoaffinity labeling probes.

  6. Anthracycline photoaffinity labeling of a mitochondrial polypeptide in P388 murine leukemic cell lines

    SciTech Connect

    Averbuch, S.D.; Glover, C.J.; Felsted, R.L.

    1986-12-01

    N-(p-Azido(3,5-/sup 3/H)benzoyl)daunorubicin ((/sup 3/H)NABD), a radioactive photoactive anthracycline analogue, was used to photoaffinity label anthracycline binding polypeptides in P388 murine leukemic cell lines. Whole cell homogenates were mixed with 6 X 10(-8) M (/sup 3/H)NABD, exposed to ultraviolet light, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for radiolabel incorporation. Autoradiofluorography showed incorporation of radioactivity into a Mr 18,000 component independent of polypeptides prominently stained with Coomassie blue. Photolabeling of subcellular fractions showed predominant mitochondrial localization of the Mr 18,000 radiolabel. The protein composition of the photolabeled constituents was confirmed by treatment with proteinase K, DNase and RNase, or by lipid extraction with organic solvent. (/sup 3/H)NABD photolabeling of homogenates from anthracycline sensitive and resistant cells resulted in Mr 18,000 radiolabel incorporation of 3966 +/- 355 and 6487 +/- 533 dpm per 50 micrograms cellular protein for anthracycline sensitive and resistant cells, respectively (P less than 0.005). These studies characterize the photoaffinity labeling of a low molecular weight mitochondrial polypeptide using a photoactive anthracycline analogue. The role for this polypeptide as a mediator of anthracycline activity remains to be determined.

  7. Juvenile hormone receptors in insect larval epidermis: Identification by photoaffinity labeling

    SciTech Connect

    Palli, S.R.; Osir, E.O.; Edwards, M.; Hiruma, K.; Riddiford, L.M. ); Eng, W.; Boehm, M.F.; Kulscar, P.; Ujvary, I.; Prestwich, G.D. )

    1990-01-01

    Tritiated photoaffinity analogs of the natural lepidopteran juvenile hormones, JH I and II (epoxy({sup 3}H)bishomofarnesyl diazoacetate (({sup 3}H)EHDA) and epoxy({sup 3}H)homofarnesyl diazoacetate (({sup 3}H)EHDA)), and of the JH analog methoprene (({sup 3}H)methoprene diazoketone (({sup 3}H)MDK)) were synthesized and used to identify specific JH binding proteins in the larval epidermis of the tobacco hornworm (Manduca sexta). EBDA and EHDA specifically photolabeled a 29-kDa nuclear protein (pI 5.8). This protein and a second 29-kDa protein (pI 6.0) were labeled by MDK, but excess unlabeled methoprene or MDK only prevented binding to the latter. These 29-kDa proteins are also present in larval fat body but not in epidermis from either wandering stage or allatectomized larvae, which lack high-affinity JH binding sites. A 29-kDa nuclear protein with the same developmental specificity as this JH binder bound the DNA of two larval endocuticle genes. A 38-kDa cytosolic protein was also specifically photolabeled by these photoaffinity analogs. The 29-kDa nuclear protein is likely the high-affinity receptor for JH that mediates its genomic action, whereas the 38-kDa cytosolic protein may serve as an intracellular carrier for these highly lipophilic hormones and hormone analogs.

  8. Juvenile hormone receptors in insect larval epidermis: identification by photoaffinity labeling.

    PubMed Central

    Palli, S R; Osir, E O; Eng, W; Boehm, M F; Edwards, M; Kulcsar, P; Ujvary, I; Hiruma, K; Prestwich, G D; Riddiford, L M

    1990-01-01

    Tritiated photoaffinity analogs of the natural lepidopteran juvenile hormones, JH I and II [epoxy[3H]bishomofarnesyl diazoacetate ([3H]EBDA) and epoxy[3H]homofarnesyl diazoacetate ([3H]EHDA)], and of the JH analog methoprene [[3H]methoprene diazoketone ([3H]MDK)] were synthesized and used to identify specific JH binding proteins in the larval epidermis of the tobacco hornworm (Manduca sexta). EBDA and EHDA specifically photolabeled a 29-kDa nuclear protein (pI 5.8). This protein and a second 29-kDa protein (pI 6.0) were labeled by MDK, but excess unlabeled methoprene or MDK only prevented binding to the latter. These 29-kDa proteins are also present in larval fat body but not in epidermis from either wandering stage or allatectomized larvae, which lack high-affinity JH binding sites. A 29-kDa nuclear protein with the same developmental specificity as this JH binder bound the DNA of two larval endocuticle genes. A 38-kDa cytosolic protein was also specifically photolabeled by these photoaffinity analogs. The 29-kDa nuclear protein is likely the high-affinity receptor for JH that mediates its genomic action, whereas the 38-kDa cytosolic protein may serve as an intracellular carrier for these highly lipophilic hormones and hormone analogs. Images PMID:11607060

  9. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 1. Photoaffinity labeling of murine L1210 dihydrofolate reductase and amino acid sequence of the binding region

    SciTech Connect

    Price, E.M.; Smith, P.L.; Klein, T.E.; Freisheim, J.H.

    1987-07-28

    N/sup ..cap alpha../-(4-Amino-4-deoxy-10-methylpteroyl)-N/sup epsilon/-(4-azido-5-(/sup 125/I)iodosalicylyl)-L-lysine, a photoaffinity analogue of methotrexate, is only 2-fold less potent than methotrexate in the inhibition of murine L1210 dihydrofolate reductase. Irradiation of the enzyme in the presence of an equimolar concentration of the /sup 125/I-labeled analogue ultimately leads to an 8% incorporation of the photoprobe. A 100-fold molar excess of methotrexate essentially blocks this incorporation. Cyanogen bromide digestion of the labeled enzyme, followed by high-pressure liquid chromatography purification of the generated peptides, indicates that greater than 85% of the total radioactivity is incorporated into a single cyanogen bromide peptide. Sequence analysis revealed this peptide to be residues 53-111, with a majority of the radioactivity centered around residues 63-65 (Lys-Asn-Arg). These data demonstrate that the photoaffinity analogue specifically binds to dihydrofolate reductase and covalently modifies the enzyme following irradiation and is therefore a photolabeling agent useful for probing the inhibitor binding domain of the enzyme.

  10. Identification of CRALBP ligand interactions by photoaffinity labeling, hydrogen/deuterium exchange, and structural modeling.

    PubMed

    Wu, Zhiping; Hasan, Azeem; Liu, Tianyun; Teller, David C; Crabb, John W

    2004-06-25

    Cellular retinaldehyde-binding protein (CRALBP) functions in the retinal pigment epithelium (RPE) as an acceptor of 11-cis-retinol in the isomerization step of the rod visual cycle and as a substrate carrier for 11-cis-retinol dehydrogenase. Toward a better understanding of CRALBP function, the ligand binding cavity in human recombinant CRALBP (rCRALBP) was characterized by photoaffinity labeling with 3-diazo-4-keto-11-cis-retinal and by high resolution mass spectrometric topological analyses. Eight photoaffinity-modified residues were identified in rCRALBP by liquid chromatography tandem mass spectrometry, including Tyr(179), Phe(197), Cys(198), Met(208), Lys(221), Met(222), Val(223), and Met(225). Multiple different adduct masses were found on the photolabeled residues, and the molecular identity of each modification remains unknown. Supporting the specificity of photo-labeling, 50% of the modified residues have been associate with retinoid interactions by independent analyses. In addition, topological analysis of apo- and holo-rCRALBP by hydrogen/deuterium exchange and mass spectrometry demonstrated residues 198-255 incorporate significantly less deuterium when the retinoid binding pocket is occupied with 11-cis-retinal. This hydrophobic region encompasses all but one of the photo-labeled residues. A structural model of CRALBP ligand binding domain was constructed based on the crystal structures of three homologues in the CRAL-TRIO family of lipid-binding proteins. In the model, all of the photolabeled residues line the ligand binding cavity except Met(208), which appears to reside in a flexible loop at the entrance/exit of the ligand cavity. Overall, the results expand to 12 the number of residues proposed to interact with ligand and provide further insight into CRALBP ligand and protein interactions.

  11. Photoaffinity labeling of opioid receptor with morphine-7,8-oxide (morphine epoxide)

    SciTech Connect

    Takayanagi, I.; Shibata, R.; Miyata, N.; Hirobe, M.

    1982-05-01

    The opioid receptor mediating inhibitory action of morphine in the electrically stimulated guinea pig ileum was irreversibly photoinactivated by morphine epoxide (3 X 10(-6) M). Morphine epoxide (up to 3 X 10(-5) M) did not influence the responses of rat vas deferens (epsilon-receptor) or rabbit vas deferens (kappa-receptor) to electrical stimulation. Effective concentrations of morphine epoxide were much lower in the guinea pig ileum (mu-receptor) than in the mouse vas deference (delta-receptor). The inhibitory action of (Met)-enkephalin on the twitch responses of the rat vas deferens and mouse vas deferens to electrical stimulation were not influenced after irradiation in the presence of morphine epoxide (3 X 10(-6) M). Therefore, morphine epoxide is probably a useful probe for photoaffinity labeling of the mu-receptor in vitro.

  12. Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling.

    PubMed

    Wu, Yuteng; Olsen, Lasse B; Lau, Yu Heng; Jensen, Claus Hatt; Rossmann, Maxim; Baker, Ysobel R; Sore, Hannah F; Collins, Súil; Spring, David R

    2016-04-15

    Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo-crosslinking motif and a peptide stapling reagent. Using double-click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo-crosslinking amino acid. When applied to a p53-derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull-down assays, and can be used to investigate the target selectivity of stapled peptides.

  13. Development of a Multifunctional Benzophenone Linker for Peptide Stapling and Photoaffinity Labelling

    PubMed Central

    Wu, Yuteng; Olsen, Lasse B.; Lau, Yu Heng; Jensen, Claus Hatt; Rossmann, Maxim; Baker, Ysobel R.; Sore, Hannah F.; Collins, Súil

    2016-01-01

    Abstract Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo‐crosslinking motif and a peptide stapling reagent. Using double‐click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo‐crosslinking amino acid. When applied to a p53‐derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull‐down assays, and can be used to investigate the target selectivity of stapled peptides. PMID:26919579

  14. Photoaffinity labeling of a bacterial sialidase with an aryl azide derivative of sialic acid

    SciTech Connect

    van der Horst, G.T.; Mancini, G.M.; Brossmer, R.; Rose, U.; Verheijen, F.W. )

    1990-07-05

    A photoreactive radioiodinatable derivative of 2-deoxy-2,3-didehydro-5-N-acetylneuraminic acid (NeuAc2en), 5-N-acetyl-9-(4-azidosalicoylamido)-2-deoxy-2,3-didehydroneuram inic acid (ASA-NeuAc2-en) has been synthesized and used to label the active site of Clostridium perfringens sialidase. Like NeuAc2en, its aryl azide derivative is a strong competitive inhibitor of sialidase (Ki approximately 15 microM). The absorbance spectrum of ASA-NeuAc2en shows a characteristic aryl azide peak, which disappears upon photolysis with UV light. When its radioiodinated counterpart 5-N-acetyl-9-(4-iodoazidosalicoylamido)-2-deoxy-2,3-didehydrone uraminic acid (({sup 125}I)IASA-NeuAc2en) was photolyzed in the presence of C. perfringens sialidase a 72-kDa protein was labeled. Labeling occurred specifically in the active site since it was inhibited in the presence of NeuAc2en. Chemical cleavage of the photoaffinity-labeled 72-kDa protein demonstrates that specifically labeled peptides involved in the formation of the active site can easily be determined. ASA-NeuAc2en is a valuable new tool for the identification and structural/functional analysis of sialidases and other proteins, recognizing this sialic acid derivative.

  15. Membrane penetration of Sendai virus glycoproteins during the early stages of fusion with liposomes as determined by hydrophobic photoaffinity labeling

    SciTech Connect

    Novick, S.L.; Hoekstra, D.

    1988-10-01

    The hydrophobic photoaffinity label 3-(trifluoromethyl)-3-(m-(/sup 125/I)iodophenyl)diazirine was used to label Sendai virus proteins during fusion with cardiolipin and phosphatidylserine liposomes. Preferential labeling of the viral fusion protein during the initial stages of fusion demonstrated that this protein interacts with the hydrophobic core of the target membrane as an initiating event of virus-liposome fusion. Labeling showed time, temperature, and pH dependence consistent with earlier fluorescent measurements of fusion kinetics. The present method provides conclusive evidence supporting the hypothesis that hydrophobic interaction of the fusion protein with the target bilayer is an initial event in the fusion mechanism of viral membranes.

  16. Photoaffinity labelling of the rat liver nuclear thyroid hormone receptor with (/sup 125/I)triiodothyronine

    SciTech Connect

    David-Inouye, Y.; Somack, R.; Nordeen, S.K.; Apriletti, J.W.; Baxter, J.D.; Eberhardt, N.L.

    1982-11-01

    (/sup 125/I)Triiodothyronine (T3) was used as a photoreactive probe for the thyroid hormone nuclear receptor in photoaffinity labelling experiments. Autoradiograms of photolysis products electrophoresed on either one or two-dimensional gels showed that (/sup 125/I)T3 covalently, but nonspecifically, labelled many proteins in the partially purified receptor preparations used. However, one of these proteins with an estimated molecular weight of 47,000 and an isoelectric point of approximately 6.2 +/- 0.5 pH units appears to be the thyroid hormone receptor, since, in contrast to the other proteins, its photoinduced labelling was blocked by concentrations of T3 and thyroxine (T4) similar to those that inhibit binding of (/sup 125/I)T3 by the receptor in equilibrium binding assays. In addition, the isoelectric point of the photolabelled protein agrees with that determined in separate equilibrium isoelectric focusing studies. These results indicate that (/sup 125/)T3 can serve as a photoreactive probe for the thyroid hormone nuclear receptor, and they suggest that this receptor is a single polypeptide chain of molecular weight 47,000 with an isoelectric point of 6.2 +/- 0.5 pH units.

  17. Photoaffinity labelling of the rat liver nuclear thyroid hormone receptor with (/sup 125/I)triiodothyronine

    SciTech Connect

    David-Inouye, Y.; Somack, R; Nordeen, S.K.; Apriletti, J.W.; Baxter, J.D.; Eberhardt, N.L.

    1982-11-01

    (/sup 125/I)Triiodothyronine (T/sub 3/) was used as a photoreactive probe for the thyroid hormone nuclear receptor in photoaffinity labelling experiments. Autoradiograms of photolysis products electrophoresed on either one or two-dimensional gels showed that (/sup 125/I)T/sub 3/ covalently, but nonspecifically, labelled many proteins in the partially purified receptor preparations used. However, one of these proteins with an estimated molecular weight of 47,000 and an isoelectric point of approximately 6.2 +/- 0.5 pH units appears to be the thyroid hormone receptor, since, in contrast to the other proteins, its photoinduced labelling was blocked by concentrations of T/sub 3/ and thyroxine (T/sub 4/) similar to those that inhibit binding of (/sup 125/I)T/sub 3/ by the receptor in equilibrium binding assays. In addition, the isoelectric point of the photolabelled protein agrees with that determined in separate equilibrium isoelectric focusing studies. These results indicate that (/sup 125/I)T/sub 3/ can serve as a photoreactive probe for the thyroid hormone nuclear receptor, and they suggest that this receptor is a single polypeptide chain of molecular weight 47,000 with an isoelectric point of 6.2 +/- 0.5 pH units.

  18. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    SciTech Connect

    Hicks, G.R.; Rayle, D.L.; Jones, A.M.; Lomax, T.L. )

    1989-07-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-(7-{sup 3}H)IAA(({sup 3}H)N{sub 3}IAA), in a manner similar to the accumulation of ({sup 3}H)IAA. The association of the ({sup 3}H)N{sub 3}IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of ({sup 3}H)N{sub 3}IAA to plasma membrane vesicles prior to exposure to UV light and detected by subsequent NaDodSO{sub 4}/PAGE and fluorography. When the reaction temperature was lowered to {minus}196{degree}C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  19. Dopamine transport sites selectively labeled by a novel photoaffinity probe: 125I-DEEP

    SciTech Connect

    Grigoriadis, D.E.; Wilson, A.A.; Lew, R.; Sharkey, J.S.; Kuhar, M.J. )

    1989-08-01

    The dopamine transporter was labeled using a photosensitive compound related to GBR-12909, {sup 125}I-1-(2-(diphenylmethoxy)ethyl)-4-(2- (4-azido-3-iodophenyl)ethyl)piperazine ({sup 125}I-DEEP). {sup 125}I-DEEP bound reversibly and with high affinity to the dopamine transport protein in the absence of light and could be covalently attached to the protein following exposure to UV light. In rat striatal homogenates, {sup 125}I-DEEP was found to incorporate covalently into a protein with apparent molecular weight of 58,000 Da. The properties of this binding protein were characteristic of the dopamine transporter since covalent attachment could be inhibited by dopamine-uptake blockers with the proper pharmacological rank order of potencies. Covalent binding was also inhibited in a stereospecific manner by (+) and (-) cocaine, as well as other cocaine analogs. The protein was not found in the cerebellum. The dopamine transporter appears to exist in a glycosylated form since photoaffinity-labeled transport sites could adsorb to wheat germ-agglutinin and could be specifically eluted from the column by beta-N-acetylglucosamine.

  20. Photoaffinity labeling the. beta. -adrenergic receptor with an iodoazido derivative of norepinephrine

    SciTech Connect

    Resek, J.F.

    1989-01-01

    The {beta}-adrenergic receptor is an integral membrane protein coupled to adenylate cyclase by the guanine nucleotide binding protein, Gs. Agonist binding to the receptor results in coupling the receptor to Gs, increased adenylate cyclase activity, and receptor desensitization. In contrast, antagonists bind but do not activate the receptor or result in desensitization. To study the structure and regulation of the {beta}-adrenergic receptor in the membrane, it is useful to develop ligands which covalently label the binding site. In this thesis the synthesis and characterization of the first agonist photolabel for the {beta}-adrenergic receptor is presented. The agonist photoaffinity label, N-(p-azido-m-iodophenethylamidoisobutyl)-norepinephrine (NAIN), was synthesized in non-radioactive and radioactive carrier-free forms with {sup 125}I (2,200 Ci/mmole). NAIN was chemically characterized by TLC mobility, melting point, NMR, IR, and Mass Spectroscopy. NAIN was shown to be competitive with the {beta}-adrenergic ligand ({sup 125}I)-ICYP in several membranes containing {beta}-adrenergic receptors. Binding data indicated that NAIN coupled the receptor to Gs and had an affinity for the receptor which was similar to isoproterenol. NAIN stimulated adenylate cyclase activity in guinea pig lung and S49 WT mouse lymphoma cell membranes with a K{sub act} and V max similar to isoproterenol while in frog erythrocyte ghosts, NAIN produced 77% of the maximally stimulated adenylate cyclase activity of isoproterenol. These data show that NAIN is an agonist for the {beta}-adrenergic receptor.

  1. Photoaffinity site-specific covalent labeling of human corticosteroid-binding globulin.

    PubMed Central

    Marver, D; Chiu, W; Wolff, M E; Edelman, I S

    1976-01-01

    A method was developed for the synthesis of high-specific-activity 21-diazo-21-[6,7-(3)H]deoxycorticosterone, an analog of corticosterone. This analog was used as a photoaffinity label of a high affinity steroid-binding protein, human corticosteroid-binding globulin. Based on direct binding studies and crosscompetition experiments, this diazo derivative exhibited the requisite affinity (within a factor of 1.5 times that of corticosterone) and site specificity to qualify as an affinity labeling legand. Irradiation of corticosteroid-binding globulin with the 21-diazo derivative resulted in irreversible binding to corticosteroid-binding globulin, identified by polyacrylamide gel electrophoresis. Specificity of covalent binding to corticosteroid-binding globulin was established by competition analysis with various steroids. Irreversibility of photodependent binding was shown by persistence of the complex on electrophoresis (in contrast to the noncovalently linked complex), and resistance to exchange with corticosterone or pregnanediol and to solvent extraction. Site specificity of covalent binding was inferred from the effects of a scavenger, Tris-HC1, and fluorescence quenching of a neighboring tryptophan. PMID:1069998

  2. Most drugs that reverse multidrug resistance also inhibit photoaffinity labeling of P-glycoprotein by a vinblastine analog

    SciTech Connect

    Akiyama, S.; Cornwell, M.M.; Kuwano, M.; Pastan, I.; Gottesman, M.M.

    1988-02-01

    Multidrug-resistant human KB carcinoma cells express a 170,000-dalton membrane glycoprotein (P-glycoprotein) that can be photoaffinity labeled with the vinblastine analog N-(p-azido-(3-/sup 125/I)salicyl)-N'-(beta-aminoethyl)vindesine. Several agents that suppress the multidrug-resistant phenotype, including N-solanesyl-N,N'-bis(3,4-dimethylbenzyl)ethylenediamine, cepharanthine, quinidine, and reserpine, were found to inhibit photolabeling of P-glycoprotein at doses comparable to those that reverse multidrug resistance. However, the phenothiazines chlorpromazine and trifluoperazine, which also effectively reverse multidrug resistance, were poor inhibitors of the photoaffinity labeling of P-glycoprotein. Chloroquine, propranolol, or atropine, which only partially reversed the drug resistance, also did not inhibit photolabeling. Naphthalene sulfonamide calmodulin inhibitors, W7 and W5, as well as many other drugs that did not circumvent multidrug resistance, did not inhibit photolabeling. These studies suggest that most, but not all, agents that phenotypically suppress multidrug resistance also inhibit drug binding to a site on P-glycoprotein with which a photoaffinity analog of vinblastine interacts.

  3. Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling

    SciTech Connect

    Winder, B.S.

    1988-01-01

    Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ({sup 3}H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of {sup 3}H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked {sup 3}H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked {sup 3}H-EFDA in toluene alone, and of the protein-linked {sup 3}H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) for binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III.

  4. Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues.

    PubMed

    Tulloch, Lindsay B; Menzies, Stefanie K; Fraser, Andrew L; Gould, Eoin R; King, Elizabeth F; Zacharova, Marija K; Florence, Gordon J; Smith, Terry K

    2017-09-05

    Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photoaffinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the FoF1-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or

  5. Photoaffinity labeling of cytochrome P4501A1 with azidocumene: identification of cumene hydroperoxide binding region.

    PubMed

    Cvrk, T; Strobel, H W

    1998-01-01

    Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which cumene hydroperoxide binds, we prepared an analog of cumene hydroperoxide for use as a photoaffinity label. p-Azido-isopro-pylbenzene (azidocumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [3H]-azidocumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492-503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494-512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501-504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501-L502-K503, is the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A

  6. Photoaffinity labeling of Ras converting enzyme using peptide substrates that incorporate benzoylphenylalanine (Bpa) residues: improved labeling and structural implications.

    PubMed

    Kyro, Kelly; Manandhar, Surya P; Mullen, Daniel; Schmidt, Walter K; Distefano, Mark D

    2011-12-15

    Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-l-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design.

  7. Photoaffinity Labeling of Ras Converting Enzyme using Peptide Substrates that Incorporate Benzoylphenylalanine (Bpa) Residues: Improved Labeling and Structural Implications

    PubMed Central

    Kyro, Kelly; Manandhar, Surya P.; Mullen, Daniel; Schmidt, Walter K.; Distefano, Mark D.

    2012-01-01

    Rce1p catalyzes the proteolytic trimming of C-terminal tripeptides from isoprenylated proteins containing CAAX-box sequences. Because Rce1p processing is a necessary component in the Ras pathway of oncogenic signal transduction, Rce1p holds promise as a potential target for therapeutic intervention. However, its mechanism of proteolysis and active site have yet to be defined. Here, we describe synthetic peptide analogues that mimic the natural lipidated Rce1p substrate and incorporate photolabile groups for photoaffinity-labeling applications. These photoactive peptides are designed to crosslink to residues in or near the Rce1p active site. By incorporating the photoactive group via p-benzoyl-L-phenylalanine (Bpa) residues directly into the peptide substrate sequence, the labeling efficiency was substantially increased relative to a previously-synthesized compound. Incorporation of biotin on the N-terminus of the peptides permitted photolabeled Rce1p to be isolated via streptavidin affinity capture. Our findings further suggest that residues outside the CAAX-box sequence are in contact with Rce1p, which has implications for future inhibitor design. PMID:22079863

  8. Photoaffinity analogues of methotrexate as folate antagonist binding probes. 2. Transport studies, photoaffinity labeling, and identification of the membrane carrier protein for methotrexate from murine L1210 cells

    SciTech Connect

    Price, E.M.; Freisheim, J.H.

    1987-07-28

    A membrane-derived component of the methotrexate/one-carbon-reduced folate transport system in murine L1210 cells has been identified by using a photoaffinity analogue of methotrexate. The compound, a radioiodinated 4-azidosalicylyl derivative of the lysine analogue of methotrexate, is transported into murine L1210 cells in a temperature-dependent, sulfhydryl reagent inhibitable manner with a K/sub t/ of 506 +/- 79 nM and a V/sub max/ of 17.9 +/- 4.2 pmol min/sup -1/ (mg of total cellular protein)/sup -1/. Uptake of the iodinated compound at 200 nM is inhibited by low amounts of methotrexate. The parent compounds of the iodinated photoprobe inhibit (/sup 3/H)methotrexate uptake, with the uniodinated 4-azidosalicylyl derivative exhibiting a K/sub i/ of 66 +/- 21 nM. UV irradiation, at 4 /sup 0/C, of a cell suspension that had been incubated with the probe results in the covalent modification of a 46K-48K protein. This can be demonstrated when the plasma membranes from the labeled cells are analyzed via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Labeling of this protein occurs half-maximally at a reagent concentration that correlates with the K/sub t/ for transport of the iodinated compound. Protection against labeling of this protein by increasing amounts of methotrexate parallels the concentration dependence of inhibition of photoprobe uptake by methotrexate. Evidence that, in the absence of irradiation and at 37/sup 0/C, the iodinated probe is actually internalized is demonstrated by the labeling of two soluble proteins (M/sub r/ 38K and 21K) derived from the cell homogenate supernatant.

  9. Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam

    SciTech Connect

    Thomassin, J.; Tephly, T.R. )

    1990-09-01

    Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP-glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of (3H)flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with (3H) flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000).

  10. Binding Ensemble PROfiling with (F)photoaffinity Labeling (BEProFL) Approach: Mapping the Binding Poses of HDAC8 Inhibitors

    PubMed Central

    He, Bai; Velaparthi, Subash; Pieffet, Gilles; Pennington, Chris; Mahesh, Aruna; Holzle, Denise L.; Brunsteiner, Michael; van Breemen, Richard; Blond, Sylvie Y.; Petukhov, Pavel A.

    2009-01-01

    A Binding Ensemble PROfiling with (F)photoaffinity Labeling (BEProFL) approach that utilizes photolabeling of HDAC8 with a probe containing a UV-activated aromatic azide, mapping the covalent modifications by liquid chromatography-tandem mass-spectrometry, and a computational method to characterize the multiple binding poses of the probe is described. Using the BEProFL approach two distinct binding poses of the HDAC8 probe were identified. The data also suggest that an “upside-down” pose with the surface binding group of the probe bound in an alternative pocket near the catalytic site may contribute to the binding. PMID:19886628

  11. Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling.

    PubMed

    Dong, Maoqing; Miller, Laurence J

    2013-08-01

    Direct analysis of mode of peptide docking using intrinsic photoaffinity labeling has provided detailed insights for the molecular basis of cholecystokinin (CCK) interaction with the type 1 CCK receptor. In the current work, this technique has been applied to the closely related type 2 CCK receptor that also binds the natural full agonist peptide, CCK, with high affinity. A series of photolabile CCK analog probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogs that possessed full biological activity utilized in photoaffinity labeling. The position 29 probe, incorporating a photolabile benzoyl-phenylalanine in that position, was shown to bind with high affinity and to be a full agonist, with potency not different from that of natural CCK, and to covalently label the type 2 CCK receptor in a saturable, specific and efficient manner. Using proteolytic peptide mapping, mutagenesis, and radiochemical Edman degradation sequencing, this probe was shown to establish a covalent bond with type 2 CCK receptor residue Phe¹²⁰ in the first extracellular loop. This was in contrast to its covalent attachment to Glu³⁴⁵ in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors.

  12. Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand

    SciTech Connect

    Nikolics, K.; Szonyi, E.; Ramachandran, J.

    1988-03-08

    Photoreactive derivatives of GnRH and its analogues were prepared by incorporation of the 2-nitro-4(5)-azidophenylsulfenyl (2,4(5)-NAPS) group into amino acid residues at position 1, 3, 6, or 8 of the decapeptide sequence. The modification of Trp/sup 3/ by the 2,4-NAPS group led to a complete loss of the luteinizing hormone (LH) releasing as well as LH-release-inhibiting activity of the peptide. The (D-Lys(2,4-NAPS))/sup 6/ analog was a very potent agonist that, after covalent attachment by photoaffinity labeling, caused prolonged LH secretion at a submaximal rate. (Orn(2,4-NAPS))/sup 8/-GnRH, a full agonist with a relative potency of 7% of GnRH, after photoaffinity labeling caused prolonged maximal LH release from cultured pituitary cells. In contrast, (Orn(2,5-NAPS))/sup 8/-GnRH, although being equipotent with the 2,4-NAPS isomer in terms of LH releasing ability, was unable to cause prolonged LH release after photoaffinity labeling. Thus, (Orn(2,4-NAPS))/sup 8/GnRH is very effective photolabeling ligand of the functionally significant pituitary GnRH receptor. Based on this compound, a pituitary peptidase resistant derivative, D-Phe/sup 6/, (Orn(2,4-NAPS))/sup 8/-GnRH-(1-9)-ethylamide, was synthesized. This derivative showed high-affinity binding to pituitary membranes with a K/sub d/ comparable to those of other GnRH analogues. A radioiodinated form of this peptide was used for pituitary GnRH-receptor labeling. This derivative labeled 59- and 57-kDa proteins in rat and 58- and 56-kDa proteins in bovine pituitary membrane preparations, respectively. This peptide also labeled pituitary GnRH receptors in the solubilized state and therefore appears to be a suitable ligand for the isolation and further characterization of the receptor.

  13. Photoaffinity labeling of the human erythrocyte glucose transporter with /sup 4/H-labelled forskolin

    SciTech Connect

    Shanahan, M.F.; Edwards, B.M.; Morris, D.P.

    1986-05-01

    Forskolin, a potent activator of adenylate cyclase, is also known to inhibit glucose transport in a number of cells. The authors have investigated photoincorporation of (/sup 3/H)forskolin into erythrocyte membrane proteins using a technique they previously developed for photolabeling the erythrocyte glucose transporter with cytochalasin B (CB). A 30-40s irradiation of erythrocyte ghosts in the presence of (/sup 3/H)forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into all of the major membrane protein bands. However, most of the incorporation occurred in only three regions of the gel. Peak 1 was a sharp peak near the top of the gel in the region corresponding to spectrin, peak 2 appeared to be associated with band 3 (approx. 90kDa), and the third region labeled was between 41-60 kDa which corresponds to the region of the glucose transporter. This region appeared to contain several overlapping peaks with the largest incorporation of label occurring around 45 kDa in the area of red cell actin. When photolabeling was performed in the presence of 400 ..mu..M cytochalasin B (8.0 ..mu..M forskolin) the labeling in the 41-60 kDa region was totally inhibited while labeling of the 90 kDa peak was partially blocked. CB had no effect on the photolabeling of peak 1 by forskolin.

  14. Subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ

    SciTech Connect

    Smith, R.A.; Branca, A.A.; Reichert, L.E. Jr.

    1985-11-15

    Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs.

  15. Photoaffinity labeling of human platelet and rabbit kidney. cap alpha. -adrenoceptors with (/sup 3/H)SKF 102229

    SciTech Connect

    Regan, J.W.; Raymond, J.R.; Lefkowitz, R.J.; DeMarinis, R.M.

    1986-06-13

    A newly developed ..cap alpha../sub 2/-adrenergic photoaffinity ligand, 3-methyl-6-chloro-9-azido-1H-2,3,4,5-tetrahydro-3-benzazepine (SKF 102229), has been radiolabeled with tritium to a specific activity of approx. 80 Ci/mmol. Using membranes prepared from human platelets and from rabbit kidney, ..cap alpha../sub 2/-adrenoceptors have been covalently labeled following photolysis in the presence of (/sup 3/H)SKF 102229. As determined by SDS-PAGE, the apparent molecular weight of ..cap alpha../sub 2/-adrenoceptors from both of these tissues was 64,000. The yield of covalent insertion of (/sup 3/H)SKF 102229 into the ..cap alpha../sub 2/-adrenoceptor was very good. Thus, following photolysis up to 90% of the ..cap alpha../sub 2/-adrenoceptors could be irreversibly labeled with (/sup 3/H)SKF 102229.

  16. Synthesis of iodine-125 labeled (+/-)-15-(4-azidobenzyl)carazolol: a potent beta-adrenergic photoaffinity probe

    SciTech Connect

    Heald, S.L.; Jeffs, P.W.; Lavin, T.N.; Nambi, P.; Lefkowitz, R.J.; Caron, M.G.

    1983-06-01

    (+/-)-15-(4-Azidobenzyl)carazolol (2), a potent beta-adrenergic photoaffinity ligand has been radioiodinated to theoretical specific activity (2175 Ci/mmol) and shown to label covalently beta-adrenergic receptor peptides in avian and amphibian erythrocyte membrane preparations. The radioiodinated analogues of the desired compound (2) were optimally prepared by two synthetic steps from (+/-)-15-(4-aminobenzyl)carazolol (8). The latter was iodinated with carrier-free Na/sup 125/I and chloramine T to yield two major isotopomers (the monoiodinated derivatives 9 and 10), which were separated by thin-layer chromatography and converted via diazonium salt formation to their respective 4-azides, 12 and 6. These azides can be used interchangeably in ligand binding or photoaffinity labeling experiments. Compound 8 was obtained by catalytic reduction of the nitro derivative (7), which was arrived at by direct reaction of 1,1-dimethyl-2-(4-nitrophenyl)ethylamine (3) with 4-(2,3-epoxypropoxy)carbazole (5). Of the desired isomers, (+/-)-15-(4-azido-3-iodobenzyl)carazolol (6) could be synthesized from 1,1-dimethyl-2-(4-azido-3-iodophenyl)ethylamine (4) by direct reaction with 5. This and the preceding sequence of reactions were carried out by using nonradioactive materials, and separation and purification of products were accomplished by high-performance liquid chromatography. The compounds described have been shown to be potent beta-adrenergic antagonistsec The photoactive azide derivatives of these compounds (6 and 12) have been shown to covalently incorporate into the beta-adrenergic receptor binding subunit of frog and turkey erythrocyte membrane preparations. Incorporation of the ligands into these polypeptides can be blocked specifically by both beta-adrenergic agonists and antagonists.

  17. Novel Benzodiazepine Photoaffinity Probe Stereoselectively Labels a Site Deep Within the Membrane-spanning Domain of the Cholecystokinin Receptor

    PubMed Central

    Hadac, Elizabeth M.; Dawson, Eric S.; Darrow, James W.; Sugg, Elizabeth E.; Lybrand, Terry P.; Miller, Laurence J.

    2008-01-01

    An understanding of the molecular basis of drug action provides opportunities for refinement of drug properties and for development of more potent and selective molecules that act at the same biological target. In this work, we have identified the active enantiomers in racemic mixtures of structurally related benzophenone derivatives of 1,5-benzodiazepines, representing both antagonist and agonist ligands of the type A cholecystokinin receptor. The parent compounds of the 1,5-benzodiazepine CCK receptor photoaffinity ligands were originally prepared in an effort to develop orally active drugs. The enantiomeric compounds reported in this study selectively photoaffinity-labeled the CCK receptor, resulting in the identification of a site of attachment for the photolabile moiety of the antagonist probe deep within the receptor’s membrane-spanning region at Leu88, a residue within transmembrane segment two. In contrast, the agonist probe labeled a region including extracellular loop one and a portion of transmembrane segment three. The antagonist covalent attachment site to the receptor served as a guide in the construction of theoretical three-dimensional molecular models for the antagonist-receptor complex. These models provided a means for visualization of physically plausible ligand-receptor interactions in the context of all currently available biological data that address small molecule interactions with the CCK receptor. Our approach, featuring the use of novel photolabile compounds targeting the membrane-spanning receptor domain to probe the binding site region, introduces powerful tools and a strategy for direct and selective investigation of non-peptidyl ligand binding to peptide receptors. PMID:16451051

  18. Identification of a third form of NaK-ATPase catalytic subunit in rat brain by photoaffinity labeling

    SciTech Connect

    Lowndes, J.M.; Millan, N.M.; Ruoho, A.E.; Hokin-Neaverson, M.

    1987-05-01

    Using photoaffinity labeling, they have found a form of the NaK-ATPase catalytic subunit, ..cap alpha..(-), in the rat brain that is distinct from the ..cap alpha.. and ..cap alpha..(+) forms. Strong radiolabeling of ..cap alpha..(-) was obtained with (/sup 125/I)azido-iodophenethylamido-succinyl-cymarin (AISC). AISC is a new cardiotonic steroid photolabel which they have synthesized and characterized chemically and biochemically. This compound labels ..cap alpha..(-) better than the photolabels that they have previously reported. SDS-PAGE (5%) of photolabeled rat brain microsomes showed that ..cap alpha..(-) migrated with faster mobility than the dog kidney ..cap alpha.. subunit. The ..cap alpha..(-) appears to have different specificity for different cardiotonic steroids than either ..cap alpha..(+) or ..cap alpha... The radiolabeling of rat brain ..cap alpha..(+) and dog kidney ..cap alpha.. with (/sup 125/I)AISC was protectable by ouabain; in contrast, 1 mM ouabain did not reduce the (/sup 125/I)AISC-labeling of ..cap alpha..(-), although the labeling was protected with 200 ..mu..M cymarin or AISC. The results indicate that the ..cap alpha..(-) form of the NaK-ATPase in rat brain binds cymarin and its derivative but has little affinity for ouabain. It is possible that ..cap alpha..(-) may be the translation product of the rat brain ..cap alpha..(III) mRNA which has recently been described.

  19. Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate.

    PubMed

    Kyro, Kelly; Manandhar, Surya P; Mullen, Daniel; Schmidt, Walter K; Distefano, Mark D

    2010-08-01

    Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras converting enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site.

  20. Photoaffinity Labeling of Ras Converting Enzyme 1 (Rce1p) using a Benzophenone-Containing Peptide Substrate

    PubMed Central

    Kyro, Kelly; Manandhar, Surya P.; Mullen, Daniel; Schmidt, Walter K.; Distefano, Mark D.

    2010-01-01

    Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras Converting Enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site. PMID:20619662

  1. The effect of structural differences in the reducing terminus of sugars on the binding affinity of carbohydrates and proteins analyzed using photoaffinity labeling.

    PubMed

    Ohtsuka, Isao; Sadakane, Yutaka; Higuchi, Mari; Hada, Noriyasu; Hada, Junko; Kakiuchi, Nobuko; Sakushima, Akiyo

    2011-01-15

    Because carbohydrates and proteins bind with such low affinity, the nature of their interactions is not clear. Photoaffinity labeling with diazirin groups is useful for elucidating the roles of carbohydrates in these binding processes. However, when carbohydrate probes are synthesized according to this conventional method, the reducing terminus of the sugar is opened to provide an acyclic structure. Because greater elucidation of carbohydrate-protein interactions requires a closed-ring carbohydrate in addition to the photoreactive group, we synthesized new molecular tools. The carbohydrate ligands were synthesized in three steps (glycosylation with allyl alcohol, deprotection, and ozonolysis). Specific binding proteins for carbohydrate ligands were obtained by photoaffinity labeling. Closed ring-type carbohydrate ligands, in which the reducing sugar is closed, bound to lectins more strongly than open ring-type sugars. Carbohydrate to protein binding was observed using AFM.

  2. Adrenocortical nuclear progesterone-binding protein: Identification by photoaffinity labeling and evidence for deoxyribonucleic acid binding and stimulation by adrenocorticotropin

    SciTech Connect

    Demura, T.; Driscoll, W.J.; Lee, Y.C.; Strott, C.A. )

    1991-01-01

    Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinct from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.

  3. Interaction of P-aminobenzoic acid with normal and sickel erythrocyte membrane: photoaffinity labelling of the binding sites

    SciTech Connect

    Premachandra, B.R.

    1986-03-05

    Electron microscopic studies revealed that P-Amino benzoic acid (PABA) could prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right side out membrane vesicles (ROV) revealed a similar number of binding sites (1.2-1.4 ..mu..mol/mg) and Kd (1.4-1.6 mM) values for both normal and sickle cell membranes. /sup 14/C-Azide analogue of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface since a 20 fold excess of azide inhibited PABA binding in a linear fashion. The azide was covalently incorporated into the membrane components only upon irradiation (52-35% of the label found in the proteins and the rest in lipids). Electrophoretic analysis of photolabelled ROV revealed that the azide interacts chiefly with Band 3 protein. PABA inhibited both high and low affinity calcium (Ca) binding sites situated on either surface of the membrane in a non-competitive manner; however, Ca binding stimulated by Mg-ATP was not affected. Ca transport into inside out vesicles was inhibited by PABA; but it did not affect the calcium ATP-ase activity. The authors studies suggest that the mechanism of action of PABA is mediated by its interaction with Band 3 protein (anion channel), calcium channel and calcium binding sites of erythrocyte membrane.

  4. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    SciTech Connect

    Middleton, R.E.; Cohen, J.B. )

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.

  5. Regulation of the GLUT1 glucose transporter in cultured myocytes: total number and subcellular distribution as determined by photoaffinity labelling.

