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

International Nuclear Information System (INIS)

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 ( 3 H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of 3 H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked 3 H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked 3 H-EFDA in toluene alone, and of the protein-linked 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

Design of photoaffinity labeling probes derived from 3,4,5-trimethylfuran-2(5H)-one for mode of action elucidation

Czech Academy of Sciences Publication Activity Database

Pošta, Martin; Soós, V.; Beier, Petr

2016-01-01

Roč. 72, 27/28 (2016), s. 3809-3817 ISSN 0040-4020 Institutional support: RVO:61388963 Keywords : photoaffinity labeling probes * protein cross-linking * smoke * butenolides Subject RIV: CC - Organic Chemistry Impact factor: 2.651, year: 2016

Photoaffinity labeling of myosin subfragment-one-with 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate

International Nuclear Information System (INIS)

Mahmood, R.

1985-01-01

The photoaffinity analogue 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz 2 ATP) contains the photoreactive benzophenone group esterified at the 2' or 3' hydroxyl groups of ribose. MgBz 2 ADP has a single binding site on skeletal myosin chymotryptic subfragment-one (SF 1 ) with a binding constant of 3.2 x 10 5 M -1 . Bz 2 ATP is also a substrate for the ATPase activity of SF 1 in the presence of different cations. The irradiation of SF 1 with [ 3 H]Bz 2 ATP photoinactivates the ATPase activity with concomitant incorporation of the analogue into the enzyme. Polyacrylamide gel electrophoresis of photolabeled SF 1 after milk trypsin digestion shows that all three tryptic peptides, 25 K, 50K, and 20 K, and both light chains are labeled. The presence of ATP during irradiation reduces labeling of the 50 K peptide only indicating that the other peptides are non-specifically labeled. To reduce the non-specific labeling [ 3 H]Bz 2 ATP is trapped on SF 1 by cross-linking the two reactive thiols, SH 1 and SH 2 , by N,N'-p-phenylene dimaleimide or Co(II)/Co(III) phenanthroline complexes. The Co(II)/Co(III) phenanthroline modified [ 14 C]Bz 2 ATP-SF 1 , after proteolytic digestion, yields five labeled peptides which were purified by gel filtration and high performance liquid chromatography

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

OpenAIRE

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 a...

Photoaffinity labeling of [3H]flunitrazepam- and [3H]Ro15-4513-bound pellets in rat cerebral cortex and cerebellum

International Nuclear Information System (INIS)

Chiu, T.H.; Yu, Onnfoh; Rosenberg, H.C.

1989-01-01

Irreversible incorporation of [ 3 H]flunitrazepam and [ 3 H]Ro15-4513 into GABA/benzodiazepine receptor subunits was studied by UV/irradiation using ligand-bound membrane pellets from rat cerebral cortical and cerebellar synaptic membranes. Specific incorporation for [ 3 H]flunitrazepam was greater in the pellet than in the suspension. The incorporation was identical for [ 3 H]Ro15-4513 in both pellet and suspension. With the ligand-bound pellets, 50% of the available binding sites were photolabeled by both ligands in cortex and cerebellum. SDS polyacrylamide gel electrophoresis and fluorography of [ 3 H]flunitrazepam photo-labeled receptor revealed the same number of major sites in both brain regions. In contrast, [ 3 H]Ro15-4513 appears to label fewer sites in cortex and cerebellum. Photoaffinity labeling with [ 3 H]flunitrazepam in ligand-bound membrane pellet provides a more selective and reliable method for studying the subunit structure of GABA/benzodiazepine receptor complex

Synthesis of 25-hydroxyvitamin D3 3β-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D3: Photoaffinity labeling of rat serum vitamin D binding protein

International Nuclear Information System (INIS)

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

1991-01-01

Vulnerability of 25-hydroxy-[26,27- 3 H]vitamin D 3 3β-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D 3 (25-OH-D 3 ) toward standard conditions of carboxymethylationin promoted the authors to synthesize 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D 3 , and 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitro-[3,5- 3 H]phenyl)amino]propyl ether ( 3 H-25-ANE), the radiolabeled counterpart of 25-ANE competes for the 25-OH-D 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 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 3 . These results provide strong evidence for the covalent labeling of the 25-OH-D 3 binding site in rDPB by 3 H-25-ANE

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

International Nuclear Information System (INIS)

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

1987-01-01

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

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

International Nuclear Information System (INIS)

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

1989-01-01

The photoaffinity probe 5-azido-uridine 5'-β-[ 32 P]-diphosphate glucose was used to identify the major UDPG-binding polypeptide of red beet (1,3)-β-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

Recent Advances in Target Characterization and Identification by Photoaffinity Probes

Directory of Open Access Journals (Sweden)

Sang J. Chung

2013-08-01

Full Text Available Target identification of biologically active molecules such as natural products, synthetic small molecules, peptides, and oligonucleotides mainly relies on affinity chromatography, activity-based probes, or photoaffinity labeling (PAL. Amongst them, activity-based probes and PAL have offered great advantages in target identification technology due to their ability to form covalent bonds with the corresponding targets. Activity-based probe technology mainly relies on the chemical reactivity of the target proteins, thereby limiting the majority of the biological targets to enzymes or proteins which display reactive residues at the probe-binding site. In general, the probes should bear a reactive moiety such as an epoxide, a Michael acceptor, or a reactive alkyl halide in their structures. On the other hand, photoaffinity probes (PAPs are composed of a target-specific ligand and a photoactivatable functional group. When bound to the corresponding target proteins and activated with wavelength-specific light, PAPs generate highly reactive chemical species that covalently cross-link proximal amino acid residues. This process is better known as PAL and is widely employed to identify cellular targets of biologically active molecules. This review highlights recent advances in target identification by PAL, with a focus on the structure and chemistry of the photoaffinity probes developed in the recent decade, coupled to the target proteins identified using these probes.

Equivalent molecular mass of cytosolic and nuclear forms of Ah receptor from Hepa-1 cells determined by photoaffinity labeling with 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin

International Nuclear Information System (INIS)

Prokipcak, R.D.; Okey, A.B.