    PubMed Central

    el-Kebbi, I M; Roser, S; Pollet, R J; Cushman, S W; Wilson, C M

    1994-01-01

    We have used the impermeant photoaffinity label 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-[2-3H] 1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-[2-3H]BMPA) to identify and quantify the glucose transporters on the surface of BC3H-1 cells, a continuously cultured skeletal-muscle cell line lacking the MyoD transcription factor required for cell fusion. ATB-[2-3H]BMPA was used in combination with immunoprecipitation of the GLUT1 glucose transporter, the only isoform expressed in these cells. The total cellular GLUT1 content was also determined by photolabelling and immunoprecipitation after cell permeabilization with digitonin (0.025%). In glucose-starved cells, 85% of the glucose transporters were present at the cell surface in the basal state, with little change in response to insulin (200 nM), correlating with lack of additional 2-deoxyglucose uptake in response to insulin. Feeding the cells with glucose (25 mM) for 24 h resulted in an 80% decrease in the total GLUT1 content relative to starved cells, of which only 25% were present on the cell surface. This was associated with an 85% decrease in 2-deoxyglucose uptake. In addition, acute stimulation of the fed cells with insulin or phorbol 12-myristate 13-acetate (PMA) led to an increase in GLUT1 at the cell surface, and, in correspondence, an increase in 2-deoxyglucose uptake by approx. 2- and 4-fold respectively. We conclude that exofacial photoaffinity labelling of glucose transporters with ATB-[2-3H]BMPA in the presence and absence of digitonin, followed by specific immunoprecipitation, provides an accurate measure of total and cell-surface glucose transporters in differentiated BC3H-1 muscle cells. This technique demonstrates that glucose pre-feeding (1) decreases the total number of GLUT1 and (2) redistributes the majority of the remaining transporters to an intracellular site, where they can now be translocated to the cell surface in response to insulin and PMA. PMID:8037688

  6. Imaging of neuroendocrine tumours with gamma-emitting radiopharmaceuticals.

    PubMed

    Bombardieri, E; Coliva, A; Maccauro, M; Seregni, E; Orunesu, E; Chiti, A; Lucignani, G

    2010-02-01

    Nuclear medicine can image some tumors by means of receptor specific radiopharmaceuticals, and offers the possibility to characterize cancer through the detection of its receptor expression. This is the case of neuroendocrine tumours (NETs), that are visualized by different radiolabelled somatostatin analogues that bind 5 distinct somatostatin receptor types (named sstr1-5) that show different tissue distribution. The subtypes sstr2 and sstr5 are the most commonly expressed in NETs. Until now the most widely used radiolabelled somatostatin analogue for planar and single photon emission computed tomography (SPECT) has been [(111)In]pentetreotide, because of its commercial availability. Other analogues labelled with gamma emitting radionuclides are [(99m)Tc]EDDA/HYNIC-TOC, [(99m)Tc]P829, [(111)In]DOTA-lanreotide, [(111)In]DOTA-NOC-ATE, [(111)In]DOTA-BOC-ATE. However, these compounds have not been successful for the routine use. Moreover, NETs express various receptors that can be depicted by different radiopharmaceuticals, such as [(123)I]VIP and [(111)In]GLP-1. Besides this, some precursors of the catecholamines metabolism, as meta-iodo-benzyl-guanidine (MIBG), labelled with (123)I or (131)I, accumulates in neuroendocrine tissues, in particular those of sympathoadrenal lineage. MIBG scintigraphy is currently indicated for neuroblastoma, paraganglioma and phaeocromocitoma. An impressive technological progress has been achieved recently with PET and, in particular, with the development of hybrid instrumentations (PET/CT) combining nuclear imaging with radiological imaging providing both functional and morphologic information. Among positron emitting tracers, the [(18)F]FDG is the most diffuse in oncology, but other more effective tracers are available for NETs, such as the analogues labelled with 68Ga. The diagnostic sensitivity and accuracy of these technology is superior to that of gamma emitting radiopharmaceuticals, but the fact that they are not still registered

  7. Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling.

    PubMed

    Lapinsky, David J; Velagaleti, Ranganadh; Yarravarapu, Nageswari; Liu, Yi; Huang, Yurong; Surratt, Christopher K; Lever, John R; Foster, James D; Acharya, Rejwi; Vaughan, Roxanne A; Deutsch, Howard M

    2011-01-01

    In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this knowledge gap, ligands were synthesized in which the piperidine nitrogen of 3- and 4-iodomethylphenidate was substituted with a benzyl group bearing a photoreactive azide. Analog (±)-3a demonstrated modest DAT affinity and a radioiodinated version was shown to bind covalently to rat striatal DAT and hDAT expressed in cultured cells. Co-incubation of (±)-3a with nonradioactive d-(+)-methylphenidate or (-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (β-CFT, WIN-35,428, a cocaine analog) blocked DAT labeling. Compound (±)-3a represents the first successful example of a DAT photoaffinity ligand based on the methylphenidate scaffold. Such ligands are expected to assist in mapping non-tropane ligand-binding pockets within plasma membrane monoamine transporters.

  8. Azido-Iodo-N-Benzyl Derivatives of threo-Methylphenidate (Ritalin, Concerta): Rational Design, Synthesis, Pharmacological Evaluation, and Dopamine Transporter Photoaffinity Labeling

    PubMed Central

    Lapinsky, David J.; Velagaleti, Ranganadh; Yarravarapu, Nageswari; Liu, Yi; Huang, Yurong; Surratt, Christopher K.; Lever, John R.; Foster, James D.; Acharya, Rejwi; Vaughan, Roxanne A.; Deutsch, Howard M.

    2010-01-01

    In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this knowledge gap, ligands were synthesized in which the piperidine nitrogen of 3- and 4-iodomethylphenidate was substituted with a benzyl group bearing a photoreactive azide. Analog (±)-3a demonstrated modest DAT affinity and a radioiodinated version was shown to bind covalently to rat striatal DAT and hDAT expressed in cultured cells. Co-incubation of (±)-3a with nonradioactive D-(+)-methylphenidate or (−)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (β-CFT, WIN-35,428, a cocaine analog) blocked DAT labeling. Compound (±)-3a represents the first successful example of a DAT photoaffinity ligand based on the methylphenidate scaffold. Such ligands are expected to assist in mapping non-tropane ligand-binding pockets within plasma membrane monoamine transporters. PMID:21129986

  9. Identification of a 23 kDa protein from maize photoaffinity-labelled with 5-azido-[7-3H]indol-3-ylacetic acid.

    PubMed Central

    Feldwisch, J; Zettl, R; Campos, N; Palme, K

    1995-01-01

    A 23 kDa protein (p23) was identified in microsomal extracts from maize coleoptiles by photoaffinity labelling with 5-azido-[7-3H]indol-3-ylacetic acid ([3H]N3IAA). Labelling of p23 was blocked by unlabelled IAA, N3IAA, indol-3-ylbutyric acid and indol-3-yl-lactate. In addition, labelling was efficiently decreased by tryptophan, as well as by the scavenger p-aminobenzoic acid. Labelling was, however, not affected by synthetic auxins such as 1-naphthylacetic acid or 2,4-dichlorophenoxyacetic acid. Competition data suggest that the label was probably bound via the indole ring, and hence labelling was not specific for auxins. The 23 kDa protein was solubilized from crude microsomes by extraction with Triton X-100 and purified to homogeneity by ion-exchange, size-exclusion and reversed-phase chromatography. After electroblotting, the amino acid sequences of the p23 N-terminus as well as the several tryptic peptides were obtained. Database comparisons revealed sequence identity with a maize manganese superoxide dismutase. We conclude that photoaffinity labelling of p23 was pseudo-affinity, and therefore the binding site for IAA is not specific. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7848285

  10. Photoaffinity labeling of the dopamine reuptake carrier protein with 3-azido sup 3 H GBR-12935

    SciTech Connect

    Berger, S.P.; Martenson, R.E.; Laing, P.; Thurkauf, A.; Decosta, B.; Rice, K.C.; Paul, S.M. )

    1991-04-01

    A high affinity tritiated azido-diphenylpiperazine derivative, 3-azido {sup 3}H GBR-12935, was synthesized as a potential photoaffinity probe of the dopamine transporter. Initially, the reversible binding of 3-azido {sup 3}H GBR-12935 to crude synaptosomal membranes from the rat striatum was characterized. Specific binding was sodium dependent and inhibited by a variety of drugs that are known to potently inhibit dopamine uptake. Other neurotransmitter uptake inhibitors, as well as cis-flupenthixol, a potent inhibitor of {sup 3}H GBR-12935 binding to piperazine binding sites, failed to inhibit specific binding at concentrations of less than or equal to 10 microM. A good correlation was observed between the relative potencies of these drugs in inhibiting dopamine uptake into synaptosomes and in inhibiting specific 3-azido {sup 3}H GBR-12935 binding to rat striatal membranes. These data suggest that 3-azido {sup 3}H GBR-12935, like other diphenylpiperazines such as {sup 3}H GBR-12935 and {sup 3}H GBR-12909, binds primarily to the dopamine transporter under defined assay conditions. After UV photolysis of crude synaptosomal membranes preincubated with 3-azido {sup 3}H GBR-12935 (1-2 nM), a single radiolabeled polypeptide with an apparent molecular mass of 80 kDa was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Photoincorporation of 3-azido {sup 3}H GBR-12935 into this polypeptide was inhibited selectively by compounds that inhibit the uptake of dopamine and was completely dependent on the presence of Na+. No photolabeled proteins were observed when cerebellar membranes were substituted for striatal membranes. Essentially complete adsorption of the radiolabeled 80-kDa polypeptide to wheat germ agglutinin and elution with N-acetyl-D-glucosamine strongly suggest that the dopamine transporter polypeptide photolabeled by 3-azido {sup 3}H GBR-12935 is glycosylated.

  11. Recycling of photoaffinity-labeled insulin receptors in rat adipocytes. Dissociation of insulin-receptor complexes is not required for receptor recycling

    SciTech Connect

    Huecksteadt, T.; Olefsky, J.M.; Brandenberg, D.; Heidenreich, K.A.

    1986-07-05

    We have used an iodinated, photoreactive analog of insulin, /sup 125/I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, to covalently label insulin receptors on the cell surface of isolated rat adipocytes. Following internalization of the labeled insulin-receptor complexes at 37/sup 0/C, we measured the rate and extent of recycling of these complexes using trypsin to distinguish receptors on the cell surface from those inside the cell. The return of internalized photoaffinity-labeled receptors to the cell surface was very rapid at 37/sup 0/C proceeding with an apparent t 1/2 of 6 min. About 95% of the labeled receptors present in the cell 20 min after the initiation of endocytosis returned to the cell surface by 40 min. Recycling was slower at 25 and 16/sup 0/C compared to 37/sup 0/C and essentially negligible at 12/sup 0/C or in the presence of energy depleters. Addition of excess unlabeled insulin had no effect on the recycling of photoaffinity-labeled insulin receptor complexes, whereas monensin, chloroquine, and Tris partially inhibited this process. These data indicate that dissociation of insulin from internalized receptors is not necessary for insulin receptor recycling. Furthermore, agents which have been shown to prevent vesicular acidification inhibit the recycling of insulin receptors by a mechanism other than prevention of ligand dissociation.

  12. The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: carbohydrate-protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar.

    PubMed

    Ohtsuka, Isao; Sadakane, Yutaka; Hada, Noriyasu; Higuchi, Mari; Atsumi, Toshiyuki; Kakiuchi, Nobuko

    2014-08-01

    Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate-protein interactions. We investigated whether carbohydrate-lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp-GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.

  13. Human kidney thiopurine methyltransferase (TPMT): Photoaffinity labeling with ( sup 3 H-methyl)- S-adenosyl-L-methionine ( sup 3 H-ado-met)

    SciTech Connect

    Van Loon, J.A.; Weinshilboum, R.M. )

    1990-02-26

    TPMT (EC 2.1.1.67) catalyzes the S-methylation of 6-mercaptopurine (6-MP) and other heterocyclic and aromatic thiol drugs. TPMT activity and immunoreactive protein in humans are regulated by a common genetic polymorphism. Human kidney contains two isozymes of TPMT which can be separated by ion exchange chromatography. Partially purified isozymes of human kidney TPMT were exposed to UV light in the presence of {sup 3}H-Ado-Met. After photolysis, these preparations were subjected to SDS-polyacrylamide gel electrophoresis. Autoradiography of gels revealed that a 34 kDa protein was the predominant species that was radioactively labeled for both isozymes. Photoaffinity labeling of TPMT was inhibited in a concentration-dependent fashion by the methyl acceptor substrate, 6-MP, and by three TPMT inhibitors, 3,4-dimethoxy-5-hydroxybenzoic acid, 6-methylmercaptopurine and S-adenosyl-L-homocysteine. Of three wavelengths tested, 254, 300, and 350 nm, labeling was the greatest at 254 nm, near the absorption maximum for Ado-Met. Photoaffinity labeling of TPMT with {sup 3}H-Ado-Met should make it possible to determine the amino acid sequence of the active site and may make it possible to define the molecular basis of the genetic polymorphism for this important drug-metabolizing enzyme.

  14. Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.

    PubMed

    Walseth, Timothy F; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S; Slama, James T

    2012-01-20

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.

  15. Photoaffinity labeling of the Torpedo californica nicotinic acetylcholine receptor with an aryl azide derivative of phosphatidylserine

    SciTech Connect

    Blanton, M.P.; Wang, H.H. )

    1990-02-06

    A photoactivatable analogue of phosphatidylserine, {sup 125}I-labeled 4-azidosalicylic acid-phosphatidylserine ({sup 125}I ASA-PS), was used to label both native acetylcholine receptor (AchR)-rich membranes from Torpedo californica and AchR membranes affinity purified from Torpedo reconstituted into asolectin vesicles. The radioiodinated arylazido group attaches directly to the phospholipid head group and thus probes for regions of the AchR structure in contact with the negatively charged head group of phosphatidylserine. All four subunits of the AchR incorporated the label, with the {alpha} subunit incorporating approximately twice as much as each of the other subunits on a per mole basis. The regions of the AchR {alpha} subunit that incorporated {sup 125}I ASA-PS were mapped by Staphylococcus aureus V8 protease digestion. The majority of label incorporated into fragments representing a more complete digestion of the {alpha} subunit was localized to 11.7- and 10.1-kDa V8 cleavage fragments, both beginning at Asn-339 and of sufficient length to contain the hydrophobic region M4. An 18.7-kDa fragment beginning at Ser-173 and of sufficient length to contain the hydrophobic regions M1, M2, and M3 was also significantly labeled. In contrast, V8 cleavage fragments representing roughly a third of the amino-terminal portion of the {alpha} subunit incorporated little or no detectable amount of probe.

  16. Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

    SciTech Connect

    Morris, D.I.; Robbins, J.D.; Ruoho, A.E.; Sutkowski, E.M.; Seamon, K.B. )

    1991-07-15

    Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of (3H)forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited (3H)forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP {gamma} S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolin but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP {gamma} S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.

  17. Photoaffinity labeling of alpha- and beta- scorpion toxin receptors associated with rat brain sodium channel.

    PubMed

    Darbon, H; Jover, E; Couraud, F; Rochat, H

    1983-09-15

    Azido nitrophenylaminoacetyl [125I]iodo derivative of toxin II from Centruroides suffusus suffusus, a beta-toxin, and azido nitrophenylaminoacetyl [125I]iodo derivative of toxin V from Leiurus quinquestriatus quinquestriatus, an alpha-toxin, have been covalently linked after binding to their receptor sites that are related to the voltage sensitive sodium channel present in rat brain synaptosomes. Both derivatives labeled two polypeptides of 253000 +/- 20000 and 35000 +/- 2000 mol. wt. Labeling was blocked for each derivative by a large excess of the corresponding native toxin but no cross inhibition was obtained. These results suggest that both alpha - and beta - scorpion toxin receptors are located on or near the same two membrane polypeptides which may be part of the voltage dependent sodium channel.

  18. Photoaffinity labeling of opiate receptors using intrinsically photoactive /sup 3/H-opiates

    SciTech Connect

    Kooper, G.N.; Levinson, N.R.; Copeland, C.F.; Bowen, W.D.

    1988-03-01

    Opiate receptors in rat and cow brain membranes have been labeled irreversibly using the intrinsic photolability of 3H-opiates. Membranes were incubated with 3H-ligand and then irradiated with UV light of 254 nm. Nonspecific binding was determined in the presence of 10 microM unlabeled levallorphan. Irreversible binding was defined as binding which survived heat or acid denaturation of membranes. Specific incorporation of label into denatured samples was observed only when unbound or loosely bound 3H-ligand was washed free from the membranes prior to irradiation. There was a general correlation between photosensitivity of the 3H-ligand and its ability to photolabel receptors. Hence, photolabeling presumably results by covalent attachment of highly reactive species generated during photochemical decomposition of ligand. With 3H-etorphine, optimal irradiation time was 5 min. In addition to 3H-etorphine, receptors could be labeled irreversibly with 3H-oxymorphone, 3H-dihydromorphine, and 3H-ethylketocyclazocine. Of the specific binding present in irradiated, nondenatured samples, 45-60% remained attached to receptors upon denaturation. 3H-Ethylketocyclazocine exhibited an 86% yield of incorporation. Signal-to-noise levels of 50-80% could be achieved in denatured samples. Therefore, this method provides a means of covalently labeling opiate receptors in high yield and with high signal-to-noise ratios. The opioid peptides, 3H-D-Ala2,D-Leu5-enkephalin, 3H-D-Ser2,Leu5,Thr6-enkephalin, 3H-D-Ala2,Met5-enkephalin amide, and 3H-D-Ala2,N-MePhe4,Gly-ol5-enkephalin, as well as the benzomorphan, 3H-bremazocine, apparently lack the structural characteristics which allow photolabeling.

  19. Identification of UDPG-binding polypeptides and purified (1,3)-. beta. -glucan synthase by photoaffinity labelling with 5-azido-UDPG

    SciTech Connect

    Frost, D.J.; Wu, A.; Read, S.M.; Wasserman, B.P. ); Drake, R.R.; Haley, B.E. )

    1989-04-01

    The photoaffinity probe 5-azido-uridine 5{prime}-{beta}-({sup 32}P)-diphosphate glucose was used to identify the major UDPG-binding polypeptide of red beet (1,3)-{beta}-glucan synthase. Glucan synthase was purified from plasma membranes by sequential solubilization with CHAPS followed by product entrapment. Two major polypeptides at 72 and 54 kD were labelled by probe. Labelling of both was abolished with increasing levels of cold UDPG. However, labelling of the 54 kD polypeptide was dependent upon the presence of divalent cations. These data suggest that the 54 kD polypeptide is a substrate-binding and cation-regulated component of the glucan synthase complex.

  20. /sup 3/H)forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter

    SciTech Connect

    Shanahan, M.F.; Morris, D.P.; Edwards, B.M.

    1987-05-05

    Irradiation of erythrocyte ghosts in the presence of (/sup 3/H)forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of the glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of (/sup 3/H)cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin.

  1. Using mass spectrometry to study the photo-affinity labeling of protein tyrosine phosphatase 1B

    NASA Astrophysics Data System (ADS)

    Leriche, Tammy; Skorey, Kathryn; Roy, Patrick; McKay, Dan; Bateman, Kevin P.

    2004-11-01

    Protein tyrosine phosphatase 1B (PTP1B) is a potential target for the treatment of Type II diabetes and several companies are developing small molecule inhibitors of this enzyme. Part of the characterization of these compounds as PTP1B inhibitors is the understanding of how they bind in the enzyme active site. The use of photo-activated inhibitors that target the active site can provide such insight. This paper describes the characterization of a photoprobe directed at the active site of PTP1B. Mass spectrometry revealed the specific binding of the probe to the intact protein. Digestion of the labeled protein followed by LC-MS and LC-MS/MS was used to show that the photoprobe binds to a specific active site amino acid. This was confirmed by comparison with the X-ray structure of PTP1B with a PTP1B inhibitor. The probe labels a conserved acidic residue (Asp) that is required for catalytic activity. This photoprobe may prove to be a useful tool for the development of a PTP1B inhibitor or for the study of PTPs in general.

  2. Photoaffinity labeling of the 5-hydroxytryptamine 1A receptor in rat hippocampus.

    PubMed

    Ransom, R W; Asarch, K B; Shih, J C

    1986-10-01

    1-[2-(4-Azidophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (p-azido-PAPP) inhibits [3H]5-hydroxytryptamine [( 3H]5-HT) binding to 5-HT1A and 5-HT1B sites in rat brain with equilibrium dissociation constants (KD) of 0.9 nM and 230 nM, respectively. [3H]p-Azido-PAPP was synthesized and its reversible and irreversible binding properties to the hippocampal 5-HT1A site characterized. [3H]p-Azido-PAPP labeled a single class of sites in rat hippocampal membranes with a KD of 1 nM and a maximal binding density of 370 fmol/mg protein. The pharmacological profile of [3H]p-azido-PAPP binding was consistent with the radioligand's selective interaction with the 5-HT1A receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes preincubated with [3H]p-azido-PAPP and irradiated showed a major band of incorporation of radioactivity at approximately 55,000 daltons. This incorporation could be blocked when membranes were incubated with 1 microM of several agents that have high affinity for 5-HT1A sites [5-HT, 8-hydroxy-2-(di-n-propylamino)tetraline, TVX Q 7821, spiperone, buspirone, d-lysergic acid diethylamide, metergoline]. The results indicate that on photolysis [3H]p-azido-PAPP irreversibly labels a polypeptide that is, or is a subunit of, the 5-HT1A receptor in rat hippocampus.

  3. Photoaffinity labeling of opiate receptors with /sup 3/H-etorphine: possible species differences in glycosylation

    SciTech Connect

    Bowen, W.D.; Kooper, G.

    1986-01-01

    Opiate receptors from whole rat brain (minus cerebellum) and cow striatum were labeled irreversibly using the intrinsic photolability of /sup 3/H-etorphine. After incubation with 2 nM /sup 3/H-etorphine and centrifugal washing, membranes were irradiated with light of 254 nm. Non-specific binding was determined by carrying out incubations in presence and absence of 10 microM levallorphan. Specific binding in photolabeled membranes was 75-80%, with a photo-incorporation yield of approximately 50%. Photolabeled membranes were extracted with CHAPS/Lubrol and unbound /sup 3/H-etorphine was removed by dialysis and passage over Sephadex G-25. Solubilized proteins were then subjected to chromatography on wheat germ agglutinin, and retained proteins were eluted with N-acetyl D-glucosamine (NAG). Protein profiles from rat brain and cow striatum were identical, with 89% of the total protein flowing through unretained and 11% eluted by NAG. However, the profile of radioactivity was markedly different in the two species. With rat, the specific activity (cpm/A280) was the same for flow-through and NAG-eluate. With cow, the specific activity of the NAG-eluate was 17 times greater than the flow-through. These results indicate that cow striatum and rat whole brain contain populations of opiate receptors which are glycosylated differently.

  4. Efficient photoaffinity labeling of the rat V1a vasopressin receptor using a linear azidopeptidic antagonist.

    PubMed

    Carnazzi, E; Aumelas, A; Phalipou, S; Mouillac, B; Guillon, G; Barberis, C; Seyer, R

    1997-08-01

    We have synthesized and fully characterized by fast-atom-bombardment-mass, NMR and ultraviolet spectroscopies the vasopressin antagonist 3-azidophenylpropionyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr(3I )-NH2. Easily radioiodinatable just before use, it has a high affinity for the natural rat liver V1a receptor [dissociation constant (Kd) = 54 +/- 20 pM; Carnazzi, E., Aumelas, A., Barberis, C., Guillon, G. & Seyer, R. (1994) J. Med. Chem. 37, 1841-1849] and for both the rat vasopressin V1a receptor expressed in Spodoptera frugiperda 9 cells (Sf9 cells, Kd = 688 +/- 35 pM) and in COS-7 cells (Kd = 320 +/- 20 pM). This probe labels specifically the V1a receptors in an ultraviolet-dependent manner, and binds covalently to about 12% of the receptors with high stability over several days, even in dissociation or solubilization conditions. SDS/PAGE studies and autoradiographic analyses of the photolabeled receptors reveal a single band (49.5 kDa) and two bands (63 kDa and 93.6 kDa) for receptor-probe associations obtained in Sf9 and COS-7 cells respectively. These molecular masses are consistent with non-glycosylated and highly glycosylated forms of the receptor, according to each expression system. In rat liver membranes, we have identified apparent molecular masses of about 32, 45 and more than 67 kDa. We finally demonstrated a proteolysis of the receptor that appeared to be Zn2+ and leupeptin sensitive. The high potency of this ligand is promising for the monitoring of the purification of the V1a receptor and for mapping its antagonist-binding site.

  5. Photoaffinity labelling of MSH receptors on Anolis melanophores: irradiation technique and MSH photolabels for irreversible stimulation

    SciTech Connect

    Eberle, A.N.

    1984-01-01

    Excised dorsal skin of Anolis carolinensis was exposed to high intensity UV-irradiation in the presence of different photoreactive alpha-MSH derivatives. The resulting covalent binding of the hormone to its receptor induced irreversible pigment dispersion. The duration of the longlasting response depended on the type and length of irradiation; it was maximal after two 5 min irradiation phases with a light intensity of approximately 180 mW/cm/sup 2/ and a spectrum from 310 to 550 nm, fresh hormone being added after the first phase. (N alpha-(4-Azidophenylacetyl-serine1)-alpha-MSH (I), (2'-(2-nitro-4-azidophenylsulphenyl)-tryptophan/sub 9/)-alpha-MSH (II) and (p-azidophenylalanine/sub 13/)-alpha-MSH (III) all inserted into the receptor to about the same extent, as judged from the persistence of the longlasting signal. In contrast, (D-alanine1, p-azidophenylalanin2/sub 2/, norvaline/sub 4/)-alpha-MSH (IV) and (N alpha-(4-azidophenylacetyl)-serine1, leucine/sub 9/)-alpha-MSH (V) gave much less insertion and (leucine/sub 9/, p-azidophenylalanine/sub 13/)-alpha-MSH (VI) hardly any insertion when applied in the same relative excess (5-fold the concentration inducing a maximal response). Covalent attachment of the cleavable photolabel (N alpha-(4-azidophenyl)-1, 3'-dithio-propionyl-serine1)-alpha-MSH (VII) and subsequent washing of the skin in buffer containing 1% beta-mercaptoethanol released the peptide from the receptor. Insertion of the C-terminal photolabel (p-azidophenylalanine/sub 13/)-alpha-MSH was reduced by the weak antagonist H-Phe-Ala-Trp-Gly-Gly-Pro-Val-NH/sub 2/. These experiments prove that hormone receptors can be covalently labelled in tissue with very limited light transparency.

  6. Photoaffinity Labeling of Mouse Fibroblast Enzymes by a Base Excision Repair Intermediate: New Evidence on the Role of PARP-1 in DNA Repair

    SciTech Connect

    Lavrik, Olga I.; Prasad, Rajendra; Sobol, Robert W.; Horton, Julie K.; Ackerman, Eric J. ); Wilson, Samuel H.

    2001-07-06

    To examine mammalian base excision repair (BER) enzymes interacting with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast (MEF) crude extract. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by AP endonuclease creating a nick with 3' hydroxyl and 5' reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was introduced at the 3' hydroxyl group. With near UV-light exposure (312nm) of the extract-probe mixture, only six proteins were strongly labeled, including poly (ADP-ribose) polymerase (PARP-1) and the well-known BER participants flap endonuclease (FEN-1), DNA polymerase b (b-pol), and AP endonuclease (APE). The amount of probe crosslinked to PARP-1 was greater than that crosslinked to the other proteins. The specificity of PARP-1 labeling was examined by competition experiments involving various oligonucleotide competitors; competition of labeling by the probe was much greater for the BER intermediates tested than for normal double-stranded DNA. The specificity of PARP-1 labeling also was examined using DNA probes with alternate structures; PARP-1 labeling was stronger with a DNA oligomer representing a BER intermediate than with a molecule representing a nick in double-stranded DNA. These results identifying interaction of PARP-1 with a BER intermediate are discussed in light of PARP-1's role in mammalian BER.

  7. Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage

    PubMed Central

    1993-01-01

    Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue. PMID:8478607

  8. [3H]Azidodantrolene photoaffinity labeling, synthetic domain peptides and monoclonal antibody reactivity identify the dantrolene binding sequence on RyR1

    SciTech Connect

    Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manju B.; Ma, Jianjie; Ikemoto, Noriaki; Jimenez, Leslie S.; Morimoto, Hiromi; Williams, Philip G.; Parness, Jerome

    2002-06-14

    Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum in normal skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Though its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in sarcoplasmic reticulum, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene(1). Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene,indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1, previously shown to affect RyR1 function in vitro and in vivo, were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2, peptide s containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody which recognizes RyR1 and its 1400 amino acid N-terminal fragment, recognizes DP1 and DP1-2 in both Western blots and immunoprecipitation assays, and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in sarcoplasmic reticulum. Our results indicate that synthetic domain peptides can mimic a native, ligand binding conformation in vitro, and that the dantrolene binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino-acids 590-609.

  9. Direct photoaffinity labeling of gizzard myosin with ( sup 3 H)uridine diphosphate places Glu185 of the heavy chain at the active site

    SciTech Connect

    Garabedian, T.E.; Yount, R.G. )

    1990-12-25

    The active site of chicken gizzard myosin was labeled by direct photoaffinity labeling with ({sup 3}H)UDP. ({sup 3}H) UDP was stably trapped at the active site by addition of vanadate (Vi) and Co{sup 2+}. The extraordinary stability of the myosin.Co2+.(3H)UDP.Vi complex (t1/2 greater than 5 days at 0{degrees}C) allowed it to be purified free of extraneous ({sup 3}H)UDP before irradiation began. Upon UV irradiation, greater than 60% of the trapped ({sup 3}H)UDP was photoincorporated into the active site. Only the 200-kDa heavy chain was labeled, confirming earlier results using ({sup 3}H)UTP. Extensive tryptic digestion of photolabeled myosin subfragment 1 followed by high performance liquid chromatography separations and removal of nucleotide phosphates by treatment with alkaline phosphatase allowed two labeled peptides to be isolated. Sequencing of the labeled peptides and radioactive counting showed that Glu185 was the residue labeled. Since UDP is a zero-length cross-linker, Glu185 is located at the purine-binding pocket of the active site of smooth myosin and adjacent to the glycine-rich loop which binds the polyphosphate portion of ATP. This Glu residue is conserved in smooth and nonmuscle myosins and is the same residue identified previously by ({sup 3}H)UTP photolabeling in Acanthamoeba myosin II.

  10. Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase. Structural alterations in the beta-adrenergic receptor revealed by photoaffinity labeling.

    PubMed

    Stadel, J M; Nambi, P; Lavin, T N; Heald, S L; Caron, M G; Lefkowitz, R J

    1982-08-25

    Preincubation of turkey erythrocytes with isoproterenol results in an impaired ability of beta-adrenergic agonists to stimulate adenylate cyclase in membranes prepared from these cells. The biochemical basis for this agonist-induced desensitization was investigated using the new beta-adrenergic antagonist photoaffinity label [125I]p-azidobenzylcarazolol ([125I]PABC). Exposure of [125I]PABC-labeled turkey erythrocyte membranes to high intensity light leads to specific covalent incorporation of the labeled compound into two polypeptides, Mr approximately equal to 38,000 and 50,000, as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Incorporation of [125I]PABC into these two polypeptides is completely blocked by a beta-adrenergic agonist and antagonist consistent with covalent labeling of the beta-adrenergic receptor. After desensitization of the turkey erythrocyte by preincubation with 10(-5) M isoproterenol, the beta-adrenergic receptor polypeptides specifically labeled by [125I]PABC in membranes prepared from desensitized erythrocytes were of larger apparent molecular weight (Mr approximately equal to 42,000 versus 38,000, and 53,000 versus 50,000) compared to controls. When included during the preincubation of the erythrocytes with isoproterenol, the antagonist propranolol (10(-5) M) inhibited both agonist-promoted desensitization of the adenylate cyclase and the altered mobility of the [125I]PABC-labeled receptor polypeptides. These data indicate that structural alterations in the beta-adrenergic receptor accompany the desensitization process in turkey erythrocytes.

  11. Identification of the binding sites for ubiquinone and inhibitors in the Na+-pumping NADH-ubiquinone oxidoreductase from Vibrio cholerae by photoaffinity labeling

    PubMed Central

    Ito, Takeshi; Ninokura, Satoshi; Kitazumi, Yuki; Mezic, Katherine G.; Cress, Brady F.; Koffas, Mattheos A. G.; Morgan, Joel E.; Barquera, Blanca; Miyoshi, Hideto

    2017-01-01

    The Na+-pumping NADH-quinone oxidoreductase (Na+-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae. The V. cholerae Na+-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [125I]PAD-1 and [125I]PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu-32–Met-39 and Phe-131–Lys-138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg-43–Lys-54 and Trp-23–Gly-89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap one another. Unexpectedly, although the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. G140A and E144C), the binding reactivities of [125I]PAD-1 and [125I]PAD-2 to the mutated enzymes were unchanged compared with those of the wild-type enzyme. We also found that photoaffinity labeling by [125I]PAD-1 and [125I]PAD-2, rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na+-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work. PMID:28298441

  12. Identification of the Binding Sites for Ubiquinone and Inhibitors in the Na(+)-Pumping NADH-Ubiquinone Oxidoreductase of Vibrio cholerae by Photoaffinity Labeling.

    PubMed

    Ito, Takeshi; Murai, Masatoshi; Ninokura, Satoshi; Kitazumi, Yuki; Mezic, Katherine G; Cress, Brady F; Koffas, Mattheos A G; Morgan, Joel E; Barquera, Blanca; Miyoshi, Hideto

    2017-03-15

    The Na(+)-pumping NADH-quinone oxidoreductase (Na(+)-NQR) is the first enzyme of the respiratory chain and the main ion transporter in many marine and pathogenic bacteria, including Vibrio cholerae The V. cholerae Na(+)-NQR has been extensively studied, but its binding sites for ubiquinone and inhibitors remain controversial. Here, using a photoreactive ubiquinone PUQ-3 as well as two aurachin-type inhibitors [(125)I]PAD-1 and [(125)I]PAD-2 and photoaffinity labeling experiments on the isolated enzyme, we demonstrate that the ubiquinone ring binds to the NqrA subunit in the regions Leu32-Met39 and Phe131-Lys138, encompassing the rear wall of a predicted ubiquinone-binding cavity. The quinolone ring and alkyl side chain of aurachin bound to the NqrB subunit in the regions Arg43-Lys54 and Trp23-Gly89, respectively. These results indicate that the binding sites for ubiquinone and aurachin-type inhibitors are in close proximity but do not overlap with one another. Unexpectedly, while the inhibitory effects of PAD-1 and PAD-2 were almost completely abolished by certain mutations in NqrB (i.e. Gly140Ala and Glu144Cys), the binding reactivities of [(125)I]PAD-1 and [(125)I]PAD-2 to the mutated enzymes were unchanged compared to those of the wild-type enzyme. We also found that photoaffinity labeling by [(125)I]PAD-1 and [(125)I]PAD-2 rather than being competitively suppressed in the presence of other inhibitors, is enhanced under some experimental conditions. To explain these apparently paradoxical results, we propose models for the catalytic reaction of Na(+)-NQR and its interactions with inhibitors on the basis of the biochemical and biophysical results reported here and in previous work.

  13. I. Structural studies on rhodopsin by photoaffinity labeling. II. Bioorganic studies on a rhodopsin analog from a retinal containing a 9-membered ring in the side chain

    SciTech Connect

    Sastry, L.

    1989-01-01

    Photoaffinity labeling of rhodopsin was carried out to identify the amino acids in the binding sites of the retinal and thereby arrive at a possible helical arrangement of the protein. A retinal analog, with a radioactive photolabel (diazoacetate) at position 3 was used for these studies. {sup 14}C label 3S-diazoacetoxy-9-cis-retinal bound to bovine opsin and regenerated a chromophore with {lambda}{sub max} at 465 nm. Photolysis of the complex at 254 nm resulted in covalent crosslinking of the retinal analog to the protein in 18-20% yield. Proteolytic cleavage (V8 protease) of the crosslinked protein and determination of the distribution of radioactivity indicated that both fragments V8-L (Met{sub 1}-Glu{sub 239}) and V8-S (Ser{sub 240}-Clu{sub 341}) were labeled. Further cleavage of labeled V8-S with CNBr (in HCOOH) showed that the major crosslinking sites were contained in a 51 residue peptide in helix 6, CNBr c+d (Val{sub 258}-Met{sub 308}). An attempt was made to construct a chemical model for bathorhodopsin, the primary photochemical intermediate in the bleaching sequence of rhodopsin.

  14. Differential photoaffinity labeling of catalytic subunits of NaK-ATPase with carrier-free /sup 125/I-cardiac glycosides

    SciTech Connect

    Lowndes, J.; Hokin-Neaverson, M.; Ruoho, A.

    1986-05-01

    The authors have obtained evidence for structural differences in the cardiac glycoside binding site between the ..cap alpha.. and ..cap alpha..(+) forms of the catalytic subunit of NaK-ATPase, using three closely related photoaffinity derivatives of the cardiotonic steroid, digitoxigenin. (/sup 125/I)N-(p-azido-m-iodo-o-hydroxybenzoyl)-4-amino-4,6-dideoxy-galactosyl digitoxigenin (IA-GaD), (/sup 125/I)N-(3-(p-azido-m-iodophenyl)-propionyl)-4-amino-4,6-dideoxy-ga-lactosyl digitoxigenin (AIPP-GaD) and (/sup 125/I)N-(3-(p-azido-m-iodophenyl)-propionyl)-4-amino-4,6-dideoxy-glucosyl digitoxi-genin (AIPP-GluD) were synthesized. AIPP-GaD and AIPP-GluD are stereoisomers. Eel electroplax and dog kidney NaK-ATPase (..cap alpha.. form) and rat brain synaptosomes (rich in ..cap alpha..(+) form) were photolabelled and then analyzed by SDS-PAGE and autoradiography. Photolysis with either carrier-free IA-GaD or AIPP-GluD gave ouabain-protectable labelling of NaK-ATPase catalytic subunit from all three tissues. However, photolysis with AIPP-GaD showed protectable labelling of the enzyme from eel and kidney but not from brain. This suggests a structural difference in the ..cap alpha..(+) form which results in either an inability to bind AIPP-GaD, or, perhaps more likely, an absence of a photoinsertion site in the correct location in the ..cap alpha..(+) form, as compared with the ..cap alpha.. form. It is of interest that the labelling pattern of the enzyme in the human erythrocyte resembles that of the brain enzyme.

  15. 6-Amidopyrene as a label-assisted laser desorption/ionization (LA-LDI) enhancing tag: development of photoaffinity pyrene derivative

    NASA Astrophysics Data System (ADS)

    Yoneda, Kozo; Hu, Yaping; Kita, Masaki; Kigoshi, Hideo

    2015-12-01

    Pyrene-conjugated compounds are detected by label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) without matrixes. We found that 6-amidopyrene derivatives were highly detectable by the LDI MS instrument equipped with a 355 nm laser. In a certain case of a 6-amidopyrene derivative, a molecular ion peak [M]+• and a characteristic fragment ion peak [M-42]+• were detected in an amount of only 10 fmol. The latter peak, corresponding to the 6-aminopyrene fragment, might be generated in situ by the removal of ketene (CH2=C=O) from the parent molecule. A photoaffinity amidopyrene derivative of an antitumor macrolide aplyronine A (ApA-PaP) was synthesized, which showed potent cytotoxicity and actin-depolymerizing activity. In an LDI MS analysis of the MeOH- and water-adducts of ApA-PaP, oxime N-O bonds as well as amidopyrene N-acetyl moieties were preferentially cleaved, and their internal structures were confirmed by MS/MS analysis. Amidopyrene moiety might enhance fragmentation and stabilize the cleaved fragments by intramolecular or intermolecular weak interactions including hydrogen bonding. Our chemical probe methods might contribute to a detailed analysis of binding modes between various ligands and target biomacromolecules that include multiple and weak interactions.

  16. 6-Amidopyrene as a label-assisted laser desorption/ionization (LA-LDI) enhancing tag: development of photoaffinity pyrene derivative

    PubMed Central

    Yoneda, Kozo; Hu, Yaping; Kita, Masaki; Kigoshi, Hideo

    2015-01-01

    Pyrene-conjugated compounds are detected by label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) without matrixes. We found that 6-amidopyrene derivatives were highly detectable by the LDI MS instrument equipped with a 355 nm laser. In a certain case of a 6-amidopyrene derivative, a molecular ion peak [M]+• and a characteristic fragment ion peak [M–42]+• were detected in an amount of only 10 fmol. The latter peak, corresponding to the 6-aminopyrene fragment, might be generated in situ by the removal of ketene (CH2=C=O) from the parent molecule. A photoaffinity amidopyrene derivative of an antitumor macrolide aplyronine A (ApA–PaP) was synthesized, which showed potent cytotoxicity and actin-depolymerizing activity. In an LDI MS analysis of the MeOH- and water-adducts of ApA–PaP, oxime N–O bonds as well as amidopyrene N-acetyl moieties were preferentially cleaved, and their internal structures were confirmed by MS/MS analysis. Amidopyrene moiety might enhance fragmentation and stabilize the cleaved fragments by intramolecular or intermolecular weak interactions including hydrogen bonding. Our chemical probe methods might contribute to a detailed analysis of binding modes between various ligands and target biomacromolecules that include multiple and weak interactions. PMID:26667050

  17. Photoaffinity labeling via nitrenium ion chemistry: protonation of the nitrene derived from 4-amino-3-nitrophenyl azide to afford reactive nitrenium ion pairs.

    PubMed

    Voskresenska, Valentyna; Wilson, R Marshall; Panov, Maxim; Tarnovsky, Alexander N; Krause, Jeanette A; Vyas, Shubham; Winter, Arthur H; Hadad, Christopher M

    2009-08-19

    Phenyl azides with powerful electron-donating substituents are known to deviate from the usual photochemical behavior of other phenyl azides. They do not undergo ring expansion but form basic nitrenes that protonate to form nitrenium ions. The photochemistry of the widely used photoaffinity labeling system 4-amino-3-nitrophenyl azide, 5, has been studied by transient absorption spectroscopy from femtosecond to microsecond time domains and from a theoretical perspective. The nitrene generation from azide 5 occurs on the S(2) surface, in violation of Kasha's rule. The resulting nitrene is a powerful base and abstracts protons extremely rapidly from a variety of sources to form a nitrenium ion. In methanol, this protonation occurs in about 5 ps, which is the fastest intermolecular protonation observed to date. Suitable proton sources include alcohols, amine salts, and even acidic C-H bonds such as acetonitrile. The resulting nitrenium ion is stabilized by the electron-donating 4-amino group to afford a diiminoquinone-like species that collapses relatively slowly to form the ultimate cross-linked product. In some cases in which the anion is a good hydride donor, cross-linking is replaced by reduction of the nitrenium ion to the corresponding amine.

  18. Photoaffinity Labeling via Nitrenium Ion Chemistry: Protonation of the Nitrene Derived from a 4-Amino-3-nitrophenylazide to Afford Reactive Nitrenium Ion Pairs

    PubMed Central

    Voskresenska, Valentyna; Wilson, R. Marshall; Panov, Maxim; Tarnovsky, Alexander N.; Krause, Jeanette A.; Vyas, Shubham; Winter, Arthur H.; Hadad, Christopher M.

    2009-01-01

    Phenyl azides with powerful electron-donating substituents are known to deviate from the usual photochemical behavior of other phenyl azides. They do not undergo ring expansion, but form basic nitrenes that protonate to form nitrenium ions. The photochemistry of the widely used photoaffinity labeling system 4-amino-3-nitrophenyl azide, 5, has been studied by transient absorption spectroscopy from femtosecond to microsecond time domains and from a theoretical perspective. The nitrene generation from azide 5 occurs on the S2 surface, in violation of Kasha's rule. The resulting nitrene is a powerful base and abstracts protons extremely rapidly from a variety of sources to form a nitrenium ion. In methanol, this protonation occurs in about 5 ps, which is the fastest intermolecular protonation observed to date. Suitable proton sources include alcohols, amine salts, and even acidic C-H bonds such as acetonitrile. The resulting nitrenium ion is stabilized by the electron-donating 4-amino group to afford a diiminoquinone-like species that collapses relatively slowly to form the ultimate cross-linked product. In some cases in which the anion is a good hydride donor, cross-linking is replaced by reduction of the nitrenium ion to the corresponding amine. PMID:19624129

  19. Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases.