1990-01-01

The structure of the Ah receptor previously has been extensively characterized by reversible binding of the high affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin. We report the use of [ 3 H]2,3,7,8-tetrachlorodibenzo-p-dioxin as a photoaffinity ligand for Ah receptor from the mouse hepatoma cell line Hepa-1c1c9. Both cytosolic and nuclear forms of Ah receptor could be specifically photoaffinity-labeled, which allowed determination of molecular mass for the two forms under denaturing conditions. After analysis by fluorography of polyacrylamide gels run in the presence of sodium dodecyl sulfate, molecular mass for the cytosolic form of Ah receptor was estimated at 92,000 +/- 4,300 and that for the nuclear form was estimated at 93,500 +/- 3,400. Receptor in mixture of cytosol and nuclear extract (each labeled separately with [ 3 H]2,3,7,8-tetrachlorodibenzo-p-dioxin) migrated as a single band. These results are consistent with the presence of a common ligand-binding subunit of identical molecular mass in both cytosolic and nuclear complexes

Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

International Nuclear Information System (INIS)

Raymond, J.R.; Fargin, A.; Lohse, M.J.; Regan, J.W.; Senogles, S.E.; Lefkowitz, R.J.; Caron, M.G.

1989-01-01

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor

Photoaffinity labeling the substance P receptor using a derivative of substance P containing para-benzoylphenylalanine

International Nuclear Information System (INIS)

Boyd, N.D.; White, C.F.; Leeman, S.E.; Cerpa, R.; Kaiser, E.T.

1991-01-01

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe 8 residue of SP. [Phe 8 (OpBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125 I-labeled Bolton-Hunter conjugate of [Phe 8 (pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites with an affinity K D = 0.4 nM. The binding of 125 I-[Phe 8 (pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125 I-[Phe 8 (pBz)]SP underwent covalent linkage to two polypeptides of M r = 53 000 and 46 000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125 I-[Phe 8 (pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies. The highly specific and remarkably efficient photolabeling achieved with 125 I-[Phe 8 (pBz)]SP suggests that this photoaffinity probe will be of considerable value for the characterization of the molecular structure of the SP receptor

Affinity purification of human granulocyte macrophage colony-stimulating factor receptor alpha-chain. Demonstration of binding by photoaffinity labeling

International Nuclear Information System (INIS)

Chiba, S.; Shibuya, K.; Miyazono, K.; Tojo, A.; Oka, Y.; Miyagawa, K.; Takaku, F.

1990-01-01

The human granulocyte macrophage colony-stimulating factor (GM-CSF) receptor alpha-chain, a low affinity component of the receptor, was solubilized and affinity-purified from human placenta using biotinylated GM-CSF. Scatchard analysis of 125 I-GM-CSF binding to the placental membrane extract disclosed that the GM-CSF receptor had a dissociation constant (Kd) of 0.5-0.8 nM, corresponding to the Kd value of the GM-CSF receptor alpha-chain on the intact placental membrane. Affinity labeling of the solubilized protein using a photoreactive cross-linking agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), demonstrated a single specific band of 70-95 kDa representing a ligand-receptor complex. Approximately 2 g of the placental membrane extract was subjected to a biotinylated GM-CSF-fixed streptavidin-agarose column, resulting in a single major band at 70 kDa on a silver-stained sodium dodecyl sulfate gel. The radioiodination for the purified material disclosed that the purified protein had an approximate molecular mass of 70 kDa and a pI of 6.6. Binding activity of the purified material was demonstrated by photoaffinity labeling using HSAB- 125 I-GM-CSF, producing a similar specific band at 70-95 kDa as was demonstrated for the crude protein

A novel photoaffinity ligand for the phencyclidine site of the N-methyl-D-aspartate receptor labels a Mr 120,000 polypeptide

International Nuclear Information System (INIS)

Sonders, M.S.; Barmettler, P.; Lee, J.A.; Kitahara, Y.; Keana, J.F.; Weber, E.

1990-01-01

A radiolabeled photoaffinity ligand has been developed for the N-methyl-D-aspartate (NMDA)-preferring excitatory amino acid receptor complex. [3H]3-Azido-(5S, 10R)(+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine [3H]3-azido-MK-801 demonstrated nearly identical affinity, density of binding sites, selectivity, pH sensitivity, and pharmacological profile in reversible binding assays with guinea pig brain homogenates to those displayed by its parent compound, MK-801. When employed in a photo-labeling protocol designed to optimize specific incorporation, [3H]3-azido-MK-801 labeled a single protein band which migrated in sodium dodecyl sulfate-polyacrylamide gels with Mr = 120,000. Incorporation of tritium into this band was completely inhibited when homogenates and [3H]3-azido-MK-801 were coincubated with 10 microM phencyclidine. These data suggest that the phencyclidine site of the NMDA receptor complex is at least in part comprised of a Mr = 120,000 polypeptide

Photoaffinity labeling of opiate (enkephalin) receptor of rat brain plasma membranes with 125I(D-Ala2, p-N3-Phe4-Met5)-enkephalin

International Nuclear Information System (INIS)

Yeung, C.W.T.

1986-01-01

A photoreactive (D-Ala 2 , p-N 3 -Phe 4 -Met 5 )enkephalin derivative was prepared, iodinated with carrier free 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

Photoaffinity labeling of the human erythrocyte monosaccharide transporter with an aryl azide derivative of D-glucose

International Nuclear Information System (INIS)

Shanahan, M.F.; Wadzinski, B.E.; Lowndes, J.M.; Ruoho, A.E.

1985-01-01

A photoreactive, radioiodinated derivative of glucose, N-(4-iodoazidosalicyl)-6-amido-6-deoxyglucopyranose (IASA-glc), has been synthesized and used as a photoaffinity label for the human erythrocyte monosaccharide transporter. Photoinactivation and photoinsertion are both light-dependent and result in a marked decrease in the absorption spectra of the compound. When [ 125 I]IASA-glc was photolyzed with erythrocyte ghost membranes, photoinsertion of radiolabel was observed in three major regions, spectrin, band 3, and a protein of 58,000 daltons located in the zone 4.5 region. Of the three regions which were photolabeled, only labeling of polypeptides in the zone 4.5 region was partially blocked by D-glucose. In the non-iodinated form, N-(4-azidosalicyl)-6-amido-6-deoxy-glucopyranose inhibited the labeling of the transporter by [ 125 I]IASA-glc more effectively than D-glucose. The ability to synthesize this [ 125 I]containing photoprobe for the monosaccharide transporter at carrier-free levels offers several new advantages for investigating the structure of this transport protein in the erythrocyte

Photoaffinity labeling of the progesterone receptor from human endometrial carcinoma

International Nuclear Information System (INIS)

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

1985-01-01

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- 3 H]promegestone ([ 3 H]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 [ 3 H]R5020 was observed in EnCa 101 grown in the absence of estrogen, nor was any observed in EnCa K or EnCa V

Sex pheromone receptor proteins. Visualization using a radiolabeled photoaffinity analog

International Nuclear Information System (INIS)

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

1988-01-01

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-[ 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

A V1-vascular vasopressin antagonist suitable for radioiodination and photoaffinity labeling

International Nuclear Information System (INIS)

Thibonnier, M.; Chehade, N.; Hinko, A.