    PubMed Central

    Hanna, M M; Zhang, Y; Reidling, J C; Thomas, M J; Jou, J

    1993-01-01

    A new photocrosslinking CTP analog that functioned as a substrate during transcription was synthesized and used to photoaffinity label E. coli and bacteriophage T7 RNA polymerases. This analog, 5-((4-azidophenacyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP during synthesis of RNA by both polymerases. Analog was placed at the 3' end or internally within RNA. Both polymerases inefficiently incorporated two 5-APAS-CMP molecules sequentially, as was found for the related 5-APAS-UMP. Analog was placed at the 3' end of RNA in transcription complexes paused at the site of Q-modification of E. coli RNA polymerase, downstream of the lambda PR' promoter (+16), a pause that requires specific DNA sequences but no apparent RNA hairpin. Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase. Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier results involving a NusA-enhanced pause site downstream from an RNA hairpin. Images PMID:7684833

  20. Synthesis of 25-hydroxyvitamin D sub 3 3. beta. -3 prime -(N-(4-azido-2-nitrophenyl)amino)propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D sub 3 : Photoaffinity labeling of rat serum vitamin D binding protein

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Van Baelen, H. )

    1991-05-14

    Vulnerability of 25-hydroxy-(26,27-{sup 3}H)vitamin D{sub 3} 3{beta}-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D{sub 3} (25-OH-D{sub 3}) toward standard conditions of carboxymethylationin promoted the authors to synthesize 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D{sub 3}, and 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE), the radiolabeled counterpart of 25-ANE competes for the 25-OH-D{sub 3} binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with {sup 3}H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D{sub 3}. These results provide strong evidence for the covalent labeling of the 25-OH-D{sub 3} binding site in rDPB by {sup 3}H-25-ANE.

  1. Photoaffinity labeling of ribulose-1,5-bisphosphate carboxylase/oxygenase activase with ATP gamma-benzophenone. Identification of the ATP gamma-phosphate binding domain.

    PubMed

    Salvucci, M E; Rajagopalan, K; Sievert, G; Haley, B E; Watt, D S

    1993-07-05

    The phosphate-binding domain of the ATP-binding site of tobacco Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) activase was elucidated by photo-affinity labeling with a monoanhydride of ADP with N-(4-(benzoyl)phenylmethyl)phosphoramide ([gamma-32P]ATP gamma BP). Covalent incorporation of [gamma-32P]ATP gamma BP into the 42-kDa Rubisco activase subunit was dependent upon irradiation with ultraviolet light. Photolabelling of Rubisco activase with ATP gamma BP exhibited saturation kinetics; the apparent Kd for photolabeling was 5 microM. Two lines of evidence showed that ATP gamma BP modified Rubisco activase at the ATP-binding domain. First, physiological concentrations of ATP and ADP afforded complete protection against photolabeling of Rubisco activase by ATP gamma BP. Second, photolysis of Rubisco activase in the presence of ATP gamma BP decreased both the ATPase and the Rubisco activating activities. Inactivation of enzyme activity was dependent on ATP gamma BP concentration and could be prevented by including ADP during photolabeling. The region of Rubisco activase that was modified by ATP gamma BP was identified by isolating photolabeled peptides. Sequence analysis showed that ATP gamma BP modified Rubisco activase in two distinct regions; one region, S117-A136, is adjacent to the P-loop and the other region, V223-T234, exhibits homology to a region of adenylate kinase that ligates the essential metal ion. Photolabeling of these two regions of Rubisco activase was consistent with modification of the ATP gamma-phosphate-binding domain of Rubisco activase with ATP gamma BP.

  2. 5-Azido-2-aminopyridine, a new nitrene/nitrenium ion photoaffinity labeling agent that exhibits reversible intersystem crossing between singlet and triplet nitrenes.

    PubMed

    Panov, Maxim S; Voskresenska, Valentyna D; Ryazantsev, Mikhail N; Tarnovsky, Alexander N; Wilson, R Marshall

    2013-12-26

    The photochemistry of a new photoaffinity labeling (PAL) agent, 5-azido-2-(N,N-diethylamino)pyridine, was studied in aprotic and protic solvents using femtosecond-to-microsecond transient absorption and product analysis, in conjunction with ab initio multiconfigurational and multireference quantum chemical calculations. The excited singlet S1 state is spectroscopically dark, whereas photoexcitation to higher-lying singlet excited S2 and S3 states drives the photochemical reaction toward a barrierless ultrafast relaxation path via two conical intersections to S1, where N2 elimination leads to the formation of the closed-shell singlet nitrene. The singlet nitrene undergoes intersystem crossing (ISC) to the triplet nitrene in aprotic and protic solvents as well as protonation to form the nitrenium ion. The ISC rate constants in aprotic solvents increase with solvent polarity, displaying a "direct" gap effect, whereas an "inverse" gap effect is observed in protic solvents. Transient absorption actinometry experiments suggest that a solvent-dependent fraction from 20% to 50% of nitrenium ions is generated on a time scale of a few tens of picoseconds. The closed-shell singlet and triplet nitrene are separated by a small energy gap in protic solvents. As a result, the unreactive triplet state nitrene undergoes delayed, thermally activated reverse ISC to reform the reactive closed-shell singlet nitrene, which subsequently protonates, forming the remaining fraction of nitrenium ions. The product studies demonstrate that the resulting nitrenium ion stabilized by the electron-donating 4-amino group yields the final cross-linked product with high, almost quantitative efficiency. The enhanced PAL function of this new azide with respect to the widely applied 4-amino-3-nitrophenyl azide is discussed.

  3. Insights into interactions between the alpha-helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labeling.

    PubMed

    Pham, Vi; Dong, Maoqing; Wade, John D; Miller, Laurence J; Morton, Craig J; Ng, Hooi-Ling; Parker, Michael W; Sexton, Patrick M

    2005-08-05

    Fish-like calcitonins (CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Two analogues of the antagonist sCT(8-32) containing a single photolabile p-benzoyl-l-phenylalanine (Bpa) residue in position 8 or 19 were used. Both analogues retained high affinity for the CT receptor and potently inhibited agonist-induced cAMP production. The [Bpa(19)]sCT(8-32) analogue cross-linked to the receptor at or near the equivalent cross-linking site of the full-length peptide, within the fragment Cys(134)-Lys(141) (within the amino terminus of the receptor, adjacent to transmembrane 1) (Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720-6729). In contrast, proteolytic mapping and mutational analysis identified Met(49) as the cross-linking site for [Bpa(8)]sCT(8-32). This site differed from the previously identified cross-linking site of the agonist [Bpa(8)]human CT (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 31177-31182) and may provide evidence for conformational differences between interaction with active and inactive state receptors. Molecular modeling suggests that the difference in cross-linking between the two Bpa(8) analogues can be accounted for by a relatively small change in peptide orientation. The model was also consistent with cooperative interaction between the receptor amino terminus and the receptor core.

  4. Direct photoaffinity labeling by nucleotides of the apparent catalytic site on the heavy chains of smooth muscle and Acanthamoeba myosins

    SciTech Connect

    Maruta, H.; Korn, E.D.

    1981-01-10

    The heavy chains of Acanthamoeba myosins, IA, IB and II, turkey gizzard myosin, and rabbit skeletal muscle myosin subfragment-1 were specifically labeled by radioactive ATP, ADP, and UTP, each of which is a substrate or product of myosin ATPase activity, when irradiated with uv light at 0/sup 0/C. With UTP, as much as 0.45 mol/mol of Acanthamoeba myosin IA heavy chain and 1 mol/mol of turkey gizzard myosin heavy chain was incorporated. Evidence that the ligands were associated with the catalytic site included the observations that reaction occurred only with nucleotides that are substrates or products of the ATPase activity; that the reaction was blocked by pyrophosphate which is an inhibitor of the ATPase activity; that ATP was bound as ADP; and that label was probably restricted to a single peptide following limited subtilisin proteolysis of labeled Acanthamoeba myosin IA heavy chain and extensive cleavage with CNBr and trypsin of labeled turkey gizzard myosin heavy chain.

  5. Specific labeling and permanent activation of the retinal rod cGMP-activated channel by the photoaffinity analog 8-p-azidophenacylthio-cGMP.

    PubMed Central

    Brown, R L; Gerber, W V; Karpen, J W

    1993-01-01

    Cyclic nucleotide-gated ion channels play a key role in visual excitation and a variety of other signaling pathways. The photoaffinity probe 8-p-azidophenacylthio-cGMP (APT-cGMP) has been developed for structural and functional studies of the cGMP-activated channel of retinal rod outer segments. Using this analog, we have demonstrated both specific labeling of the channel in a partially purified biochemical preparation from bovine rod outer segments and permanent activation of the channel current in excised membrane patches from salamander outer segments. After UV illumination, a 32P-labeled version of APT-cGMP was shown by SDS/PAGE and autoradiography to be covalently attached to the 63-kDa channel subunit. This incorporation was significantly reduced by 8-Br-cGMP but was not reduced by 5'-GMP. In patch-clamp experiments APT-cGMP was a potent activator of the channel; APT-cGMP typically opened half of the channels in a patch at a 10-fold lower concentration than cGMP. Exposure of membrane patches to UV light in the presence of APT-cGMP resulted in a persistent current observed in the absence of bath-applied nucleotide. This current increased with repeated exposure of the patch to both UV light and fresh APT-cGMP, approaching the maximum current originally evoked by saturating (500 microM) cGMP. At this point, addition of 500 microM cGMP caused a negligible increase in current. The persistent current had several other properties expected of current through cGMP-activated channels: it was outwardly rectifying; outward current was blocked > 90% by 2 mM internal Mg2+, whereas inward current was blocked much less efficiently; a low concentration of cGMP caused a larger increase in current atop a half-maximal persistent current than it did originally. We conclude that the persistent current was caused by the covalent tethering of cGMP moieties to channel binding sites, resulting in irreversible channel activation. APT-cGMP should prove useful for further studies of

  6. Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

    SciTech Connect

    Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

    1987-06-30

    3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3.

  7. Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavage of the labeled protein: Identification of an 11. 5-kDa peptide containing the putative 25-hydroxyvitamin D sub 3 binding site

    SciTech Connect

    Ray, R.; Holick, M.F. ); Bouillon, R.; Baelen, H.V. )

    1991-07-30

    In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitrophenyl)amino)propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D{sub 3} 3{beta}-3{prime}-(N-(4-azido-2-nitro-(3,5-{sup 3}H)phenyl)amino)propyl ether ({sup 3}H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D{sub 3} for the binding site of the latter in hDBP and (2) {sup 3}H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with {sup 3}H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D{sub 3}.

  8. Identification of the glucose transporter in mammalian cell membranes using an /sup 125/(I)-forskolin photoaffinity label

    SciTech Connect

    Ruoho, A.; Wadzinski, B.; Shanahan, M.

    1987-05-01

    The glucose transporter has been identified in a variety of mammlian cell membranes using a carrier-free photoactivatable radioiodinated derivative of forskolin, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin, (I-125)IAPS-Fsk, at 1-10 nM. The membranes which have been photolabeled with (I-125)IAPS-Fsk are: rat cardiac sarcolemmal membranes, rat cortex and cerebellum synaptic membranes, human placental membranes, and wild type S49 lymphoma cell membranes. The glucose transporter in rat cardiac sarcolemmal membranes and rat cortex and cerebellum synaptic membranes was determined to be 45 kDa by SDS-PAGE. Photolysis of human placental membranes and S49 lymphoma membranes with (I-125)IAPS-Fsk followed by SDS-PAGE indicated specific derivatization of a broad band (45-55 kDa) in placental membranes and a narrower band (45 kDa) in the S49 lymphoma membranes. Digestion of the (I-125)IPAS-Fsk labelled placental and S49 lymphoma membranes with endo-B-galactosidase showed a reduction in the apparent molecular weight of the radiolabelled band to 40 kDa. Trypsinization of labelled placental and lymphoma membranes produced an 18 kDa radiolabelled proteolytic fragment. (I-125)IAPS-Fsk is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.

  9. Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1.

    PubMed

    Budelier, Melissa M; Cheng, Wayland W L; Bergdoll, Lucie; Chen, Zi-Wei; Janetka, James W; Abramson, Jeff; Krishnan, Kathiresan; Mydock-McGrane, Laurel; Covey, Douglas F; Whitelegge, Julian P; Evers, Alex S

    2017-06-02

    Voltage-dependent anion channel-1 (VDAC1) is a highly regulated β-barrel membrane protein that mediates transport of ions and metabolites between the mitochondria and cytosol of the cell. VDAC1 co-purifies with cholesterol and is functionally regulated by cholesterol, among other endogenous lipids. Molecular modeling studies based on NMR observations have suggested five cholesterol-binding sites in VDAC1, but direct experimental evidence for these sites is lacking. Here, to determine the sites of cholesterol binding, we photolabeled purified mouse VDAC1 (mVDAC1) with photoactivatable cholesterol analogues and analyzed the photolabeled sites with both top-down mass spectrometry (MS), and bottom-up MS paired with a clickable, stable isotope-labeled tag, FLI-tag. Using cholesterol analogues with a diazirine in either the 7 position of the steroid ring (LKM38) or the aliphatic tail (KK174), we mapped a binding pocket in mVDAC1 localized to Thr(83) and Glu(73), respectively. When Glu(73) was mutated to a glutamine, KK174 no longer photolabeled this residue, but instead labeled the nearby Tyr(62) within this same binding pocket. The combination of analytical strategies employed in this work permits detailed molecular mapping of a cholesterol-binding site in a protein, including an orientation of the sterol within the site. Our work raises the interesting possibility that cholesterol-mediated regulation of VDAC1 may be facilitated through a specific binding site at the functionally important Glu(73) residue. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Photoaffinity labeling of opiate (enkephalin) receptor of rat brain plasma membranes with /sup 125/I(D-Ala/sup 2/, p-N/sub 3/-Phe/sup 4/-Met/sup 5/)-enkephalin

    SciTech Connect

    Yeung, C.W.T.

    1986-05-01

    A photoreactive (D-Ala/sup 2/, p-N/sub 3/-Phe/sup 4/-Met/sup 5/)enkephalin derivative was prepared, iodinated with carrier free /sup 125/I and then purified by high performance liquid chromatography. The purified radioactive photoprobe was monoiodinated at the amino terminal tyrosine residue. This radioactive photoprobe was used to photoaffinity label plasma membranes prepared from rat brain, spinal cord and cerebellum. The photolabeled plasma membranes were analyzed by sodium dodecyl sulfate gel electrophoresis. A 46,000-daltons band was specifically photolabeled in the plasma membranes of brain and spinal cord but not in the plasma membranes from cerebellum. The photolabeling of this band was inhibited by peptides related to enkephalin by not but substance P or gastrin tetrapeptide. These data demonstrate that the labeled 46,000-daltons band is a protein of the opiate (enkephalin)receptor.

  11. pH-Dependent Changes in Photoaffinity Labeling Patterns of the H1 Influenza Virus Hemagglutinin by Using an Inhibitor of Viral Fusion

    PubMed Central

    Cianci, Christopher; Yu, Kuo-Long; Dischino, Douglas D.; Harte, William; Deshpande, Milind; Luo, Guangxiang; Colonno, Richard J.; Meanwell, Nicholas A.; Krystal, Mark

    1999-01-01

    The hemagglutinin (HA) protein undergoes a low-pH-induced conformational change in the acidic milieu of the endosome, resulting in fusion of viral and cellular membranes. A class of compounds that specifically interact with the HA protein of H1 and H2 subtype viruses and inhibit this conformational change was recently described (G. X. Luo et al., Virology 226:66–76, 1996, and J. Virol. 71:4062–4070, 1997). In this study, purified HA trimers (bromelain-cleaved HA [BHA]) are used to examine the properties and binding characteristics of these inhibitors. Compounds were able to inhibit the low-pH-induced change of isolated trimers, as detected by resistance to digestion with trypsin. Protection from digestion was extremely stable, as BHA-inhibitor complexes could be incubated for 24 h in low pH with almost no change in BHA structure. One inhibitor was prepared as a radiolabeled photoaffinity analog and used to probe for specific drug interactions with the HA protein. Analysis of BHA after photoaffinity analog binding and UV cross-linking revealed that the HA2 subunit of the HA was specifically radiolabeled. Cross-linking of the photoaffinity analog to BHA under neutral (native) pH conditions identified a stretch of amino acids within the α-helix of HA2 that interact with the inhibitor. Interestingly, cross-linking of the analog under acidic conditions identified a different region within the HA2 N terminus which interacts with the photoaffinity compound. These attachment sites help to delineate a potential binding pocket and suggest a model whereby the BHA is able to undergo a partial, reversible structural change in the presence of inhibitor compound. PMID:9971755

  12. 3'-Phosphoadenosine-5'-phosphosulfate: Photoaffinity ligand for sulfotransferase enzymes

    SciTech Connect

    Otterness, D.M.; Powers, S.P.; Miller, L.J.; Weinshilboum, R.M. )

    1991-01-01

    Sulfation is an important pathway in the biotransformation of many drugs, xenobiotic compounds, neurotransmitters, and hormones. The sulfate donor for these reactions is 3'-phosphoadenosine-5'-phosphosulfate (PAPS). We set out to determine whether PAPS might serve as a photoaffinity ligand for sulfotransferase enzymes. UV irradiation of (35S)PAPS with partially purified human liver thermostable (TS) phenol sulfotransferase (PST) radioactively labeled a protein with a molecular mass of 35 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoaffinity labeling of TS PST with (35S) PAPS did not require the presence of a phenolic substrate but rather was inhibited by p-nitrophenol, a sulfate acceptor substrate for TS PST. Inhibitors of TS PST enzymatic activity, including 3'-phosphoadenosine-5'-phosphate, ATP, ADP, and 2,6-dichloro-4-nitrophenol, also inhibited photoaffinity labeling of the 35-kDa protein with (35S)PAPS, in a concentration-dependent fashion, with IC50 values of 14 microM, 2.1 mM, 7.7 mM, and 91 microM, respectively. The 35-kDa protein that was radioactively labeled by (35S)PAPS in the presence of UV light coeluted with TS PST enzymatic activity during gel filtration high performance liquid chromatography. (35S)PAPS was then used to photoaffinity label another sulfotransferase enzyme, the thermolabile (TL) form of PST partially purified from human liver. Therefore, (35S)PAPS appears to be a photoaffinity ligand that could be used to study a variety of PAPS-dependent sulfotransferases. Photoaffinity labeling of TS and TL PST, as well as other PAPS-dependent sulfotransferases, should enhance our ability to purify this important group of enzymes and to determine amino acid sequences at or near their active sites.

  13. Design and synthesis of a new class of fluorescent photoaffinity label with specific reference to 4-benzoyl-1-benzamidofluorescein: a new photolabel for adenine nucleotide binding domains on enzymes

    NASA Astrophysics Data System (ADS)

    Rosen, Jane E.

    1993-05-01

    Benzophenone was used as the photoreactive moiety in the synthesis of several water soluble, fluorescent photoaffinity labels. The following compounds were synthesized: 5-(2-(p- benzoylbenzamido)ethylamino-1-napthalenesulfonate (BzEDANS); 5-(2-(p- benzoylbenzamido)hexylamino-1-napthalenesulfonate (BzHDANS); and 4-benzoyl-1- benzamidofluorescein (BzAF). BzEDANS and BzHDANS were found to be unsuitable for use as photochemical probes. They were incapable of photoinduced covalent binding to methylene carbon due to intramolecular triplet-triplet energy transfer. BzAF was synthesized because its fluorescent moiety, fluorescein, is an inefficient acceptor for intramolecular quenching of the benzophenone triplet state diradical intermediate. BzAF was found to be a suitable and efficient photolabel and is presently a prototype for a new class of fluorescent photolabel.

  14. Characterizing the Epothilone Binding Site on β-Tubulin by Photoaffinity Labeling: Identification of β-Tubulin Peptides TARGSQQY and TSRGSQQY as Targets of an Epothilone Photoprobe for Polymerized Tubulin.

    PubMed

    Ranade, Adwait R; Higgins, LeeAnn; Markowski, Todd W; Glaser, Nicole; Kashin, Dmitry; Bai, Ruoli; Hong, Kwon Ho; Hamel, Ernest; Höfle, Gerhard; Georg, Gunda I

    2016-04-14

    Photoaffinity labeling with an epothilone A photoprobe led to the identification of the β-tubulin peptides TARGSQQY and TSRGSQQY as targets of the photoprobe for polymerized tubulin. These peptides represent residues 274-281 in different β-tubulin isotypes. Placing the carbene producing 21-diazo/triazolo moiety of the photoprobe in the vicinity of the TARGSQQY peptide in a homology model of TBB3 predicted a binding pose and conformation of the photoprobe that are very similar to the ones reported for 1) the high resolution cocrystal structure of epothilone A with an α,β-tubulin complex and for 2) a saturation transfer difference NMR and transferred NOESY NMR study of dimeric and polymerized tubulin. Our findings thus provide additional support for these models as physiologically the most relevant among several modes of binding that have been proposed for epothilone A in the taxane pocket of β-tubulin.

  15. Design, synthesis, and characterization of a novel, 4-[2-(diphenylmethoxy)ethyl]-1-benzyl piperidine-based, dopamine transporter photoaffinity label.

    PubMed

    Dutta, A K; Fei, X S; Vaughan, R A; Gaffaney, J D; Wang, N; Lever, J R; Reith, M E

    2001-03-09

    The dopamine transporter (DAT) has been implicated strongly in cocaine's reinforcing effects. Many derivatives of piperidine analogs of GBR 12909 have been developed and were found to be quite potent and selective for the DAT. In this regard, most of these derivatives were found to be much more selective for the DAT than conventional GBR compounds e.g. GBR 12909 when their selectivity was compared with the serotonin transporter (SERT). A brief structure-activity relationship (SAR) study has been carried out in the development of a novel photoaffinity ligand which illustrated the effect of the presence of a sterically bulky iodine atom next to the azido group in activity and selectivity for the DAT. This SAR study also led to the development of the compound 4 which is one of the most potent and selective blockers for the DAT known today. The photoaffinity ligand [125I]AD-96-129 was incorporated into the DAT molecule as was demonstrated by immunoprecipitation with serum 16 which is specific for DAT. This photolabeling was antagonized by DAT-specific blockers and was unaffected by specific SERT and norepinephrine transporter (NET) blockers indicating interaction of this novel ligand with the DAT.

  16. Photoaffinity labeling of rat steroid 5 alpha-reductase (isozyme-1) by a benzophenone derivative of a 4-methyl-4-azasteroid.

    PubMed

    Taylor, M F; Bhattacharyya, A K; Rajagopalan, K; Hiipakka, R; Liao, S; Collins, D C

    1996-05-01

    [1,2-3H]N-4(Benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha-androstane-17 beta-carboxamide ([3H]-4MABP) has been synthesized as a photoaffinity probe of the steroid-binding domain of rat steroid 5 alpha-reductase isozyme-1 (5 alpha R-1). Reversible binding of the probe to 5 alpha R-1 in microsomal preparations yielded a reversible dissociation constant (Kd) of -3 nM, whereas inhibition experiments indicated that the probe had a 50% inhibition concentration of 4.4 nM and was a competitive inhibitor of the enzyme (Ki approximately 3 nM) with respect to testosterone. SDS-PAGE analysis of microsomal, detergent-solubilized, and (6.5%) polyethylene glycol-precipitated fractions of 5 alpha R-I photolyzed with [3H]4MABP in the presence of NADPH showed that the radioactivity was incorporated into a single protein band with a mass of 26 kDa (apparent molecular weight of 5 alpha R-1). UV photolysis was accompanied by an irreversible loss in enzyme activity, consistent with its covalent modification. Increasing the time of UV irradiation and concentration of [3H]4MABP indicated that the half-life and apparent Kd for its photo insertion were approximately 3 min and 7.5 nM, respectively. Photolysis in the presence of a 20-fold excess of N,N-diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide or the 3-carboxysteroid SKF-105111 resulted in partial protection of 5 alpha R-1 from the probe, whereas minimal incorporation of radioactivity was observed in the absence of NADPH or in the presence of NADP+. The results indicate that [3H]4MABP is an effective probe of the steroid (D-ring) binding domain of 5 alpha R-1.

  17. Photoaffinity labeling of the thymidine triphosphate binding domain in Escherichia coli DNA polymerase I: identification of histidine-881 as the site of cross-linking

    SciTech Connect

    Pandey, V.N.; Williams, K.R.; Stone, K.L.; Modak, M.J.

    1987-12-01

    Using the technique of ultraviolet-mediated cross-linking of substrate deoxynucleoside triphosphates (dNTPs) to their acceptor site, the authors have labeled the Klenow fragment of Escherichia coli DNA polymerase I (Pol I) with (..cap alpha..-/sup 32/P)dTTP. Covalent cross-linking of (..cap alpha..-/sup 32/P)dTTP to the Klenow fragment is shown to be at the substrate binding site by the following criteria; (a) the cross-linking reaction requires dTTP in its metal chelate form; (b) dTTP is readily competed out by other dNTPs as well as by substrate binding site directed reagents; (c) labeling with dTTP occurs at a single site as judged by peptide mapping. Under optimal conditions, a modification of approximately 20% of the enzyme was achieved. Following tryptic digestion of the (..cap alpha..-/sup 32/P)dTTP-labeled Klenow fragment, reverse-phase high-performance liquid chromatography demonstrated that 80% of the radioactivity was contained within a single peptide. The amino acid composition and sequence of this peptide identified it as the peptide spanning amino acid residues 876-890 in the primary sequence of E. coli Pol I. Chymotrypsin and Staphylococcus aureus V8 protease digestion of the labeled tryptic peptide in each case yielded a single smaller fragment that was radioactive. Amino acid analysis and sequencing of these small peptides further narrowed the dTTP cross-linking site to within the region spanning residues 876-883. They concluded that histidine-881 is the primary attachment site for dTTP in E. coli DNA Pol I, since during amino acid sequencing analysis of all three radioactive peptides loss of the histidine residue at the expected cycle is observed.

  18. Photoaffinity labeling by ( sup 3 H)-N5-methyl-N5-isobutylamiloride of proteins which cofractionate with Na+/H+ antiport activity

    SciTech Connect

    Wu, J.S.; Lever, J.E. )

    1989-04-04

    N5-Methyl-N5-isobutylamiloride (MIA) is one of a series of 5-N-substituted amiloride analogues which exhibit high affinity and specificity for inhibition of Na+/H+ antiport. Amiloride-sensitive ({sup 3}H)MIA binding to renal brush border membranes exhibited a Kd of 250 nM and a Bmax of 8.6 pmol/mg of protein. Specific binding was optimal at pH 7.5 and inhibited in the presence of Na+ and Li+. Inhibition by amiloride exhibited biphasic kinetics. After resolution of solubilized membranes by high-pressure liquid chromatography, MIA binding activity cofractionated together with Na+/H+ antiport activity, measured after reconstitution in asolectin vesicles, into a major and a minor peak. When fractions containing the major peak of Na+/H+ antiport activity were incubated with ({sup 3}H)MIA and then photolyzed with a mercury arc lamp, covalent incorporation of label into polypeptides of apparent molecular mass 81 and 107 kDa was observed. These photolabeled bands were also observed in intact brush border membranes in addition to labeled polypeptides of apparent molecular mass 60 and 46 kDa, respectively. Labeling was inhibited by amiloride, reduced in the presence of Na+, and not observed in the absence of photolysis. These data point to the 81- and 107-kDa polypeptides as candidates for identification as components of a Na+/H+ antiport system in renal brush border membranes.

  19. Evidence from photoaffinity labelling studies for coupling of the alpha/sub 1/-adrenergic receptor to a guanine-nucleotide (G) binding protein

    SciTech Connect

    Graham, R.M.; Sena, L.; Schwarz, K.R.; Homcy, C.J.

    1986-05-01

    In contrast to ..beta..- and ..cap alpha../sub 2/-adrenergic receptors the role of a G-protein in signal transduction at ..cap alpha..-adrenergic receptors has been difficult to define. Using rat hepatic membranes prepared to avoid retention of endogenous nucleotides and activation of Ca/sup 2 +/-sensitive proteases, a Gpp(NH)p shift in agonist ((-)epinephrine) affinity from an IC/sub 50/ 10/sup -6/ to 5 x 10/sup -5/ M was readily demonstrable in competition studies with the ..cap alpha../sub 1/-specific radioligand (/sup 3/H)prazosin, but was not observed in membranes prepared without protease inhibitors (PIs). Labelling of these membranes with the photolabile prazosin analog, (/sup 125/I)CP65,526, followed by SDS-PAGE/autoradiography revealed a predominant, specifically labelled protein of M/sub r/ = 80,000, whereas a M/sub r/ = 59,000 peptide was evident with membranes prepared in the absence of PIs. The IC/sub 50/ for inhibition of labelling of the M/sub r/ = 80,000 peptide by (-)epinephrine, as determined by radiochromatogram scanning of autoradiographs of the photolabelled receptor, shifted from 10/sup -7/ to 10/sup -6/ in the presence of Gpp(NH)p. However, no shift in agonist affinity at the M/sub r/ = 59,000 peptide was evident in membranes prepared without PIs. This approach provides visual evidence for a G-protein-mediated shift in agonist affinity at the ..cap alpha../sub 1/-adrenergic receptor and allows a correlation between subunit size analysis and ligand binding.

  20. Photoaffinity labeling of the human red-blood-cell urea-transporter polypeptide components. Possible homology with the Kidd blood group antigen.

    PubMed

    Neau, P; Degeilh, F; Lamotte, H; Rousseau, B; Ripoche, P

    1993-12-01

    The tritiated urea analogue 1-(3-azido-4-chlorophenyl)-3methyl-2-thiourea ([3H]MeACPTU) was used as a probe to photolabel the human red-blood-cell membrane facilitated urea transporter. On irradiation, [3H]MeACPTU incorporated irreversibly into white ghost membranes. SDS/gel electrophoresis of membranes revealed radioactive incorporation in five major bands of 200, 110, 60, 40 and 14 kDa. The labeling of the 40-kDa and 60-kDa bands was partly prevented by the presence of a high concentration of other urea analogues such as thiourea and 1-(3,4-dichlorophenyl) 2-thiourea (DCPTU). The photolabeling pattern obtained with white ghosts of the Kidd blood-group type Jk(a-,b-) showed no labeling of the 40-kDa polypeptide. Protecting experiments carried out with anti-Jka, anti-Jkb and anti-Jk3 sera prevented radioactive incorporation in the 60-kDa band and in the 110-kDa band. Urea permeability of pink ghosts of blood type Jk(a+,b+) measured in the presence of Jk3 antibodies was 19% lower than the control values. However, urea permeability of frog urinary bladder epithelial cells was not affected by the presence of Jk-reactive antibodies. These results support the hypothesis that the Kidd antigen and the facilitated urea transporter are the same protein. Our estimation of the number of copies in each cell is close to that of the previously published value of 14000.

  1. Molecular structure of rat brain apamin receptor: differential photoaffinity labeling of putative K/sup +/ channel subunits and target size analysis

    SciTech Connect

    Seagar, M.J.; Labbe-Jullie, C.; Granier, C.; Goll, A.; Glossmann, H.; Rietschoten, J.V.; Couraud, F.

    1986-07-01

    Two photoreactive apamin derivatives were prepared with an aryl azide group coupled at different positions on the neurotoxin molecule. These ligands were used to identify membrane components in the environment of the neuronal binding site that is associated with a Ca/sup 2 +/-activated K/sup +/ channel. /sup 125/I-(..cap alpha..-ANPAA-Cys/sub 1/)apamin labeled a single M/sub r/ 86,000 chain in cultured neurons whereas two bands corresponding to M/sub r/ 86,000 and 59,000 were detected in synaptic membrane preparations, suggesting that the M/sub r/ 59,000 polypeptide may be a degradation product. Randomly modified /sup 125/I-ANPAA-apamin gave a cross-linking profile equivalent to the sum of those obtained with the two defined derivatives. The apamin binding site seems to be located at the frontier between three or more putative K/sup +/ channel subunits which are only accessible from limited regions of the receptor-associated photoprobe. Irradiation of frozen rat brain membranes with high-energy electrons led to a reduction in /sup 125/I-apamin receptor capacity, yielding a target size for the functional binding unit of M/sub r/ 84,000-115,000, which could be constituted by the M/sub r/ 86,000 subunit alone or by the M/sub r/ 86,000 subunit in conjunction with one of the two smaller subunits.

  2. Synthesis of 5-nitro-2-(N-3-(4-azidophenyl)-propylamino)-benzoic acid: Photoaffinity labeling of human red blood cell ghosts with a 5-nitro-2-(3-phenylpropylamino)-benzoic acid analog

    SciTech Connect

    Branchini, B.R.; Murtiashaw, M.H.; Egan, L.A. )

    1991-04-15

    A photoaffinity analog of the potent epithelial chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid has been synthesized and characterized. In the dark, this reagent, 5-nitro-2-(N-3-(4-azidophenyl)-propylamino)-benzoic acid, and the parent compound reversibly inhibited chloride efflux in human red blood cell ghosts. Irradiation of ghost membranes with 350 microM arylazide analog reduced the rate of chloride efflux to 33% of the control value. The photoinactivation process was not reversed by exhaustive washing of ghost membranes. Covalent incorporation of the photoaffinity reagent was supported by difference ultraviolet spectroscopy, which indicated the attachment of the substituted 2-amino-5-nitrobenzoic acid chromophore to ghost membranes. The novel photolabeling agent described here should be a useful structural probe for chloride channels in erythrocyte membranes and epithelial cells.

  3. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    SciTech Connect

    Ho, L.T.; Nie, Z.M.; Mende, T.J.; Richardson, S.; Chavan, A.; Kolaczkowska, E.; Watt, D.S.; Haley, B.E.; Ho, R.J. )

    1989-01-01

    Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by (125I)PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that (a) no other AC-regulatory proteins are known to be of this size, (b) the catalytic unit of bovine brain enzyme is in the same range and (c) this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.

  4. Comparison of CAP88 and MCNP for Overhead Gamma-emitting Plumes

    SciTech Connect

    Mcnaughton, Michael; Gillis, Jessica Mcdonnel; McClory, Aysha Reede; Whicker, Jeffrey Jay; Fuehne, David Patrick

    2016-01-08

    The purpose of this paper is to use the Monte Carlo N-Particle Code (MCNP) to investigate the dose from gamma-emitting radionuclides such as Carbon-11 when a plume passes overhead. MCNP results are compared with results from the EPA program, CAP88. In some cases, typically near the source during stable conditions, the CAP88 results are less than the MCNP results. However, in the case of a receptor 800 m from a source at the Los Alamos Neutron Science Center (LANSCE), the CAP88 result is greater than the MCNP result.

  5. Photoaffinity labeling of the rat plasma vitamin D binding protein with (26,27-3H)-25-hydroxyvitamin D3 3 beta-(N-(4-azido-2-nitrophenyl)glycinate)

    SciTech Connect

    Ray, R.; Holick, S.A.; Hanafin, N.; Holick, M.F.

    1986-08-26

    It is well recognized that the vitamin D binding protein (DBP) is important for the transport of vitamin D, 25-hydroxyvitamin D (25-OH-D), and its metabolites. In an attempt to better understand the molecular-binding properties of this ubiquitous protein, we designed and synthesized a photoaffinity analogue of 25-OH-D3 and its radiolabeled counterpart. This analogue, 25-hydroxyvitamin D3 3 beta-(N-(4-azido-2-nitrophenyl)glycinate) (25-OH-D3-ANG), was recognized by the rat DBP and was about 10 times less active than 25-OH-D3 in terms of binding. Incubation of (/sup 3/H)25-OH-D3 or (/sup 3/H)25-OH-D3-ANG with rat DBP revealed that both compounds were specifically bound to a protein with a sedimentation coefficient of 4.1 S. Each was displaced with a 500-fold excess of 25-OH-D3. When (/sup 3/H)25-OH-D3-ANG was exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3, there was no displacement of tritium from the 4.1S peak. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of (/sup 3/H)25-OH-D3-ANG exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3 revealed one major band with a molecular weight of 52 000. These data provide strong evidence that (/sup 3/H)25-OH-D3-ANG was covalently linked to the rat DBP. This photoaffinity probe should provide a valuable tool for the analysis of the binding site on this transport protein.

  6. Photoaffinity ligands in the study of cytochrome p450 active site structure.

    PubMed

    Gartner, Carlos Augusto

    2003-04-01

    While photoaffinity ligands have been widely used to probe the structures of many receptors and nucleic acid binding proteins, their effective use in the study of cytochrome p450 structure is less established. Nevertheless, significant advances in this field have been made since the technique was first applied to p450cam in 1979. In several cases, especially studies involving p450s of the 1A and 2B families, peptides covalently modified with photoaffinity ligands have been isolated and characterized. Some of these peptides were predicted by molecular modeling to line substrate binding regions of the enzymes. Other data obtained from such studies were more difficult to reconcile with theory. This review addresses the status of photoaffinity labeling as a tool for studying cytochrome p450 structure. In addition, potential future directions in this field are discussed, including the development of heme-directed agents and validation of their effectiveness as photoaffinity ligands using sperm whale myoglobin as a test protein. The potential for hydroxyaromatic compounds to serve as photoactivated probes of active site nucleophiles is also discussed. This class of compounds and its derivatives has long been known in the fields of photochemistry and photophysics to be precursors of reactive radicals and quinone methides that are likely to serve as effective active site probes of the p450s.

  7. Gamma-emitting natural and anthropogenic radionuclides in the terrestrial environment of Kongsfjord, Svalbard.

    PubMed

    Dowdall, M; Gerland, S; Lind, B

    2003-04-15

    This paper presents results obtained from a radiometric survey, conducted by the Norwegian Radiation Protection Authority, into the levels of gamma-emitting radionuclides, both anthropogenic and natural, in the terrestrial environment of Kongsfjorden, which lies on the North-Western Coast of Spitsbergen in the Arctic archipelago of Svalbard (79 degrees N, 12 degrees E). Samples of terrestrial matrices were taken during field campaigns conducted between 2000 and 2002 and analysed for a range of gamma-emitting radionuclides. The objectives of this study included an assessment of the levels of gamma-emitting radionuclides in the terrestrial environment of the region, identification of processes and activities that influence the accumulation and redistribution of such nuclides within the region and elucidation of the behaviour of such radionuclides within a high arctic environment. Results indicate a quite homogenous spatial distribution of such radionuclides within the study area and highlight the relatively low levels of contamination by the anthropogenic radionuclide, 137Cs, on Svalbard. Average values and ranges of the radionuclides activities in surface soils (0-3 cm) were: 238U 42 Bq/kg (17-134), 226Ra 43 Bq/kg (12-137), 232Th 21 Bq/kg (4-52), 40K 283 Bq/kg (31-564), 137Cs 35 Bq/kg (1-146). Average levels of these nuclides in avian faecal materials were 238U 63 Bq/kg, 226Ra 54 Bq/kg, 232Th 19 Bq/kg, 40K 365 Bq/kg, 137Cs 78 Bq/kg. Enrichment of radionuclides is apparent in soils taken from locations close to bird colonies in the locale, maximum levels of the radionuclides being found in samples associated with such colonies. The results indicate that this is due to concentration of such radionuclides within the faecal material of the birds and subsequent enrichment of the nearby soils either via direct incorporation of the faeces into the soil or by leaching processes. The results indicate that this process may result in contamination of non-related species, such

  8. Monte Carlo simulation of indoor external exposure due to gamma-emitting radionuclides in building materials

    NASA Astrophysics Data System (ADS)

    Deng, Jun; Cao, Lei; Su, Xu

    2014-10-01

    The use of building materials containing naturally occurring radionuclides, such as 40K, 238U, 232Th and their progeny, could lead to external exposures to the residents of such buildings. In this paper, a set of models are constructed to calculate the specific effective dose rates (the effective dose rate per Bq/kg of 40K, the 238U series, and the 232Th series) imposed on residents by building materials with the MCNPX code. The effect of chemical composition, position concerned in the room and thickness as well as density of material is analyzed. In order to facilitate more precise assessment of indoor external dose due to gamma-emitting radionuclides in building materials, three regressive expressions are proposed and validated by measured data to calculate specific effective rates for 40K. the 238U series and the 232Th series, respectively.

  9. Inter-laboratory comparisons of short-lived gamma-emitting radionuclides in nuclear reactor water.

    PubMed

    Klemola, S K

    2008-01-01

    Inter-laboratory comparisons of gamma-emitting nuclides in nuclear power plant coolant water have been carried out in Finland since 1994. The reactor water samples are taken and prepared by one of the two nuclear power plants and delivered to the participants. Since all the participants get their sample within just a few hours it has been possible to analyse and compare results of nuclides with half-lives shorter than 1h. The total number of short-lived nuclides is 26. All the main nuclides are regularly identified and the activities have been obtained with reasonable accuracy throughout the years. The overall deviation of the results has decreased in 13 years. The effects of true coincidence summing and discrepancies in nuclear data have been identified as potential sources of remaining discrepancies. All the participants have found this type of comparison very useful.

  10. Toxoids of Pseudomonas aeruginosa exotoxin-A: photoaffinity inactivation of purified toxin and purified toxin derivatives.

    PubMed Central

    Callahan, L T; Martinez, D; Marburg, S; Tolman, R L; Galloway, D R

    1984-01-01

    For the preparation of greatly detoxified but highly immunogenic toxoids, two enzymatically active, low-toxicity derivatives of Pseudomonas aeruginosa exotoxin-A were further inactivated by photoaffinity labeling. These derivatives were formed during toxin purification, when a relatively crude toxin preparation was concentrated by ammonium sulfate precipitation and subsequently dialyzed. These derivatives, designated peak-1 protein (PK-1) and peak-2 protein (PK-2) were antigenically indistinguishable from native toxin, but had isoelectric points (5.00 and 4.90, respectively) that were different from that of the native toxin (4.95). Although the enzymatic activities and molecular weights of PK-1 and PK-2 were similar to those of native toxin, their toxicities were greatly reduced (ca. 500-fold). Photoaffinity labeling of fully active toxin-A, purified by a process which limits the formation of these derivatives, decreased its enzymatic activity (ca. 30-fold) and toxicity (ca. 100-fold). Likewise, photoaffinity labeling of purified PK-1 and PK-2 decreased their enzymatic activities and toxicities (ca. 30-fold and 100-fold, respectively) and, thus, yielded toxoids that were ca. 50,000-fold less toxic than unpurified native toxin. These toxoids were irreversibly detoxified and highly immunogenic during 9 months of storage at 4 degrees C. Images PMID:6321348

  11. Sex pheromone receptor proteins. Visualization using a radiolabeled photoaffinity analog

    SciTech Connect

    Vogt, R.G.; Prestwich, G.D.; Riddiford, L.M.

    1988-03-15

    A tritium-labeled photoaffinity analog of a moth pheromone was used to covalently modify pheromone-selective binding proteins in the antennal sensillum lymph and sensory dendritic membranes of the male silk moth, Antheraea polyphemus. This analog, (E,Z)-6,11-(/sup 3/H)hexadecadienyl diazoacetate, allowed visualization of a 15-kilodalton soluble protein and a 69-kilodalton membrane protein in fluorescence autoradiograms of electrophoretically separated antennal proteins. Covalent modification of these proteins was specifically reduced when incubation and UV irradiation were conducted in the presence of excess unlabeled pheromone, (E,Z)-6,11-hexadecadienyl acetate. These experiments constitute the first direct evidence for a membrane protein of a chemosensory neuron interacting in a specific fashion with a biologically relevant odorant.

  12. Transport and distribution of artificial gamma-emitting radionuclides in the River Yenisei and its sediment.

    PubMed

    Semizhon, Tatiana; Röllin, Stefan; Spasova, Yana; Klemt, Eckehard

    2010-05-01

    Discharges from the Krasnoyarsk Mining and Chemical Industrial Complex (KMCIC) near Krasnoyarsk resulted in radioactive contamination of sediments of the River Yenisei. Between 1999 and 2006, 16 sediment cores were collected at different positions 15-1500 km downstream from the discharge point. The concentration of artificial gamma-emitting radionuclides ((137)Cs, (60)Co, (152)Eu, and (241)Am) was determined with the objective to analyze the migration processes leading to the transport of these radionuclides along the river and to their vertical distribution within the sediment. In cores taken in the vicinity of the reactors, the average activity concentration of (137)Cs, (152)Eu, and (60)Co was about 1000 Bq kg(-1), and the activity concentration of (241)Am was about 20 Bq kg(-1). Contamination levels of artificial radionuclides were decreasing with increasing distance downstream the KMCIC: The fastest decrease of average activity by a factor of 10 over a distance of 300 km was observed for (241)Am, whereas for (137)Cs this decrease occurred over a distance of 1100 km. Sequential extraction experiments revealed that in all depths and at all distances the studied radionuclides were tightly bound to the sediment. To investigate the mechanisms of transport of the (137)Cs and (60)Co contamination, mathematical models have been used to describe the contamination in the river water and within the sediments. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  13. Gamma-emitting radionuclides in the shallow marine sediments off the Sindh coast, Arabian Sea.

    PubMed

    Akram, M; Qureshi, Riffat M; Ahmad, Nasir; Solaija, Tariq Jamal

    2006-01-01

    Determination of gamma emitting radionuclides in shallow marine sediments off the Sindh coast has been carried out using a gamma spectrometry technique. The activity concentration measured in various sediment samples off the Sindh coast has been found to vary from 15.93 +/- 5.22 to 30.53 +/- 4.70 Bq kg(-1) for 226Ra, from 11.72 +/- 1.22 to 33.94 +/- 1.86 Bq kg(-1) for 228Ra and from 295.22 +/- 32.83 to 748.47 +/- 28.75 Bq kg(-1) for 40K. The calculated mean values of radium equivalent activity, absorbed dose rate and effective dose are 98 Bq kg(-1), 49 nGy h(-1) and 0.06 mSv y(-1), respectively. No artificial radionuclide was detected in the samples measured from the study area. As no data on radioactivity of the coastal environment of Pakistan are available, the data presented here will serve as baseline information on radionuclide concentration in shallow sea sediments off the Sindh coast. The data will also be useful for tracking pollution inventories from unusual radiological events (if any) in the territorial waters of the study area. Further, the information presented will contribute to modelling of a regional radioactivity database from the perspectives of the International Atomic Energy Agency's Asia-Pacific Marine Radioactivity Database and Global Marine Radioactivity Database.

  14. Identification of the binding subunit of the sigma-type opiate receptor by photoaffinity labeling with 1-(4-azido-2-methyl(6-/sup 3/H)phenyl)-3-(2-methyl(4,6-/sup 3/H)phenyl)guanidine

    SciTech Connect

    Kavanaugh, M.P.; Tester, B.C.; Scherz, M.W.; Keana, J.F.W.; Weber, E.

    1988-04-01

    The sigma-type opiate receptor is a distinct binding site in the brain that may mediate some of the psychotomimetic effects caused by benzomorphan opiates and phencyclidine in humans. The authors have developed a synthetic, highly selective ligand for this receptor, 1,3-di-o-tolylguanidine (DTG). To identify the binding protein(s) of the sigma receptor, they have now synthesized a radiolabeled azide derivative of DTG, ((/sup 3/H)N/sub 3/DTG). In guinea pig brain membrane binding assays conducted in the dark, (/sup 3/H)N/sub 3/DTG bound reversibly, selectively, and with high affinity to sigma receptors. The drug specificity profile of reversible (/sup 3/H)-N/sub 3/DTG binding was identical to that of (/sup 3/H)DTG and /sup 3/H-labeled (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding indicating that (/sup 3/H)N/sub 3/DTG is a selective sigma receptor ligand. Guinea pig brain membranes were photoaffinity-labeled with (/sup 3/H)N/sub 3/DTG. NaDodSO/sub 4//PAGE of detergent-solubilized membrane extract identified a single 29-kDa radioactive band. Sepharose Cl-6B gel chromatography of photolabeled brain membranes solubilized with the nondenaturing detergent sodium cholate showed a radioactive complex with a Stoke's radius of 4.6 nm (M/sub r/, 150,000) that may represent the intact sigma receptor complex. NaDodSO/sub 4//PAGE of this complex showed the radiolabeled material was a 29-kDa polypeptide that may be binding subunit of the sigma receptor.

  15. Evaluation of α-pyrones and pyrimidones as photoaffinity probes for affinity-based protein profiling.

    PubMed

    Battenberg, Oliver A; Nodwell, Matthew B; Sieber, Stephan A

    2011-08-05

    α-Pyrones and pyrimidones are common structural motifs in natural products and bioactive compounds. They also display photochemistry that generates high-energy intermediates that may be capable of protein reactivity. A library of pyrones and pyrimidones was synthesized, and their potential to act as photoaffinity probes for nondirected affinity-based protein profiling in several crude cell lysates was evaluated. Further "proof-of-principle" experiments demonstrate that a pyrimidone tag on an appropriate scaffold is equally capable of proteome labeling as a benzophenone.

  16. A photoaffinity probe designed for host-specific signal flavonoid receptors in phytopathogenic Peronosporomycete zoospores of Aphanomyces cochlioides.

    PubMed

    Sakihama, Yasuko; Shimai, Takashi; Sakasai, Mitsuyoshi; Ito, Toshiaki; Fukushi, Yukiharu; Hashidoko, Yasuyuki; Tahara, Satoshi

    2004-12-15

    Aphanomyces cochlioides zoospores show chemotaxis to cochliophilin A (5-hydroxy-6,7-methylenedioxyflavone, 1), a host derived attractant, and also respond to 5,7-dihydroxyflavone (2) known as an equivalent chemoattractant. To investigate the chemotactic receptors in the zoospores, we designed photoaffinity probes 4'-azido-5,7-dihydroxyflavone (3) and 4'-azido-7-O-biotinyl-5-hydroxyflavone (4) considering chemical structure of 2. Both 3 and 4 had zoospore attractant activity which was competitive with that of 1. When zoospores were treated with the biotinylated photoaffinity probe followed by UV irradiation and streptavidin-gold or peroxidase-conjugated streptavidin, probe-labeled proteins were detected on the cell membrane. This result indicated that the 1-specific-binding proteins, a candidate for hypothetical cochliophilin A receptor, were localized on the cell membrane of the zoospores. This is the first experimental evidence of flavonoid-binding proteins being present in zoospores, using chemically synthesized azidoflavone as photoaffinity-labeling reagent.

  17. The potential for gamma-emitting radionuclides to contribute to an understanding of erosion processes in South Africa

    NASA Astrophysics Data System (ADS)

    Foster, Ian D. L.; Boardman, John; Collins, Adrian L.; Copeland-Phillips, Ruth; Kuhn, Nikolaus J.; Mighall, Tim M.; Pulley, Simon; Rowntree, Kate M.

    2017-03-01

    Several research projects undertaken by the authors and others over the last 14 years have used fallout and geogenic radionuclides for understanding erosion processes and sediment yield dynamics in South Africa over the last 100-200 years as European settlers colonised the interior plains and plateaux of the country and imported new livestock and farming techniques to the region. These projects have used two fallout radionuclides (210Pb and 137Cs) to date sediments accumulating in reservoirs, farm dams, wetlands, alluvial fans and floodouts and have used other fallout nuclides (7Be) and long-lived geogenic radionuclides (e.g. 40K, 235U) as part of a composite fingerprint exploring contemporary sediment sources and changes to sources through time. While successful in many parts of the world, applying these techniques in Southern Africa has posed a number of challenges often not encountered elsewhere. Here we explore some of the benefits and challenges in using gamma-emitting radionuclides, especially 137Cs, in these landscapes. Benefits include the potential for discriminating gully sidewall from topsoil sources, which has helped to identify contemporary gully systems as sediment conduits, rather than sources, and for providing a time-synchronous marker horizon in a range of sedimentary environments that has helped to develop robust chronologies. Challenges include the spatial variability in soil cover on steep rocky hillslopes, which is likely to challenge assumptions about the uniformity of initial fallout nuclide distribution, the paucity of stable (non-eroding) sites in order to estimate atmospheric fallout inventories, and the limited success of 210Pb dating in some rapidly accumulating high altitude catchments where sediments often comprise significant amounts of sand and gravel. Despite these challenges we present evidence suggesting that the use of gamma-emitting radionuclides can make a significant contribution to our understanding of erosion processes and

  18. Photoaffinity antigens for human gammadelta T cells.

    PubMed

    Sarikonda, Ghanashyam; Wang, Hong; Puan, Kia-Joo; Liu, Xiao-hui; Lee, Hoi K; Song, Yongcheng; Distefano, Mark D; Oldfield, Eric; Prestwich, Glenn D; Morita, Craig T

    2008-12-01

    Vgamma2Vdelta2 T cells comprise the major subset of peripheral blood gammadelta T cells in humans and expand during infections by recognizing small nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate, an endogenous isoprenoid intermediate. Recognition of these nonpeptide Ags is mediated by the Vgamma2Vdelta2 T cell Ag receptor. Several findings suggest that prenyl pyrophosphates are presented by an Ag-presenting molecule: contact between T cells and APC is required, the Ags do not bind the Vgamma2Vdelta2 TCR directly, and Ag recognition is abrogated by TCR mutations in CDRs distant from the putative Ag recognition site. Identification of the putative Ag-presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate Ags with the presenting molecule. In this study, we show that photoaffinity analogues of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C(5)-OPP), can crosslink to the surface of tumor cell lines and be presented as Ags to gammadelta T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, beta(2)-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C(5)-OPP. Finally, pulsing of BZ-(C)-C(5)-OPP is inhibited by isopentenyl pyrophosphate and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide Ags are presented by a novel-Ag-presenting molecule that is widely distributed and nonpolymorphic, but not classical MHC class I, MHC class II, or CD1.

  19. Structure-Activity Relationship of Semicarbazone EGA Furnishes Photoaffinity Inhibitors of Anthrax Toxin Cellular Entry.

    PubMed

    Jung, Michael E; Chamberlain, Brian T; Ho, Chi-Lee C; Gillespie, Eugene J; Bradley, Kenneth A

    2014-04-10

    EGA, 1, prevents the entry of multiple viruses and bacterial toxins into mammalian cells by inhibiting vesicular trafficking. The cellular target of 1 is unknown, and a structure-activity relationship study was conducted in order to develop a strategy for target identification. A compound with midnanomolar potency was identified (2), and three photoaffinity labels were synthesized (3-5). For this series, the expected photochemistry of the phenyl azide moiety is a more important factor than the IC50 of the photoprobe in obtaining a successful photolabeling event. While 3 was the most effective reversible inhibitor of the series, it provided no protection to cells against anthrax lethal toxin (LT) following UV irradiation. Conversely, 5, which possessed weak bioactivity in the standard assay, conferred robust irreversible protection vs LT to cells upon UV photolysis.

  20. Activity Concentrations and Dose Assessment of Gamma Emitting Radionuclides in Canned Tuna and Sardines Produced after the Fukushima Nuclear Accident.

    PubMed

    Ababneh, Zaid Q; Al-Masoud, Fahad I; Ababneh, Anas M

    2016-01-01

    The aim of the present work was to investigate the radioactivity concentrations of gamma emitting radionuclides in canned tuna and sardines that were produced after the Fukushima nuclear accident and to assess the resulting radiation doses to the public. Fifty-eight brands of canned tuna and sardines consumed in the Middle East and produced from different parts of the world were analyzed using a germanium detector. Cesium-137 (137Cs) was not detected above the minimum detectable activity in any of the samples. Natural radionuclides 40K, 226Ra and 228Ra were detected with wide activity concentration ranges and with average values of (in Bq kg(-1) wet weight): 68 ± 36, 0.31 ± 0.45, 0.34 ± 0.25, respectively, in tuna samples and with averages of 129 ± 67, 0.20 ± 0.33, 0.60 ± 0.31 in sardine samples. The results of the activity concentrations of 40K and 226Ra showed some regional dependence. Tuna samples produced in Europe have almost twice the concentration of 40K and half the concentration of 226Ra as compared to samples produced in either East or South Asia and North America. Moreover, sardine samples produced in North Africa and Europe have almost twice the concentrations of 40K and 226Ra as those produced in East or South Asia and North America. Dose assessment due to ingestion of canned seafood was also performed, and the committed effective dose was found to be well within the worldwide average.

  1. Photoaffinity approaches to determining the sequence selectivities of DNA-small molecule interactions: actinomycin D and ethidium.

    PubMed Central

    Marsch, G A; Graves, D E; Rill, R L

    1995-01-01

    The DNA photoaffinity ligands, 7-azidoactinomycin D and 8-azidoethidium, form DNA adducts that cause chain cleavage upon treatment with piperidine. Chemical DNA sequencing techniques were used to detect covalent binding. The relative preferences for modifications of all possible sites defined by a base pair step (e.g. GC) were determined within all quartet contexts such as (IGCJ). These preferences are described in terms of 'effective site occupations', which express the ability of a ligand to covalently modify some base in the binding site. Ideally, the effective site occupations measured for photoaffinity agents can also be related to site-specific, non-covalent association constants of the ligand. The sites most reactive with 7-azidoactinomycin D were those preferred for non-covalent binding of unsubstituted actinomycin D. GC sites were most reactive, but next-nearest neighbors exerted significant influences on reactivity. GC sites in 5'-(pyrimidine)GC(purine)-3' contexts, particularly TGCA, were most reactive, while reactivity was strongly suppressed for GC sites with a 5'-flanking G, or a 3'-flanking C. High reactivities were also observed for bases in the first (5') GG steps in TGGT, TGGG and TGGGT sequences recently shown to bind actinomycin D with high affinity. Pyrimidine-3',5'-purine steps and GG steps flanked by a T were most preferred by 8-azidoethidium, in agreement with the behavior of unsubstituted ethidium. The good correspondence between expected and observed covalent binding preferences of these two azide analogs demonstrates that photoaffinity labeling can identify highly preferred sites of non-covalent DNA binding by small molecules. PMID:7739904

  2. Characterisation of Photoaffinity-Based Chemical Probes by Fluorescence Imaging and Native-State Mass Spectrometry.

    PubMed

    Teruya, Kanae; Rankin, Gregory M; Chrysanthopoulos, Panagiotis K; Tonissen, Kathryn F; Poulsen, Sally-Ann

    2017-04-18

    Chemical probes are small-molecule reagents used by researchers for labelling and detection of biomolecules. We present the design, synthesis, and characterisation of a panel of 11 structurally diverse photoaffinity labelling (PAL) probes as research tools for labelling the model enzyme carbonic anhydrase (CA) in challenging environments, including in protein mixtures and cell lysates. We targeted the ubiquitous CA II as well as the two cancer-associated CAs (CA IX and CA XII) that are of high priority as potential biomarkers of aggressive and/or multidrug-resistant cancer. We utilise an atypical biophysical approach, native state mass spectrometry, to monitor the initial protein-probe binding and subsequent UV crosslinking efficiency of the protein:probe complex. This mass spectrometry methodology represents a new approach for chemical probe optimisation and development that might have broader applications to chemical probe characterisation beyond this study. This also represents one of the first studies, to the best of our knowledge, in which a comprehensive set of PAL probes has been used to establish the relationship between probe structure, noncovalent protein-probe binding, and covalent protein-probe crosslinking efficiency. Our results demonstrate the benefits of a comprehensive analysis of chemical probe structure-activity relationships to support the development of optimum chemical probes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Response-surface models for deterministic effects of localized irradiation of the skin by discrete {beta}/{gamma} -emitting sources

    SciTech Connect

    Scott, B.R.

    1995-12-01

    Individuals who work at nuclear reactor facilities can be at risk for deterministic effects in the skin from exposure to discrete {Beta}- and {gamma}-emitting ({Beta}{gamma}E) sources (e.g., {Beta}{gamma}E hot particles) on the skin or clothing. Deterministic effects are non-cancer effects that have a threshold and increase in severity as dose increases (e.g., ulcer in skin). Hot {Beta}{gamma}E particles are {sup 60}Co- or nuclear fuel-derived particles with diameters > 10 {mu}m and < 3 mm and contain at least 3.7 kBq (0.1 {mu}Ci) of radioactivity. For such {Beta}{gamma}E sources on the skin, it is the beta component of the dose that is most important. To develop exposure limitation systems that adequately control exposure of workers to discrete {Beta}{gamma}E sources, models are needed for systems that adequately control exposure of workers to discrete {Beta}{gamma}E sources, models are needed for evaluating the risk of deterministic effects of localized {Beta} irradiation of the skin. The purpose of this study was to develop dose-rate and irradiated-area dependent, response-surface models for evaluating risks of significant deterministic effects of localized irradiation of the skin by discrete {Beta}{gamma}E sources and to use modeling results to recommend approaches to limiting occupational exposure to such sources. The significance of the research results as follows: (1) response-surface models are now available for evaluating the risk of specific deterministic effects of localized irradiation of the skin; (2) modeling results have been used to recommend approaches to limiting occupational exposure of workers to {Beta} radiation from {Beta}{gamma}E sources on the skin or on clothing; and (3) the generic irradiated-volume, weighting-factor approach to limiting exposure can be applied to other organs including the eye, the ear, and organs of the respiratory or gastrointestinal tract and can be used for both deterministic and stochastic effects.

  4. Methionine acts as a "magnet" in photoaffinity crosslinking experiments.

    PubMed

    Wittelsberger, Angela; Thomas, Beena E; Mierke, Dale F; Rosenblatt, Michael

    2006-03-20

    Photoaffinity crosslinking has been utilized to probe the nature of the ligand-receptor interface for a number of G protein-coupled receptor systems. Often the photoreactive benzophenone moiety incorporated in the ligand is found to react with a methionine in the receptor. We introduced methionines one-at-a-time into the region 163-176 of the parathyroid hormone receptor, and find that crosslinking occurs to the side-chain of methionine over a range of 11 amino acids. We call this the "Magnet Effect" of methionine. Hence, crosslinking contact points can be significantly shifted by the presence of methionine in a receptor domain.

  5. Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

    SciTech Connect

    Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L. )

    1989-09-01

    A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-(4-(2-(2-((4- aminophenyl)methylcarbonylamino)ethylaminocarbonyl)- ethyl)phenyl)ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-((R)-1-methyl- 2-phenylethyl)adenosine (R-PIA) greater than (+)-N6-((S)-1-methyl-2- phenylethyl)adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-(beta, gamma-imido)triphosphate.

  6. Sediment studies at Bikini Atoll part 3. Inventories of some long-lived gamma-emitting radionuclides associated with lagoon surface sediments

    SciTech Connect

    Noshkin, V.E.

    1997-12-01

    Surface sediment samples were collected during 1979 from 87 locations in the lagoon at Bikini Atoll. The collections were made to better define the concentrations and distribution of long-lived radionuclides associated with the bottom material and to show what modifications occurred to the composition of the surface sediment from the nuclear testing program conducted by the United States at the Atoll between 1946 and 1958. This is the last of three reports on Bikini sediment studies. In this report, we discuss the concentrations and inventories of the residual long-lived gamma-emitting radionuclides in sediments from the lagoon. The gamma-emitting radionuclides detected most frequently in sediments collected in 1979, in addition to Americium-241 ({sup 241}Am) (discussed in the second report of this series), included Cesium-137 ({sup 137}Cs), Bismuth-207 ({sup 207}Bi), Europium-155 ({sup 155}Eu), and Cobalt-60 ({sup 60}Co). Other man-made, gamma-emitting radionuclides such as Europium-152,154 ({sup 152,154}Eu), Antimony-125 ({sup 125}Sb), and Rhodium-101,102m ({sup 101,102m}Rh) were occasionally measured above detection limits in sediments near test site locations. The mean inventories for {sup 137}Cs, {sup 207}Ei, {sup 155}Eu, and {sup 60}Co in the surface 4 cm of the lagoon sediment to be 1.7, 0.56, 7.76, and 0.74 TBq, respectively. By June 1997, radioactive decay would reduce these values to 1.1, 0.38, 0.62, and 0.07 TBq, respectively. Some additional loss results from a combination of different processes that continuously mobilize and return some amount of the radionuclides to the water column. The water and dissolved constituents are removed from the lagoon through channels and exchange with the surface waters of the north equatorial Pacific Ocean. Highest levels of these radionuclides are found in surface deposits lagoonward of the Bravo Crater. Lowest concentrations and inventories are associated with sediment lagoonward of the eastern reef. The quantities in

  7. [New reagents for affinity modification of biopolymers. Photoaffinity modification of Tte-DNA polymerase].

    PubMed

    Kolpashchikov, D M; Zakharenko, A L; Dezhurov, S V; Rechkunova, N I; Khodyreva, S N; Degtiarev, S Kh; Litvak, V V; Lavrik, O I

    1999-02-01

    Arylazides N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-beta-alanine (Ia) and N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-glycine (Ib) were synthesized and covalently attached to 5-(3-aminopropenyl-1)-dUTP through the amino group to give 5'-triphosphate (IIa) and 5'-triphosphate (IIb). The resulting azides were subjected to photolysis in aqueous solution. The spectral and photochemical characteristics of azides (I) and (II) imply that their use for the modification of biopolymers holds promise. Compounds (IIa, b) effectively substituted dTTP in DNA polymerization catalyzed by thermostable DNA polymerase from Thermus thermophilus B-35 (Tte DNA polymerase). Photoaffinity modification of Tte DNA polymerase was carried out by dTTP analogues (IIa, b) and by earlier obtained 5-[N-(5-azido-2-nitrobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine 5'-triphosphate (III) and 5-[N-(4-azido-2,3,5,6-tetrafluorobenzyol)-trans-3- aminopropenyl-1]deoxyuridine 5'-triphosphate (IV) using two variants of labeling. All four dTTP analogues were shown to modify Tte DNA polymerase.

  8. Reduced Uncertainties in the Supernova Production of the Gamma Emitting Nuclei 26Al, 44Ti, and 60Fe Using Effective Helium Burning Rates

    NASA Astrophysics Data System (ADS)

    Austin, Sam M.; West, Christopher; Heger, Alexander

    2016-09-01

    Uncertainties in the helium burning reaction rates caused large uncertainties in previous predictions of the production of the gamma emitting nuclei 26Al and (especially) 60Fe in core collapse supernovae. This precluded a meaningful comparison of the predictions with observed gamma ray intensities. We present results using a newly developed effective reaction rate (ERR) for the helium burning reactions to predict the yields of 26Al, 44Ti, and 60Fe. The resulting yield uncertainties using the ERR are much smaller than obtained previously, and smaller than other uncertainties. The yield ratio, 60Fe/26Al, had variations of less than 20 percent and appears to be the most robust observable related to the production of these nuclei. We also estimated the effects of failed supernovae on the yields by using a compactness filter. This substantially reduced the three yields but the ratio, 60Fe/26Al, was little affected. Research supported by US NSF and DOE, and by ARC.

  9. Novel photoaffinity ligands for the GA-receptor

    SciTech Connect

    Suttle, J.C.; Hultstrand, J.F.; Tanaka, F.S. )

    1990-05-01

    Previous studies from this laboratory have shown that certain N-substituted phthalimides (NSPs) exhibit GA-like activity in a range of specific bioassays and that bioactive NSPs compete with ({sup 3}H)-GA{sub 4} for soluble binding sites in cucumber homogenates. As such, these compounds may prove useful in the purification and characterization of GA receptor proteins. To this end, five azido-NSPs have been synthesized and are currently being screened for biological activity and photochemical stability. Three azido-NSPs elicit {alpha}-amylase production in barley half-seeds and stimulate tissue elongation in d{sub 5} maize, lettuce, sunflower, and soybean. Further evaluations are in progress and these data as well as the utility of these compounds as photo-affinity ligands will be discussed.

  10. A procedure to calculate the self-shielding and detection efficiency for a gamma-emitting disk and sodium iodide crystal

    NASA Astrophysics Data System (ADS)

    Arcipiani, Biagio; Pedretti, Edmondo

    1980-07-01

    This paper reports on a procedure to correct for the detector efficiency and radiation self-absorption the number of counts tallied when the activity of a gamma-emitting thick foil is measured by means of a sodium iodide crystal. A model is set up whereby, after ideally dividing the disk into a large number of slices, it is shown how to separate for each slice the role of radiation detection from that of the absorption in the material between the slice and the crystal. While the former is accounted for by using an available Monte Carlo code, the latter is reduced to the calculation of suitable geometrical factors. Formulas for these factors are derived and were coded for an electronic computer. The Fortran IV program is available. Numerical results of the geometrical factors are shown for a 14 mm radius and 2.07 mm thick indium foil irradiated in a plasma focus machine, and these are compared with those obtained by a crude approximation reported elsewhere.

  11. Synthesis and characterization of photoaffinity probes that target the 5-HT3 receptor.

    PubMed

    Jack, Thomas; Ruepp, Marc-David; Thompson, Andrew J; Mühlemann, Oliver; Lochner, Martin

    2014-01-01

    The 5-HT3 receptor is one of several ion channels responsible for the transmission of nerve impulses in the peripheral and central nervous systems. Until now, it has been difficult to characterize transmembrane receptors with classical structural biology approaches like X-ray crystallography. The use of photoaffinity probes is an alternative approach to identify regions in the protein where small molecules bind. To this end, we present two photoaffinity probes based on granisetron, a well known antagonist of the 5-HT3 receptor. These new probes show nanomolar binding affinity for the orthosteric binding site. In addition, we investigated their reactivity using irradiation experiments.

  12. Probing the active site of a diels-alderase ribozyme by photoaffinity cross-linking.

    PubMed

    Wombacher, Richard; Jäschke, Andres

    2008-07-09

    The active site of a Diels-Alderase ribozyme is located in solution by photoaffinity cross-linking using a productlike azidobenzyl probe. Two key nucleotides are identified that contact the Diels-Alder product in a conformation-dependent fashion. The design of such probes does not require knowledge of the three-dimensional structure of the ribozyme, and the technique yields both static and dynamic structural information. This work establishes photoaffinity cross-linking as an empirical approach that is applied here for the first time to an artificial ribozyme.

  13. Preparation of radioactive ''mixed'' waste samples for measurement of RCRA (Resource Conservation and Recovery Act) organic compounds. [Mixed waste containing alpha-, beta-, or gamma-emitting radionuclides

    SciTech Connect

    Tomkins, B.A.; Caton, J.E.

    1987-01-01

    A radioactive ''mixed'' waste typically contains alpha-, beta-, or gamma-emitting radionuclides and varying quantities of semivolatile or volatile organic species, some or all of which may be named specifically by the Resource Conservation and Recovery Act (RCRA). Because there are no acceptable means available currently for disposing of these mixed wastes, they are presently stored above-ground in sealed drums. For this reason, analytical procedures which can determine RCRA organics in radioactive waste are necessary for deciding the proper approach for disposal. An important goal of this work is the development of methods for preparing mixed waste samples in a manner which allows the RCRA organics to be measured in conventional organic analysis laboratories without special precautions. Analytical procedures developed for handling mixed waste samples must satisfy not only the usual constraints present in any trace-level organic chemical determination, but also those needed to insure the protection of the operator from radioactive contamination. Consequently, procedures should be designed to use the least amount of radioactive sample commensurate with achieving acceptable sensitivity with the RCRA analytical methods. Furthermore, the unusual laboratory glassware which would normally be used should be replaced with disposable materials wherever possible, in order to reduce the ''clean-up'' time required, and thereby reduce the operator's exposure to radioactivity. Actual sample handling should be reduced to the absolute minimum. Finally, the final isolate must exhibit a sufficiently low level of alpha, beta, or gamma activity to permit detailed characterization in a conventional organic analysis laboratory. 4 refs., 5 tabs.

  14. Photoaffinity antigens for human γδ T cells1

    PubMed Central

    Sarikonda, Ghanashyam; Wang, Hong; Puan, Kia-Joo; Liu, Xiao-hui; Lee, Hoi K.; Song, Yongcheng; Distefano, Mark D.; Oldfield, Eric; Prestwich, Glenn D.; Morita, Craig T.

    2009-01-01

    Vγ2Vδ2 T cells comprise the major subset of peripheral blood γ δ T cells in humans and expand during infections by recognizing small, nonpeptide prenyl pyrophosphates. These molecules include (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP), a microbial isoprenoid intermediate, and isopentenyl pyrophosphate (IPP), an endogenous isoprenoid intermediate. Recognition of these nonpeptide antigens is mediated by the Vγ2Vδ2 T cell antigen receptor (TCR). Several findings suggest that prenyl pyrophosphates are presented by an antigen presenting molecule: contact between T cells and APCs is required; the antigens do not bind the Vγ2Vδ2 TCR directly; and antigen recognition is abrogated by TCR mutations in CDRs distant from the putative antigen recognition site. Identification of the putative antigen presenting molecule, however, has been hindered by the inability to achieve stable association of nonpeptide prenyl pyrophosphate antigens with the presenting molecule. In this study, we show that photoaffinity analogs of HMBPP, meta/para-benzophenone-(methylene)-prenyl pyrophosphates (m/p-BZ-(C)-C5-OPP), can cross-link to the surface of tumor cell lines and be presented as antigens to γ δ T cells. Mutant tumor cell lines lacking MHC class I, MHC class II, β2-microglobulin, and CD1, as well as tumor cell lines from a variety of tissues and individuals, will all crosslink to and present m-BZ-C5-OPP. Finally, pulsing of BZ-(C)-C5-OPP is inhibited by IPP and an inactive analog, suggesting that they bind to the same molecule. Taken together, these results suggest that nonpeptide antigens are presented by a novel antigen presenting molecule that is widely distributed, non-polymorphic, but not classical MHC class I, MHC class II, or CD1. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript

  15. Studies of serotonin uptake and serotonin/sub 1A/ receptor using photoaffinity probes

    SciTech Connect

    Lee, J.D.

    1986-01-01

    The serotonergic system in the central nervous system (CNS) has been implicated in many physiological functions. The objectives of this study are: (1) to develop a new photoaffinity probe that can be used to identify the protein associated with the substrate binding site of the 5-HT uptake carrier, (2) to investigate the existence of different conformational states of the 5-HT carrier, (3) to solubilize the 5-HT/sub 1a/ receptor proteins from bovine hippocampus, and (4) to identify the 5-HT/sub 1a/ receptor proteins using a new photoaffinity probe, i.e., 1-(2-(4-aminophenyl)ethyl)4-(3-trifluoromethylphenyl)piperazine ((/sup 3/H)p-azido-PAPP), with high affinity for 5-HT/sub 1a/ receptor.

  16. Affinity labeling of the galactose/N-acetylgalactosamine-specific receptor of rat hepatocytes: preferential labeling of one of the subunits

    SciTech Connect

    Lee, R.T.; Lee, Y.C.

    1987-10-06

    The galactose/N-acetylgalactosamine-specific receptor (also known as asialoglycoprotein receptor) of rat hepatocytes consists of three subunits, one of which (43 kilodalton (kDa)) exists in a greater abundance (up to 70% of total protein) over the two minor species (52 and 60 kDa). When the receptor on the hepatocyte membranes was photoaffinity labeled with an /sup 125/I-labeled high-affinity reagent the labeling occurred mainly (51-80%) on one of the minor bands (52 kDa). Similarly, affinity-bound, N-acetylgalactosamine-modified lactoperoxidase radioiodinated the same 52-kDa band preferentially. In contrast, both the photoaffinity labeling and lactoperoxidase-catalyzed iodination of the purified, detergent-solubilized receptor resulted in a distribution of the label that is comparable to the Coomassie blue staining pattern of the three bands; i.e., the 43-kDa band was the major band labeled. These and other experimental results suggest that the preferential labeling of the minor band and inefficient labeling of the major band on the hepatocyte membrane resulted from a specific topological arrangement of these subunits on the membranes. The authors postulate that in the native, membrane-bound state of the receptor, the 52-kDa minor band is topologically prominent, while the major (43 kDa) band is partially masked. This partial masking may result from a tight packing of the receptor subunits on the membranes to form a lattice work.

  17. Photoaffinity labeling of the tetrodotoxin binding component of Electrophorus electricus electroplax.

    PubMed

    Uehara, S; Uyemura, K

    1985-08-01

    Radioactive azide derivatives of tetrodotoxin (TTX) were synthesized using 2-nitro-4-azidephenyl-[3H]beta alanine for the purpose of photolabeling of the Na channel. Three azide derivatives, N1, N2 and N3, were separated by ion exchange chromatography on Bio-Rex 70 resin and reversed phase high performance liquid chromatography. N3 was more stable and obtained at a higher yield than the other two derivatives. Bioactivity of N3 was one-twentieth of that of TTX. N3 showed reversible binding to membranes of Electrophorus electricus electroplax in the dark with Kd = 30 nM and B max = 5.2 pmol/mg protein. By photoirradiation, irreversible binding of N3 to the membranes was observed. A N3 binding component was solubilized by lubrol PX and partially purified from the electroplax membranes by Sephadex G25 and Sepharose 6B column chromatography. The component, purified 500 fold from the starting membranes, showed molecular weight of 10,000.

  18. Identification of nuclear proteins that interact with platinum-modified DNA by photoaffinity labeling.

    PubMed

    Zhang, Christiana Xin; Chang, Pamela V; Lippard, Stephen J

    2004-06-02

    Interactions between cellular proteins and cisplatin-modified DNA are important in determining the anticancer activity of the drug. To develop a general approach for identifying proteins that mediate cellular responses to cisplatin, photoreactive cisplatin analogues having a tethered benzophenone moiety were prepared and used to form the major 1,2-intrastrand platinum-DNA cross-links. Upon irradiation of the platinated DNA dissolved in a HeLa nuclear extract, the appended photolabile benzophenone group generates a highly reactive species that binds irreversibly to cellular proteins that interact with the probe. Several DNA-protein cross-linked adducts were identified that may function in the cellular processing of cisplatin-DNA adducts. Of these, PARP-1 had not previously been demonstrated directly to contact Pt-DNA cross-links in human cells.

  19. Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148.

    PubMed Central

    Carroll, S F; McCloskey, J A; Crain, P F; Oppenheimer, N J; Marschner, T M; Collier, R J

    1985-01-01

    Irradiation of mixtures of diphtheria toxin fragment A and [carbonyl-14C]NAD with UV light (253.7 nm) is known to induce efficient transfer of the radiolabel to position 148, corresponding to glutamic acid in the unmodified protein. Here we report the structure of the photoproduct at position 148, as determined by chemical and photochemical methods, fast-atom-bombardment mass spectrometry, and nuclear magnetic resonance. The photoproduct [an alpha-amino-gamma-(6-nicotin-amidyl)butyric acid residue] contains the entire nicotinamide moiety of NAD linked via its number 6 carbon to the decarboxylated gamma-methylene carbon of Glu-148. No portion of the ADP-ribosyl group of NAD is present. These findings are consistent with the idea that Glu-148 lies at or near the catalytic center of diphtheria toxin. PMID:3864158

  20. Photoaffinity labeling of diphtheria toxin fragment A with NAD: structure of the photoproduct at position 148.

    PubMed

    Carroll, S F; McCloskey, J A; Crain, P F; Oppenheimer, N J; Marschner, T M; Collier, R J

    1985-11-01

    Irradiation of mixtures of diphtheria toxin fragment A and [carbonyl-14C]NAD with UV light (253.7 nm) is known to induce efficient transfer of the radiolabel to position 148, corresponding to glutamic acid in the unmodified protein. Here we report the structure of the photoproduct at position 148, as determined by chemical and photochemical methods, fast-atom-bombardment mass spectrometry, and nuclear magnetic resonance. The photoproduct [an alpha-amino-gamma-(6-nicotin-amidyl)butyric acid residue] contains the entire nicotinamide moiety of NAD linked via its number 6 carbon to the decarboxylated gamma-methylene carbon of Glu-148. No portion of the ADP-ribosyl group of NAD is present. These findings are consistent with the idea that Glu-148 lies at or near the catalytic center of diphtheria toxin.

  1. Photoaffinity Labeling Reveals Nuclear Proteins that Uniquely Recognize Cisplatin-DNA Interstrand Cross-Links

    PubMed Central

    Zhu, Guangyu; Lippard, Stephen J.

    2009-01-01

    The DNA-binding inorganic compound cisplatin is one of the most successful anticancer drugs. The detailed mechanism by which cells recognize and process of cisplatin-DNA damage is of great interest. Although the family of proteins that bind cisplatin 1,2- and 1,3-intrastrand cross-links has been identified, much less is known about cellular protein interactions with cisplatin interstrand cross-links (ICLs). In order to address this question, a photoreactive analogue of cisplatin, PtBP6, was used to construct a DNA duplex containing a site-specific platinum ICL. This DNA probe was characterized and used in photo-cross-linking experiments to separate and identify nuclear proteins that bind to the ICL by peptide mass fingerprint analysis. Several such proteins were discovered, including PARP-1, hMutSβ, DNA ligase III, XRCC1, and PNK. The photo-cross-linking approach was independently validated by an electrophoretic mobility shift assay demonstrating hMutSβ binding to a cisplatin ICL. Proteins that recognize the platinum ICL were also identified in cisplatin resistant cells, cells halted at various phases of the cell cycle, and in different carcinoma cells. Nuclear proteins that bind to the platinum ICL differ from those binding to intrastrand cross-links, indicating different mechanisms for disruption of cellular functions. PMID:19364127

  2. Reducing Uncertainties in the Production of the Gamma-emitting Nuclei 26Al, 44Ti, and 60Fe in Core-collapse Supernovae by Using Effective Helium Burning Rates

    NASA Astrophysics Data System (ADS)

    Austin, Sam M.; West, Christopher; Heger, Alexander

    2017-04-01

    We have used effective reaction rates (ERRs) for the helium burning reactions to predict the yield of the gamma-emitting nuclei 26Al, 44Ti, and 60Fe in core-collapse supernovae (SNe). The variations in the predicted yields for values of the reaction rates allowed by the ERR are much smaller than obtained previously, and smaller than other uncertainties. A “filter” for SN nucleosynthesis yields based on pre-SN structure was used to estimate the effect of failed SNe on the initial mass function averaged yields; this substantially reduced the yields of all these isotopes, but the predicted yield ratio 60Fe/26Al was little affected. The robustness of this ratio is promising for comparison with data, but it is larger than observed in nature; possible causes for this discrepancy are discussed.

  3. Comparison of labels for Carafate in a gastric ulcer model

    SciTech Connect

    Knight, L.C.; Fisher, R.S.; Malmud, L.S.

    1984-01-01

    The purpose of this study was to evaluate three radiolabels for the drug Carafate (basic aluminum sucrose octasulfate), which, when ingested orally, is believed to coat gastric ulcers and protect them from digestive enzymes to promote healing. In order to study the mode of action and residence time in the stomach using external imaging, a gamma-emitting label which is truly bound to the molecule is needed. Carafate has been radiolabeled with Se-75, In-111 (both chemically incorporated into the molecule) and with Tc-99m-HSA which physically adheres to Carafate. In the presence of stomach acid, Carafate polymerizes; when the labeled Carafates were mixed in vitro with 0.1N HCl, >90% of the radio-activity was associated with the polymer in the case of Se-75 and Tc-99m, but the In-111 label was less stable (25-35% bound to polymer). The three labeled preparations were administered orally to rats with gastric ulcers, and the transit of each was followed by gamma camera imaging. Gamma camera images confirmed radioactivity remaining at the ulcer site after unbound material had emptied from the stomach, and the focal activity persisted for >5 hours. The stomachs were then removed, washed and dissected at 5.5 hours and in vitro measurements of ulcer crater: normal stomach tissue radioactivity ratios averaged 15.4, 6.3, and 5.6 for the Se-75, In-111, and Tc-99m-HSA labels, respectively. Biodistribution studies of oral Se-75-Carafate in rats and pigs indicated that very little is absorbed from the GI tract and the distribution is similar to that of C-14-Carafate. It is concluded that Se-75 is the best marker for Carafate of these three gamma-emitting labels and Se-75-Carafate is suitable for studying the kinetics of the drug Carafate in human subjects.

  4. Photoaffinity ligand for dopamine D2 receptors: azidoclebopride

    SciTech Connect

    Niznik, H.B.; Guan, J.H.; Neumeyer, J.L.; Seeman, P.

    1985-02-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind (/sup 3/H)spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol.

  5. Affinity Labeling of Membrane Receptors Using Tissue-Penetrating Radiations

    PubMed Central

    Wong, Franklin C.; Boja, John; Ho, Beng; Kuhar, Michael J.; Wong, Dean F.

    2013-01-01

    Photoaffinity labeling, a useful in vivo biochemical tool, is limited when applied in vivo because of the poor tissue penetration by ultraviolet (UV) photons. This study investigates affinity labeling using tissue-penetrating radiation to overcome the tissue attenuation and irreversibly label membrane receptor proteins. Using X-ray (115 kVp) at low doses (<50 cGy or Rad), specific and irreversible binding was found on striatal dopamine transporters with 3 photoaffinity ligands for dopamine transporters, to different extents. Upon X-ray exposure (115 kVp), RTI-38 and RTI-78 ligands showed irreversible and specific binding to the dopamine transporter similar to those seen with UV exposure under other conditions. Similarly, gamma rays at higher energy (662 keV) also affect irreversible binding of photoreactive ligands to peripheral benzodiazepine receptors (by PK14105) and to the dopamine (D2) membrane receptors (by azidoclebopride), respectively. This study reports that X-ray and gamma rays induced affinity labeling of membrane receptors in a manner similar to UV with photoreactive ligands of the dopamine transporter, D2 dopamine receptor (D2R), and peripheral benzodiazepine receptor (PBDZR). It may provide specific noninvasive irreversible block or stimulation of a receptor using tissue-penetrating radiation targeting selected anatomic sites. PMID:23936811

  6. Characterization of histamine H/sub 1/-receptor binding peptides in guinea pig brain using (/sup 125/I)iodoazidophenpyramine, an irreversible specific photoaffinity probe

    SciTech Connect

    Ruat, M.; Koerner, M.; Garbarg, M.; Gros, C.; Schwartz, J.C.; Tertiuk, W.; Ganellin, C.R.

    1988-04-01

    Aminophenpyramine, a derivative of mepyramine (pyrilamine), a typical antagonists of histamine at its H/sub 1/ receptor was synthesized and converted into (/sup 125/I)iodoazidophenpyramine, a potential photoaffinity probe for the H/sub 1/ receptor. In the dark, reversible binding of this probe to cerebellar membranes occurred with a K/sub d/ of 1.2 x 10/sup -11/ M and a B/sub max/ of 240 fmol/mg of protein and was inhibited by various H/sub 1/-receptor antagonists with the expected potencies. These features establish the compound as one of the most potent H/sub 1/-receptor antagonists known so far. Upon IV irradiation, 5% of the bound radioactivity was covalently incorporated into cerebellar membrane polypeptides as shown by standard NaDodSO/sub 4//PAGE. Two bands of 47 and 56 kDa were consistently labeled, labeling being prevented by various H/sub 1/-receptor antagonists with the expected potencies and stereoselectivity. In the presence of protease inhibitors, labeling of the 56-kDa peptide increased at the expense of the 47-kDa peptide, suggesting that the latter was produced by hydrolysis of the former under the action of membrane proteases. In the absence of 2-mercaptoethanol, a band of 350-400 kDa appeared, apparently at the expense of the lighter bands, suggesting that the latter might be linked by one or more disulfide bridges to a higher molecular mass complex. The authors propose that at least part of the ligand binding domain of the histamine H/sub 1/ receptor resides within a subunit of apparent molecular mass 56,000.

  7. Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin Transporter Based on the Selective Serotonin Reuptake Inhibitor (S)-Citalopram

    PubMed Central

    2015-01-01

    Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (Ki = 24–227 nM) for hSERT, as assessed by [3H]5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with Ki values ranging from 3.8 to 9.9 μM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [125I]15 compared to that by [125I]22 and [125I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug–protein binding interactions for (S)-citalopram at hSERT. PMID:26153715

  8. Specificity of binding of beta-glucoside activators of ryegrass (1-->3)-beta-glucan synthase and the synthesis of some potential photoaffinity activators.