1990-01-01

We have previously characterized the V1-vascular arginine vasopressin (AVP) receptors of human platelets. We now report on a radiomonoiodinated and photoreactive V1-vascular AVP antagonist (V1-ag) to be used for the purification of human V1-vascular AVP receptors. The V1-ag, d(CH2)5Tyr(Me)Tyr(NH2)AVP was modified by radiomonoiodination of d(CH2)5-Tyr(Me)Tyr(NH2)AVP with the Iodogen technique, and derivatization of d(CH2)5Tyr(Me)Tyr[125I](NH2)-AVP with the photoreactive crosslinker, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) (each step included HPLC purification). In competition experiments, the affinity of these V1-ag for the human platelet AVP receptors remained excellent. Irreversible photoaffinity labeling of the platelet V1-vascular AVP receptor was successfully achieved by UV lamp exposure (365 nm, 20 min). Thus, AzBz-d(CH2)5Tyr(Me)Tyr[125I](NH2)AVP is a promising tool to use for the purification of human V1-vascular AVP receptors

[3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

International Nuclear Information System (INIS)

Benke, D.; Honer, M.; Mohler, H.; Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C.

1999-01-01

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [ 3 H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [ 3 H]CGP 61594 labeled with high affinity (K D =23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [ 3 H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [ 3 H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [ 3 H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

[{sup 3}H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

Energy Technology Data Exchange (ETDEWEB)

Benke, D.; Honer, M.; Mohler, H. [Institute of Pharmacology, ETH and University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich (Switzerland); Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C. [NS Research, Novartis Pharma AG, CH-4002 Basle (Switzerland)

1999-02-01

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [{sup 3}H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [{sup 3}H]CGP 61594 labeled with high affinity (K{sub D}=23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [{sup 3}H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [{sup 3}H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [{sup 3}H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

(/sup 3/H)Clonazepam, like (/sup 3/H)flunitrazepam, is a photoaffinity label for the central type of benzodiazepine receptors

Energy Technology Data Exchange (ETDEWEB)

Sieghart, W. (Vienna Univ. (Austria)); Moehler, H. (Hoffmann-La Roche (F.) and Co., Basel (Switzerland))

1982-06-16

(/sup 3/H)Clonazepam, like (/sup 3/H)flunitrazepam, is irreversibly bound to membrane proteins of brain tissue when exposed to UV light. In polyacrylamide gel electrophoresis followed by fluorography, the same pattern of photolabelled proteins was obtained in cerebellum and in hippocampus when either (/sup 3/H)clonazepam or (/sup 3/H)flunitrazepam was used as photoaffinity label. Since (/sup 3/H)clonazepam does not interact with the peripheral type of benzodiazepine binding site present in the brain, these results confirm previous evidence that the proteins photolabelled with (/sup 3/H)flunitrazepam are associated with the central type of benzodiazepine receptor.

Photoaffinity labeling of the lumenal K+ site of the gastric (H+ + K+)-ATPase

International Nuclear Information System (INIS)

Keeling, D.J.; Fallowfield, C.; Lawrie, K.M.; Saunders, D.; Richardson, S.; Ife, R.J.

1989-01-01

A photoaffinity label for the lumenal K+ site of the gastric (H+ + K+)-ATPase has been identified. Seven azido derivatives based upon the reversible K+ site inhibitor SCH 28080 were studied, one of which, m-ATIP (8-(3-azidophenylmethoxy)-1,2,3-trimethylimidazo[1,2-a] pyridinium iodide), was subsequently synthesized in radiolabeled form. In the absence of UV irradiation, m-ATIP inhibited K+ -stimulated ATPase activity in lyophilized gastric vesicles competitively with respect to K+, with a Ki value of 2.4 microM at pH 7.0. Irradiation of lyophilized gastric vesicles at pH 7.0 with [ 14 C]m-ATIP in the presence of 0.2 mM ATP resulted in a time-dependent inactivation of ATPase activity that was associated with an incorporation of radioactivity into a 100-kDa polypeptide representing the catalytic subunit of the (H+ + K+)-ATPase. Both inactivation and incorporation were blocked in the presence of 10 mM KCl but not with 10 mM NaCl, consistent with interaction at the K+ site. The level of incorporation required to produce complete inhibition of ATPase activity was 1.9 +/- 0.2 times the number of catalytic phosphorylation sites in the same preparation. Tryptic digestion of gastric vesicle membranes, labeled with [ 14 C]m-ATIP, failed to release the radioactivity from the membranes suggesting that the site of interaction was close to or within the membrane-spanning sections of this ion pump

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

Energy Technology Data Exchange (ETDEWEB)

Thomassin, J.; Tephly, T.R. (Univ. of Iowa, Iowa City (USA))

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).

Affinity Labeling of Membrane Receptors Using Tissue-Penetrating Radiations

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Franklin C. Wong

2013-01-01

Full Text Available 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.

Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes

International Nuclear Information System (INIS)

Liang, T.; Cheung, A.H.; Reynolds, G.F.; Rasmusson, G.H.

1985-01-01

21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a K/sub i/ value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [ 3 H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([ 3 H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2- 3 H]Diazo-MAPD binds to a single high affinity site in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000

Photoaffinity labeling of cAMP-dependent protein kinase by 4-azido-2-nitrophenyladenylyl pyrophosphate

International Nuclear Information System (INIS)

Johnson, D.R.; Ho, H.T.; Wong, S.S.