    PubMed Central

    Ng, K; Johnson, E; Stone, B A

    1996-01-01

    Structure-activity relationships among glycoside activators of ryegrass (Lolium multiflorum) (1-->3)-beta-glucan synthase were investigated using a number of natural and synthetic glycosides, including some carrying photoaffinity functions. There is an absolute requirement for a beta-D-glycosyl moiety in the activator, both S- and N-glucosides are active, and the position of the glucosidic linkage in beta-glucose disaccharides has a significant effect on the affinity of binding. However, the binding requirement does not extend beyond a single beta-D-glucosyl residue, and beta-D-oligoglucosides are less effective than disaccharides. The nature of the aglycon has a major influence on the binding affinity. Hydrophobic aglycons lower the concentration required for half-maximal stimulation of the enzyme obtained from an Eadie-Hofstee plot of kinetic data (Ka) for activation, but charge aglycons increase Ka. Relative to methyl-beta-D-glucoside and cellobiose (Ka 1.1 mM), the most potent compounds tested were N-[4-(benzoyl)benzoyl]-beta-D-glucosylamine and 2'-[4-azidosalicylamino]ethyl-1-thio-beta-D-glucoside with K(a)s of approximately 30 microM. The latter also was tested for its potential to specifically label the beta-glucoside-binding site on the synthase, but under the conditions used the binding was found to be nonspecific. PMID:8756503

  9. (±)-2-(N-tert-Butylamino)-3'-[(125)I]-iodo-4'-azidopropiophenone: a dopamine transporter and nicotinic acetylcholine receptor photoaffinity ligand based on bupropion (Wellbutrin, Zyban).

    PubMed

    Lapinsky, David J; Aggarwal, Shaili; Nolan, Tammy L; Surratt, Christopher K; Lever, John R; Acharya, Rejwi; Vaughan, Roxanne A; Pandhare, Akash; Blanton, Michael P

    2012-01-01

    Towards addressing the knowledge gap of how bupropion interacts with the dopamine transporter (DAT) and nicotinic acetylcholine receptors (nAChRs), a ligand was synthesized in which the chlorine of bupropion was isosterically replaced with an iodine and a photoreactive azide was added to the 4'-position of the aromatic ring. Analog (±)-3 (SADU-3-72) demonstrated modest DAT and α4β2 nAChR affinity. A radioiodinated version was shown to bind covalently to hDAT expressed in cultured cells and affinity-purified, lipid-reincorporated human α4β2 neuronal nAChRs. Co-incubation of (±)-[(125)I]-3 with non-radioactive (±)-bupropion or (-)-cocaine blocked labeling of these proteins. Compound (±)-[(125)I]-3 represents the first successful example of a DAT and nAChR photoaffinity ligand based on the bupropion scaffold. Such ligands are expected to assist in mapping bupropion-binding pockets within plasma membrane monoamine transporters and ligand-gated nAChR ion channels. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Novel Azido-Iodo Photoaffinity Ligands for the Human Serotonin Transporter Based on the Selective Serotonin Reuptake Inhibitor (S)-Citalopram.

    PubMed

    Kumar, Vivek; Yarravarapu, Nageswari; Lapinsky, David J; Perley, Danielle; Felts, Bruce; Tomlinson, Michael J; Vaughan, Roxanne A; Henry, L Keith; Lever, John R; Newman, Amy Hauck

    2015-07-23

    Three photoaffinity ligands (PALs) for the human serotonin transporter (hSERT) were synthesized based on the selective serotonin reuptake inhibitor (SSRI), (S)-citalopram (1). The classic 4-azido-3-iodo-phenyl group was appended to either the C-1 or C-5 position of the parent molecule, with variable-length linkers, to generate ligands 15, 22, and 26. These ligands retained high to moderate affinity binding (K(i) = 24-227 nM) for hSERT, as assessed by [(3)H]5-HT transport inhibition. When tested against Ser438Thr hSERT, all three PALs showed dramatic rightward shifts in inhibitory potency, with Ki values ranging from 3.8 to 9.9 μM, consistent with the role of Ser438 as a key residue for high-affinity binding of many SSRIs, including (S)-citalopram. Photoactivation studies demonstrated irreversible adduction to hSERT by all ligands, but the reduced (S)-citalopram inhibition of labeling by [(125)I]15 compared to that by [(125)I]22 and [(125)I]26 suggests differences in binding mode(s). These radioligands will be useful for characterizing the drug-protein binding interactions for (S)-citalopram at hSERT.

  11. Immunophotoaffinity labeling of the binding proteins for 1-methyladenine, an oocyte maturation-inducing hormone of starfish.

    PubMed

    Kida, Tetsuo; Matsuda, Shinjiro; Kuyama, Atsushi; Toraya, Tetsuo

    2014-01-01

    Starfish oocytes are naturally arrested at the prophase stage of the first meiotic division and resume meiosis in response to 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative receptors for 1-MeAde have not yet been characterized biochemically, although the specific binding of 1-MeAde to the isolated cortices of starfish oocytes was reported so far. Based on the structure-activity relationship of 1-MeAde analogs, we have designed a photoaffinity labeling reagent. The photoaffinity labeling of oocyte membrane fractions, followed by immunoblotting analysis with anti-1-MeAde antibody, results in the detection of an almost single protein band. This 1-MeAde-binding protein might be a possible candidate of the maturation-inducing hormone receptor of starfish.

  12. Fluorescein Derivatives as Bifunctional Molecules for the Simultaneous Inhibiting and Labeling of FTO Protein.

    PubMed

    Wang, Tianlu; Hong, Tingting; Huang, Yue; Su, Haomiao; Wu, Fan; Chen, Yi; Wei, Lai; Huang, Wei; Hua, Xiaoluan; Xia, Yu; Xu, Jinglei; Gan, Jianhua; Yuan, Bifeng; Feng, Yuqi; Zhang, Xiaolian; Yang, Cai-Guang; Zhou, Xiang

    2015-11-04

    The FTO protein is unequivocally reported to play a critical role in human obesity and in the regulation of cellular levels of m(6)A modification, which makes FTO a significant and worthy subject of study. Here, we identified that fluorescein derivatives can selectively inhibit FTO demethylation, and the mechanisms behind these activities were elucidated after we determined the X-ray crystal structures of FTO/fluorescein and FTO/5-aminofluorescein. Furthermore, these inhibitors can also be applied to the direct labeling and enrichment of FTO protein combined with photoaffinity labeling assay.

  13. Insecticidal quinazoline derivatives with (trifluoromethyl)diazirinyl and azido substituents as NADH:ubiquinone oxidoreductase inhibitors and candidate photoaffinity probes.

    PubMed

    Latli, B; Wood, E; Casida, J E

    1996-03-01

    Two candidate photoaffinity probes are designed from 4-substituted quinazolines known to be potent insecticides/acaricides and NADH:ubiquinone oxidoreductase inhibitors acting at or near the rotenone site. 4-(11-Azidoundecyl-2-amino)quinazoline, based on the undecylamino analog SAN 548A as a prototype, was synthesized in 18% overall yield from ethyl 10-undecenoate by oxidation of the terminal double bond, reductive amination, coupling to 4-chloroquinazoline, and functional group manipulation of the terminal ethyl ester to an alcohol, a mesylate and finally nucleophilic displacement with azide ions. 4-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)phenethoxy)quinaz oline [the (trifluoromethyl)diazirinyl analog of fenazaquin insecticide/acaricide] was prepared from 4-bromophenethyl alcohol in 31% overall yield by first introducing the trifluoromethylketone moiety followed by its conversion to the (trifluoromethyl)-diazirine and finally coupling to 4-chloroquinazoline as above. Both candidate photoaffinity probes have the inhibitory potency of rotenone (IC50 of 3-4 nM in each case). The azidoundecylamino compound has inadequate photoreactivity whereas that of the (trifluoromethyl)diazirinyl analog is ideal at 350 nm. Radiosynthesis of the latter photoaffinity ligand included introduction of the diazirinyl moiety as the carbene precursor, oxidation of (trifluoromethyl)diazirinylphenethyl alcohol to the corresponding acid with Jones' reagent, and reduction of the phenacetyl chloride intermediate with sodium borotritide to incorporate tritium.

  14. Characterization of a phosphate binding domain on the alpha-subunit of chloroplast ATP synthase using the photoaffinity phosphate analogue 4-azido-2-nitrophenyl phosphate.

    PubMed

    Groth, G; Mills, D A; Christiansen, E; Richter, M L; Huchzermeyer, B

    2000-11-14

    The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated chloroplast coupling factor 1 (CF(1)). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bound CF(1) by ANPP by exposing thylakoid membranes to increasing concentrations of the reagent. ANPP exhibited saturable binding to two sites on CF(1), one on the beta-subunit and one on the alpha-subunit. Labeling by ANPP resulted in the complete inhibition of both ATP synthesis and ATP hydrolysis by the membrane-bound enzyme. Labeling of both sites by ANPP was reduced by more than 80% in the presence of P(i) (> or = 10 mM) and ATP (> or = 0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of approximately 35% inhibition at concentrations > or = 2 mM. ANPP-labeled tryptic peptides of the alpha-subunit were isolated and sequenced. The majority of the probe was contained in three peptides corresponding to residues Gln(173) to Arg(216), Gly(217) to Arg(253), and His(256) to Arg(272) of the alpha-subunit. In the mitochondrial F(1) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamma-phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposite direction to the adenine binding site with reference to the phosphate binding site on the alpha-subunit. The study has confirmed that ANPP is a bona fide phosphate analogue and suggests that it specifically targets the gamma-phosphate binding site within the nucleotide binding pockets on the alpha- and beta

  15. Gamma emitting radioactive materials in household dinnerware

    NASA Astrophysics Data System (ADS)

    Khalil, Fawzia Ahmad

    A variety of commonly available household and tableware items and some specialty glass materials commonly found in everyday life were examined for their radioactivity content with two different detection and measurement methods. Dinnerware is produced mainly from clay and sand at high temperatures. Therefore, it should be expected to have some degree of radioactivity. It is also stored in confined places, which permits radon accumulation. The natural radioactivity due to the presence of 238U, 232Th and 40K in dinnerware used in houses was measured. Many dinnerware items from various origins that are sold on the open market were studied. Measurements of specific activities of 238U, 232Th, 40K and 137Cs radionuclide for the samples were carried out. The measurements were made by gamma-ray spectrometry having a high-purity germanium (HpGe) detector connected to a multichannel analyzer and a computer system. The average values of specific activities were (6.03 ± 0.54 to 223.67 ± 22.37 for 238U; 2.87 ± 0.14 to 513.85 ± 15.42 for 232Th; 28.67 ± 2.01 to 2726.70 ± 54.53 for 40K; and 0.592 ± 0.037 to 3.549 ± 0.248 for 137Cs) Bq kg-1, respectively. The glazed samples seemed to contribute most of the activity, although also unglazed samples showed some activity. The absorbed dose rates, radium equivalent and external hazard index were also calculated and tabulated. CR-39 solid-state nuclear track detectors were used to measure the radon track density, exhalation rate and effective radium content for the investigated samples. The exhalation rate was found to vary from 4.376 to 8.144 Bq m-2 d-1. It appears that foreign ceramic products, especially Chinese ones with high uranium content, eventually enter the country. The results from the two methods are compared and their combined uncertainties were estimated from the relation of relative combined variance. In Egypt, no special regulations exist concerning radioactivity in glazed earthenware. On the basis of the previous considerations, it appears that more specific regulations for dinnerware imports are necessary.

  16. Facile synthesis of diazido-functionalized biaryl compounds as radioisotope-free photoaffinity probes by Suzuki-Miyaura coupling.

    PubMed

    Hosoya, Takamitsu; Inoue, Atsushi; Hiramatsu, Toshiyuki; Aoyama, Hiroshi; Ikemoto, Takaaki; Suzuki, Masaaki

    2009-03-15

    Suzuki-Miyaura coupling of 3-azido-5-(azidomethyl)phenylboronic acid pinacol ester with various aryl bromides affords corresponding diazido-functionalized biaryl compounds in good yields. This approach provides an easy access to radioisotope-free photoaffinity probes possessing biaryl structure. By using this method, we prepared a novel diazido-functionalized dantrolene analog, which showed selective inhibitory effect on physiological Ca(2+) release (PCR) from sarcoplasmic reticulum (SR) in mouse skeletal muscle without affecting Ca(2+)-induced Ca(2+) release (CICR).

  17. Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2

    PubMed Central

    Vaidya, Aditya Sudheer; Karumudi, Bhargava; Mendonca, Emma; Madriaga, Antonett; Abdelkarim, Hazem; van Breemen, Richard B.; Petukhov, Pavel A.

    2012-01-01

    The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs. PMID:22771007

  18. Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling.

    PubMed

    Head, Sarah A; Liu, Jun O

    2016-09-20

    Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

  19. Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling

    SciTech Connect

    Lohse, M.J.; Klotz, K.N.; Schwabe, U.

    1991-04-01

    It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine ((R)-AHPIA) into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling.

  20. LNP 906, the first high-affinity photoaffinity ligand selective for I1 imidazoline receptors

    PubMed Central

    Dragan, Urosevic; Stephan, Schann; Jean-Daniel, Ehrhardt; Pascal, Bousquet; Hugues, Greney

    2004-01-01

    The hypotensive effect of imidazoline-like drugs, such as clonidine, was attributed both to α2-adrenergic receptors and nonadrenergic imidazoline receptors, which are divided into I1, I2 and I3 subtypes. We have recently synthesized a derivative of (2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), the first high-affinity and selective ligand for I1 receptors (I1R), with a photoactivable function (LNP 906). This work aims to test whether this derivative retained the binding properties of LNP 911 and bound irreversibly to I1R. Binding studies showed that LNP 906 exhibited nanomolar affinity for I1R and was selective for I1R over I2 receptors and α2-adrenergic receptors (α2Ars). Upon exposure to u.v. light, LNP 906 irreversibly blocked the binding of [125I]-paraiodoclonidine (PIC) to I1R, time- and dose-dependently, on PC12 cell membranes and interacted with I1R in a reversible and competitive manner in the absence of light. Pharmacological studies showed that this blockade was prevented by the concomitant presence of rilmenidine (a well-known I1 agonist), but not by rauwolscine (an α2 antagonist). Finally, LNP 906 clearly antagonized the decrease in forskolin-stimulated cAMP level induced by rilmenidine, but not by melatonin. These results indicate that LNP 906 is the first high-affinity and selective photoaffinity ligand for I1R and that it behaves as an I1R antagonist. PMID:15178642

  1. Photoaffinity Ligand for the Inhalational Anesthetic Sevoflurane Allows Mechanistic Insight into Potassium Channel Modulation.

    PubMed

    Woll, Kellie A; Peng, Wesley; Liang, Qiansheng; Zhi, Lianteng; Jacobs, Jack A; Maciunas, Lina; Bhanu, Natarajan; Garcia, Benjamin A; Covarrubias, Manuel; Loll, Patrick J; Dailey, William P; Eckenhoff, Roderic G

    2017-04-03

    Sevoflurane is a commonly used inhaled general anesthetic. Despite this, its mechanism of action remains largely elusive. Compared to other anesthetics, sevoflurane exhibits distinct functional activity. In particular, sevoflurane is a positive modulator of voltage-gated Shaker-related potassium channels (Kv1.x), which are key regulators of action potentials. Here, we report the synthesis and validation of azisevoflurane, a photoaffinity ligand for the direct identification of sevoflurane binding sites in the Kv1.2 channel. Azisevoflurane retains major sevoflurane protein binding interactions and pharmacological properties within in vivo models. Photoactivation of azisevoflurane induces adduction to amino acid residues that accurately reported sevoflurane protein binding sites in model proteins. Pharmacologically relevant concentrations of azisevoflurane analogously potentiated wild-type Kv1.2 and the established mutant Kv1.2 G329T. In wild-type Kv1.2 channels, azisevoflurane photolabeled Leu317 within the internal S4-S5 linker, a vital helix that couples the voltage sensor to the pore region. A residue lining the same binding cavity was photolabeled by azisevoflurane and protected by sevoflurane in the Kv1.2 G329T. Mutagenesis of Leu317 in WT Kv1.2 abolished sevoflurane voltage-dependent positive modulation. Azisevoflurane additionally photolabeled a second distinct site at Thr384 near the external selectivity filter in the Kv1.2 G329T mutant. The identified sevoflurane binding sites are located in critical regions involved in gating of Kv channels and related ion channels. Azisevoflurane has thus emerged as a new tool to discover inhaled anesthetic targets and binding sites and investigate contributions of these targets to general anesthesia.

  2. Biotinylated Quercetin as an Intrinsic Photoaffinity Proteomics Probe for the Identification of Quercetin Target Proteins

    PubMed Central

    Wang, Rongsheng E.; Hunt, Clayton R.; Chen, Jiawei; Taylor, John-Stephen

    2012-01-01

    Quercetin is a flavonoid natural product that is found in many foods and has been found to have a wide range of medicinal effects. Though a number of quercetin binding proteins have been identified, there has been no systematic approach to identifying all potential targets of quercetin. We describe an O7- biotinylated derivative of quercetin (BioQ) that can act as a photoaffinity proteomics reagent for capturing quercetin binding proteins, which can then be identified by LC MS/MS. BioQ was shown to inhibit heat induction of HSP70 with almost the same efficiency as quercetin, and to both inhibit and photocrosslink to CK2 kinase, a known target of quercetin involved in activation of the heat shock transcription factor. BioQ was also able to pull down a number of proteins from unheated and heated Jurkat cells following UV-irradiation that could be detected by both silver staining and Western blot analysis with an anti-biotin antibody. Analysis of the protein bands by trypsinization and LC MS/MS led to the identification of heat shock proteins HSP70 and HSP90 as possible quercetin target proteins, along with ubiquitin-activating enzyme, a spliceosomal protein, RuvB-like 2 ATPases, and eukaryotic translation initiation factor 3. In addition, a mitochondrial ATPase was identified that has been previously shown to be a target of quercetin. Most of the proteins identified have also been previously suggested to be potential anticancer targets, suggesting that quercetin's antitumor activity may be due to its ability to inhibit multiple target proteins. PMID:21798748

  3. Genetically encoded protein photocrosslinker with a transferable mass spectrometry-identifiable label

    PubMed Central

    Yang, Yi; Song, Haiping; He, Dan; Zhang, Shuai; Dai, Shizhong; Lin, Shixian; Meng, Rong; Wang, Chu; Chen, Peng R.

    2016-01-01

    Coupling photocrosslinking reagents with mass spectrometry has become a powerful tool for studying protein–protein interactions in living systems, but it still suffers from high rates of false-positive identifications as well as the lack of information on interaction interface due to the challenges in deciphering crosslinking peptides. Here we develop a genetically encoded photo-affinity unnatural amino acid that introduces a mass spectrometry-identifiable label (MS-label) to the captured prey proteins after photocrosslinking and prey–bait separation. This strategy, termed IMAPP (In-situ cleavage and MS-label transfer After Protein Photocrosslinking), enables direct identification of photo-captured substrate peptides that are difficult to uncover by conventional genetically encoded photocrosslinkers. Taking advantage of the MS-label, the IMAPP strategy significantly enhances the confidence for identifying protein–protein interactions and enables simultaneous mapping of the binding interface under living conditions. PMID:27460181

  4. (±)-2-(N-tert-Butylamino)-3′-[125I]-iodo-4′-azidopropiophenone: a dopamine transporter and nicotinic acetylcholine receptor photoaffinity ligand based on bupropion (Wellbutrin, Zyban)

    PubMed Central

    Lapinsky, David J.; Aggarwal, Shaili; Nolan, Tammy L.; Surratt, Christopher K.; Lever, John R.; Acharya, Rejwi; Vaughan, Roxanne A.; Pandhare, Akash; Blanton, Michael P.

    2011-01-01

    Towards addressing the knowledge gap of how bupropion interacts with the dopamine transporter (DAT) and nicotinic acetylcholine receptors (nAChRs), a ligand was synthesized in which the chlorine of bupropion was isosterically replaced with an iodine and a photoreactive azide was added to the 4′-position of the aromatic ring. Analog (±)-3 (SADU-3-72) demonstrated modest DAT and α4β2 nAChR affinity. A radioiodinated version was shown to bind covalently to hDAT expressed in cultured cells and affinity-purified, lipid-reincorporated human α4β2 neuronal nAChRs. Co-incubation of (±)-[125I]-3 with non-radioactive (±)-bupropion or (−)-cocaine blocked labeling of these proteins. Compound (±)-[125I]-3 represents the first successful example of a DAT and nAChR photoaffinity ligand based on the bupropion scaffold. Such ligands are expected to assist in mapping bupropion-binding pockets within plasma membrane monoamine transporters and ligand-gated nAChR ion channels. PMID:22119468

  5. Food Labels

    MedlinePlus

    ... Loss Surgery? A Week of Healthy Breakfasts Shyness Food Labels KidsHealth > For Teens > Food Labels Print A ... have at least 95% organic ingredients. continue Making Food Labels Work for You The first step in ...

  6. Azido Push–Pull Fluorogens Photoactivate to Produce Bright Fluorescent Labels

    PubMed Central

    Lord, Samuel J.; Lee, Hsiao-lu D.; Samuel, Reichel; Weber, Ryan; Liu, Na; Conley, Nicholas R.; Thompson, Michael A.; Twieg, Robert J.; Moerner, W. E.

    2009-01-01

    Dark azido push–pull chromophores have the ability to be photoactivated to produce bright fluorescent labels suitable for single-molecule imaging. Upon illumination, the aryl azide functionality in the fluorogens participates in a photochemical conversion to an aryl amine, thus restoring charge-transfer absorption and fluorescence. Previously, we reported that one compound, DCDHF-V-P-azide, was photoactivatable. Here, we demonstrate that the azide-to-amine photoactivation process is generally applicable to a variety of push–pull chromophores, and we characterize the photophysical parameters including photoconversion quantum yield, photostability, and turn-on ratio. Azido push–pull fluorogens provide a new class of photoactivatable single-molecule probes for fluorescent labeling and super-resolution microscopy. Lastly, we demonstrate that photoactivated push–pull dyes can insert into bonds of nearby biomolecules, simultaneously forming a covalent bond and becoming fluorescent (fluorogenic photoaffinity labeling). PMID:19860443

  7. Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking.

    PubMed

    Wooddell, C I; Burgess, R R

    2000-11-07

    The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP. With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.

  8. Tin-117m-labeled stannic (Sn.sup.4+) chelates

    DOEpatents

    Srivastava, Suresh C.; Meinken, George E.; Richards, Powell

    1985-01-01

    The radiopharmaceutical reagents of this invention and the class of Tin-117m radiopharmaceuticals are therapeutic and diagnostic agents that incorporate gamma-emitting nuclides that localize in bone after intravenous injection in mammals (mice, rats, dogs, and rabbits). Images reflecting bone structure or function can then be obtained by a scintillation camera that detects the distribution of ionizing radiation emitted by the radioactive agent. Tin-117m-labeled chelates of stannic tin localize almost exclusively in cortical bone. Upon intravenous injection of the reagent, the preferred chelates are phosphonate compounds, preferable, PYP, MDP, EHDP, and DTPA. This class of reagents is therapeutically and diagnostically useful in skeletal scintigraphy and for the radiotherapy of bone tumors and other disorders.

  9. Microdosimetric model of astatine-211 labeled antibodies for radioimmunotherapy

    SciTech Connect

    Humm, J.L.

    1987-11-01

    Astatine-211 is an alpha-emitter with a short half-life (7.2 hr). This paper discusses the potential of /sup 211/At targeted by antibodies for tumor therapy and the possible advantage of /sup 211/At over beta- and gamma-emitting radionuclides such as /sup 131/I currently employed in the field of radioimmunotherapy. Since the longest range alpha-particle from /sup 211/At is only 67 microns and the rate of energy loss is high (track averaged linear energy transfer LT approximately 120 keV/micron), a disintegration of /sup 211/At produces a large and extremely localized deposition of energy. A Monte-Carlo model has been developed for studying the stochastic fluctuation of alpha-particle hits and energy deposition in cell nuclei in an attempt to determine the efficacy of /sup 211/At-labeled antibodies for tumor cell inactivation. Calculations have been performed for 2 extreme conditions: (a) the case of /sup 211/At retained in the capillary, and (b) for a homogeneous distribution of /sup 211/At-labeled antibody in the tumor. The results of these two calculations represent the boundary conditions between which any real solution must lie. Finally, developments to the model to include antibody transport across the capillary membrane and through the tumor tissue are discussed.

  10. Synthesis and characterization of new adenosine A/sub 1/ receptor radioligands, N/sup 6/-3-/sup 125/IODO-4-aminobenzyladenosine, and a photoaffinity probe, N/sup 6/-3-/sup 125/IODO-4-azidobenzyladenosine

    SciTech Connect

    Patel, A.P.

    1986-01-01

    Characterization of adenosine receptors in brain and fat membranes has been achieved with /sup 3/H-cyclohexyladenosine, /sup 125/Iodohydroxyphenylisopropyladenosine (/sup 125/IHPIA) and other radioligands. However, these radioligands failed to label cardiac receptors. The authors have synthesized two new radioligands, /sup 125/Iodoaminobenzyladenosine (/sup 125/IABA), and a photoaffinity probe /sup 125/Iodoazidobenzyladenosine (/sup 125/IAzBA). /sup 125/IABA bound to a single class of adenosine receptors on rat ventricle membranes with a K/sub D/ equivalent to that of /sup 125/IHPIA and Bmax of 15.2 fmol/mg protein but with one-sixth the nonspecific binding of the other radioligand. In brain membranes, /sup 125/IABA bound with a higher affinity (K/sub D/ 1.93 nM) than in the heart membranes (K/sub D/ 11.6 nM). Iodination of aminobenzyladenosine increased affinity for the adenosine receptor by 22-fold; from studies on adenylate cyclase in adipocytes and chick heart cells, the new ligand was found to be a full agonist at an A/sub 1/ adenosine receptor. /sup 125/Iodoazidobenzyladenosine (/sup 125/IAzBA) bound with high affinity (K/sub D/-1nM); to receptors on cortical membranes from dog, rat, guinea pig and pig. On photolysis, /sup 125/IAzBA on the adenosine receptor was covalently incorporated into a receptor binding subunit with a Mr of 34 kDa. /sup 125/IAzBA specifically photoincorporated into 34 kDa peptide in dog, guinea pig, rat, chick and pig cortical membranes; and in rat fat membranes. These results suggest that the A/sub 1/ adenosine receptor binding subunit in various mammalian species and tissues resides on a 34 kDa protein.

  11. The Ligand Binding Region of the Sigma-1 Receptor: Studies Utilizing Photoaffinity Probes, Sphingosine and N-Alkylamines

    PubMed Central

    Ruoho, Arnold E.; Chu, Uyen B.; Ramachandran, Subramaniam; Fontanilla, Dominique; Mavlyutov, Timur; Hajipour, Abdol R.

    2015-01-01

    The sigma-1 receptor is a 26 kDa endoplasmic reticulum resident membrane protein that has been shown to have chaperone activity in addition to its promiscuous binding to pharmacological agents. Ligand binding domain(s) of the sigma-1 receptor have been identified using the E. coli expressed and purified receptor protein and novel radioiodinated azido photoaffinity probes followed by pro-teolytic and chemical cleavage strategies. The outcome of these experiments indicates that the sigma-1 receptor ligand binding regions are formed primarily by juxtaposition of its second and third hydrophobic domains, regions where the protein shares considerable homology with the fungal enzyme, sterol isomerase that is essential for the biosynthesis of ergosterol. Data indicate that these hydrophobic steroid binding domain like (SBDL) regions on the sigma-1 receptor are likely to interact selectively with N-alkyl amines such as the endogenous sphingolipids and with synthetic N-alkylamines and N-aralkylamines derivatives. A proposed model for the sigma-1 receptor is presented. PMID:22288412

  12. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the Food and Drug Administration (FDA) and the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

  13. Derivatization and photolysis of a photoaffinity reagent for probing protein and cell surface interactions

    SciTech Connect

    Murphy, H.; Harris, H.W. Jr.

    1986-05-01

    The synthesis of the novel, heterobifunctional, cleavable, photoactivable crosslinking reagent, N-(4-(p-azido-m-(/sup 125/I) iodophenylazo)benzoyl)-3-aminopropyl-N'-oxysulfosuccinimide has been described by Denny and Blobel. This reagent is desirable because after photolysis and azo bond cleavage the /sup 125/I is transferred from the reagent to the crosslinked molecule. The authors demonstrate that using the reported synthesis 99% of the desired reagent is destroyed during the chloramine-T iodination step. They report a synthesis revision which produces high yields of the uniodinated (U) reagent. The derivatized reagent may be used in its iodinated (I) or U forms. To study the U reagent, a horseradish peroxidase (HRP) molecule is derivatized with nine reagent molecules. The derivatized HRP has 70% of its original enzymatic activity. After photolysis, 14% of this activity is retained and SDS-PAGE electrophoresis shows a crosslinked complex of HRP molecules. After endocytosis by cells, photolysis attaches the soluble derivatized HRP to membranes allowing them to be traced in the electron microscope. To study the I reagent, an amino-dextran (MW 73-400) molecule is derivatized with three U reagent molecules. The U reagent molecules are then iodinated by the chloramine-T method. With photolysis and cleavage, the /sup 125/I labeled reagent on dextran transfers its label to bovine serum albumin or ovalbumin. The authors conclude this reagent is a versatile probe for study of protein or cell surface topography.

  14. Synthesis of Arylazide‐ and Diazirine‐Containing CrAsH‐EDT2 Photoaffinity Probes

    PubMed Central

    Syeda, Shameem S.; Rice, Daren; Hook, Derek J.; Heckert, Leslie L.

    2016-01-01

    Two photo‐crosslinking biarsenical (CrAsH‐EDT2)‐modified probes were synthesized that are expected to be useful tools for tetracysteine‐labeled proteins to facilitate the co‐affinity purification of their DNA binding sequences and interacting proteins. In addition, improvements for the synthesis of CrAsH‐EDT2 and N 1‐(4‐azido‐2‐nitrophenyl)hexane‐1,6‐diamine are reported. Both photoprobes effectively entered HeLa cells (and the nucleus) and were dependent on the tetracysteine motif in recombinant DMRT1 (doublesex and Mab3‐related transcription factor) to induce fluorescence, suggesting that their crosslinking abilities can be exploited for the identification of nucleic acids and proteins associated with a protein of interest. PMID:26948688

  15. Identification of the binding subunit of the D/sub 1/-dopamine receptor by photoaffinity crosslinking

    SciTech Connect

    Amlaiky, N.; Berger, J.G.; Chang, W.; McQuade, R.D.; Caron, M.G.

    1986-03-01

    A derivative of the potent D/sub 1/ antagonist SCH-23390 has been synthesized. This compound, (R,S)-1-(m-aminophenyl)-7-chloro-8-hydroxy-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH-38548), has been radioiodinated by a chloramine T procedure yielding 3 radioiodinated products. One of these separated isomers (R/sub f/ = 0.35; CH/sub 2/Cl/sub 2/:MEOH:TEA; 82.5:17.5:0.1 on TLC) binds reversibly to rat striatal membranes with high affinity (K/sub D/ approx. 80 pM) appropriate stereoselectivity and D/sub 1/-dopaminergic specificity. (/sup 125/I)SCH-38548 can be covalently incorporated into a peptide of M/sub r/ approx. 72,000 using the heterobifunctional crosslinking reagent N-succinimidyl-6-(4'-azido-2'-nitro-phenylamino) hexanoate. Covalent incorporation of (/sup 125/I) SCH-38548 into the M/sub r/ approx. 72,000 peptide can be blocked by dopaminergic agents with D/sub 1/-dopaminergic specificity (for agonists: SKF 38393 > apomorphine > dopamine; for antagonists: SCH-23390 > cis-flupentixol >>> trans-flupentixol). The D/sub 1/-dopaminergic selectivity and specificity of the labeling was further demonstrated by the fact that other antagonists such as domperidone, ketanserin, phentolamine and alprenolol did not compete for the covalent labeling of the M/sub r/ approx. 72,000 peptide. This new radioligand should be useful in the molecular characterization of the D/sub 1/-dopaminergic receptor from various sources.

  16. Tin-117m-labeled stannic (Sn/sup 4 +/) chelate of diethylenetriamine pentaacetic acid (DTPA) for application in diagnosis and therapy

    DOEpatents

    Srivastava, S.C.; Meinken, G.E.; Richards, P.

    1983-08-25

    The radiopharmaceutical reagents of this invention and the class of Tin-117m radiopharmaceuticals are therapeutic and diagnostic agents that incorporate gamma-emitting nuclides that localize in bone after intravenous injection in mammals (mice, rats, dogs, and rabbits). Images reflecting bone structure or function can then be obtained by a scintillation camera that detects the distribution of ionizing radiation emitted by the radioactive agent. Tin-117m-labeled chelates of stannic tin localize almost exclusively in cortical bone. Upon intravenous injection of the reagent, the preferred chelates are phosphonate compounds, preferable, PYP, MDP, EHDP, and DTPA. This class of reagents is therapeutically and diagnostically useful in skeletal scintigraphy and for the radiotherapy of bone tumors and other disorders.

  17. Food labeling

    MedlinePlus

    ... States Food and Drug Administration (FDA) has proposed making changes to the food labels that may correct these problems. AMOUNTS PER SERVING The total calories and the calories from fat are listed. These numbers help consumers make decisions about fat intake. The list of nutrients includes ...

  18. Two and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

    SciTech Connect

    Suhadolnik, R.J.; Kariko, K.; Sobol, R.W. Jr.; Li, S.W.; Reichenbach, N.L.; Haley, B.E.

    1988-11-29

    The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from (..gamma..-/sup 32/P)2-azidoATP and (..cap alpha..-/sup 32/P)8-azidoAPT by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p/sub 3/A/sub 4/(/sup 32/P)pCp from RNase L with affinity equivalent to p/sub 3/A/sub 3/. The 8-azido photoprobe also activates RNase L to hydrolyze poly(U)(/sup 32/P)pCp 50% at 7 /times/ 10/sup /minus/9/ M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p/sub 3/A/sub 3/ activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10/sup /minus/9/ M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The (..gamma..-/sup 32/P)2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the (..cap alpha..-/sup 32/P)8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p/sub 3/A/sub 3/ allosteric binding site was obtained.

  19. An auxin-binding protein is localized to the plasma membrane of maize coleoptile cells: Identification by photoaffinity labeling and purification of a 23-kDa polypeptide

    SciTech Connect

    Feldwisch, J.; Zettl, R.; Hesse, F.; Schell, J.; Palme, K. )

    1992-01-15

    Plasma membrane vesicles were isolated from maize (Zea mays L.) coleoptile tissue by aqueous two-phase partitioning and assayed for homogeneity by the use of membrane-specific enzymatic assays. Using 5-azido-(7-{sup 3}H)indole-3-acetic acid (({sup 3}H)N{sub 3}IAA), the authors identified several IAA-binding proteins with the molecular masses of 60 kDa (pm60), 58 kDa (pm58), and 23 kDa (pm23). Using Triton X-114, they were able to selectively extract pm23 from the plasma membrane. They show that auxins and functional analogues compete with ({sup 3}H)N{sub 3}IAA for binding to pm23. They found that PAB130, a polyclonal antibody raised against auxin-binding protein 1 (ABP-1), recognized ABP-1 as well as pm23. This suggests that pm23 shares common epitopes with ABP-1. In addition, they identified an auxin-binding protein with a molecular mass of 24 kDa (pm24), which was detected in microsomal but not in plasma membrane vesicle preparations. Like pm23 this protein was extracted from membrane vesicles with Triton X-114. They designed a purification scheme allowing simultaneous purification of pm23 and pm24. Homogeneous pm23 and pm24 were obtained from coleoptile extracts after 7,000-fold purification.

  20. Bupropion Binds to Two Sites in the Torpedo Nicotinic Acetylcholine Receptor Transmembrane Domain: A Photoaffinity Labeling Study with the Bupropion Analog [125I]-SADU-3-72

    PubMed Central

    Pandhare, Akash; Hamouda, Ayman K.; Staggs, Brandon; Aggarwal, Shaili; Duddempudi, Phaneendra K.; Lever, John R.; Lapinsky, David J.; Jansen, Michaela; Cohen, Jonathan B.; Blanton, Michael P.

    2012-01-01

    Bupropion, a clinically-used antidepressant and smoking-cessation drug, acts as a noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). To identify its binding site(s) in nAChRs, we developed a photoreactive bupropion analog, (±)-2-(N-tert-butylamino)-3′-[125I]-iodo-4′-azidopropiophenone (SADU-3-72). Based upon inhibition of [125I]SADU-3-72 binding, SADU-3-72 binds with high affinity (IC50 = 0.8 μM) to the Torpedo nAChR in the resting (closed channel) state and in the agonist-induced desensitized state, and bupropion binds to that site with three-fold higher affinity in the desensitized (IC50 = 1.2 μM) than in the resting state. Photolabeling of Torpedo nAChRs with [125I]SADU-3-72 followed by limited in-gel digestion of nAChR subunits with endoproteinase Glu-C established the presence of [125I]SADU-3-72 photoincorporation within nAChR subunit fragments containing M1-M2-M3 helices (αV8-20K, βV8-22/23K and γV8-24K) or M1-M2 helices (δV8-14). Photolabeling within βV8-22/23K, γV8-24K and δV8-14 was reduced in the desensitized state and inhibited by ion channel blockers selective for the resting (tetracaine) or desensitized (thienycyclohexylpiperidine (TCP)) state, and this pharmacologically specific photolabeling was localized to the M2-9 leucine ring (δLeu265, βLeu257) within the ion channel. In contrast, photolabeling within the αV8-20K was enhanced in the desensitized state and not inhibited by TCP, but was inhibited by bupropion. This agonist-enhanced photolabeling was localized to αTyr213 in αM1. These results establish the presence of two distinct bupropion binding sites within the Torpedo nAChR transmembrane domain: a high affinity site at the middle (M2-9) of the ion channel and a second site near the extracellular end of αM1 within a previously described halothane (general anesthetic) binding pocket. PMID:22394379

  1. Photoaffinity labeling of the sigma-1 receptor with N-[3-(4-nitrophenyl)propyl]-N-dodecylamine: evidence of receptor dimers.

    PubMed

    Chu, Uyen B; Ramachandran, Subramaniam; Hajipour, Abdol R; Ruoho, Arnold E

    2013-02-05

    The sigma-1 receptor is a ligand-regulated endoplasmic reticulum (ER) resident chaperone involved in the maintenance of cellular homeostasis. Coupling of the sigma-1 receptor with various ER and/or plasma membrane ion channels is associated with its ability to regulate the locomotor activity and cellular proliferation produced in response to sigma-1 receptor ligands. A number of endogenous small molecules bind to the sigma-1 receptor and have been shown to regulate its activity; these include progesterone, N,N-dimethyltryptamine, d-erythro-sphingosine, and/or other endogenous lipids. We previously reported the synthesis of long chain N-alkylamine derivatives and the characterization of the structure-activity relationship between the chain length of N-alkylamine and affinities at the sigma-1 receptor. Here, we present data demonstrating the photoincorporation of one of these N-alkylamine derivatives, N-[3-(4-nitrophenyl)propyl]-N-dodecylamine (4-NPPC12), to the sigma-1 receptor. Matrix-assisted laser desorption ionization time-of-flight and tandem mass spectrometry showed that 4-NPPC12 photoinserted at histidine 154 of the derivatized population of the sigma-1 receptor. Interestingly, light-dependent photoinsertion of 4-NPPC12 resulted in an enhanced electrophoretic mobility of only 50% of the derivatized receptor molecules as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proposed binding and reactivity of 4-NPPC12 evoke a ligand binding model for the sigma-1 receptor that likely involves a receptor dimer and/or oligomer.

  2. Identification of the reactive intermediates produced upon photolysis of p-azidoacetophenone and its tetrafluoro analogue in aqueous and organic solvents: implications for photoaffinity labeling.

    PubMed

    Cline, Meredith R; Mandel, Sarah M; Platz, Matthew S

    2007-02-20

    Photolysis of p-azidoacetophenone (1a) or 2,3,5,6-tetrafluoro-p-azidoacetophenone (1b) releases the corresponding singlet nitrenes 2a and 2b. In aqueous solutions singlet nitrenes relax (1.1 ps and 43 ns, respectively) to the lower energy triplet nitrenes 3a and 3b, intermediates which do not react to form cross-links or adducts with typical amino acids and nucleic acids. In a hydrophobic environment singlet nitrene 2a partitions between forming triplet nitrene 3a and an acyl-substituted didehydroazepine 4a, which can be detected by LFP and time-resolved IR spectroscopy. The absolute rate constant of reaction of didehydroazepine 4a with water, in acetonitrile, was determined (3.5 x 10(4) M-1 s-1) by laser flash photolysis (LFP) techniques with IR detection at ambient temperature. Photolysis of tetrafluoro azide 1b releases singlet nitrene 2b, which has a lifetime of 172 ns in benzene and can readily be intercepted by pyridine to form ylide 10b (lambdamax = 415 nm). Singlet nitrene 2b reacts with the unactivated CH bonds of cyclohexane to form adduct 8b in 46% yield. Absolute rate constants of reaction of 1b with N-methylimidazole, phenol, dibutyl sulfide, indole, methanol, and dimethyl sulfoxide were determined using the pyridine ylide probe method. It is concluded that photolysis of p-azidoacetophenone (1a) will not lead to cross-link formation but that tetrafluorinated azide 1b can form useful singlet nitrene derived adducts upon photolysis.

  3. Development of Gamma-Emitting Receptor Binding Radiopharmace

    SciTech Connect

    Reba, Richard

    2003-02-20

    The long-term objective is to develop blood-brain barrier (BBB) permeable m2-selective (relative to m1, m3, and m4) receptor-binding radiotracers and utilize these radiotracers for quantifying receptor concentrations obtained from PET or SPECT images of human brain. In initial studies, we concluded that the lipophilicity and high affinity prevented (R,S)-I-QNB from reaching a flow-independent and receptor-dependent state in a reasonable time. Thus, it was clear that (R,S)-I-QNB should be modified. Therefore, during the last portion of this funded research, we proposed that more polar heterocycles should help accomplish that. Since reports of others concluded that radiobromination and radiofluorination of the unactivated phenyl ring is not feasible (Newkome et al,,1982), we, therefore, explored during this grant period a series of analogues of (R)-QNB in which one or both of the six-membered phenyl rings is replaced by a five-membered thienyl (Boulay et al., 1995), or furyl ring. The chemistry specific aims were to synthesize novel compounds designed to be m2-selective mAChR ligands capable of penetrating into the CNS, and develop methods for efficient radiolabeling of promising m2-selective muscarinic ligands. The pharmacology specific aims were to determine the affinity and subtype-selectivity of the novel compounds using competition binding studies with membranes from cells that express each of the five muscarinic receptor subtypes, to determine the ability of the promising non-radioactive compounds and radiolabeled novel compounds to cross the BBB, to determine the biodistribution, in-vivo pharmacokinetics, and in-vitm kinetics of promising m2-selective radioligands and to determine the distribution of receptors for the novel m2-selective radioligands using quantitative autoradiography of rat brain, and compare this distribution to the distribution of known m2-selective compounds.

  4. Introduction to Pesticide Labels

    EPA Pesticide Factsheets

    Pesticide product labels provide critical information about how to safely and legally handle and use pesticide products. Unlike most other types of product labels, pesticide labels are legally enforceable. Learn about pesticide product labels.

  5. Immunophotoaffinity labeling of binders of 1-methyladenine, the oocyte maturation-inducing hormone of starfish.

    PubMed

    Toraya, Tetsuo; Kida, Tetsuo; Kuyama, Atsushi; Matsuda, Shinjiro; Tanaka, Seiichi; Komatsu, Yo; Tsurukai, Taro

    2017-03-25

    Starfish oocytes are arrested at the prophase stage of the first meiotic division in the ovary and resume meiosis by the stimulus of 1-methyladenine (1-MeAde), the oocyte maturation-inducing hormone of starfish. Putative 1-MeAde receptors on the oocyte surface have been suggested, but not yet been biochemically characterized. Immunophotoaffinity labeling, i.e., photoaffinity labeling combined with immunochemical detection, was attempted to detect unknown 1-MeAde binders including putative maturation-inducing hormone receptors in starfish oocytes. When the oocyte crude membrane fraction or its Triton X-100/EDTA extract was incubated with N(6)-[6-(5-azido-2-nitrobenzoyl)aminohexyl]carboxamidomethyl-1-methyladenine and then photo-irradiated, followed by western blotting with antibody that was raised against a 1-MeAde hapten, a single band with Mr of 47.5 K was detected. The band was lost when extract was heated at 100 °C. A similar 47.5 K band was detected in the crude membrane fraction of testis as well. Upon labeling with whole cells, this band was detected in immature and maturing oocytes, but only faintly in mature oocytes. As judged from these results, this 1-MeAde binder might be a possible candidate of the starfish maturation-inducing hormone receptors. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Pesticide Label Review Training

    EPA Pesticide Factsheets

    This training will help ensure that reviewers evaluate labels according to four core principles. It also will help pesticide registrants developing labels understand what EPA expects of pesticide labels, and what the Agency generally finds acceptable.

  7. Characterization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein: Sequence analysis and identification of a single affinity-labeled histidine residue by mass spectrometry

    SciTech Connect

    Brinegar, A.C.; Cooper, G.; Stevens, A.; Hauer, C.R.; Shabanowitz, J.; Hunt, D.F.; Fox, J.E. )

    1988-08-01

    A wheat embryo cytokinin-binding protein was covalently modified with the radiolabeled photoaffinity ligand 2-azido-N{sup 6}-({sup 14}C)benzyladenine. A single labeled peptide was obtained after proteolytic digestion and isolation by reversed-phase and anion-exchange HPLC. Sequencing by classical Edman degradation identified 11 of the 12 residues but failed to identify the labeled amino acid. Analysis by laser photodissociation Fourier-transform mass spectrometry of 10 pmol of the peptide independently confirmed the Edman data and also demonstrated that the histidine residue nearest the C terminus (underlined) was modified by the reagent in the sequence Ala-Phe-Leu-Gln-Pro-Ser-His-His{und His}-Asp-Ala-Asp-Glu.

  8. Deep Label Distribution Learning With Label Ambiguity

    NASA Astrophysics Data System (ADS)

    Gao, Bin-Bin; Xing, Chao; Xie, Chen-Wei; Wu, Jianxin; Geng, Xin

    2017-06-01

    Convolutional Neural Networks (ConvNets) have achieved excellent recognition performance in various visual recognition tasks. A large labeled training set is one of the most important factors for its success. However, it is difficult to collect sufficient training images with precise labels in some domains such as apparent age estimation, head pose estimation, multi-label classification and semantic segmentation. Fortunately, there is ambiguous information among labels, which makes these tasks different from traditional classification. Based on this observation, we convert the label of each image into a discrete label distribution, and learn the label distribution by minimizing a Kullback-Leibler divergence between the predicted and ground-truth label distributions using deep ConvNets. The proposed DLDL (Deep Label Distribution Learning) method effectively utilizes the label ambiguity in both feature learning and classifier learning, which help prevent the network from over-fitting even when the training set is small. Experimental results show that the proposed approach produces significantly better results than state-of-the-art methods for age estimation and head pose estimation. At the same time, it also improves recognition performance for multi-label classification and semantic segmentation tasks.

  9. /sup 125/I-labeled crosslinking reagent that is hydrophilic, photoactivatable, and cleavable through an azo linkage

    SciTech Connect

    Denny, J.B.; Blobel, G.

    1984-09-01

    A radioactive crosslinking reagent, N-(4-(p-azido-m-(/sup 125/I)iodophenylazo)benzoyl)-3-aminopropyl-N'-oxysulfosuccinimide ester, has been synthesized. The reagent is photoactivatable, water-soluble, cleavable through an azo linkage, and labeled with /sup 125/I at the carrier-free specific activity of 2000 Ci/mmol. Any protein derivatized with the reagent is thus converted into an /sup 125/I-labeled photoaffinity probe. Crosslinks are formed following photolysis with 366-nm light, and cleavage by sodium dithionite results in the donation of radioactivity to the distal partner in crosslinked complexes. The newly labeled proteins are then analyzed by gel electrophoresis and autoradiography. The compound was prepared by iodination of N-(4-(p-aminophenylazo)benzoyl)-3-aminopropionic acid using carrier-free Na/sup 125/I and chloramine-T, followed by azide formation and conversion to the water-soluble sulfosuccinimide ester. As a model system, protein A-Sepharose was derivatized with the reagent under subdued light. Each derivatized protein A molecule contained only one crosslinker. The derivatized protein A-Sepharose was then photolyzed in the presence of human serum and subsequently treated with sodium dithionite. Analysis of the serum by gel electrophoresis revealed that 1.1% of the radioactive label originally present on the protein A-Sepharose was transferred to the heavy chain of IgG, which was the most intensely labeled protein in the gel. The next most intensely labeled protein was IgG light chain, which incorporated radioactivity that was lower by a factor of 3.6 than that of the heavy chain. 36 references, 3 figures.

  10. Mental Labels and Tattoos

    ERIC Educational Resources Information Center

    Hyatt, I. Ralph

    1977-01-01

    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  11. Pesticide Labeling Questions & Answers

    EPA Pesticide Factsheets

    Pesticide manufacturers, applicators, state regulatory agencies, and other stakeholders raise questions or issues about pesticide labels. The questions on this page are those that apply to multiple products or address inconsistencies among product labels.

  12. Soil Fumigant Labels - Chloropicrin

    EPA Pesticide Factsheets

    Search by EPA registration number, product name, or company name, and follow the link to the Pesticide Product Label System (PPLS) for details on each fumigant. Updated labels include new safety requirements for buffer zones and related measures.

  13. Soil Fumigant Labels - Dazomet

    EPA Pesticide Factsheets

    Updated labels include new safety requirements for buffer zones and related measures. Find information from the Pesticide Product Labeling System (PPLS) for products such as Basamid G, manufactured by Amvac.

  14. Mental Labels and Tattoos

    ERIC Educational Resources Information Center

    Hyatt, I. Ralph

    1977-01-01

    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  15. Soil Fumigant Labels

    EPA Pesticide Factsheets

    The 2012 updated pesticide labels include new safety requirements for buffer zones and related measures. Find labels for each different type of fumigant: chloropicrin, dazomet, dimethyl disulfide, metam sodium/potassium, and methyl bromide.

  16. Electronic Submission of Labels

    EPA Pesticide Factsheets

    Pesticide registrants can provide draft and final labels to EPA electronically for our review as part of the pesticide registration process. The electronic submission of labels by registrants is voluntary but strongly encouraged.

  17. The Labelling of Chemicals.

    ERIC Educational Resources Information Center

    Education in Science, 1979

    1979-01-01

    Describes the impact on chemistry laboratories and teachers in the United Kingdom of the Packaging and Labelling of Dangerous Substances Regulations 1978. These regulations require suppliers to label containers in particular ways. (HM)

  18. Semiotic labelled deductive systems

    SciTech Connect

    Nossum, R.T.

    1996-12-31

    We review the class of Semiotic Models put forward by Pospelov, as well as the Labelled Deductive Systems developed by Gabbay, and construct an embedding of Semiotic Models into Labelled Deductive Systems.

  19. Label Review Training: Module 1: Label Basics, Page 16

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the importance of labels and the role in enforcement.

  20. Label Review Training: Module 1: Label Basics, Page 14

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about positive effects from proper labeling.

  1. Label Review Training: Module 1: Label Basics, Page 15

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the consequences of improper labeling.

  2. Label Review Training: Module 1: Label Basics, Page 21

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about types of labels.

  3. Label Review Training: Module 1: Label Basics, Page 19

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section covers supplemental distributor labeling.

  4. Label Review Training: Module 1: Label Basics, Page 17

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See an overview of the importance of labels.

  5. Label Review Training: Module 1: Label Basics, Page 22

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about what labels require review.

  6. Label Review Training: Module 1: Label Basics, Page 27

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See examples of mandatory and advisory label statements.

  7. Label Review Training: Module 1: Label Basics, Page 26

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about mandatory and advisory label statements.

  8. Label Review Training: Module 1: Label Basics, Page 24

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is about which labels require review.

  9. Label Review Training: Module 1: Label Basics, Page 18

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section discusses the types of labels.

  10. Label Review Training: Module 1: Label Basics, Page 23

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Lists types of labels that do not require review.

  11. Sample Pesticide Label for Label Review Training

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  12. Pesticide Product Label System

    EPA Pesticide Factsheets

    The Pesticide Product Label System (PPLS) provides a collection of pesticide product labels (Adobe PDF format) that have been approved by EPA under Section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). New labels were added to PPLS on November 21, 2014. Pesticide product labels provide critical information about how to safely handle and use registered pesticide products. An approved pesticide product label represents the full content of EPAs registration decision regarding that product. Pesticide labels contain detailed information on the use, storage, and handling of a product. This information will be found on EPA stamped-approved labels and, in some cases, in subsequent related correspondence, which is also included in PPLS. You may need to review several PDF files for a single product to determine the complete current terms of registration.

  13. Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein

    NASA Technical Reports Server (NTRS)

    Kutner, A.; Link, R. P.; Schnoes, H. K.; DeLuca, H. F.

    1986-01-01

    3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

  14. Photoactivable analogs for labeling 25-hydroxyvitamin D3 serum binding protein and for 1,25-dihydroxyvitamin D3 intestinal receptor protein

    NASA Technical Reports Server (NTRS)

    Kutner, A.; Link, R. P.; Schnoes, H. K.; DeLuca, H. F.

    1986-01-01

    3-Azidobenzoates and 3-azidonitrobenzoates of 25-hydroxyvitamin D3 as well as 3-deoxy-3-azido-25-hydroxyvitamin D3 and 3-deoxy-3-azido-1,25-dihydroxyvitamin D3 were prepared as photoaffinity labels for vitamin D serum binding protein and 1,25-dihydroxyvitamin D3 intestinal receptor protein. The compounds prepared were easily activated by short- or long-wavelength uv light, as monitored by uv and ir spectrometry. The efficacy of the compounds to compete with 25-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 for the binding site of serum binding protein and receptor, respectively, was studied to evaluate the vitamin D label with the highest affinity for the protein. The presence of an azidobenzoate or azidonitrobenzoate substituent at the C-3 position of 25-OH-D3 significantly decreased (10(4)- to 10(6)-fold) the binding activity. However, the labels containing the azido substituent attached directly to the vitamin D skeleton at the C-3 position showed a high affinity, only 20- to 150-fold lower than that of the parent compounds with their respective proteins. Therefore, 3-deoxy-3-azidovitamins present potential ligands for photolabeling of vitamin D proteins and for studying the structures of the protein active sites.

  15. Labeling of Patient Specimens

    DTIC Science & Technology

    2011-01-26

    printers in each clinic to print labels .JDI Capt Cutter Research compatible printer, Cost, Time Frame Develop standard training for all clinics...Standardize label content, automate with inkless printers once process is proven c . Place visual reminders for providers and support staff 2. Event

  16. Labeling and Delinquency.

    ERIC Educational Resources Information Center

    Adams, Mike S.; Robertson, Craig T.; Gray-Ray, Phyllis; Ray, Melvin C.

    2003-01-01

    Index comprised of six contrasting descriptive adjectives was used to measure incarcerated youths' perceived negative labeling from the perspective of parents, teachers, and peers. Results provided partial support for hypothesis that juveniles who choose a greater number of negative labels will report more frequent delinquent involvement. Labeling…

  17. Labeling and Delinquency.

    ERIC Educational Resources Information Center

    Adams, Mike S.; Robertson, Craig T.; Gray-Ray, Phyllis; Ray, Melvin C.

    2003-01-01

    Index comprised of six contrasting descriptive adjectives was used to measure incarcerated youths' perceived negative labeling from the perspective of parents, teachers, and peers. Results provided partial support for hypothesis that juveniles who choose a greater number of negative labels will report more frequent delinquent involvement. Labeling…

  18. Government perspective: food labeling.

    PubMed

    Philipson, Tomas

    2005-07-01

    The Food and Drug Administration acknowledges the severity of the obesity epidemic. The Food and Drug Administration recognizes the importance of food labeling as a vehicle for dietary messages and, thus, enforces stringent guidelines to maintain the integrity of the food label. As food labels await another upgrade to make them more effective and easier to understand, the Food and Drug Administration considers what information will be most useful for consumers to make healthy choices. The causal relationship between food labels and subsequent diet choice is not well understood; more research in this area is needed. The Commissioner of the Food and Drug Administration has recently appointed an Obesity Working Group to develop proposals on pertinent topics of obesity, including the role of food labeling as a dietary guide.

  19. Mining Multi-label Data

    NASA Astrophysics Data System (ADS)

    Tsoumakas, Grigorios; Katakis, Ioannis; Vlahavas, Ioannis

    A large body of research in supervised learning deals with the analysis of single-label data, where training examples are associated with a single label λ from a set of disjoint labels L. However, training examples in several application domains are often associated with a set of labels Y ⊆ L. Such data are called multi-label.

  20. Label Review Training: Module 1: Label Basics, Page 29

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is a quiz on Module 1.

  1. Label Review Training: Module 1: Label Basics, Page 25

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review: clarity, accuracy, consistency with EPA policy, and enforceability.

  2. Soil Fumigant Labels - Methyl Bromide

    EPA Pesticide Factsheets

    Search soil fumigant pesticide labels by EPA registration number, product name, or company, and follow the link to The Pesticide Product Label System (PPLS) for details. Updated labels include new safety requirements for buffer zones and related measures.

  3. Off-Label Drug Use

    MedlinePlus

    ... their drugs for off-label uses. Off-label marketing is very different from off-label use. Why ... Employment Become a Supplier Report Fraud or Abuse Global Health ACS CAN Sign Up for Email Policies ...

  4. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1983-07-15

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  5. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1985-11-12

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label. 5 figs.

  6. Capacitive label reader

    DOEpatents

    Arlowe, H. Duane

    1985-01-01

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  7. DNA polymerase photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate labels an Escherichia coli DNA polymerase I Klenow fragment substrate binding site.

    PubMed

    Moore, B M; Jalluri, R K; Doughty, M B

    1996-09-10

    The nucleotide photoprobe 2-[(4-azidophenacyl)thio]-2'-deoxyadenosine 5'-triphosphate (1) was evaluated as a photoaffinity label of the DNA polymerase I Klenow fragment. Photolabel [3H]-1 covalently labeled the Klenow fragment with photolysis at 300 nm, reaching saturation at an approximate 1:1 mole ratio at 5.7 microM and with an EC50 (the effective concentration at 50% maximum photoincorporation) of about 0.74 microM. Saturating concentrations of poly(dA).(T)10 protect the Klenow fragment from [3H]-1 photoincorporation, and TTP at a concentration approximately equal to its KD for the free enzyme form shifts the dose-response curve for photoincorporation of [3H]-1 into the Klenow fragment by a factor of 2, indicating a competitive relationship between TTP and 1. Additionally, the photoincorporation of [3H]-1 into the Klenow fragment has an absolute requirement for magnesium, with no significant photoincorporation observed at concentrations of 1 up to 10 microM in the absence of magnesium. These results demonstrate that, as designed, photoprobe 1 binds to both the dNTP and a portion of the template-primer binding sites on the Klenow fragment. Photoaffinity labeling of the Klenow fragment by 1 yielded a single radiolabeled tryptic fragment which was isolated by HPLC; sequence analysis identified Asp732 in the peptide fragment Asp732-Ile733-His734-Arg735 as the site of covalent modification. Molecular modeling and complementary NMR analysis of the conformation of 1 indicated preferred C3'-exo and C2'-exo-C3'-endo symmetrical twist furanose ring puckers, with a high antibase conformation and a +sc C-5 torsional angle. Docking studies using Asp732 as an anchor point for the azide alpha-nitrogen on the photolabel indicate that the dNTP binding site is at the edge of the DNA binding cleft opposite the exonuclease site and that the template binding site includes helix O in the finger motif of the Klenow fragment.

  8. Like your labels?

    PubMed

    Field, Michele

    2010-01-01

    The descriptive “conventions” used on food labels are always evolving. Today, however, the changes are so complicated (partly driven by legislation requiring disclosures about environmental impacts, health issues, and geographical provenance) that these labels more often baffle buyers than enlighten them. In a light-handed manner, the article points to how sometimes reading label language can be like deciphering runes—and how if we are familiar with the technical terms, we can find a literal meaning, but still not see the implications. The article could be ten times longer because food labels vary according to cultures—but all food-exporting cultures now take advantage of our short attention-span when faced with these texts. The question is whether less is more—and if so, in this contest for our attention, what “contestant” is voted off.

  9. Label Review Training - Resources

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  10. Routing and Label Space Reduction in Label Switching Networks

    NASA Astrophysics Data System (ADS)

    Solano, Fernando; Caro, Luis Fernando; Stidsen, Thomas; Papadimitriou, Dimitri

    This chapter is devoted to the analysis and modeling of some problems related to the optimal usage of the label space in label switching networks. Label space problems concerning three different technologies and architectures - namely Multi-protocol Label Switching (MPLS), Ethernet VLAN-Label Switching (ELS) and All-Optical Label Switching (AOLS) - are discussed in this chapter. Each of these cases yields to different constraints of the general label space reduction problem. We propose a generic optimization model and, then, we describe some adaptations aiming at modeling each particular case. Simulation results are briefly discussed at the end of this chapter.

  11. Full-contact domain labeling: identification of a novel phosphoinositide binding site on gelsolin that requires the complete protein.

    PubMed

    Feng, L; Mejillano, M; Yin, H L; Chen, J; Prestwich, G D

    2001-01-30

    Gelsolin, an actin and phosphoinositide binding protein, was photoaffinity labeled using a variety of benzophenone-containing phosphoinositide polyphosphate analogues. The N-terminal half and the C-terminal half of gelsolin showed synergy in the binding of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Competitive displacement experiments with dibutyryl, dioctanoyl, or dipalmitoyl derivatives of PtdIns(4,5)P(2) suggested that, in addition to the inositol headgroup, a diacylglyceryl moiety was important for binding; these analogues also inhibited the gelsolin-severing activity of F-actin. In addition to the previously identified PtdIns(4,5)P2 binding site in the N-terminal half of gelsolin, a new binding site was identified in the C-terminal half by mapping the photocovalently modified peptide fragments. Moreover, increasing concentrations of Ca(2+) decreased the binding of the photolabile analogues to the C-terminal phosphoinositide binding site on gelsolin. A molecular model of the binding of PtdIns(4,5)P2 within two folded repeats of gelsolin has been calculated using these data.

  12. Nanostructured luminescently labeled nucleic acids.

    PubMed

    Kricka, Larry J; Fortina, Paolo; Park, Jason Y

    2017-03-01

    Important and emerging trends at the interface of luminescence, nucleic acids and nanotechnology are: (i) the conventional luminescence labeling of nucleic acid nanostructures (e.g. DNA tetrahedron); (ii) the labeling of bulk nucleic acids (e.g. single-stranded DNA, double-stranded DNA) with nanostructured luminescent labels (e.g. copper nanoclusters); and (iii) the labeling of nucleic acid nanostructures (e.g. origami DNA) with nanostructured luminescent labels (e.g. silver nanoclusters). This review surveys recent advances in these three different approaches to the generation of nanostructured luminescently labeled nucleic acids, and includes both direct and indirect labeling methods. Copyright © 2016 John Wiley & Sons, Ltd.

  13. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... QUALITY SYSTEM REGULATION Labeling and Packaging Control § 820.120 Device labeling. Each manufacturer shall establish and maintain procedures to control labeling activities. (a) Label integrity. Labels... accuracy including, where applicable, the correct expiration date, control number, storage instructions...

  14. Omega 3 (peripheral type benzodiazepine binding) site distribution in the rat immune system: an autoradiographic study with the photoaffinity ligand (/sup 3/H)PK 14105

    SciTech Connect

    Benavides, J.; Dubois, A.; Dennis, T.; Hamel, E.; Scatton, B.

    1989-04-01

    The anatomical distribution of omega 3 (peripheral type benzodiazepine binding) sites in the immune system organs of the rat has been studied autoradiographically at both macroscopic and microscopic levels of resolution using either reversible or irreversible (UV irradiation) labeling with (/sup 3/H)PK 14105. In thymus sections, (/sup 3/H)PK 14105 labeled with high affinity (Kd, derived from saturation experiments = 10.8 nM) a single population of sites which possessed the pharmacological characteristics of omega 3 sites. In the thymus gland, higher omega 3 site densities were detected in the cortex than in the medulla; in these subregions, silver grains were associated to small (10-18 microns diameter) cells. In the spleen, omega 3 sites were more abundant in the white than in the red pulp. In the white pulp, silver grains were denser in the marginal zone than in the vicinity of the central artery and labeling was, as in the thymus, associated to small cytoplasm-poor cells. In the red pulp, omega 3 site associated silver grains were observed mainly in the Bilroth cords. In the lymph nodes, the medullary region showed a higher labeling than the surrounding follicles and paracortex. A significant accumulation of silver grains was observed in the lymph node medullary cords. In the intestine, Peyer patches were particularly enriched in omega 3 sites (especially in the periphery of the follicles). The distribution of omega 3 sites in the immune system organs suggests a preferential labeling of cells of T and monocytic lineages. This is consistent with the proposed immunoregulatory properties of some omega 3 site ligands.

  15. Selective T-cell Ablation with Bismuth-213 Labeled Anti-TCR Alpha Beta as Nonmyeloablative Conditionaing for Allogeneic Canine Marrow Transplantion

    SciTech Connect

    Bethge, W. A.; Wilbur, D. Scott; Storb, R.; Hamlin, Donald K.; Santos, E. B.; Brechbiel, M. W.; Fisher, Darrell R.; Sandmaier, B. M.

    2003-06-15

    Two major immunological barriers, the host versus graft (HVG) and the graft versus host (GVH) reaction, must be overcome for successful allogeneic hematopoietic stem cell transplantation. T-cells are involved in these barriers in the major histocompatibility complex-identical settings. We hypothesized that selective ablation of T-cells using radioimmunotherapy, together with postgrafting immunosuppression, would ensure stable allogeneic engraftment. We developed a canine model of nonmyeloablative marrow transplantation in which host immune reactions are impaired by a single dose of 2 Gy total body irradiation (TBI), and where both GVH and residual HVG reactions are controlled by postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP). We substituted the alpha-emitter bismuth-213 linked to a monoclonal antibody against TCR(alpha,beta)using the metal-binding chelate CHX-A”-DTPA, for 2 Gy TBI. Biodistribution studies using a gamma-emitting indium-111-labeled anti-TCR mAb showed uptake primarily in blood, marrow, lymph nodes, spleen and liver. In a dosimetry study, 4 dogs were treated with 0.13-0.46 mg/kg TCR mAb labeled with 3.7-5.6 mCi/kg (137-207 MBq/kg) Bi-213. The treatment was administered in 6 injections on days -3 and -2 followed by transplantion of dog leukocyte antigen-identical marrow on day 0 and postgrafting immunosuppression with MMF and CSP. Therapy was well tolerated except for elevations of transaminases, which were transient in all but one dog. No other organ toxicities or signs of graft-versus-host-disease were noted. The dogs had prompt allogeneic hematopoietic engraftment and achieved stable mixed donor-host hematopoietic chimerism with donor contributions ranging from 5-55 % with >30 weeks follow up.

  16. A Deceiving Label?

    ERIC Educational Resources Information Center

    Lum, Lydia

    2009-01-01

    The author reports on the growing debate among educators on whether the umbrella Asian Pacific Islander label conceals disparities among Asian American students or provides political power in numbers. Nationally, experts say that support services aimed at not only Southeast Asians, but all Asian Pacific Islander students, remain scarce in higher…

  17. A Deceiving Label?

    ERIC Educational Resources Information Center

    Lum, Lydia

    2009-01-01

    The author reports on the growing debate among educators on whether the umbrella Asian Pacific Islander label conceals disparities among Asian American students or provides political power in numbers. Nationally, experts say that support services aimed at not only Southeast Asians, but all Asian Pacific Islander students, remain scarce in higher…

  18. From Labels to Opportunities

    ERIC Educational Resources Information Center

    Wolter, Deborah

    2017-01-01

    The author argues that to truly help young students who struggle with reading and writing--including those with identified disabilities or conditions that effect building literacy--teachers should avoid the approach of focusing on a student's deficits and creating labels for him or her (dyslexic, English language learner, and so on). A rush to…

  19. Covalent labeling of hydrosmotic toad bladder receptors with an antagonist of vasotocin

    SciTech Connect

    Eggena, P.; Buku, A.; Ma, C.L.; Somoza, L.I.; Wyssbrod, H.R.; Schwartz, I.L.; Glass, J.D.

    1987-06-01

    A photoreactive analogue of vasotocin, (1-desamino,4-lysine(azidobenzoyl),8-arginine)vasotocin (4-N3-AVT), has been examined in the isolated toad urinary bladder for biological activity and binding to hormonal receptors. Although 4-N3-AVT induced only a small increase in bladder permeability to water, it behaved as a potent inhibitor of hydrosmotic action of (8-arginine)vasotocin (AVT) and (8-arginine)vasopressin (AVP). The inhibitory action of 4-N3-AVT was readily reversed on removal of the analogue from the serosal bathing solution. On the other hand, when bladders were exposed to 4-N3-AVT in the presence of long wavelength UV light (365 nm), the inhibition by 4-N3-AVT was not reversed on washout of the analogue. The dose of vasopressin required for a half-maximal response (ED50 value) was increased from 5 X 10(-9) to 1.3 X 10(-7) M in bladders photolabeled with 4-N3-AVT and the maximal response capacity of the tissue (intrinsic activity) was reduced to 79% of nonphotolabeled controls. A crude membrane preparation derived from bladders photolabeled with 4-N3-AVT contained 72 fmol of specific binding sites for tritium-labeled vasopressin per milligram protein, whereas nonphotolabeled controls had 136 fmol of specific binding sites per milligram protein. These observations suggest that 4-N3-AVT forms a covalent bond with hydrosmotic receptors in the presence of UV light. This is the first antagonistic photoaffinity analogue observed in the toad bladder and it may serve as a useful tool for analyzing the cellular mechanism of action of antidiuretic hormone.

  20. Label Review Training: Module 1: Label Basics, Page 7

    EPA Pesticide Factsheets

    Page 7, Label Training, Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human he

  1. "Minimalist" cyclopropene-containing photo-cross-linkers suitable for live-cell imaging and affinity-based protein labeling.

    PubMed

    Li, Zhengqiu; Wang, Danyang; Li, Lin; Pan, Sijun; Na, Zhenkun; Tan, Chelsea Y J; Yao, Shao Q

    2014-07-16

    Target identification of bioactive compounds within the native cellular environment is important in biomedical research and drug discovery, but it has traditionally been carried out in vitro. Information about how such molecules interact with their endogenous targets (on and off) is currently highly limited. An ideal strategy would be one that recapitulates protein-small molecule interactions in situ (e.g., in living cells) and at the same time enables enrichment of these complexes for subsequent proteome-wide target identification. Similarly, small molecule-based imaging approaches are becoming increasingly available for in situ monitoring of a variety of proteins including enzymes. Chemical proteomic strategies for simultaneous bioimaging and target identification of noncovalent bioactive compounds in live mammalian cells, however, are currently not available. This is due to a lack of photoaffinity labels that are minimally modified from their parental compounds, yet chemically tractable using copper-free bioorthogonal chemistry. We have herein developed novel minimalist linkers containing both an alkyl diazirine and a cyclopropene. We have shown chemical probes (e.g., BD-2) made from such linkers could be used for simultaneous in situ imaging and covalent labeling of endogenous BRD-4 (an important epigenetic protein) via a rapid, copper-free, tetrazine-cyclopropene ligation reaction (k2 > 5 M(-1) s(-1)). The key features of our cyclopropenes, with their unique C-1 linkage to BRD-4-targeting moiety, are their tunable reactivity and solubility, relative stability, and synthetic accessibility. BD-2, which is a linker-modified analogue of (+)-JQ1 (a recently discovered nanomolar protein-protein-interaction inhibitor of BRD-4), was subsequently used in a cell-based proteome profiling experiment for large-scale identification of potential off-targets of (+)-JQ1. Several newly identified targets were subsequently confirmed by preliminary validation experiments.

  2. Use the Nutrition Facts Label

    MedlinePlus

    ... Features Spokespeople News Archive eNewsletters Calendar Use the Nutrition Facts Label You can help your family eat ... to some of their favorite foods. Use the Nutrition Facts label found on food packages to make ...

  3. Decode the Sodium Label Lingo

    MedlinePlus

    ... For Preschooler For Gradeschooler For Teen Decode the Sodium Label Lingo Published January 24, 2013 Print Email Reading food labels can help you slash sodium. Here's how to decipher them. "Sodium free" or " ...

  4. Labeling lake water with tritium

    USGS Publications Warehouse

    Frederick, B.J.

    1963-01-01

    A method of packaging tritiated water in a manner that facilitates safe handling in environmental labeling operations, and procedures followed in labeling a large body of water with a small volume of tritiated water are described. ?? 1963.

  5. Collective Multi-Label Classification

    DTIC Science & Technology

    2005-01-01

    there is one output random variable . We begin by de- scribing this traditional classifier, then we describe its common ex- tension to the multi- label ...dependencies among the output variables . In addition to having feature for each label -term pair, CML main- tains features accounting for label co...over all possible multi- labelings — that is, over all subsets of Y . This method is intuitively appealing: it is easy to explain, and it is informative

  6. Microgravity Science Glovebox - Labels

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Labels are overlaid on a photo (0003837) of the Microgravity Science Glovebox (MSG). The MSG is being developed by the European Space Agency (ESA) and NASA are developing the MSG for use aboard the International Space Station (ISS). Scientists will use the MSG to carry out multidisciplinary studies in combustion science, fluid physics and materials science. The MSG is managed by NASA's Marshall Space Flight Center (MSFC). Photo Credit: NASA/MSFC

  7. Food Labels Tell the Story!

    MedlinePlus

    ... My World From the Label to the Table! Food Labels Tell the Story! What is in food? Food provides your body with all of the ... your food choices. Nutrition Facts—the Labels on Food Products Beginning in 1994, the US government began ...

  8. Learning with imperfectly labeled patterns

    NASA Technical Reports Server (NTRS)

    Chittineni, C. B.

    1979-01-01

    The problem of learning in pattern recognition using imperfectly labeled patterns is considered. The performance of the Bayes and nearest neighbor classifiers with imperfect labels is discussed using a probabilistic model for the mislabeling of the training patterns. Schemes for training the classifier using both parametric and non parametric techniques are presented. Methods for the correction of imperfect labels were developed. To gain an understanding of the learning process, expressions are derived for success probability as a function of training time for a one dimensional increment error correction classifier with imperfect labels. Feature selection with imperfectly labeled patterns is described.

  9. Review of nutrition labeling formats.

    PubMed

    Geiger, C J; Wyse, B W; Parent, C R; Hansen, R G

    1991-07-01

    This article examines nutrition labeling history as well as the findings of nine research studies of nutrition labeling formats. Nutrition labeling regulations were announced in 1973 and have been periodically amended since then. In response to requests from consumers and health care professionals for revision of the labeling system, the Food and Drug Administration initiated a three-phase plan for reform of nutrition labeling in 1990. President Bush signed the Nutrition Labeling and Education Act in November 1990. Literature analysis revealed that only nine studies with an experimental design have focused on nutrition labeling since 1971. Four were conducted before 1975, which was the year that nutrition labeling was officially implemented, two were conducted in 1980, and three were conducted after 1986. Only two of the nine studies supported the traditional label format mandated by the Code of Federal Regulations, and one study partially supported it. Four of the nine studies that evaluated graphic presentations of nutrition information found that consumer comprehension of nutrition information was improved with a graphic format for nutrition labeling: three studies supported the use of bar graphs and one study supported the use of a pie chart. Full disclosure (ie, complete nutrient and ingredient labeling) was preferred by consumers in two of the three studies that examined this variable. The third study supported three types of information disclosure dependent upon socioeconomic class. In those studies that tested graphics, a bar graph format was significantly preferred and showed better consumer comprehension than the traditional format.

  10. Map labeling and its generalizations

    SciTech Connect

    Doddi, S. |; Marathe, M.V.; Mirzaian, A.; Moret, B.M.E.; Zhu, B. |

    1997-01-01

    Map labeling is of fundamental importance in cartography and geographical information systems and is one of the areas targeted for research by the ACM Computational Geometry Impact Task Force. Previous work on map labeling has focused on the problem of placing maximal uniform, axis-aligned, disjoint rectangles on the plane so that each point feature to be labeled lies at the corner of one rectangle. Here, we consider a number of variants of the map labeling problem. We obtain three general types of results. First, we devise constant-factor polynomial-time-approximation algorithms for labeling point features by rectangular labels, where the feature may lie anywhere on the boundary of its label region and where labeling rectangles may be placed in any orientation. These results generalize to the case of elliptical labels. Secondly, we consider the problem of labeling a map consisting of disjoint rectilinear fine segments. We obtain constant-factor polynomial-time approximation algorithms for the general problem and an optimal algorithm for the special case where all segments are horizontal. Finally, we formulate a bicriteria version of the map-labeling problem and provide bicriteria polynomial- time approximation schemes for a number of such problems.

  11. Supplementing national menu labeling.

    PubMed

    Hodge, James G; White, Lexi C

    2012-12-01

    The US Food and Drug Administration's forthcoming national menu labeling regulations are designed to help curb the national obesity epidemic by requiring calorie counts on restaurants' menus. However, posted calories can be easily ignored or misunderstood by consumers and fail to accurately describe the healthiness of foods. We propose supplemental models that include nutritional information (e.g., fat, salt, sugar) or specific guidance (e.g., "heart-healthy" graphics). The goal is to empower restaurant patrons with better data to make healthier choices, and ultimately to reduce obesity prevalence.

  12. 49 CFR 583.5 - Label requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... of the fuel economy label required by 15 U.S.C. 2006, or a separate label. A separate label may... case of a label that is included as part of the Monroney price information label or fuel economy label... motor vehicle equipment and that, to the best of the requester's knowledge, the outside supplier is...

  13. Food labels: a critical assessment.

    PubMed

    Temple, Norman J; Fraser, Joy

    2014-03-01

    Foods sold in packages have both front-of-package (FOP) labels and back-of-package (BOP) labels. The aim of this review is to determine the role they play in informing consumers as to the composition of foods in order to help select a healthy diet. Recent literature was evaluated and findings combined with assessments made by the authors of food labels used in the United States and Canada. Research shows that most consumers have difficulty understanding the information provided by both FOP and BOP food labels used in the United States and Canada. Research has evaluated the merits of alternative designs. FOP labels should be based on a clear and simple design. They should present information on key nutrients (total fat, saturated fat, sugar, and sodium or salt) and also energy value. They should have color and words that indicate "high," "medium," and "low" levels. Labels can also state quantity per serving. The traffic light system is the best example of this design. An extra traffic light indicating the overall health value of the food should be added. A clearer BOP label also is needed. Implementation of a new food labeling system will probably be opposed by the food industry. More research is needed into which food label designs are most effective, especially for persuading consumers to select healthier food. Both FOP and BOP food labels used in the United States and Canada need to be redesigned using a traffic light system. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Gamma-tracking and sensitivity to gamma-emitting backgrounds in SuperNEMO

    NASA Astrophysics Data System (ADS)

    Calvez, Steven

    2017-09-01

    SuperNEMO, successor to the NEMO3 experiment, is looking for the neutrinoless double beta decay. Its unique design, combining both tracking and calorimetry techniques, provides essential topological informations. Indeed, fully reconstructing the event kinematics allows a powerful background discrimination, would discriminate between the several hypothesized underlying mechanisms, but also gives access to a variety of event topologies which can be used to measure the different background contributions. The SuperNEMO software relies on a range of algorithms to ensure a faithful event reconstruction. The improved detector performance for γ detection coupled to new γ-reconstruction algorithms, based on geometrical and Time-of-Flight criteria, will not only improve the measurements of the γ-emitter backgrounds (208Tl, 214Bi...) but also increase the sensitivity for the search of ββ-decays to the excited states.

  15. Gamma-emitting radionuclide measurements at the US geological survey national water quality laboratory, Denver, Colorado

    USGS Publications Warehouse

    In, Che Yang; Ambats, E.

    1982-01-01

    Like sediment samples from Scofield Resevior in Utah were analyzed for 210Pb by the gamma-ray spectrometric method. The top 10 cm of surface sediment yielded excess 210Pb activity (excluding in situ 226Ra supported 210Pb) of 1.05 pCi/g dry weight and decreased to 0.25 pCi/g at a depth of 25 cm. Based on these data, sedimentation rate was approximately 0.49 cm/y for a total of 30 cm of lake sediment and a lake history of approximately 60 y. An alternative method of 210Pb measurements using wet chemical procedures followed by beta counting gave equivalent results. ?? 1982.

  16. (Development of gamma emitting, receptor-binding, radiotracers for imaging the brain and pancreas)

    SciTech Connect

    Not Available

    1989-01-01

    This progress report covers the period from March 1, 1987 to Feb. 28, 1988. In studies to better understand the nature of the m-AChR receptor subtypes, we have generated a manuscript which has been submitted for publication in Life sciences entitled: The effect of chronic atropine and diisopropylfluorophosphate on rat brain muscarinic acetylcholine receptor subtype concentrations. We have also developed a more direct synthesis of 3-quinuclidinyl 4-iodobenzilate and its analogues. During this contract period, we have been involved with the synthesis of analogues 3-quinuclidinyl benzilate (QNB). We have determined the affinity constants of various compounds synthesized this year for the muscarinic receptor from rat corpus striatum. We have continued our investigation of the m-AChR in pancreas. 25 refs., 3 figs., 5 tabs.

  17. Gauge for assessing gamma-emitting contaminants in pipes -- a feasibility study

    SciTech Connect

    Wilson, R.D.

    1996-12-31

    A computer modeling study was used to evaluate a new method for assessing the gamma-ray activity inside pipes that results from accumulation of naturally occurring radioactive material (NORM) or from industrial processes involving man-made radionuclides. Experiments conducted with a physical model of an analogue to the measurement problem verified aspects of the computer simulations. This new assessment method that incorporates a pipe scale gauge appears to be feasible and to have broad application for solving environmental remediation problems and for meeting certain needs of the petroleum industry.

  18. Development of gamma-emitting, receptor binding radiotracers for imaging the brain and pancreas

    SciTech Connect

    Reba, R.C.

    1990-01-01

    This progress report covers period from Nov. 1, 1989 to Aug. 31, 1990. The long term objective was to develop receptor-binding radiotracers for SPECT or PET imaging of CNS or peripheral nervous system. The specific chemistry aims, as understood on the basis of past findings, were: to synthesize and develop a more polar analogs of 4IQNB, possessing similar binding characteristics but eliminated more rapidly from the surrounding tissues and the target organ, to design a method of introducing a technetium chelating group onto a molecule or cholinergic agent without drastic lowering of its apparent affinity, to synthesize and develop radiotracers based on m-AChR antagonists selective for one of the subtypes of the receptor. The chemistry service aims were to prepare and characterize (R,R)- and (R,S)-4IQNB and derivatives, to provide the triazene intermediate to other investigators, and to provide ({sup 123}I)4IQNB for in vivo imaging. The biochemistry aims were to characterize the vitro and in vivo properties of novel compounds and to perform the pharmacokinetic studies. 3 refs., 5 tabs.

  19. Optimizing connected component labeling algorithms

    SciTech Connect

    Wu, Kesheng; Otoo, Ekow; Shoshani, Arie

    2005-01-16

    This paper presents two new strategies that can be used to greatly improve the speed of connected component labeling algorithms. To assign a label to a new object, most connected component labeling algorithms use a scanning step that examines some of its neighbors. The first strategy exploits the dependencies among them to reduce the number of neighbors examined. When considering 8-connected components in a 2D image, this can reduce the number of neighbors examined from four to one in many cases. The second strategy uses an array to store the equivalence information among the labels. This replaces the pointer based rooted trees used to store the same equivalence information. It reduces the memory required and also produces consecutive final labels. Using an array instead of the pointer based rooted trees speeds up the connected component labeling algorithms by a factor of 5 {approx} 100 in our tests on random binary images.

  20. Optimizing connected component labeling algorithms

    NASA Astrophysics Data System (ADS)

    Wu, Kesheng; Otoo, Ekow; Shoshani, Arie

    2005-04-01

    This paper presents two new strategies that can be used to greatly improve the speed of connected component labeling algorithms. To assign a label to a new object, most connected component labeling algorithms use a scanning step that examines some of its neighbors. The first strategy exploits the dependencies among them to reduce the number of neighbors examined. When considering 8-connected components in a 2D image, this can reduce the number of neighbors examined from four to one in many cases. The second strategy uses an array to store the equivalence information among the labels. This replaces the pointer based rooted trees used to store the same equivalence information. It reduces the memory required and also produces consecutive final labels. Using an array instead of the pointer based rooted trees speeds up the connected component labeling algorithms by a factor of 5 ~ 100 in our tests on random binary images.

  1. Principles of protein labeling techniques.

    PubMed

    Obermaier, Christian; Griebel, Anja; Westermeier, Reiner

    2015-01-01

    Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.

  2. Label Structured Cell Proliferation Models

    DTIC Science & Technology

    2010-06-16

    variable as a mass-like quantity. The specific model for the dynamics of life and death processes of a population of cells labeled with CFSE is proposed in... variables = + where < 0 is label degradation velocity. Because we really don’t understand completely the degradation process (there appears to be...little agreement as to what variables on which this velocity might depend) and to allow for generality (other labels that might be used may well

  3. Label Ranking Algorithms: A Survey

    NASA Astrophysics Data System (ADS)

    Vembu, Shankar; Gärtner, Thomas

    Label ranking is a complex prediction task where the goal is to map instances to a total order over a finite set of predefined labels. An interesting aspect of this problem is that it subsumes several supervised learning problems, such as multiclass prediction, multilabel classification, and hierarchical classification. Unsurprisingly, there exists a plethora of label ranking algorithms in the literature due, in part, to this versatile nature of the problem. In this paper, we survey these algorithms.

  4. GEO label: The General Framework for Labeling and Certification

    NASA Astrophysics Data System (ADS)

    Bye, B. L.; McCallum, I.; Maso, J.