1986-01-01

A photoaffinity analogue of ATP, 4-azido-2-nitrophenyl-adenylyl pyrophosphate (ANAP) has been synthesized to investigate the topographical interaction between the catalytic and the regulatory subunits of the bovine heart type II cAMP-dependent protein kinase. The synthesis involves coupling of 4-azido-2-nitrophenyl phosphate with adenosine 5'-monophosphomorpholidate. ANAP has an absorption maximum at 260 nm (molar absorptivity = 35.4 x 10 3 M -1 cm -1 ) and a shoulder at 320 nm. Kinetically, ANAP inhibits the enzyme competitively against ATP with a Ki of 0.37 mM. The catalytic subunit is inactivated by ANAP upon photolysis in the presence of magnesium ion. ATP protects the enzyme from photoinactivation but the regulatory subunit does not. Gel electrophoretic analysis of the enzyme labeled by [ 14 C]ANAP shows that the photoincorporated ANAP is associated mainly with the catalytic subunit, even when the regulator dimer is in twelve fold excess. Little or no ANAP is found incorporated into the regulator subunit. The data suggest that the photoreactive portion of ANAP does not lie within reach of the regulatory protein when the analogue is bound to the catalytic subunit

Active site labeling of the guanine-7-methyltransferase

International Nuclear Information System (INIS)

Streaker, E.; Sitz, T.O.

1992-01-01

Studies on the guanine-7-methyltransferase have defined three domains in the active site: the S-adenosylmethionine (SAM) region, the cap region (GpppG), and the RNA binding domain (--NpNpNpNpNp---). The authors attempted to label the SAM binding domain by a photoaffinity label using 8-azido-SAM and another method using 3 H-SAM and long exposures to uv-light. Neither method was successful. The next approach was to attempt to label the cap-RNA binding domain (GpppGpNpNpNpNpN) by synthesizing RNA containing 8-azido-Ap using an in vitro transcription system and T7 RNA polymerase. The 8-azido-ATP inhibited the T7 RNA polymerase preventing the synthesis of RNA. As they were unable to synthesize the photoaffinity label, they next tried to synthesize an end labeled RNA and directly label by long exposures to uv-light. When the enzyme was incubated with 32 P-labeled RNA for 15 min at 37 degrees and then exposed to a germicidal lamp for various times at O degrees, optimal labeling occurred after 45 min. Various enzyme preparations were labeled by this method and two polypeptides were found to specifically bind the non-methylated mRNA analog. This labeling method should allow characterization of the subunit structure and generate information about the nature of the RNA binding domain

Photoaffinity labeling of bacteriorhodopsin

International Nuclear Information System (INIS)

Ding, Weidong; Tsipouras, Athanasios; Ok, Hyun; Yamamoto, Toshihiro; Gawinowicz, M.A.; Nakanishi, Koji

1990-01-01

14 C-Labeled optically pure 3S- and 3R-(diazoacetoxy)-all-trans-retinals were incorporated separately into bacterioopsin to reconstitute functional bacteriorhodopsin (bR) analogues, 3S- and 3R-diazo-bRs. UV irradiation at 254 nm generated highly reactive carbenes, which cross-linked the radiolabeled retinals to amino acid residues in the vicinity of the β-ionone ring. The 3S- and 3R-diazo analogues were found to cross-link, respectively, to cyanogen bromide fragments CN 7/CN 9 and CN 8/CN 9. More specifically, Thr121 and Gly122 in fragment CN 7 were found to be cross-linked to the 3S-diazo analogue. The identification of cross-linked residues and fragments favors assignments of the seven helices A-G-F-E-D-C-B or B-C-D-E-F-G-A to helices 1-2-3-4-5-6-7 in the two-dimensional electron density map. The present results show that the chromophore chain is oriented with the ionone ring inclined toward the outside of the membrane (the 9-methyl group also faces the extracellular side of the membrane)

Understanding the mechanism of sweet taste: synthesis of tritium labeled guanidineacetic acids

Energy Technology Data Exchange (ETDEWEB)

Nagarajan, S.; Kellogg, M.S.; DuBois, G.E. (NutraSweet Company, Mt. Prospect, IL (United States)); Williams, D.S. (Amersham International plc, Cardiff (United Kingdom). Cardiff Labs.); Gresk, C.J.; Markos, C.S. (Searle Research and Development, Skokie, IL (United States))

1992-08-01

Syntheses of tritium labeled guanidineacetic acid sweetener and a tritiated photoaffinity labeling reagent via the catalytic hydrogenation of the dibromo intermediates are described. These labeled compounds were required for the investigation of sweet taste mechanism. (author).

Understanding the mechanism of sweet taste: synthesis of tritium labeled guanidineacetic acids

International Nuclear Information System (INIS)

Nagarajan, S.; Kellogg, M.S.; DuBois, G.E.; Williams, D.S.

1992-01-01

Syntheses of tritium labeled guanidineacetic acid sweetener and a tritiated photoaffinity labeling reagent via the catalytic hydrogenation of the dibromo intermediates are described. These labeled compounds were required for the investigation of sweet taste mechanism. (author)

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 D3 binding site

International Nuclear Information System (INIS)

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

1991-01-01

In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitro-[3,5- 3 H]phenyl)amino]propyl ether ( 3 H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D 3 for the binding site of the latter in hDBP and (2) 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 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 3

Photoaffinity labeling of the oxysterol binding protein

International Nuclear Information System (INIS)

Taylor, F.R.; Kandutsch, A.A.; Anzalone, L.; Spencer, T.A.

1986-01-01

A cytosolic receptor protein for oxygenated sterols, that is thought to be involved in the regulation of HMG-CoA reductase and cholesterol biosynthesis, can be labeled covalently by the photoactivated affinity compound [5,6- 3 H]-7,7'-azocholestane-3β,25-diol (I). Several other compounds were tested including 25-hydroxycholesta-4,6-dien-3-one, 25-azido-27-norcholest-5-en-3β-ol,3β,25-dihydroxycholest-5-en-7-one and 3β-hydroxycholesta-8(14),9(11)-dien-15-one. However, these sterols either did not bind to the receptor with adequate affinity or did not react covalently with the receptor during photolysis. Compound I binds to the receptor with very high affinity (K/sub d/ = 30 nM). After activation with long wavelength UV, two tritium labeled proteins, M/sub r/ approximately 95K and 65K daltons, are found upon SDS gel electrophoresis. No labeling occurs when the binding reaction is carried out in the presence of a large excess of 25-hydroxycholesterol. It is possible that the smaller polypeptide is a degradation product. Under the reaction conditions investigated so far labeling is relatively inefficient (< 1% of bound sterol). These results are generally consistent with previous information suggesting that the M/sub r/ of the receptor subunit is 97,000. Covalent labeling of the receptor should greatly facilitate its further purification and characterization

Covalent labeling of the beta-adrenergic ligand-binding site with para-(bromoacetamidyl)benzylcarazolol. A highly potent beta-adrenergic affinity label

International Nuclear Information System (INIS)

Dickinson, K.E.; Heald, S.L.; Jeffs, P.W.; Lefkowitz, R.J.; Caron, M.G.