    2012-04-01

    The Group on Earth Observations (GEO) is coordinating efforts to build a Global Earth Observation System of Systems, or GEOSS. As part of a strategy to increase the involvement of the science and technology community in GEOSS, both as users and developers of GEOSS itself, GEO decided to develop a GEO label concept related to the scientific relevance, quality, acceptance and societal needs for services and data sets of GEOSS. The development of a GEO label is included in the GEO work plan and several projects address the challenges of developing a GEO label concept. Within the different projects developing the GEO label, various perspectives and approaches are being applied. In order to arrive at a generally accepted GEO label concept, a common understanding and basic knowledge of labeling is necessary. Assessment of quality of internationally standardized Earth observation data products implies possible certification. A general understanding of the framework for international standards and certification will also contribute to a more coherent discussion and more efficient development of a GEO label. We will describe the general labeling and certification framework emphasizing the relation to the three elements of the GEO label: quality, user acceptance and relevance. Based on a survey of international labels done by the EGIDA project, we have analyzed the legal framework and organization of labels and certification. We will discuss the frameworks for certification, user ratings, registration and analysis of user requirements. Quality assessment is a particular focus of the analysis and is based on the work done by the GeoViQua project. A GEO label will function both as a data distribution strategy and as a general management system for data. Through a label users can compare different data sets and get access to more information about the relevant data, including quality. A label will provide traceability of data both in the interest of users as well as data

  5. Labeling conventions in isoelectronic sequences

    SciTech Connect

    Maniak, S.T.; Curtis, L.J. )

    1990-08-01

    The isoelectronic exposition of atomic structure properties involves labeling ambiguities when more than one level of the same total angular momentum and parity is present, and an energy ordered labeling of these levels can lead to apparent isoelectronic discontinuities. For example, in the recent oscillator strength calculations for S-like ions by Saloman and Kim (Phys. Rev. A 38, 577 (1988)), abrupt changes in the rates were sometimes observed between one isoelectronic element and the next. We suggest an alternative labeling scheme that removes these discontinuities and produces a smooth isoelectronic variation. This alternative labeling offers advantages for data exposition and for semiempirical interpolation and extrapolation.

  6. Labeled Cocaine Analogs

    DOEpatents

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-03-30

    Novel methods for positron emission tomography or single photon emission spectroscopy using tracer compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)napthyl Y in .beta. configuration is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, The compounds bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  7. Laser labeling, a safe technology to label produce

    USDA-ARS?s Scientific Manuscript database

    Laser labeling of fruits and vegetables is an alternative means to label produce. Low energy CO2 laser beams etch the surface showing the contrasting underlying layer. These etched surfaces can promote water loss and potentially allow for entry of decay organisms. The long-term effects of laser labe...

  8. Laser labeling, a safe technology to label produce

    USDA-ARS?s Scientific Manuscript database

    Labeling of the produce has gained marked attention in recent years. Laser labeling technology involves the etching of required information on the surface using a low energy CO2 laser beam. The etching forms alphanumerical characters by pinhole dot matrix depressions. These openings can lead to wat...

  9. Nutrition Marketing on Food Labels

    ERIC Educational Resources Information Center

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

  10. Nutrition Marketing on Food Labels

    ERIC Educational Resources Information Center

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

  11. Health claims on food labels.

    PubMed

    Tollefson, L

    1994-03-01

    Food and drug law requires that the ingredients in most foods be disclosed on their labels, but until recently there was no requirement that nutrition information be provided. The Nutrition Labeling and Education Act of 1990 (NLEA), passed on November 8, 1990, mandated the Food and Drug Administration to establish regulations requiring most foods to have a uniform nutrition label showing the amount of calories, calories from fat, total fat, saturated fatty acids, cholesterol, total carbohydrates, complex carbohydrates, sugars, fiber, protein, and sodium. The Act also establishes the circumstances under which content claims and disease claims may be made about nutrients in food. This paper briefly discusses recent changes in the food label brought about by the NLEA and focuses on health claims on food labels.

  12. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium content...

  13. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium content...

  14. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium content...

  15. 16 CFR 1633.12 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... the Standard shall bear a permanent, conspicuous, and legible label(s) containing the following... with black text. The label text shall comply with the following format requirements: (1) All... as needed for varying information. The label must be white with black text. The label shall contain...

  16. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of...

  17. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of...

  18. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of...

  19. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  20. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium content...

  1. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium content...

  2. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium content...

  3. Labeled Cocaine Analogs

    DOEpatents

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-01-26

    Novel compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)naphthyl Y in .beta. configuration is Y.sub.1 or Y.sub.2, where Y.sub.1 is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, and Y.sub.2 is 2-methanesulfonyloxy ethoxy, 3-methanesulfonyloxy propoxy, 4-methanesulfonyloxy butoxy, 2-methanesulfonyloxy cyclopropoxy, 2 or 3-methanesulfonyloxy cyclobutoxy, 1'methanesulfonyloxy isopropoxy, 1'-fluoro, 3'-methanesulfonyloxy isopropoxy, 1'-methanesulfonyloxy, 3'-fluoro isopropoxy, 1'-methanesulfonyloxy isobutoxy, or 4'-methanesulfonyloxy isobutoxy bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  4. Synthesis Of Labeled Metabolites

    DOEpatents

    Martinez, Rodolfo A.; Silks, III, Louis A.; Unkefer, Clifford J.; Atcher, Robert

    2004-03-23

    The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

  5. Algorithms for Labeling Focus Regions.

    PubMed

    Fink, M; Haunert, Jan-Henrik; Schulz, A; Spoerhase, J; Wolff, A

    2012-12-01

    In this paper, we investigate the problem of labeling point sites in focus regions of maps or diagrams. This problem occurs, for example, when the user of a mapping service wants to see the names of restaurants or other POIs in a crowded downtown area but keep the overview over a larger area. Our approach is to place the labels at the boundary of the focus region and connect each site with its label by a linear connection, which is called a leader. In this way, we move labels from the focus region to the less valuable context region surrounding it. In order to make the leader layout well readable, we present algorithms that rule out crossings between leaders and optimize other characteristics such as total leader length and distance between labels. This yields a new variant of the boundary labeling problem, which has been studied in the literature. Other than in traditional boundary labeling, where leaders are usually schematized polylines, we focus on leaders that are either straight-line segments or Bezier curves. Further, we present algorithms that, given the sites, find a position of the focus region that optimizes the above characteristics. We also consider a variant of the problem where we have more sites than space for labels. In this situation, we assume that the sites are prioritized by the user. Alternatively, we take a new facility-location perspective which yields a clustering of the sites. We label one representative of each cluster. If the user wishes, we apply our approach to the sites within a cluster, giving details on demand.

  6. Label Review Training: Module 1: Label Basics, Page 5

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  7. 76 FR 75809 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ... Protection Reference Center was launched as a Web page in February 1999. The Web page includes a PowerPoint presentation titled ``Labeling 101,'' which is used by the Agency as a teaching tool at workshops on meat and...

  8. Label Review Training: Module 1: Label Basics, Page 2

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  9. Label Review Training: Module 1: Label Basics, Page 9

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  10. Label Review Training: Module 1: Label Basics, Page 8

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human he

  11. Label Review Training: Module 1: Label Basics, Page 6

    EPA Pesticide Factsheets

    Page 6, Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment

  12. Label Review Training: Module 1: Label Basics, Page 4

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  13. Label Review Training: Module 1: Label Basics, Page 3

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  14. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling..., muscle weakness, and osteomalacia. (b) Professional labeling for an antacid-antiflatulent combination...

  15. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling..., muscle weakness, and osteomalacia. (b) Professional labeling for an antacid-antiflatulent combination...

  16. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling..., muscle weakness, and osteomalacia. (b) Professional labeling for an antacid-antiflatulent combination...

  17. Mobile Application for Pesticide Label Matching

    EPA Pesticide Factsheets

    The label matching application will give inspectors the ability to instantly compare pesticide product labels against state and federal label databases via their cell phone, tablet or other mobile device.

  18. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  19. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  20. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  1. 49 CFR 172.442 - CORROSIVE label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.442 CORROSIVE label. (a) Except for size and color, the CORROSIVE label...

  2. 49 CFR 172.442 - CORROSIVE label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.442 CORROSIVE label. (a) Except for size and color, the CORROSIVE label...

  3. 49 CFR 172.442 - CORROSIVE label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.442 CORROSIVE label. (a) Except for size and color, the CORROSIVE label...

  4. Soil Fumigant Labels - Dimethyl Disulfide (DMDS)

    EPA Pesticide Factsheets

    Search by EPA registration number, product name, or company and follow the link to the Pesticide Product Labeling System (PPLS) for label details. Updated labels include new safety requirements for buffer zones and related measures.

  5. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 9 2011-04-01 2011-04-01 false Location and size of symbol on label and labeling... AND PACKAGING REQUIREMENTS FOR CONTROLLED SUBSTANCES § 1302.04 Location and size of symbol on label and labeling. The symbol shall be prominently located on the label or the labeling of the commercial...

  6. 78 FR 24211 - Draft Guidance for Industry on Safety Considerations for Container Labels and Carton Labeling...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-24

    ... Container Labels and Carton Labeling Design To Minimize Medication Errors; Availability AGENCY: Food and... Labels and Carton Labeling Design to Minimize Medication Errors.'' The draft guidance focuses on safety aspects of the container label and carton labeling design for prescription drug and biological products...

  7. Approximation Algorithms for Free-Label Maximization

    NASA Astrophysics Data System (ADS)

    de Berg, Mark; Gerrits, Dirk H. P.

    Inspired by air traffic control and other applications where moving objects have to be labeled, we consider the following (static) point labeling problem: given a set P of n points in the plane and labels that are unit squares, place a label with each point in P in such a way that the number of free labels (labels not intersecting any other label) is maximized. We develop efficient constant-factor approximation algorithms for this problem, as well as PTASs, for various label-placement models.

  8. New Labeling for Neonicotinoid Pesticides

    EPA Pesticide Factsheets

    These documents, a graphic of the bee advisory box and letters to pesticide registrants, describe steps by EPA to change pesticide labels to better protect pollinators by being clearer and more precise in their directions for pesticide application.

  9. Locating the Vehicle Emissions Label

    EPA Pesticide Factsheets

    The EPA vehicle emissions label is entitled Vehicle Emission Control Information and contains the name and trademark of the manufacturer and an unconditional statement of compliance with EPA emission regulations.

  10. How to Read Drug Labels

    MedlinePlus

    ... Home > Healthy Aging > Drugs and alternative medicine Healthy Aging How to read drug labels Printer-friendly version ... html Connect with other organizations National Institute on Aging, NIH, HHS http://www.nia.nih.gov/ U.S. ...

  11. Meat and Poultry Labeling Terms

    MedlinePlus

    ... Food Safety and Inspection Service and the Agriculture Marketing Service have officially evaluated a meat product for ... refer to these factsheets from the USDA Agricultural Marketing Service: Organic Food Standards and Labels: The Facts ...

  12. "Off-Label" Drug Use

    MedlinePlus

    ... for a single ailment. This is simply the nature of both drug devel- opment and clinical medicine. ... off-label use of cancer drugs. Given the nature of cancer and cancer drugs, this approach sounds ...

  13. Relaxation labeling using modular operators

    SciTech Connect

    Duncan, J.S.; Frei, W.

    1983-01-01

    Probabilistic relaxation labeling has been shown to be useful in image processing, pattern recognition, and artificial intelligence. The approaches taken to date have been encumbered with computationally extensive summations which generally prevent real-time operation and/or easy hardware implementation. The authors present a new and unique approach to the relaxation labeling problem using modular, VLSI-oriented hierarchical complex operators. One of the fundamental concepts of this work is the representation of the probability distribution of the possible labels for a given object (pixel) as an ellipse, which may be summed with neighboring object's distribution ellipses, resulting in a new, relaxed label space. The mathematical development of the elliptical approach will be presented and compared to more classical approaches, and a hardware block diagram that shows the implementation of the relaxation scheme using vlsi chips will be presented. Finally, results will be shown which illustrate applications of the modular scheme, iteratively, to both edges and lines. 13 references.

  14. Label-Free Receptor Assays

    PubMed Central

    Fang, Ye

    2010-01-01

    Label-free biosensors offer integrated, kinetic and multi-parametric measures of receptor biology and ligand pharmacology in whole cells. Being highly sensitive and pathway-unbiased, label-free receptor assays can be used to probe the systems cell biology including pleiotropic signaling of receptors, and to characterize the functional selectivity and phenotypic pharmacology of ligand molecules. These assays provide a new dimension for elucidating receptor biology and for facilitating drug discovery. PMID:21221420

  15. Label-Free Receptor Assays.

    PubMed

    Fang, Ye

    2011-01-01

    Label-free biosensors offer integrated, kinetic and multi-parametric measures of receptor biology and ligand pharmacology in whole cells. Being highly sensitive and pathway-unbiased, label-free receptor assays can be used to probe the systems cell biology including pleiotropic signaling of receptors, and to characterize the functional selectivity and phenotypic pharmacology of ligand molecules. These assays provide a new dimension for elucidating receptor biology and for facilitating drug discovery.

  16. Electrothermal branding for embryo labeling.

    PubMed

    Wang, L; Beebe, D J; Williams, A R; Easley, K D

    1997-11-01

    A novel embryo labeling technique based on electrothermal branding is developed. Two types of micro branding irons are fabricated and tested. One utilizes 25 microns tungsten wire as the heating element. The other utilizes surface micromachining techniques to fabricate polysilicon branding irons. The thermal behavior of the branding irons and the heat distributions in the embryos are analytically modeled. Micron-scale labels on unfertilized bovine embryos are achieved.

  17. Availability of Spanish prescription labels.

    PubMed

    Sharif, Iman; Lo, Sarah; Ozuah, Philip O

    2006-02-01

    The research team conducted a cross-sectional telephone survey of all pharmacies in the Bronx, New York (99.4% participation rate) to determine availability of Spanish prescription labels. One hundred twenty five pharmacies (78%) were small independent pharmacies; 36 (22%) were large-chain pharmacies. Overall, 111 (69%) stated that they could provide prescription labels in Spanish. Overall, for all the pharmacy ZIP codes, the mean proportion of the population that was Spanish-speaking was 46.8% (range 11% to 71.6%). Seventy-eight (48%) pharmacies were located in areas where more than 50% of the population were Spanish-speaking, 48 (30%) were located in areas with 25.1-50% Spanish-speakers, and 35 (22%) were in areas with up to 25% Spanish-speakers. Small independent pharmacies were more likely than large chain pharmacies to provide prescription labels in Spanish (71% vs. 61%, p=0.25). All the pharmacists commented that a patient must specifically request a Spanish prescription label in order to receive one. Pharmacies located in areas with the highest proportion of Spanish speakers were more likely to provide prescription labels in Spanish (82% vs. 62% vs. 49%; p=.001). Of the 111 pharmacies that could provide Spanish labels, 95 (86%) used a computer program to perform the translation and 16(14%) used a lay employee. Of pharmacies using a computer program, only one had a Spanish-speaking pharmacist who could check and correct the computer translations.

  18. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 25 2014-07-01 2014-07-01 false Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  19. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 26 2012-07-01 2011-07-01 true Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  20. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  1. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  2. 40 CFR 211.104 - Label content.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 25 2011-07-01 2011-07-01 false Label content. 211.104 Section 211.104 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.104 Label content. The following data and information must be on the label of all products for...

  3. 7 CFR 70.45 - Misleading labeling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Misleading labeling. 70.45 Section 70.45 Agriculture... Misleading labeling. The use of the terms “Government Graded” and “Federal-State Graded” or terms of similar import in the labeling or advertising of any product without stating in the labeling or advertisement the...

  4. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  5. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  6. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  7. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  8. 21 CFR 225.180 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Labeling. 225.180 Section 225.180 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL CURRENT GOOD MANUFACTURING PRACTICE FOR MEDICATED FEEDS Labeling § 225.180 Labeling. Labels shall...

  9. 78 FR 2200 - Energy Labeling Rule

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-10

    ... CFR Part 305 RIN 3084-AB15 Energy Labeling Rule AGENCY: Federal Trade Commission (FTC or Commission..., clarifying testing requirements and enforcement provisions, improving online energy label disclosures, and.... Appliance Labeling Rule The Commission issued the Appliance Labeling Rule pursuant to the Energy Policy...

  10. How to Read a Nutrition Facts Label

    MedlinePlus

    ... Games, and the Internet How to Read a Nutrition Facts Label (Video) KidsHealth > For Parents > How to Read a Nutrition Facts Label (Video) Print A A A en ... nutricionales (video) Most packaged foods come with a Nutrition Facts label. These labels have a lot of ...

  11. 40 CFR 211.108 - Sample label.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Sample label. 211.108 Section 211.108 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.108 Sample label. Examples of labels conforming to the requirements...

  12. 21 CFR 610.60 - Container label.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Container label. 610.60 Section 610.60 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.60 Container label. (a) Full label. The...

  13. 75 FR 41696 - Appliance Labeling Rule

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-07-19

    ...Section 321 of the Energy Independence and Security Act of 2007 requires the Commission to consider the effectiveness of current labeling requirements for lamps (commonly referred to as light bulbs) and alternative labeling approaches. After holding a public meeting, conducting consumer research, issuing proposed changes to existing labeling requirements, and reviewing public comments, the Commission announces final amendments to the lamp labeling requirements in the Appliance Labeling Rule. The Commission also seeks further comment on several issues for consideration in any subsequent rulemaking.

  14. Nutrition Labeling Using a Computer Program

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    The 1990 Nutrition Labeling and Education Act mandated nutritional labeling of most foods. As a result, a large portion of food analysis is performed for nutritional labeling purposes. A food labeling guide and links to the complete nutritional labeling regulations are available online at http://vm.cfsan.fda.gov/˜dms/flg-toc.html. However, interpretation of these regulations and the appropriate usage of rounding rules, available nutrient content claims, reference amounts, and serving size can be difficult.

  15. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 2 2012-10-01 2012-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  16. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 2 2011-10-01 2011-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  17. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 2 2014-10-01 2014-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  18. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  19. 49 CFR 172.430 - POISON label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 2 2013-10-01 2013-10-01 false POISON label. 172.430 Section 172.430... SECURITY PLANS Labeling § 172.430 POISON label. (a) Except for size and color, the POISON label must be as follows: EC02MR91.029 (b) In addition to complying with § 172.407, the background on the POISON label...

  20. 76 FR 19237 - Food Labeling; Calorie Labeling of Articles of Food in Vending Machines

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-06

    ... 11 and 101 Food Labeling; Calorie Labeling of Articles of Food in Vending Machines; Proposed Rule #0... Labeling; Calorie Labeling of Articles of Food in Vending Machines AGENCY: Food and Drug Administration, HHS. ACTION: Proposed rule. SUMMARY: To implement the vending machine labeling provisions of the...

  1. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.

    1987-05-22

    This invention involved a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide in activators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography. 2 figs.

  2. Positron emitter labeled enzyme inhibitors

    DOEpatents

    Fowler, Joanna S.; MacGregor, Robert R.; Wolf, Alfred P.; Langstrom, Bengt

    1990-01-01

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  3. Metrics for Labeled Markov Systems

    NASA Technical Reports Server (NTRS)

    Desharnais, Josee; Jagadeesan, Radha; Gupta, Vineet; Panangaden, Prakash

    1999-01-01

    Partial Labeled Markov Chains are simultaneously generalizations of process algebra and of traditional Markov chains. They provide a foundation for interacting discrete probabilistic systems, the interaction being synchronization on labels as in process algebra. Existing notions of process equivalence are too sensitive to the exact probabilities of various transitions. This paper addresses contextual reasoning principles for reasoning about more robust notions of "approximate" equivalence between concurrent interacting probabilistic systems. The present results indicate that:We develop a family of metrics between partial labeled Markov chains to formalize the notion of distance between processes. We show that processes at distance zero are bisimilar. We describe a decision procedure to compute the distance between two processes. We show that reasoning about approximate equivalence can be done compositionally by showing that process combinators do not increase distance. We introduce an asymptotic metric to capture asymptotic properties of Markov chains; and show that parallel composition does not increase asymptotic distance.

  4. Positron emitter labeled enzyme inhibitors

    SciTech Connect

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-04-03

    This invention involves a new strategy for imaging and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography.

  5. Learning With Auxiliary Less-Noisy Labels.

    PubMed

    Duan, Yunyan; Wu, Ou

    2016-04-06

    Obtaining a sufficient number of accurate labels to form a training set for learning a classifier can be difficult due to the limited access to reliable label resources. Instead, in real-world applications, less-accurate labels, such as labels from nonexpert labelers, are often used. However, learning with less-accurate labels can lead to serious performance deterioration because of the high noise rate. Although several learning methods (e.g., noise-tolerant classifiers) have been advanced to increase classification performance in the presence of label noise, only a few of them take the noise rate into account and utilize both noisy but easily accessible labels and less-noisy labels, a small amount of which can be obtained with an acceptable added time cost and expense. In this brief, we propose a learning method, in which not only noisy labels but also auxiliary less-noisy labels, which are available in a small portion of the training data, are taken into account. Based on a flipping probability noise model and a logistic regression classifier, this method estimates the noise rate parameters, infers ground-truth labels, and learns the classifier simultaneously in a maximum likelihood manner. The proposed method yields three learning algorithms, which correspond to three prior knowledge states regarding the less-noisy labels. The experiments show that the proposed method is tolerant to label noise, and outperforms classifiers that do not explicitly consider the auxiliary less-noisy labels.

  6. 78 FR 66826 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-07

    ... product as ``organic'' or containing organic ingredients; (3) claims that are undefined in FSIS... labeling errors resulted from production mistakes, such as packaging the product in the wrong box. More... poultry products inspection regulations to expand the circumstances in which FSIS will generically...

  7. Food labeling: gluten-free labeling of foods. Final rule.

    PubMed

    2013-08-05

    The Food and Drug Administration (FDA or we) is issuing a final rule to define the term "gluten-free'' for voluntary use in the labeling of foods. The final rule defines the term "gluten-free'' to mean that the food bearing the claim does not contain an ingredient that is a gluten-containing grain (e.g., spelt wheat); an ingredient that is derived from a gluten-containing grain and that has not been processed to remove gluten (e.g., wheat flour); or an ingredient that is derived from a gluten-containing grain and that has been processed to remove gluten (e.g., wheat starch), if the use of that ingredient results in the presence of 20 parts per million (ppm) or more gluten in the food (i.e., 20 milligrams (mg) or more gluten per kilogram (kg) of food); or inherently does not contain gluten; and that any unavoidable presence of gluten in the food is below 20 ppm gluten (i.e., below 20 mg gluten per kg of food). A food that bears the claim "no gluten,'' "free of gluten,'' or "without gluten'' in its labeling and fails to meet the requirements for a "gluten-free'' claim will be deemed to be misbranded. In addition, a food whose labeling includes the term "wheat'' in the ingredient list or in a separate "Contains wheat'' statement as required by a section of the Federal Food, Drug, and Cosmetic Act (the FD&C Act) and also bears the claim "gluten-free'' will be deemed to be misbranded unless its labeling also bears additional language clarifying that the wheat has been processed to allow the food to meet FDA requirements for a "gluten-free'' claim. Establishing a definition of the term "gluten-free'' and uniform conditions for its use in food labeling will help ensure that individuals with celiac disease are not misled and are provided with truthful and accurate information with respect to foods so labeled. We are issuing the final rule under the Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA).

  8. A New Component Labelling And Merging Algorithm

    NASA Astrophysics Data System (ADS)

    Lochovsky, Amelia F.

    1987-10-01

    Component labelling is an important part of region analysis in image processing. Component labelling consists of assigning labels to pixels in the image such that adjacent pixels are given the same labels. There are various approaches to component labelling. Some require random access to the processed image; some assume special structure of the image such as a quad tree. Algorithms based on sequential scan of the image are attractive to hardware implementation. One method of labelling is based on a fixed size local window which includes the previous line. Due to the fixed size window and the sequential fashion of the labelling process, different branches of the same object may be given different labels and later found to be connected to each other. These labels are con-sidered to be equivalent and must later be collected to correctly represent one single object. This approach can be found in [F,FE,R]. Assume an input binary image of size NxM. Using these labelling algorithms, the number of equivalent pair generated is bounded by O(N*M). The number of distinct labels is also bounded by O(N*M). There is no known algorithm that merge the equivalent label pairs in time linear to the number of pairs, that is in time bounded by O(N*M). We propose a new labelling algorithm which interleaves the labelling with the merging process. The labelling and the merging are combined in one algorithm. Merged label information is kept in an equivalent table which is used to guide the labelling. In general , the algorithm produces fewer equivalent label pairs. The combined labelling and merging algorithm is O(N*M), where NxM is the size of the image. Section II describes the algorithm. Section III gives some examples We discuss implementation issues in section IV and further discussion and conclusion are given in Section V.

  9. The Labelling Approach to Deviance.

    ERIC Educational Resources Information Center

    Rains, Prudence M.; Kitsuse, John L.; Duster, Troy; Freidson, Eliot

    2003-01-01

    This reprint of one chapter from the 1975 text, "Issues in the Classification of Children" by Nicholas Hobbs and others, addresses the theoretical, methodological, and empirical issues involved in the "labeling" approach to the sociology of deviance. It examines the social process of classification, the use of classification in social agencies,…

  10. When Diagnostic Labels Mask Trauma

    ERIC Educational Resources Information Center

    Foltz, Robert; Dang, Sidney; Daniels, Brian; Doyle, Hillary; McFee, Scott; Quisenberry, Carolyn

    2013-01-01

    A growing body of research shows that many seriously troubled children and adolescents are reacting to adverse life experiences. Yet traditional diagnostic labels are based on checklists of surface symptoms. Distracted by disruptive behavior, the common response is to medicate, punish, or exclude rather than respond to needs of youth who have…

  11. How to read food labels

    MedlinePlus

    ... 24 liters) cooked. If you eat 2 cups (0.48 liters) at a meal, you are eating 2 servings. That is 2 times the amount of the calories, fats, and other items listed on the label. Calorie information tells you the number of calories in ...

  12. Revisiting Labels: "Hearing" or Not?

    ERIC Educational Resources Information Center

    Rhoades, Ellen A.

    2010-01-01

    This position paper briefly presents evidence-based findings pertaining to the language of labels for people with hearing loss that relate to stigma, expectation levels, stereotypes, and self-fulfilling prophecies. These constructs are important for auditory-based practitioners, administrators, policymakers, students, families, and persons with…

  13. The Labelling Approach to Deviance.

    ERIC Educational Resources Information Center

    Rains, Prudence M.; Kitsuse, John L.; Duster, Troy; Freidson, Eliot

    2003-01-01

    This reprint of one chapter from the 1975 text, "Issues in the Classification of Children" by Nicholas Hobbs and others, addresses the theoretical, methodological, and empirical issues involved in the "labeling" approach to the sociology of deviance. It examines the social process of classification, the use of classification in social agencies,…

  14. When Diagnostic Labels Mask Trauma

    ERIC Educational Resources Information Center

    Foltz, Robert; Dang, Sidney; Daniels, Brian; Doyle, Hillary; McFee, Scott; Quisenberry, Carolyn

    2013-01-01

    A growing body of research shows that many seriously troubled children and adolescents are reacting to adverse life experiences. Yet traditional diagnostic labels are based on checklists of surface symptoms. Distracted by disruptive behavior, the common response is to medicate, punish, or exclude rather than respond to needs of youth who have…

  15. Revisiting Labels: "Hearing" or Not?

    ERIC Educational Resources Information Center

    Rhoades, Ellen A.

    2010-01-01

    This position paper briefly presents evidence-based findings pertaining to the language of labels for people with hearing loss that relate to stigma, expectation levels, stereotypes, and self-fulfilling prophecies. These constructs are important for auditory-based practitioners, administrators, policymakers, students, families, and persons with…

  16. Psychological effectiveness of carbon labelling

    NASA Astrophysics Data System (ADS)

    Beattie, Geoffrey

    2012-04-01

    Despite the decision by supermarket-giant Tesco to delay its plan to add carbon-footprint information onto all of its 70,000 products, carbon labelling, if carefully designed, could yet change consumer behaviour. However, it requires a new type of thinking about consumers and much additional work.

  17. Reversible and irreversible labeling and autoradiographic localization of the cerebral histamine H2 receptor using ( sup 125 I)iodinated probes

    SciTech Connect

    Ruat, M.; Traiffort, E.; Bouthenet, M.L.; Schwartz, J.C.; Hirschfeld, J.; Buschauer, A.; Schunack, W. )

    1990-03-01

    Iodoaminopotentidine (I-APT)--i.e., N-(2-(4-amino-3-iodobenzamido)ethyl)-N'-cyano-N''-(3-(3- (1-piperidinylmethyl)phenoxy)propyl)guanidine--represents one of the most potent H2-receptor antagonists known so far. In membranes of guinea pig brain 125I-APT bound reversibly, selectively, and with high affinity (Kd = 0.3 nM) to a homogeneous population of sites unambiguously identified as H2 receptors by inhibition studies conducted with a large panel of antagonists. 125I-APT binding was also inhibited by histamine, and the effect was modulated by a guanyl nucleotide, which is consistent with the association of the H2 receptor with a guanine nucleotide binding regulatory protein. The low nonspecific binding of 125I-APT generated high contrast autoradiographic pictures in brain sections and established the precise distribution of H2 receptors. Their highly heterogeneous distribution and laminated pattern in some areas suggest their major association with neuronal elements. These localizations were more consistent than those of H1 receptors with the distribution of histaminergic projections, indicating that H2 receptors mediate a larger number of postsynaptic actions of histamine--e.g., in striatum. Colocalizations of H1 and H2 receptors in some areas account for their known synergistic interactions in cAMP formation induced by histamine. The distribution of 125I-APT binding sites did not strictly parallel that of the H2-receptor-linked adenylate cyclase activity, which may reflect heterogeneity among H2 receptors. After UV irradiation and SDS/PAGE analysis, (125I)iodoazidopotentidine (125I-AZPT), a photoaffinity probe derived from 125I-APT, was covalently incorporated in several peptides, among which the labeling of two peptides of 59 and 32 kDa was prevented by H2 antagonists, suggesting that they correspond to H2-receptor binding peptides or proteolysis products of the latter.

  18. The labeling debate in the United States.

    PubMed

    Marchant, Gary E; Cardineau, Guy A

    2013-01-01

    The mandatory labeling of genetically modified (GM) food has become the predominant policy issue concerning biotechnology in the United States. The controversy over GM labeling is being debated at several different levels and branches of government. At the federal level, the Food and Drug Administration, which has primary jurisdiction over food safety and labeling, has steadfastly refused to require labeling of GM foods since 1992 based on its conclusion that GM foods as a category present no unique or higher risks than other foods. Proposed legislation has been repeatedly introduced in the US. Congress over the years to mandate GM labeling, but has made very little progress. With federal labeling requirements apparently stalled, the main activity has switched to the state level, where numerous individual states are considering mandatory GM labeling, either through legislation or proposition. The debate over GM labeling, at both the federal and state levels, has focused on five issues: (1) public opinion; (2) the legality of labeling requirements; (3) the risks and benefits of GM foods; (4) the costs and burdens of GM labeling; and (5) consumer choice. While the pro-labeling forces argue that all of these factors weigh in favor of mandatory GM labeling, a more careful evaluation of the evidence finds that all five factors weigh decisively against mandatory GM labeling requirements.

  19. Soil Fumigant Labels - Metam Sodium/Potassium

    EPA Pesticide Factsheets

    Search by EPA registration number, product name, or company; and follow the link to the Pesticide Product Label System (PPLS) for details. Updated labels include new safety requirements for buffer zones and related measures.

  20. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) Display appropriate hazard warnings; (c) Use a chemical identity that permits cross-referencing between the list of hazardous chemicals, a chemical's label, and its MSDS; and (d) Include on labels for... information about the hazardous chemical....

  1. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...) Display appropriate hazard warnings; (c) Use a chemical identity that permits cross-referencing between the list of hazardous chemicals, a chemical's label, and its MSDS; and (d) Include on labels for... information about the hazardous chemical....

  2. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...) Display appropriate hazard warnings; (c) Use a chemical identity that permits cross-referencing between the list of hazardous chemicals, a chemical's label, and its MSDS; and (d) Include on labels for... information about the hazardous chemical....

  3. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) Display appropriate hazard warnings; (c) Use a chemical identity that permits cross-referencing between the list of hazardous chemicals, a chemical's label, and its MSDS; and (d) Include on labels for... information about the hazardous chemical....

  4. 30 CFR 47.42 - Label contents.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) Display appropriate hazard warnings; (c) Use a chemical identity that permits cross-referencing between the list of hazardous chemicals, a chemical's label, and its MSDS; and (d) Include on labels for... information about the hazardous chemical....

  5. WaterSense Labeled New Homes

    EPA Pesticide Factsheets

    Homes built to meet EPA’s specification can earn the WaterSense label. EPA criteria include WaterSense labeled plumbing fixtures, efficient hot water delivery systems, water-smart landscape design, and other features.

  6. Logos and Graphics on Pesticide Product Labels

    EPA Pesticide Factsheets

    There are several logos that pesticide companies can add to their labels with EPA approval. The requirements and process vary, so review the guidance carefully before applying to add a logo to a product label.

  7. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma, and...

  8. Read the Label First! Protect Your Household

    MedlinePlus

    ... is labeled for your specific pest. EPA encourages consumers to consider using EPA-registered biopesticides and products with EPA’s Safer Choice label , which are generally less harmful. Simply reading ...

  9. 27 CFR 26.39 - Labels.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... TREASURY LIQUORS LIQUORS AND ARTICLES FROM PUERTO RICO AND THE VIRGIN ISLANDS Products Coming Into the United States From Puerto Rico § 26.39 Labels. All labels affixed to bottles of liquors coming into the...

  10. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma,...

  11. 40 CFR 262.31 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.31 Labeling. Before transporting or offering hazardous waste for transportation off-site, a generator must label each package in...

  12. 40 CFR 262.31 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.31 Labeling. Before transporting or offering hazardous waste for transportation off-site, a generator must label each package in...

  13. 40 CFR 262.31 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.31 Labeling. Before transporting or offering hazardous waste for transportation off-site, a generator must label each package in...

  14. 40 CFR 262.31 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.31 Labeling. Before transporting or offering hazardous waste for transportation off-site, a generator must label each package in...

  15. 40 CFR 262.31 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... APPLICABLE TO GENERATORS OF HAZARDOUS WASTE Pre-Transport Requirements § 262.31 Labeling. Before transporting or offering hazardous waste for transportation off-site, a generator must label each package in...

  16. Requirements for Access to Pesticide Labeling Information

    EPA Pesticide Factsheets

    Employers of pesticide handlers must make sure that the handlers are given information from the pesticide labeling and have access to the labeling itself, before they do any handling task. Learn about the information employers must provide.

  17. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  18. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  19. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  20. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  1. 21 CFR 640.84 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Albumin (Human) § 640.84 Labeling. In addition to the labeling... percent albumin is administered to a patient with marked dehydration; (d) The protein...

  2. 40 CFR 205.158 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... color that contrasts with the background of the label. (5) The label must contain the following... Califfo CAL Carabela CAR Cimatti CIM Columbia COL E-Z Rider EZR Flying Dutchman FLY Foxi FOI Gadabout...

  3. 40 CFR 205.158 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... color that contrasts with the background of the label. (5) The label must contain the following... Califfo CAL Carabela CAR Cimatti CIM Columbia COL E-Z Rider EZR Flying Dutchman FLY Foxi FOI Gadabout...

  4. 40 CFR 205.158 - Labeling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... color that contrasts with the background of the label. (5) The label must contain the following... Califfo CAL Carabela CAR Cimatti CIM Columbia COL E-Z Rider EZR Flying Dutchman FLY Foxi FOI Gadabout...

  5. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma, and...

  6. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma, and...

  7. 21 CFR 640.94 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Plasma Protein Fraction (Human) § 640.94 Labeling. In addition... package labels shall contain the following information: (a) The osmotic equivalent in terms of plasma, and...

  8. 99mTc: Labeling Chemistry and Labeled Compounds

    NASA Astrophysics Data System (ADS)

    Alberto, R.; Abram, U.

    This chapter reviews the radiopharmaceutical chemistry of technetium related to the synthesis of perfusion agents and to the labeling of receptor-binding biomolecules. To understand the limitations of technetium chemistry imposed by future application of the complexes in nuclear medicine, an introductory section analyzes the compulsory requirements to be considered when facing the incentive of introducing a novel radiopharmaceutical into the market. Requirements from chemistry, routine application, and market are discussed. In a subsequent section, commercially available 99mTc-based radiopharmaceuticals are treated. It covers the complexes in use for imaging the most important target organs such as heart, brain, or kidney. The commercially available radiopharmaceuticals fulfill the requirements outlined earlier and are discussed with this background. In a following section, the properties and perspectives of the different generations of radiopharmaceuticals are described in a general way, covering characteristics for perfusion agents and for receptor-specific molecules. Technetium chemistry for the synthesis of perfusion agents and the different labeling approaches for target-specific biomolecules are summarized. The review comprises a general introduction to the common approaches currently in use, employing the N x S4-x , [3+1] and 2-hydrazino-nicotinicacid (HYNIC) method as well as more recent strategies such as the carbonyl and the TcN approach. Direct labeling without the need of a bifunctional chelator is briefly reviewed as well. More particularly, recent developments in the labeling of concrete targeting molecules, the second generation of radiopharmaceuticals, is then discussed and prominent examples with antibodies/peptides, neuroreceptor targeting small molecules, myocardial imaging agents, vitamins, thymidine, and complexes relevant to multidrug resistance are given. In addition, a new approach toward peptide drug development is described. The section

  9. Fluorescently labelled glycans and their applications.

    PubMed

    Yan, Hongbin; Yalagala, Ravi Shekar; Yan, Fengyang

    2015-11-01

    This review summarises the literature on the synthesis and applications of fluorescently labelled carbohydrates. Due to the sensitivity of fluorescent detection, this approach provides a useful tool to study processes involving glycans. A few general categories of labelling are presented, in situ labelling of carbohydrates with fluorophores, fluorescently labelled glycolipids, fluorogenic glycans, pre-formed fluorescent glycans for intracellular applications, glycan-decorated fluorescent polymers, fluorescent glyconanoparticles, and other functional fluorescent glycans.

  10. Automated labeling in document images

    NASA Astrophysics Data System (ADS)

    Kim, Jongwoo; Le, Daniel X.; Thoma, George R.

    2000-12-01

    The National Library of Medicine (NLM) is developing an automated system to produce bibliographic records for its MEDLINER database. This system, named Medical Article Record System (MARS), employs document image analysis and understanding techniques and optical character recognition (OCR). This paper describes a key module in MARS called the Automated Labeling (AL) module, which labels all zones of interest (title, author, affiliation, and abstract) automatically. The AL algorithm is based on 120 rules that are derived from an analysis of journal page layouts and features extracted from OCR output. Experiments carried out on more than 11,000 articles in over 1,000 biomedical journals show the accuracy of this rule-based algorithm to exceed 96%.

  11. Adaptive optical label packet switching

    NASA Astrophysics Data System (ADS)

    Xiao, Shilin; Liu, Zhixin; Liang, Zheng; Zhao, Zhihui; Qu, Kefeng

    2007-11-01

    This paper introduces a kind of Adaptive Optical Label Packet Switching (AOLPS) technology. Based on Optical Packet Switching (OPS), AOLPS uses optical label to achieve self-routing, and the size of optical packet is self-adaptive. At the edge nodes, IP packets are fist classified into different first-in-fist-out memories (FIFOs) according to their priority levels and destinations, and then being encapsulated into optical packets. The traffic at each FIFO is real-time monitored, and the controller in edge node employs an optimal strategy to generate suitable sized packets for transmission. Large sized packets will be adopted when traffic is heavy, and small sized packets will be used when traffic is light. This self-adaptive switching granularity can greatly improve the network performance.

  12. Labeling nuclear DNA using DAPI.

    PubMed

    Chazotte, Brad

    2011-01-01

    A number of fluorescent stains are available that label DNA and allow easy visualization of the nucleus in interphase cells and chromosomes in mitotic cells, including Hoechst, 4',6-diamidino-2-phenylindole (DAPI), ethidium bromide, propidium iodide, and acridine orange. Although not as bright as the vital Hoechst stains for DNA, DAPI has greater photostability. It is believed that DAPI associates with the minor groove of double-stranded DNA, with a preference for the adenine-thymine clusters. Cells must be permeabilized and/or fixed for DAPI to enter the cell and to bind DNA. Fluorescence increases approximately 20-fold when DAPI is bound to double-stranded DNA. This protocol describes the use of DAPI to label nuclear DNA of cells grown in culture.

  13. Pre-embedding labeling methods.

    PubMed

    Oliver, Constance

    2010-01-01

    Colloidal gold conjugates generally do not readily penetrate cells, even after permeabilization. Therefore, their use in pre-embedding immunostaining has been largely restricted to labeling cell-surface antigens for scanning or transmission electron microscopy or for tracing endocytic pathways in living cells. One nanometer gold conjugates that do penetrate cells and tissues much more readily have also been used successfully to immunolabel intracellular structures. For pre-embedding labeling, all of the immunostaining is done prior to embedding the tissue in resin or preparing the samples for scanning electron microscopy. This chapter provides methods for pre-embedding staining with unconjugated primary antibody or with primary antibody conjugated to colloidal gold. The use of colloidal gold for tracing endocytic pathways is also given.

  14. White Label Space GLXP Mission

    NASA Astrophysics Data System (ADS)

    Barton, A.

    2012-09-01

    This poster presents a lunar surface mission concept and corresponding financing approach developed by the White Label Space team, an official competitor in the Google Lunar X PRIZE. The White Label Space team's origins were in the European Space Agency's ESTEC facility in the Netherlands. Accordingly the team's technical headquarters are located just outside ESTEC in the Space Business Park. The team has active partners in Europe, Japan and Australia. The team's goal is to provide a unique publicity opportunity for global brands to land on the moon and win the prestigious Google Lunar X PRIZE. The poster presents the main steps to achieve this goal, the cost estimates for the mission, describes the benefits to the potential sponsors and supporters, and details the progress achieved to date.

  15. CD-ROM Labeling Techniques

    DTIC Science & Technology

    1992-01-06

    was allowed to dry thoroughly before application to the disc, so that the solvent used would have dispersed. Use of this, or any adhesive is risky if...the chemical composition and solvents used are not known. Some acid based adhesives have been reported to have eaten through the disc’s protective...been specially manufactured with suitable adhesive ( beeswax ) for use with CD-ROM. Both foils can be printed with customer-labeled, generic

  16. Isotope Labeling in Insect Cells

    PubMed Central

    Saxena, Krishna; Dutta, Arpana; Klein-Seetharaman, Judith

    2011-01-01

    Recent years have seen remarkable progress in applying nuclear magnetic resonance (NMR) spectroscopy to proteins that have traditionally been difficult to study due to issues with folding, posttranslational modification, and expression levels or combinations thereof. In particular, insect cells have proved useful in allowing large quantities of isotope-labeled, functional proteins to be obtained and purified to homogeneity, allowing study of their structures and dynamics by using NMR. Here, we provide protocols that have proven successful in such endeavors. PMID:22167667

  17. 40 CFR 204.55-4 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS NOISE EMISSION STANDARDS FOR CONSTRUCTION EQUIPMENT Portable Air Compressors § 204.55-4 Labeling. (a)(1) The manufacturer... label: (i) The label heading: Compressor Noise Emission Control Information; (ii) Full corporate name...

  18. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Device labeling. 820.120 Section 820.120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES QUALITY SYSTEM REGULATION Labeling and Packaging Control § 820.120 Device labeling. Each...

  19. 21 CFR 895.25 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Labeling. 895.25 Section 895.25 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... eliminated by labeling or a change in labeling, or change in advertising if the device is a restricted device...