1985-01-01

Para-(Bromoacetamidyl)benzylcarazolol (pBABC) was synthesized and found to be an extremely potent affinity label for beta-adrenergic receptors. Its interaction with mammalian (rabbit and hamster lung) and nonmammalian (turkey and frog erythrocyte) beta-adrenergic receptors was similar, displaying EC 50 values of 400-900 pM for inhibiting 125 I-cyanopindolol binding to these receptors. pBABC reduced the number of beta-adrenergic receptors in frog erythrocyte membranes, without any change in the affinity of the remaining sites for [ 125 I]iodocyanopindolol. pBABC has been radioiodinated. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this affinity probe specifically labeled the beta-adrenergic peptide of a purified preparation of hamster lung, with high efficiency (approximately 40%) and with a pharmacological specificity characteristic of an interaction at the beta 2-adrenergic receptor ligand-binding site. Comparison of the proteolyzed products derived from purified receptor labeled with [ 125 I]pBABC and with the photoaffinity agent [ 125 I]p-azidobenzylcarazolol suggested that covalent labeling of the beta-adrenergic receptor by these probes occurs at similar domains of the beta-adrenergic receptor

Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

International Nuclear Information System (INIS)

Tejedor, F.; Amils, R.; Ballesta, J.P.

1985-01-01

Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [ 125 I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis

Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

International Nuclear Information System (INIS)

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

1989-01-01

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

I. Use of m- and p-azidobenzamidines, 4-fluoro-3-nitro-phenylazide, and 3-azido-1,2,4-triazole as photoaffinity probes of tryptic binding site conformation. II. Analysis of tryptophan in proteins by an acidic reaction of 3-diazonium-1,2,4-triazole

Energy Technology Data Exchange (ETDEWEB)

DeTraglia, M.C.

1979-01-01

Meta- and para-azidobenzamidine have been prepared and evaluated as photoaffinity labels. The compounds inhibit trypsin reversibly in the dark and are competitive with substrate binding. Upon photolysis, irreversible noncompetitive inhibition is observed and is dependent upon concentration, photolysis time, and pH. Specificity of the probes is indicated by experiments in which p-tosyl-L-arginine methyl ester, a trypsin substrate, is used to protect against photoinactivation.

Photoaffinity labeling of opiate receptors using intrinsically photoactive 3H-opiates

International Nuclear Information System (INIS)

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

1988-01-01

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

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

International Nuclear Information System (INIS)

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

Novel photoaffinity ligands for the GA-receptor

International Nuclear Information System (INIS)

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

1990-01-01

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 [ 3 H]-GA 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 α-amylase production in barley half-seeds and stimulate tissue elongation in d 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

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein

International Nuclear Information System (INIS)

Lee, G.; Ronai, Z.A.; Pincus, M.R.; Brandt-Rauf, P.W.; Weinstein, I.B.; Murphy, R.B.; Delohery, T.M.; Nishimura, S.; Yamaizumi, Z.

1989-01-01

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with modified p21 protein, the cells were pulsed with [ 35 S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21 - protein complexes. By using this technique, the authors found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. They suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes

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

International Nuclear Information System (INIS)

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

1991-01-01

A high affinity tritiated azido-diphenylpiperazine derivative, 3-azido 3 H GBR-12935, was synthesized as a potential photoaffinity probe of the dopamine transporter. Initially, the reversible binding of 3-azido 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 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 3 H GBR-12935 binding to rat striatal membranes. These data suggest that 3-azido 3 H GBR-12935, like other diphenylpiperazines such as 3 H GBR-12935 and 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 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 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 3 H GBR-12935 is glycosylated

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein.

Science.gov (United States)

Lee, G; Ronai, Z A; Pincus, M R; Brandt-Rauf, P W; Murphy, R B; Delohery, T M; Nishimura, S; Yamaizumi, Z; Weinstein, I B

1989-11-01

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

Science.gov (United States)

Katz, B M; Lundquist, L J; Walsh, D A; Glass, D B

1989-06-01

PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

Membrane vesicles from multidrug-resistant human carcinoma cells contain a specific 150,000-170,000 dalton protein detected by photoaffinity labeling

International Nuclear Information System (INIS)

Cornwell, M.M.; Safa, A.R.; Felsted, R.L.; Gottesman, M.M.; Pastan, I.

1986-01-01

The authors have selected multidrug-resistant human KB carcinoma cells in high levels of colchicine (KB-C4) or vinblastine (KB-V1) which are cross-resistant to many other structurally unrelated chemotheraputic agents. To determine the mechanism of reduced drug accumulation, they measured 3 H-vinblastine ( 3 H-VBL) association with membrane vesicles made from parental drug sensitive, drug-resistant and revertant cells. Membrane vesicles from highly multidrug resistant cells exhibited increased specific and saturable binding of vinblastine, (Kd = 1 μM) that was temperature dependent and trypsin sensitive. To identify the molecules which bind vinblastine, membrane vesicles were exposed to two photo-activatable analogs of vinblastine, (N-P-(azido-3,5,-[ 3 H]-benzoyl)-N'-β-aminoethylvindisine ( 3 H-NAB) and N-P-(azido-3-[ 125 I]-solicyl)-N'-β-aminoethylvindesine ( 125 I-NASV). The specific labeling of a 150,000-170,000 dalton protein in membrane vesicles from multidrug-resistant KB-C4 and KB-V1 cells was found. 125 I-NASV labeling was inhibited by vinblastine, vincrinstine and verapamil but not by colchicine or dexamethasone. The 150,000-170,000 dalton protein may have an important role in the multidrug resistance phenotype

Comparative characterization of thyroid hormone receptors and binding proteins in rat liver nucleus, plasma membrane, and cytosol by photoaffinity labeling with L-thyroxine

International Nuclear Information System (INIS)

Dozin, B.; Cahnmann, H.J.; Nikodem, V.M.