  20. 21 CFR 895.25 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Labeling. 895.25 Section 895.25 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... eliminated by labeling or a change in labeling, or change in advertising if the device is a restricted device...

  1. 21 CFR 1271.250 - Labeling controls.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Labeling controls. 1271.250 Section 1271.250 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.250 Labeling controls. (a) General. You must establish and maintain procedures to control the labeling of HCT/Ps. You must...

  2. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  3. Obstacles to nutrition labeling in restaurants.

    PubMed

    Almanza, B A; Nelson, D; Chai, S

    1997-02-01

    This study determined the major obstacles that foodservices face regarding nutrition labeling. Survey questionnaire was conducted in May 1994. In addition to demographic questions, the directors were asked questions addressing willingness, current practices, and perceived obstacles related to nutrition labeling. Sixty-eight research and development directors of the largest foodservice corporations as shown in Restaurants & Institutions magazine's list of the top 400 largest foodservices (July 1993). P tests were used to determine significance within a group for the number of foodservices that were currently using nutrition labeling, perceived impact of nutrition labeling on sales, and perceived responsibility to add nutrition labels. Regression analysis was used to determine the importance of factors on willingness to label. Response rate was 45.3%. Most companies were neutral about their willingness to use nutrition labeling. Two thirds of the respondents were not currently using nutrition labels. Only one third thought that it was the foodservice's responsibility to provide such information. Several companies perceived that nutrition labeling would have a potentially negative effect on annual sales volume. Major obstacles were identified as menu or personnel related, rather than cost related. Menu-related obstacles included too many menu variations, limited space on the menu for labeling, and loss of flexibility in changing the menu. Personnel-related obstacles included difficulty in training employees to implement nutrition labeling, and not enough time for foodservice personnel to implement nutrition labeling. Numerous opportunities will be created for dietetics professionals in helping foodservices overcome these menu- or personnel-related obstacles.

  4. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... EMISSIONS FROM MARINE COMPRESSION-IGNITION ENGINES Certification Provisions § 94.212 Labeling. (a) General... new marine engine modified from a base engine by post-manufacture marinizers in accordance with the... shall be of a color that contrasts with the background of the label: (1) The label heading:...

  5. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS FOR HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling. (a) The labeling of the product provided to health professionals (but not to the general public): (1...

  6. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS FOR HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling. (a) The labeling of the product provided to health professionals (but not to the general public): (1...

  7. 16 CFR 306.12 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... biodiesel, biomass-based diesel, biodiesel blends, and biomass-based diesel blends. The label is 3 inches (7... the black band. Directly underneath the black band, the label shall read “contains biomass-based... the side edges of the label. (5) For biomass-based diesel blends containing more than 5 percent and no...

  8. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Device labeling. 820.120 Section 820.120 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES... shall establish and maintain procedures to control labeling activities. (a) Label integrity....

  9. 9 CFR 354.73 - Retention labels.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Retention labels. 354.73 Section 354.73 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... § 354.73 Retention labels. An inspector may use such labels, devices, and methods as may be approved...

  10. 9 CFR 354.73 - Retention labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Retention labels. 354.73 Section 354.73 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE AGENCY... § 354.73 Retention labels. An inspector may use such labels, devices, and methods as may be approved...

  11. 21 CFR 1271.250 - Labeling controls.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Labeling controls. 1271.250 Section 1271.250 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.250 Labeling controls. (a) General. You must establish and maintain procedures to control the labeling of HCT/Ps. You must design...

  12. 21 CFR 1271.250 - Labeling controls.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Labeling controls. 1271.250 Section 1271.250 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.250 Labeling controls. (a) General. You must establish and maintain procedures to control the labeling of HCT/Ps. You must design...

  13. 21 CFR 1271.250 - Labeling controls.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Labeling controls. 1271.250 Section 1271.250 Food..., AND CELLULAR AND TISSUE-BASED PRODUCTS Current Good Tissue Practice § 1271.250 Labeling controls. (a) General. You must establish and maintain procedures to control the labeling of HCT/Ps. You must design...

  14. 40 CFR 156.10 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) All required label text must: (A) Be set in 6-point or larger type; (B) Appear on a clear contrasting background; and (C) Not be obscured or crowded. (3) Language to be used. All required label or labeling text... additional text in other languages as is considered necessary to protect the public. When additional text...

  15. Do nutrition labels improve dietary outcomes?

    PubMed

    Variyam, Jayachandran N

    2008-06-01

    The disclosure of nutritional characteristics of most packaged foods became mandatory in the United States with the implementation of the Nutrition Labeling and Education Act (NLEA) in 1994. Under the NLEA regulations, a 'Nutrition Facts' panel displays information on nutrients such as calories, total and saturated fats, cholesterol, and sodium in a standardized format. By providing nutrition information in a credible, distinctive, and easy-to-read format, the new label was expected to help consumers choose healthier, more nutritious diets. This paper examines whether the disclosure of nutrition information through the mandatory labels impacted consumer diets. Assessing the dietary effects of labeling is problematic due to the confounding of the label effect with unobserved label user characteristics. This self-selection problem is addressed by exploiting the fact that the NLEA exempts away-from-home foods from mandatory labeling. Difference-in-differences models that account for zero away-from-home intakes suggest that the labels increase fiber and iron intakes of label users compared with label nonusers. In comparison, a model that does not account for self-selection implies significant label effects for all but two of the 13 nutrients that are listed on the label.

  16. Labels and Children's Perceptions of Faces

    ERIC Educational Resources Information Center

    Katz, Phyllis A.; Seavey, Carol

    1973-01-01

    The relation between type of label and perception of faces was assessed in second- and sixth-grade children. Labels associated with color increased color perception, whereas labels based on expressiveness increased differentiation of expression variations, but not color perception. (ST)

  17. 40 CFR 600.301 - Labeling requirements.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... ECONOMY AND GREENHOUSE GAS EXHAUST EMISSIONS OF MOTOR VEHICLES Fuel Economy Labeling § 600.301 Labeling... each dealer shall maintain or cause to be maintained on each automobile: (1) A general fuel economy... vehicle for which a specific label is requested which has a combined FTP/HFET-based fuel economy value,...

  18. 30 CFR 47.43 - Label alternatives.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Label alternatives. 47.43 Section 47.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.43 Label alternatives. The operator...

  19. 30 CFR 47.43 - Label alternatives.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Label alternatives. 47.43 Section 47.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.43 Label alternatives. The operator...

  20. 30 CFR 47.43 - Label alternatives.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Label alternatives. 47.43 Section 47.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.43 Label alternatives. The operator...

  1. 30 CFR 47.43 - Label alternatives.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Label alternatives. 47.43 Section 47.43 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR EDUCATION AND TRAINING HAZARD COMMUNICATION (HazCom) Container Labels and Other Forms of Warning § 47.43 Label alternatives. The operator...

  2. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label records. (a) Each licensee and permittee shall maintain a list of all approved labels currently being used... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Label records. 116.3 Section 116.3...

  3. 9 CFR 116.3 - Label records.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ..., SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS RECORDS AND REPORTS § 116.3 Label records. (a) Each licensee and permittee shall maintain a list of all approved labels currently being used... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Label records. 116.3 Section 116.3...

  4. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be...

  5. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be...

  6. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be...

  7. 21 CFR 660.28 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... panel. Blood grouping reagent Color of label paper Anti-A Blue. Anti-B Yellow. Slide and rapid tube test... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Blood Grouping Reagent § 660.28 Labeling. In... label—(1) Color coding. The final container label of all Blood Grouping Reagents shall be...

  8. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  9. 21 CFR 660.35 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.35 Labeling. In... or end of the label, oustide of the main panel. (2) If washing the cells is required by the manufacturer, the container label shall include appropriate instructions; if the cells should not be...

  10. 49 CFR 172.426 - OXIDIZER label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.426 OXIDIZER label. (a) Except for size and color, the OXIDIZER label must be as follows: EC02MR91.027 (b) In addition to complying with § 172.407, the background color on...

  11. 49 CFR 172.426 - OXIDIZER label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.426 OXIDIZER label. (a) Except for size and color, the OXIDIZER label must be as follows: EC02MR91.027 (b) In addition to complying with § 172.407, the background color on...

  12. 49 CFR 172.441 - FISSILE label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.441 FISSILE label. (a) Except for size and color, the FISSILE label must be as follows: ER26ja04.000 (b) In addition to complying with § 172.407, the background color on...

  13. 49 CFR 172.426 - OXIDIZER label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.426 OXIDIZER label. (a) Except for size and color, the OXIDIZER label must be as follows: EC02MR91.027 (b) In addition to complying with § 172.407, the background color on...

  14. 49 CFR 172.426 - OXIDIZER label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.426 OXIDIZER label. (a) Except for size and color, the OXIDIZER label must be as follows: EC02MR91.027 (b) In addition to complying with § 172.407, the background color on...

  15. 49 CFR 172.441 - FISSILE label.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.441 FISSILE label. (a) Except for size and color, the FISSILE label must be as follows: ER26ja04.000 (b) In addition to complying with § 172.407, the background color on...

  16. 49 CFR 172.441 - FISSILE label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.441 FISSILE label. (a) Except for size and color, the FISSILE label must be as follows: ER26ja04.000 (b) In addition to complying with § 172.407, the background color on...

  17. 49 CFR 172.441 - FISSILE label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.441 FISSILE label. (a) Except for size and color, the FISSILE label must be as follows: ER26ja04.000 (b) In addition to complying with § 172.407, the background color on...

  18. 40 CFR 211.105 - Label format.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Label format. 211.105 Section 211.105 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) NOISE ABATEMENT PROGRAMS PRODUCT NOISE LABELING General Provisions § 211.105 Label format. (a) Unless specified otherwise in other...

  19. Learning Words from Labeling and Directive Speech

    ERIC Educational Resources Information Center

    Callanan, Maureen A.; Akhtar, Nameera; Sussman, Lisa

    2014-01-01

    Despite the common intuition that labeling may be the best way to teach a new word to a child, systematic testing is needed of the prediction that children learn words better from labeling utterances than from directive utterances. Two experiments compared toddlers' label learning in the context of hearing words used in directive versus labeling…

  20. 76 FR 20233 - Appliance Labeling Rule

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-12

    ...). ACTION: Final rule. SUMMARY: The Commission extends the effective date for its new light bulb labeling... Commission exempts from the new label requirements incandescent bulbs that will not be produced after January... proposing to extend the effective date of new labeling rules for light bulbs to January 1, 2012.\\1\\ The new...

  1. 16 CFR 1633.12 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Practices CONSUMER PRODUCT SAFETY COMMISSION FLAMMABLE FABRICS ACT REGULATIONS STANDARD FOR THE FLAMMABILITY (OPEN FLAME) OF MATTRESS SETS Rules and Regulations § 1633.12 Labeling. (a) Each mattress set subject to the Standard shall bear a permanent, conspicuous, and legible label(s) containing the...

  2. 16 CFR 1633.12 - Labeling.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Practices CONSUMER PRODUCT SAFETY COMMISSION FLAMMABLE FABRICS ACT REGULATIONS STANDARD FOR THE FLAMMABILITY (OPEN FLAME) OF MATTRESS SETS Rules and Regulations § 1633.12 Labeling. (a) Each mattress set subject to the Standard shall bear a permanent, conspicuous, and legible label(s) containing the...

  3. 16 CFR 1633.12 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Practices CONSUMER PRODUCT SAFETY COMMISSION FLAMMABLE FABRICS ACT REGULATIONS STANDARD FOR THE FLAMMABILITY (OPEN FLAME) OF MATTRESS SETS Rules and Regulations § 1633.12 Labeling. (a) Each mattress set subject to the Standard shall bear a permanent, conspicuous, and legible label(s) containing the...

  4. Labeling Nodes Using Three Degrees of Propagation

    PubMed Central

    Mostafavi, Sara; Goldenberg, Anna; Morris, Quaid

    2012-01-01

    The properties (or labels) of nodes in networks can often be predicted based on their proximity and their connections to other labeled nodes. So-called “label propagation algorithms” predict the labels of unlabeled nodes by propagating information about local label density iteratively through the network. These algorithms are fast, simple and scale to large networks but nonetheless regularly perform better than slower and much more complex algorithms on benchmark problems. We show here, however, that these algorithms have an intrinsic limitation that prevents them from adapting to some common patterns of network node labeling; we introduce a new algorithm, 3Prop, that retains all their advantages but is much more adaptive. As we show, 3Prop performs very well on node labeling problems ill-suited to label propagation, including predicting gene function in protein and genetic interaction networks and gender in friendship networks, and also performs slightly better on problems already well-suited to label propagation such as labeling blogs and patents based on their citation networks. 3Prop gains its adaptability by assigning separate weights to label information from different steps of the propagation. Surprisingly, we found that for many networks, the third iteration of label propagation receives a negative weight. Availability The code is available from the authors by request. PMID:23284828

  5. 21 CFR 610.61 - Package label.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Package label. 610.61 Section 610.61 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.61 Package label. The following items shall...

  6. 21 CFR 610.61 - Package label.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Package label. 610.61 Section 610.61 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.61 Package label. The following items shall...

  7. 21 CFR 610.61 - Package label.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Package label. 610.61 Section 610.61 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.61 Package label. The following items shall...

  8. 21 CFR 610.61 - Package label.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Package label. 610.61 Section 610.61 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS GENERAL BIOLOGICAL PRODUCTS STANDARDS Labeling Standards § 610.61 Package label. The following items shall...

  9. Nutrition Label Viewing during a Food-Selection Task: Front-of-Package Labels vs Nutrition Facts Labels.

    PubMed

    Graham, Dan J; Heidrick, Charles; Hodgin, Katie

    2015-10-01

    Earlier research has identified consumer characteristics associated with viewing Nutrition Facts labels; however, little is known about those who view front-of-package nutrition labels. Front-of-package nutrition labels might appeal to more consumers than do Nutrition Facts labels, but it might be necessary to provide consumers with information about how to locate and use these labels. This study quantifies Nutrition Facts and front-of-package nutrition label viewing among American adult consumers. Attention to nutrition information was measured during a food-selection task. One hundred and twenty-three parents (mean age=38 years, mean body mass index [calculated as kg/m(2)]=28) and one of their children (aged 6 to 9 years) selected six foods from a university laboratory-turned-grocery aisle. Participants were randomized to conditions in which front-of-package nutrition labels were present or absent, and signage explaining front-of-package nutrition labels was present or absent. Adults' visual attention to Nutrition Facts labels and front-of-package nutrition labels was objectively measured via eye-tracking glasses. To examine whether there were significant differences in the percentages of participants who viewed Nutrition Facts labels vs front-of-package nutrition labels, McNemar's tests were conducted across all participants, as well as within various sociodemographic categories. To determine whether hypothesized factors, such as health literacy and education, had stronger relationships with front-of-package nutrition label vs Nutrition Facts label viewing, linear regression assessed the magnitude of relationships between theoretically and empirically derived factors and each type of label viewing. Overall, front-of-package nutrition labels were more likely to be viewed than Nutrition Facts labels; however, for all subgroups, higher rates of front-of-package nutrition label viewership occurred only when signage was present drawing attention to the presence and

  10. Gender, status, and psychiatric labels.

    PubMed

    Kroska, Amy; Harkness, Sarah K; Brown, Ryan P; Thomas, Lauren S

    2015-11-01

    We examine a key modified labeling theory proposition-that a psychiatric label increases vulnerability to competence-based criticism and rejection-within task- and collectively oriented dyads comprised of same-sex individuals with equivalent education. Drawing on empirical work that approximates these conditions, we expect the proposition to hold only among men. We also expect education, operationalized with college class standing, to moderate the effects of gender by reducing men's and increasing women's criticism and rejection. But, we also expect the effect of education to weaken when men work with a psychiatric patient. As predicted, men reject suggestions from teammates with a psychiatric history more frequently than they reject suggestions from other teammates, while women's resistance to influence is unaffected by their teammate's psychiatric status. Men also rate psychiatric patient teammates as less powerful but no lower in status than other teammates, while women's teammate assessments are unaffected by their teammate's psychiatric status. Also as predicted, education reduces men's resistance to influence when their teammate has no psychiatric history. Education also increases men's ratings of their teammate's power, as predicted, but has no effect on women's resistance to influence or teammate ratings. We discuss the implications of these findings for the modified labeling theory of mental illness and status characteristics theory. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Nutrition marketing on food labels.

    PubMed

    Colby, Sarah E; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Marketing strategy, nutrient label information, if the product was fruit/or milk based, and target age. Frequency distributions were computed. Forty-nine percent of all products contained nutrition marketing and of those, 48% had both nutrition marketing and were high in saturated fat, sodium and/or sugar (11%, 17%, and 31% respectively). Seventy-one percent of products marketed to children had nutrition marketing. Of those, 59% were high in saturated fat, sodium and/or sugar content, with more than half being high in sugar. The most commonly used nutrition marketing statements were "good source of calcium", "reduced/low/fat free", and "food company's health symbol". Nutrition marketing is commonly used on products high in saturated fat, sodium and/or sugar and is more often used on products marketed toward children than products marketed toward adults. Current food industry symbols may not be helping consumers select foods low in saturated fat, sodium or sugar. Published by Elsevier Inc.

  12. 40 CFR 85.530 - Vehicle/engine labels and packaging labels.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 19 2014-07-01 2014-07-01 false Vehicle/engine labels and packaging labels. 85.530 Section 85.530 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... Conversions From Tampering Prohibition § 85.530 Vehicle/engine labels and packaging labels. (a) The following...

  13. 40 CFR 85.530 - Vehicle/engine labels and packaging labels.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 19 2012-07-01 2012-07-01 false Vehicle/engine labels and packaging labels. 85.530 Section 85.530 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR... Conversions From Tampering Prohibition § 85.530 Vehicle/engine labels and packaging labels. (a) The following...

  14. Stigma of a label: educational expectations for high school students labeled with learning disabilities.

    PubMed

    Shifrer, Dara

    2013-01-01

    Poorer outcomes for youth labeled with learning disabilities (LDs) are often attributed to the student's own deficiencies or cumulative disadvantage; but the more troubling possibility is that special education placement limits rather than expands these students' opportunities. Labeling theory partially attributes the poorer outcomes of labeled persons to stigma related to labels. This study uses data on approximately 11,740 adolescents and their schools from the Education Longitudinal Survey of 2002 to determine if stigma influences teachers' and parents' educational expectations for students labeled with LDs and labeled adolescents' expectations for themselves. Supporting the predictions of labeling theory, teachers and parents are more likely to perceive disabilities in, and hold lower educational expectations for labeled adolescents than for similarly achieving and behaving adolescents not labeled with disabilities. The negative effect of being labeled with LDs on adolescents' educational expectations is partially mechanized through parents' and particularly teachers' lower expectations.

  15. Abandoning a label doesn’t make it disappear: The perseverance of labeling effects

    PubMed Central

    Foroni, Francesco; Rothbart, Myron

    2012-01-01

    Labels exert strong influence on perception and judgment. The present experiment examines the possibility that such effects may persist even when labels are abandoned. Participants judged the similarity of pairs of silhouette drawings of female body types, ordered on a continuum from very thin to very heavy, under conditions where category labels were, and were not, superimposed on the ordered stimuli. Consistent with earlier research, labels had strong effects on perceived similarity, with silhouettes sharing the same label judged as more similar than those having different labels. Moreover, when the labels were removed and no longer present, the effect of the labels, although diminished, persisted. It did not make any difference whether the labels were simply abandoned or, in addition, had their validity challenged. The results are important for our understanding of categorization and labeling processes. The potential theoretical and practical implications of these results for social processes are discussed. PMID:23105148

  16. 46 CFR 160.133-17 - Marking and labeling.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Marking and labeling. (a) Each hook body of a release mechanism must be marked with a plate or label...) The plate or label must be in English, but may also be in other languages. (c) The plate or label must...

  17. DNA Labeling Using DNA Methyltransferases.

    PubMed

    Tomkuvienė, Miglė; Kriukienė, Edita; Klimašauskas, Saulius

    2016-01-01

    DNA methyltransferases (MTases) uniquely combine the ability to recognize and covalently modify specific target sequences in DNA using the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet). Although DNA methylation plays important roles in biological signaling, the transferred methyl group is a poor reporter and is highly inert to further biocompatible derivatization. To unlock the biotechnological power of these enzymes, two major types of cofactor AdoMet analogs were developed that permit targeted MTase-directed attachment of larger moieties containing functional or reporter groups onto DNA. One such approach (named sequence-specific methyltransferase-induced labeling, SMILing) uses reactive aziridine or N-mustard mimics of the cofactor AdoMet, which render targeted coupling of a whole cofactor molecule to the target DNA. The second approach (methyltransferase-directed transfer of activated groups, mTAG) uses AdoMet analogs with a sulfonium-bound extended side chain replacing the methyl group, which permits MTase-directed covalent transfer of the activated side chain alone. As the enlarged cofactors are not always compatible with the active sites of native MTases, steric engineering of the active site has been employed to optimize their alkyltransferase activity. In addition to the described cofactor analogs, recently discovered atypical reactions of DNA cytosine-5 MTases involving non-cofactor-like compounds can also be exploited for targeted derivatization and labeling of DNA. Altogether, these approaches offer new powerful tools for sequence-specific covalent DNA labeling, which not only pave the way to developing a variety of useful techniques in DNA research, diagnostics, and nanotechnologies but have already proven practical utility for optical DNA mapping and epigenome studies.

  18. Hemoglobin Labeled by Radioactive Lysine

    DOE R&D Accomplishments Database

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  19. Consumer knowledge and attitudes toward nutritional labels.

    PubMed

    Cannoosamy, Komeela; Pugo-Gunsam, Prity; Jeewon, Rajesh

    2014-01-01

    To determine Mauritian consumers' attitudes toward nutritional labels based on the Kano model and to identify determinants of the use and understanding of nutrition labels. The researchers also used a Kano model questionnaire to determine consumers' attitudes toward nutrition labeling. Four hundred consumers residing in Mauritius. Information was elicited via a questionnaire that assessed nutritional knowledge and information about the use and understanding of nutritional labels and demographic factors. Nutritional label use and understanding, nutrition knowledge, and association of demographic factors with label use. Statistical tests performed included 1-way ANOVA and independent samples t tests. Statistically significant relationships (P < .05) were found for nutritional knowledge and nutritional label usage with demographic factors. All demographic factors with the exception of gender were significantly associated (P < .05) with nutritional label understanding. Based on the outcome of the Kano survey, calorie content, trans fat content, protein content, and cholesterol content were found to be must-be attributes: that is, attributes that, when not present, result in consumer dissatisfaction. Age, education, income, household size, and nutrition knowledge had an impact on nutritional label use. Health promoters should aim to increase the use of nutritional labels. Copyright © 2014 Society for Nutrition Education and Behavior. Published by Elsevier Inc. All rights reserved.

  20. Social determinants of diagnostic labels in depression.

    PubMed

    McPherson, Susan; Armstrong, David

    2006-01-01

    The role of diagnostic labels in medicine is usually that of labelling an illness as a means of communication. Control over labelling processes in medicine is ordinarily imposed via medical schools, textbooks, education or by diagnostic manuals. Diagnostic labels often change following new discoveries in underlying pathology such as 'consumption' being relabelled as 'TB' or 'cancer'. Sub-types of broad diagnostic labels also often emerge from such discoveries e.g. 'lung cancer' or 'throat cancer'. In mental health, underlying pathology is the subject of ongoing debate spanning ideas including the brain as a faulty organ, faulty genetics and environmental problems. With controversy over pathology comes controversy over labels and the idea that labels may be used not just for communication, but as devices of social and professional control, arising out of a social process. This study explores the codification of the diagnostic label 'depression' which emerged in the twentieth-century and has proliferated with numerous sub-types over the last 40 years. The aim is to examine its social determinants and context. Medline is used as a data source for professional label usage. A range of depression sub-type labels in professional use was identified. This exercise revealed many official and 'unofficial' terms in professional use. Citation rate plots by year were then generated for these depression sub-type labels. The rise and fall of different labels are examined in relation to social determinants and context, including publication of diagnostic manuals DSM and ICD, power shifts in psychiatry, the discovery of psychiatric drugs and the shift from inpatient to community care. Exploring the changing use of official and unofficial labels over time in this way provides a novel historical perspective on the concept of depression in the late twentieth-century.

  1. Chemical kin label in seabirds

    PubMed Central

    Célérier, Aurélie; Bon, Cécile; Malapert, Aurore; Palmas, Pauline; Bonadonna, Francesco

    2011-01-01

    Chemical signals yield critical socio-ecological information in many animals, such as species, identity, social status or sex, but have been poorly investigated in birds. Recent results showed that chemical signals are used to recognize their nest and partner by some petrel seabirds whose olfactory anatomy is well developed and which possess a life-history propitious to olfactory-mediated behaviours. Here, we investigate whether blue petrels (Halobaena caerulea) produce some chemical labels potentially involved in kin recognition and inbreeding avoidance. To overcome methodological constraints of chemical analysis and field behavioural experiments, we used an indirect behavioural approach, based on mice olfactory abilities in discriminating odours. We showed that mice (i) can detect odour differences between individual petrels, (ii) perceive a high odour similarity between a chick and its parents, and (iii) perceive this similarity only before fledging but not during the nestling developmental stage. Our results confirm the existence of an individual olfactory signature in blue petrels and show for the first time, to our knowledge, that birds may exhibit an olfactory kin label, which may have strong implications for inbreeding avoidance. PMID:21525047

  2. Label-free molecular imaging

    NASA Astrophysics Data System (ADS)

    Zhang, Junqi; Li, Qi; Fu, Rongxin; Wang, Tongzhou; Wang, Ruliang; Huang, Guoliang

    2014-03-01

    Optical microscopy technology has achieved great improvements in the 20th century. The detection limit has reached about twenty nanometers (with near-field optics, STED, PALM and STORM). But in the application areas such as life science, medical science, clinical treatment and especially in vivo dynamic measurement, mutual restrictions still exist between numeric aperture/magnification and working distance, fluorescent dependent, and between resolution and frame rate/field size, etc. This paper explores a hyperspectral scanning super-resolution label free molecules imaging method based on the white light interferometry. The vertical detection resolution was approximate to 1 nm which is the thickness of a single molecular layer and dynamic measuring range of thickness reaches to 10 μm. The spectrum-shifting algorithm is developed for robust restructure of images when the pixels are overlapped. Micro-biochip with protein binding and DNA amplification could be detected by using this spectral scanning super-resolution molecules imaging in label free. This method has several advantages as following: Firstly, the decoding and detecting steps are combined into one step. It makes tests faster and easier. Secondly, we used thickness-coded, minimized chips instead of a large microarray chip to carry the probes. This accelerates the interaction of the biomolecules. Thirdly, since only one kind of probes are attached to our thickness-coded, minimized chip, users can only pick out the probes they are interested in for a test without wasting unnecessary probes and chips.

  3. 49 CFR 172.407 - Label specifications.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... each side having a solid line inner border 5.0 to 6.3 mm (0.2 to 0.25 inches) from the edge. (2) The....7 mm (0.5 inches). (4) When text indicating a hazard is displayed on a label, the label name must be... least 5.1 mm (0.2 inches) in height. (5) The symbol on each label must be proportionate in size to...

  4. 49 CFR 172.407 - Label specifications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... each side having a solid line inner border 5.0 to 6.3 mm (0.2 to 0.25 inches) from the edge. (2) The....7 mm (0.5 inches). (4) When text indicating a hazard is displayed on a label, the label name must be... least 5.1 mm (0.2 inches) in height. (5) The symbol on each label must be proportionate in size to...

  5. 49 CFR 172.407 - Label specifications.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... each side having a solid line inner border 5.0 to 6.3 mm (0.2 to 0.25 inches) from the edge. (2) The....7 mm (0.5 inches). (4) When text indicating a hazard is displayed on a label, the label name must be... least 5.1 mm (0.2 inches) in height. (5) The symbol on each label must be proportionate in size to...

  6. 49 CFR 172.407 - Label specifications.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... each side having a solid line inner border 5.0 to 6.3 mm (0.2 to 0.25 inches) from the edge. (2) The....7 mm (0.5 inches). (4) When text indicating a hazard is displayed on a label, the label name must be... least 5.1 mm (0.2 inches) in height. (5) The symbol on each label must be proportionate in size to...

  7. Efficient Methods for Multi-Label Classification

    DTIC Science & Technology

    2015-05-22

    annotation and retrieval of music and sound effects . IEEE Transactions on Audio, Speech and Language Processing 16(2), 467–476 (2008) 19. Vens, C., Struyf...advertising [2] and music categorization [18]. In these applications there are usually tens or hundreds of thousands of labels, while the number is...Efficient Methods for Multi-label Classiffication 165 and testing efficiency, memory usage is also a bottleneck as the number of labels becoming larger

  8. Improving the accuracy of specimen labeling.

    PubMed

    Dock, Bobbi

    2005-01-01

    Accurate specimen identification is a challenge in all hospitals. A mislabeled specimen can lead to devastating consequences for a patient. In an effort to decrease the risk of potential harm caused by labeling errors, Children's Hospitals and Clinics of Minnesota successfully implemented a Zero Tolerance Laboratory Specimen Labeling process. After months of studying, charting, networking, and communicating with all stakeholders the new process led to a 75% reduction in laboratory specimen labeling errors.

  9. 16 CFR 309.17 - Labels.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... is centered. The band at the top of the label contains the name of the fuel. This band should measure 1″ (2.54 cm) deep. Spacing of the fuel name is 1/4″ (.64 cm) from the top of the label and 3/16.... “Helvetica black” type is used throughout. All type is centered. The band at the top of the label...

  10. Organic labeling influences food valuation and choice.

    PubMed

    Linder, N S; Uhl, G; Fliessbach, K; Trautner, P; Elger, C E; Weber, B

    2010-10-15

    Everyday we choose between a variety of different food items trying to reach a decision that fits best our needs. These decisions are highly dependent on the context in which the alternatives are presented (e.g. labeling). We investigate the influence of cognition on food evaluation, using an fMRI experiment in which subjects saw and bid on different foods labeled with (or without) a widely known German emblem for organically produced food. Increased activity in the ventral striatum was found for foods labeled "organic" in comparison to conventionally labeled food. Between-subject differences in activity were related to actual everyday consumption behavior of organic food.

  11. Optimal design of isotope labeling experiments.

    PubMed

    Yang, Hong; Mandy, Dominic E; Libourel, Igor G L

    2014-01-01

    Stable isotope labeling experiments (ILE) constitute a powerful methodology for estimating metabolic fluxes. An optimal label design for such an experiment is necessary to maximize the precision with which fluxes can be determined. But often, precision gained in the determination of one flux comes at the expense of the precision of other fluxes, and an appropriate label design therefore foremost depends on the question the investigator wants to address. One could liken ILE to shadows that metabolism casts on products. Optimal label design is the placement of the lamp; creating clear shadows for some parts of metabolism and obscuring others.An optimal isotope label design is influenced by: (1) the network structure; (2) the true flux values; (3) the available label measurements; and, (4) commercially available substrates. The first two aspects are dictated by nature and constrain any optimal design. The second two aspects are suitable design parameters. To create an optimal label design, an explicit optimization criterion needs to be formulated. This usually is a property of the flux covariance matrix, which can be augmented by weighting label substrate cost. An optimal design is found by using such a criterion as an objective function for an optimizer. This chapter uses a simple elementary metabolite units (EMU) representation of the TCA cycle to illustrate the process of experimental design of isotope labeled substrates.

  12. [Academic production on food labeling in Brazil].

    PubMed

    Câmara, Maria Clara Coelho; Marinho, Carmem Luisa Cabral; Guilam, Maria Cristina; Braga, Ana Maria Cheble Bahia

    2008-01-01

    To review and discuss academic production (theses and dissertations) on the topic of labeling of prepackaged foods in Brazil. A search of the database maintained by the Coordination for the Development of Higher Education Professionals (CAPES), one of the two Brazilian government research funding and support agencies, was conducted on the following keywords: "rotulagem" (labeling), "rotulagem nutricional" (food labeling) and "rótulo de alimentos" (food labels). The search covered the years 1987 (earliest year available) to 2004. We identified 49 studies on this topic. Content analysis identified three major themes: the extent to which food labels meet specific legal requirements (57.2%); the degree to which consumers understand the information on labels (22.4%); and the labeling of transgenic or genetically-modified foods (20.4%). Food labeling is a frequent topic and is adequately covered by the Brazilian academic production. In most of the studies, ineffective law enforcement appears to be the main factor in the lack of compliance with and disrespect for the food labeling rules and regulations in Brazil.

  13. Fluorescent labeling and tracking of nanoclay.

    PubMed

    Diaz, Carlos A; Xia, Yining; Rubino, Maria; Auras, Rafael; Jayaraman, Krishnamurthy; Hotchkiss, Joseph

    2013-01-07

    We report a methodology developed to detect and track stable fluorescent-labeled nanoclay, in polymer-clay nanocomposite films, and in a contact solvent after migration testing. Fluorescein-5-maleimide (fluorescein) or tetramethylrhodamine-5-maleimide (rhodamine) was covalently bonded to organically modified montmorillonite (o-MMT). Fluorescein- and rhodamine-labeled nanoclay showed good thermal stability up to 220 °C and the rhodamine-labeled nanoclay remained stable at 250 °C. Confocal laser scanning microscopy was used to confirm the tagging and to detect the fluorescent-labeled nanoclays in various systems.

  14. Component Labeling Algorithm For Video Rate Processing

    NASA Astrophysics Data System (ADS)

    Gotoh, Toshiyuki; Ohta, Yoshiyuki; Yoshida, Masumi; Shirai, Yoshio

    1987-10-01

    In this paper, we propose a raster scanning algorithm for component labeling, which enables processing under pipeline architecture. In the raster scanning algorithm, labels are provisionally assigned to each pixel of components and, at the same time, the connectivities of labels are detected at first scan. Those labels are classified into groups based on the connectivities. Finally provisional labels are updated using the result of classification and a unique label is assigned to each pixel of components. However, in the conventional algorithm, the classification process needs a vast number of operations. This prevents realizing pipeline processing. We have developed a method of preprocessing to reduce the number of provisional labels, which limits the number of label connectivities. We have also developed a new classification method whose operation is proportionate to only the number of label connectivities itself. We have made experiments with computer simulation to verify this algorithm. The experimental results show that we can process 512 x 512 x 8 bit images at video rate(1/30 sec. per 1 image) when this algorithm is implemented on hardware.

  15. Simultaneous Segmentation and Statistical Label Fusion.

    PubMed

    Asman, Andrew J; Landmana, Bennett A

    2012-02-23

    Labeling or segmentation of structures of interest in medical imaging plays an essential role in both clinical and scientific understanding. Two of the common techniques to obtain these labels are through either fully automated segmentation or through multi-atlas based segmentation and label fusion. Fully automated techniques often result in highly accurate segmentations but lack the robustness to be viable in many cases. On the other hand, label fusion techniques are often extremely robust, but lack the accuracy of automated algorithms for specific classes of problems. Herein, we propose to perform simultaneous automated segmentation and statistical label fusion through the reformulation of a generative model to include a linkage structure that explicitly estimates the complex global relationships between labels and intensities. These relationships are inferred from the atlas labels and intensities and applied to the target using a non-parametric approach. The novelty of this approach lies in the combination of previously exclusive techniques and attempts to combine the accuracy benefits of automated segmentation with the robustness of a multi-atlas based approach. The accuracy benefits of this simultaneous approach are assessed using a multi-label multi- atlas whole-brain segmentation experiment and the segmentation of the highly variable thyroid on computed tomography images. The results demonstrate that this technique has major benefits for certain types of problems and has the potential to provide a paradigm shift in which the lines between statistical label fusion and automated segmentation are dramatically blurred.

  16. Rapid screening and analysis of alpha- and gamma-emitting radionuclides in liquids using a single sample preparation procedure.

    PubMed

    Parsa, Bahman; Henitz, James B; Carter, Jennifer A

    2011-02-01

    A multifaceted radiochemical testing procedure has been developed to analyze a large number of liquid samples and measure a wide range of radionuclides in a short period of time. This method involves a single, unique and fast sample preparation procedure and allows sequential/concurrent determination of analytes with accuracy and precision. The same prepared sample can be selectively analyzed by gross alpha counting, gamma-ray spectroscopy, and alpha spectroscopy. This method is especially attractive in radiological emergency events where analytical data will be needed urgently as a basis for protective action. Given the simplicity and rapidity of the method, it may be suitable for field portable laboratories, which could save time and the cost associated with the transit of samples to a fixed laboratory. A 100 mL aliquot of sample was spiked with ¹³³Ba and ⁵⁹Fe tracers and subjected to a chemical separation procedure using a combined BaSO4 and Fe(OH)3 co-precipitation scheme. Then, the gross alpha-particle activity of the prepared sample was measured with a low-background gas-proportional counter, followed by the analysis of its photon-emitters using a gamma-ray spectroscopy system with high-purity intrinsic Ge detectors. Gamma-ray determination of ¹³³Ba and ⁵⁹Fe tracers was used to assess the chemical recoveries of BaSO4 and Fe(OH)3 fractions, respectively. Selectivity of the radionuclides for co-precipitation with either BaSO4 or Fe(OH)3 components was also investigated. Alpha mass-efficiency curves were derived using ²³⁰Th and ²⁴¹Am standards as alpha-calibration sources. Various mixtures of radionuclides, including ⁵⁴Mn, ⁵⁷Co, ⁶⁰Co, ⁸⁵Sr, ⁸⁸Y, ¹⁰⁹Cd, ¹¹³Sn, ¹³⁷Cs, ¹³⁹Ce, ²⁰³Hg, ²⁰⁹Po, ²²⁶Ra, ²²⁸Ra, ²³⁰Th, ²⁴¹Am, and natural uranium were used in this study. Most were quantitatively assayed with high chemical recoveries. Alpha-isotope identification and assessment of the prepared sample was achieved by alpha spectroscopy using passivated implanted planar silicon (PIPS) detectors. It has been shown that fission products could potentially be captured and analyzed by this method.

  17. Determination of gamma-emitting radionuclides in the inter-tidal sediments off Balochistan (Pakistan) Coast, Arabian Sea.

    PubMed

    Akram, M; Qureshi, Riffat M; Ahmad, Nasir; Solaija, Tariq Jamal

    2007-01-01

    Natural radionuclide contents of 226Ra, 228Ra and (40)K were studied for inter-tidal sediments collected from selected locations off the745 km long Balochistan Coast using HPGe detector based gamma-spectrometry system. The sampling zone extends from the beaches of Sonmiani (near Karachi metropolis) through Jiwani (close to the border of Iran). The natural radioactivity levels detected in various sediment samples range from 14.4 +/- 2.5 to 36.6 +/- 3.8 Bq kg(-1) for 226Ra, 9.8 +/- 1.2 to 35.2 +/- 2.0 Bq kg(-1) for (228)Ra and 144.6 +/- 9.4 to 610.5 +/- 23.9 Bq kg(-1) for (40)K. No artificial radionuclide was detected in any of the marine coastal sediment samples. 137Cs, (60)Co, 106Ru and 144Ce contents in sediment samples were below the limit of detection. The measured radioactivity levels are compared with those reported in the literature for coastal sediments in other parts of the world. The information presented in this paper will serve as the first ever local radioactivity database for the Balochistan/Makran Coastal belt of Pakistan. The presented data will also contribute to the IAEA's, Asia-Pacific Marine Radioactivity Database (ASPAMARD) and the Global Marine Radioactivity Database (GLOMARD).

  18. A model-based multiple-pinhole synthetic imager for stand-off range gamma-emitting objects

    NASA Astrophysics Data System (ADS)

    DeRego, Paul J.; Hecht, Adam A.; Dias da Cunha, Kenya M.; Baldez, Phoenix

    2016-09-01

    Pixelated Cadmium Zinc Telluride (CZT) detectors provide the opportunity to perform spectroscopic imaging for discriminating one radioactive material from another. Although Compton interactions provide a means for imaging high energy gamma sources, identification of materials emitting lower energy signatures are better suited to collimator imaging techniques. This paper specifically considers a multiple pinhole method for its simplicity of pinhole focusing combined with straightforward processing methods for incorporating multiple apertures to reduce photon collection time while retaining image resolution. Multiple pinhole image detections are combined using an iterative Maximum- Likelihood Expectation-Maximization (MLEM) synthetic imaging algorithm. To enable subsequent field operations, the imaging system matrix is computed using an imaging model with adjustable parameters rather than one experimentally acquired from point sources. The system model includes an object space, a multiple pinhole collimator plane, and a pixelated detection plane. The modeled object space is implemented in two dimensions to reduce image reconstruction burden since 3D imaging is not practical for single view stand-off imaging. Focusing is modeled by a function computing photon trajectory and passage through the pinhole patterned barrier plane. Results show that a MLEM processed image will achieve resolution approaching that of a single pinhole imaged onto the full detector. The multiple pinhole advantages of simple implementation with shorter focal lengths combined with the availability of portable CZT detectors would be useful in short stand-off applications. Work is currently in progress to experimentally quantify spatial resolution and imaging timelines using an eV Products D-Matrix 4x4 array of pixelated CZT modules.

  19. Development of gamma emitting receptor-binding radiotracers for imaging the brain and pancreas. Progress report, February 1983-September 1984

    SciTech Connect

    Reba, R.C.

    1984-01-01

    The possibility of measuring the change in receptor concentration as a function of disease by external imaging was investigated. The structure-binding-relationship which provides optimal localization of radiolabelled antagonist of the muscarinic acetylcholine receptors in the brain was studied. These relationships were also studied with respect to localization in the pancreas. (ACR)

  20. F-18 labeled 3-fluorodiazepam

    SciTech Connect

    Luxen, A.; Barrio, J.R.; Bida, G.T.; Satyamurthy, N.; Phelps, M.E.

    1985-05-01

    3-Fluorodiazepam is a new and potent antianxiety agent with prolonged action. The authors found that molecular fluorine (0.5% in Ne) reacts cleanly with diazepam in freon or chloroform at room temperature to produce 3-fluorodiazepam in good yields. Successful syntheses have employed 2:1 to 5:1 molar ratios diazepam: fluorine to minimize the formation of byproducts. (/sup 18/F) 3-Fluorodiazepam, a potential candidate for PET studies, (specific activity 3-5 Ci/mmol) has been synthesized from /sup 18/F-F/sub 2/ using the same procedure, followed by column chromatographic purification (Silicagel, dichloromethane: ethyl acetate, 5:1) with a radiochemical yield of 12-20% (50% maximum) and a chemical and radiochemical purity >99% as judged by reversed-phase high pressure liquid chromatography analysis (Ultrasyl octyl column, 10 ..mu.. m, 4.6 x 250 mm i.d., 60% MeOH 40% water; flow rate, 1.0 ml/min; retention time for (/sup 18/F) fluorodiazepam, 11.4 min; for diazepam, 13.5 min; radioactivity and ultraviolet detectors). Lower radiochemical yields (5-7%), and significant formation of by-products were observed when (/sup 18/F)acetylhypofluorite, prepared in the gasphase, was used as the reagent. Readily accessible routes to /sup 18/F-labeled benzodiazepines of higher specific activity were also investigated. Approaches to the synthesis of high specific activity (>200 Ci/mmol) (/sup 18/F)3-fluorodiazepam involve nucleophilic displacement at carbon-3 (e.g. from 3-chlorodiazepam) with (/sup 18/F)fluoride ion. The results presented here demonstrate the synthetic accessibility of /sup 18/F-labeled benzodiazepines for application in neurotransmitter ligand studies with PET.