1985-01-01

Photoaffinity labeling with underivatized thyroxine (T4) was used to identify and compare the T4 binding proteins in rat liver cytosol, nuclear extract, and purified plasma membrane. When these subcellular fractions were incubated with a tracer concentration of [125I]T4, irradiated with light above 300 nm, and individually analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the radioactivity profiles revealed the presence of T4 binding proteins of molecular masses of 70, 52, 43, 37, 30, and 26 kilodaltons (kDa) in cytosol, of 96, 56, 45, and 35 kDa in nuclear extract, and of 70, 44, and 30 kDa in plasma membrane. Competition experiments performed in the presence of a 1000-fold excess of unlabeled T4 demonstrated that these binding proteins display different hormone binding activities. The similar electrophoretic mobilities of some binding proteins present in the different subcellular fractions, i.e., the 70-, 43-45-, and 30-kDa proteins, suggested that these proteins might be identical. However, double-labeling experiments in which plasma membrane, nuclear extract, and cytosol were photolabeled with either [125I] or [131I]T4 and mixed, two at a time, in all possible combinations showed that from one cellular fraction to another, the radioactivity peaks corresponding to the approximately 70-, 43-45-, and 30-kDa proteins were not superimposed. Their relative positions on the gel differed by one or two slices, which indicated differences in molecular mass of 1.9-3.6 kDa. Moreover, enzymatic digestion with Staphylococcus aureus V8 protease of these three proteins, prepared from each subcellular fraction, yielded dissimilar peptide patterns

CHARACTERIZATION OF HUMAN LIVER MICROSOMAL UDP-GLYCOSYLTRANSFERASES USING PHOTOAFFINITY ANALOGS

NARCIS (Netherlands)

LITTLE, JM; DRAKE, RR; VONK, R; KUIPERS, F; LESTER, R; RADOMINSKA, A

The photoaffinity analogs [beta-P-32]5-azido-UDP-glucuronic acid ([P-32]5N3UDP-GlcUA) and [beta-P-32]5-azido-UDP-glucose ([P-32]5N(3)UDP-Glc) were used to characterize UDP-glycosyl-transferases of microsomes prepared from human liver. Photoincorporation of both probes into proteins in the 50- to

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

International Nuclear Information System (INIS)

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

1986-01-01

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 [ 3 H]25-OH-D3 or [ 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 [ 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 [ 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 [ 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

Photoaffinity labeling of undecaprenyl pyrophosphate synthetase with a farnesyl pyrophosphate analogue

International Nuclear Information System (INIS)

Baba, T.; Muth, J.; Allen, C.M.

1985-01-01

The prenyl transferase undecaprenyl pyrophosphate synthetase was partially purified from the cytosolic fraction of Escherichia coli. Its enzymic products were characterized as a family of cis-polyprenyl phosphates, which ranged in carbon number from C55 to C25. The enzyme is constituted of two subunits of approximately 30,000 molecular weight. A radiolabeled photolabile analogue of t,t-farnesyl pyrophosphate, [ 3 H]2-diazo-3-trifluoropropionyloxy geranyl pyrophosphate, was shown to label Lactobacillus plantarum and E. coli undecaprenyl pyrophosphate synthetase on UV irradiation in the presence of isopentenyl pyrophosphate and divalent cations. The only labeled polypeptide migrated on electrophoresis in a sodium dodecyl sulfate-polyacrylamide gel at a molecular weight of approximately 30,000. No protein was radiolabeled when the natural substrate, t,t-farnesyl pyrophosphate was included in the irradiation mixture. Irradiation in the presence of MgCl 2 without isopentenyl pyrophosphate gave less labeling of the polypeptide. Irradiation with only isopentenyl pyrophosphate gave little labeling of the polypeptide. When the enzyme was irradiated with 3H-photoprobe, [ 14 C]isopentenyl pyrophosphate, and MgCl 2 , the labeled polypeptide gave a ratio of 14 C/ 3 H that indicated the product must also bind to the enzyme on irradiation. These results demonstrate the ability to radiolabel the allylic pyrophosphate binding site and possibly product binding site of undecaprenyl pyrophosphate synthetase by a process which is favored when both cosubstrate and divalent cations are present

Identification of the D-1 dopamine receptor subunit in rat striatum after photoaffinity labeling

Energy Technology Data Exchange (ETDEWEB)

Kuno, T; Tanaka, C [Kobe Univ. (Japan). School of Medicine

1982-12-28

When rat striatal membranes, photolabeled with (/sup 3/H)dopamine under assay conditions similar to those used for dopamine-sensitive adenylate cyclase, were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, several radioactively labeled bands appeared. Labeling of these bands was reduced in the presence of non-radioactive dopamine during photolysis, but was unaffected by the presence of sulpiride. Haloperidol preferentially reduced the labeling of the main band which had a molecular weight of about 57,000 rather than the other weakly labeled bands. Labeling of this 57,000 dalton protein was not apparent when rat cerebellar membranes were used and was markedly eliminated by kainic acid-induced lesions that destroyed the intrastriatal nerve cell bodies. These results indicate that this 57,000 dalton protein is the binding subunit of the D-1 dopamine receptor.

Lymphocyte receptors for pertussis toxin

Energy Technology Data Exchange (ETDEWEB)

Clark, C.G.; Armstrong, G.D. (Univ. of Alberta, Edmonton (Canada))

1990-12-01

We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes.

The diageotropica mutant of tomato lacks high specific activity auxin sites

International Nuclear Information System (INIS)

Hicks, G.R.; Lomax, T.L.; Rayle, D.L.

1989-01-01

Tomato (Lycopersicum esculentum, Mill) plants homozygous for the single gene diageotropica (dgt) mutation have reduced shoot growth, abnormal vascular tissue, altered leaf morphology, and lack of lateral root branching. These and other morphological and physiological abnormalities suggest that dgt plants are unable to respond to the plant growth hormone auxin (indole-3-acetic acid, IAA). The photoaffinity auxin analogue 3 H-5N 3 -IAA specifically labels a polypeptide doublet of 40 ad 42 kD in membrane preparations from stems of the parental variety VFN8, but not from stems of dgt. In elongation tests, excised dgt roots respond in the same manner to IAA an VFN8 roots. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system which is altered in a tissue-specific manner in the mutant

Qualitative variation of photolabelled benzodiazepine receptors in different species.

Science.gov (United States)

Hebebrand, J; Friedl, W; Lentes, K U; Propping, P

1986-01-01

In order to examine whether species differences of benzodiazepine receptor subunits exist, we compared the fluorographic pattern of photoaffinity labelled subunits after SDS-PAGE in five species: fish, frog, chicken, mouse and calf. Each species showed a distinct pattern of specifically labelled proteins. We conclude that species variation of benzodiazepine receptor does indeed exist.

Characterization of cap binding proteins associated with the nucleus

International Nuclear Information System (INIS)

Patzelt, E.

1986-04-01

Eucaryotic mRNAs a carry 7-methylguanosine triphosphate residue (called cap structure) at their 5' terminus. The cap plays an important role in RNA recognition. Cap binding proteins (CBP) of HeLa cells were identified by photoaffinity labelling using the cap analogue γ-( 32 P)-(4-(benzoyl-phenyl)methylamido)-7-methylguanosine-5'-triphosphate (BP-m 7 GTP). Photoreaction of this cap analogue with HeLa cell initiation factors resulted in specific labelling of two polypeptides of Msub(r) 37000 and 26000. The latter was also labelled in crude initiation factors prepared from reticulocytes and is identical to the cap binding protein CBP I previously identified. These cap binding proteins were also affinity labelled in poliovirus infected cell extracts. Photoaffinity reaction with BP-m 7 GTP of whole HeLa cell homogenate showed three additional polypeptides with Msub(r) 120000, 89000 and 80000. These cap binding proteins were found to be associated with the nucleus and are therefore referred to as nuclear cap binding proteins, i.e. NCBP 1, NCBP 2 and NCBP 3. They were also present in splicing extracts. Photoaffinity labelling in these nuclear extracts was differentially inhibited by various cap analogues and capped mRNAs. Affinity chromatography on immobilized globin mRNA led to a partial separation of the three nuclear cap binding proteins. Chromatography on m 7 GTP-Sepharose resulted in a specific binding of NCBP 3. The different behaviour of the cap binding proteins suggests that they are functionally distinct and that they might be involved in different processes requiring cap recognition. (Author)

Preparation of high specific activity labelled triiodothyronine (T3) for radioimmunoassay

International Nuclear Information System (INIS)

Pillai, M.R.A.; Nagvekar, U.H.; Desai, C.N.; Mani, R.S.

1981-01-01

A method standardized for the preparation of high specific activity labelled triiodothyronine (T 3 ) is discussed. Iodine-125 labelled T 3 with a specific activity of 3 mCi μg was prepared by iodinating 3,5-diiodothyronine (T 2 ) and purifying it over Sephadex G-25 gel. Radochemical purity and stability evaluations were done by paper chromatography. Specific activity of the labelled T 3 prepared was estimated by the self-displacement method. The use of this high specific activity labelled T 3 in radioimmunoassay increased the sensitivity considerably. The advantage of this procedure is that the specific activity of labelled T 3 formed is independent of reaction yield and labelled T 3 yield. (author)

Cross-linking of hCG to luteal receptors

Energy Technology Data Exchange (ETDEWEB)

Ji, T.H.; Ji, I.

1985-01-01

Photoaffinity labeling of the lutropin/choriogonadotropin (LH/hCG) receptor system on porcine granulosa cells has demonstrated that both the ..cap alpha.. and ..beta.. subunits of hCG directly photoaffinity label the hormone receptor. Three new bands appear on SDS-PAGE as a consequence of photoaffinity labeling by each subunit: the molecular weights of the three bands (106K, 88K, and 83K) produced by the subunit are larger by approximately 10K than those of the three bands (96K, 76K, and 73K) labeled by the ..cap alpha.. subunit. Although it could be a coincidence that the molecular weight of the ..beta.. subunit is approximately 10K larger than that of the ..cap alpha.. subunit, the similarity in these differences suggests the possibility that both the ..cap alpha.. and ..beta.. subunits have labeled the same polypeptides.

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

Energy Technology Data Exchange (ETDEWEB)

Clagett-Dame, M.; McKelvy, J.F. (Abbott Laboratories, Abbott Park, IL (USA))

1989-10-01

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-(125I)iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide.

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

International Nuclear Information System (INIS)

Clagett-Dame, M.; McKelvy, J.F.

1989-01-01

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide

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

International Nuclear Information System (INIS)

Maruta, H.; Korn, E.D.

1981-01-01

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 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

A photoaffinity ligand for dopamine D2 receptors: azidoclebopride.

Science.gov (United States)

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 [3H]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. The degree of D2 dopamine receptor photoinduced inactivation by azidoclebopride was not significantly affected by scavengers such as p-aminobenzoic acid and dithiothreitol. Furthermore, irradiation of striatal membranes with a concentration of azidoclebopride sufficient to inactivate dopamine D2 receptors by 60% did not significantly reduce dopamine D1, serotonin (S2), benzodiazepine, alpha 1- or beta-noradrenergic receptors. This study describes the use of a novel and selective photoaffinity ligand for brain dopamine D2 receptors. The molecule, in radiolabeled form, may aid in the

Photoaffinity labeling of rat liver ribosomes by N-(2-Nitro-4-azidobenzoyl)puromycin

International Nuclear Information System (INIS)

Boehm, H.; Stahl, J.; Bielka, H.

1979-01-01

N-(2-nitro-4-azidobenzoyl)-[ 3 H]puromycin (NAB-puromycin) was synthesized as a photoreactive derivative of puromycin in order to detect ribosomal proteins located near the peptidyltransferase centre of rat liver ribosomes. Irradiation of ribosome-NAB-puromycin complexes leads to covalent attachment of the affinity label to proteins of the large ribosomal subunit, in particular to proteins L28/29, and, to a somewhat lower extent, to proteins L4, L6, L10 and L24. The results are discussed in the light of earlier studies performed with other affinity labels that attacked the peptidyltransferase region of rat liver ribosomes. (author)

Iodine-125 metaraminol: A new platelet specific labeling agent

International Nuclear Information System (INIS)

Ohmomo, Y.; Yokoyama, A.; Kawaii, K.; Horiuchi, K.; Saji, H.; Torizuka, K.

1984-01-01

In the search for a platelet specific labeling agent, Metaraminol (MA), which is a sympatomimetic amine used for the treatment of hypotension, cardiogenic shock and well recognized as a drug actively incorporated and accumulated in platelet, attracted the authors' attention. Using the classical chloramine-T iodination method, a high labeling efficiency near 98%, reaching a specific activity up to about 1000 Ci/mmole was obtained. Upon the harvest of platelet, only as platelet rich plasma (PRP), the labeling with this radiopharmaceutical was easily performed by incubation at 37 0 C for 10 min. Labeling efficiency as high as 63.0 +- 3.1% at 24 x 10/sup 8/ cells/ml was obtained. In in-vitro studies, the unaltered state of I-125 MA labeled platelet, with their cellular functions fully retained was demonstrated. Pharmacological study indicated a specific incorporation of I-125 MA by active transport system similar to that of 5-HT, along with passive diffusion. Then the in-vivo study carried out in rabbits with induced thrombi on the femoral artery, showed rather rapid disappearance of the I-125 MA labeled autologous platelet radioactivity, from circulating blood reaching as high thrombus-to-blood activity ratio as 19.8+-4.3 within 30 min post-administration. This new platelet labeling agent, I-125 MA, has many advantages over the use of IN-111 oxine and holds considerable promise for thrombus imaging with single photon emission CT upon the availability of I-123 MA

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

International Nuclear Information System (INIS)

Denny, J.B.; Blobel, G.

1984-01-01

A radioactive crosslinking reagent, N-[4-(p-azido-m-[ 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 125 I at the carrier-free specific activity of 2000 Ci/mmol. Any protein derivatized with the reagent is thus converted into an 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 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

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Combining position-specific 13C labeling with compound-specific isotope analysis: first steps towards soil fluxomics

Science.gov (United States)

Dippold, Michaela; Kuzyakov, Yakov

2015-04-01

Understanding the soil organic matter (SOM) dynamics is one of the most important challenges in soil science. Transformation of low molecular weight organic substances (LMWOS) is a key step in biogeochemical cycles because 1) all high molecular substances pass this stage during their decomposition and 2) only LMWOS will be taken up by microorganisms. Previous studies on LMWOS were focused on determining net fluxes through the LMWOS pool, but they rarely identified transformations. As LMWOS are the preferred C and energy source for microorganisms, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the biochemical pathways and its controlling factors. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools. Up to now, studies on LMWOS were nearly exclusively based on uniformly labeled organic substances i.e. all C atoms in the molecules were labeled with 13C or 14C. However, this classical approach did not allow the differentiation between use of intact initial substances in any process, or whether they were transformed to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of LMWOS and quantification of 13CO2 and 13C in bulk soil enabled following the basic metabolic pathways of soil microorganisms. However, only the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites allowed 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. So, these are the prerequisites for soil fluxomics. Our studies combining position-specific labeled glucose with amino

A convenient method to synthesize specifically labelled cholesterol with tritium

International Nuclear Information System (INIS)

Malik, S.; Kenny, M.; Ahmad, S.; Washington Univ., Seattle, WA

1992-01-01

A simple method is described to label cholesterol with tritium. Cholesterol was first oxidized to 5-cholesten-3-one which was then purified by HPLC. Its structure was established by electron impact (EI) mass spectrometry and 1 H-NMR spectroscopy. The ketone was reduced with NaB 3 H 4 to give specifically labelled cholesterol (C-3 3 H) at low specific activity. (author)

Characterization of beta-adrenergic receptors in synaptic membranes from rat cerebral cortex and cerebellum

International Nuclear Information System (INIS)

Lautens, L.

1986-01-01

Beta-adrenergic receptor ligand binding sites have been characterized in synaptic membranes from rat cerebral cortex and cerebellum using radioligand binding techniques. The equilibrium and kinetic properties of binding were assessed. The binding sites were non-interacting and exhibited two states of agonist binding which were sensitive to guanyl nucleotide. Synaptic membranes from cerebral cortex contained an equal number of beta 1 - and beta 2 -receptors; membranes from cerebellum possessed more beta 2 -than beta 1 -receptors. Photoaffinity labeling experiments revealed two different beta-adrenergic receptor polypeptides, R 1 and R 2 (and possibly a third, R 3 ) in synaptic membranes. The ratios of incorporation of photoaffinity label into R 1 : 2 were approximately 1:1 (cerebral cortex) and 5:1 (cerebellum). Photoaffinity labeling of R 1 and R 2 was inhibited equally well by both agonist and antagonist in synaptic membranes from cerebellum; whereas agonist was a less potent inhibitor in membranes from cerebral cortex. Both subtypes of beta-adrenergic receptors exhibited the same apparent molecular weight in synaptic membranes from cerebral cortex. The beta-adrenergic receptors in synaptic membranes from cerebral cortex and cerebellum were glycoproteins which exhibited the same apparent molecular weight after exposure to endoglycosidase F. The partial proteolytic digest maps of photoaffinity labeled beta-adrenergic receptors from rat cerebral cortex, cerebellum, lung and heart were compared

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

Science.gov (United States)

Peng, Tao; Hang, Howard C

2016-11-02

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Preparation of a high specific activity I-125 labeled styryl dye for leukocyte membrane labeling

International Nuclear Information System (INIS)

Lambert, C.; Mease, R.C.; Le, T.; Sabet, H.; Avren, L.I.; McAfee, J.G.

1994-01-01

The purpose of this work was to develop a high specific activity radioiodinated cell membrane probe for tracking lymphocytes in-vivo to replace the nucleus localizing, cytotoxic lipophilic chelates (In-111 oxine and Tc-99m HMPAO) currently used. Alkylation of parent dye 4-[2-[-N,N-didecylamino]phenyl]ethenyl pyridine with E-1-tributylstannyl-3-tosylpropene (prepared form E-1-tributylstannyl-1-propene-3-ol), gave a tributyltin precursor 1. Radiolabeled 3-[4-[2-[4-(N,N-didecylamino)phenyl]ethenyl]pyridino] E-[I-125]-1-iodopropene (2), was prepared from 1 using peracetic acid in acetonitrile/water. Labeling yields and specific activities achieved were 26% (∼2170 Ci/mmol), 40% (1220 Ci/mmol), and 55% (200 Ci/mmol) for nca, 0.4, and 2 nanomole carrier iodide runs respectively. Canine mixed leukocytes (0.5-1.0 x 10 8 cells) were labeled with 2 (67% and 42% yields for 200 Ci/mol and 1220 Ci/mmol preparations) and showed blood clearance similar to In 111 oxine. Radioiodinated styryl dye 2 appears to be a promising leukocyte labeling agent. Imaging studies with I-131 labeled 2 are in progress

HaloTag protein-mediated specific labeling of living cells with quantum dots

International Nuclear Information System (INIS)

So, Min-kyung; Yao Hequan; Rao Jianghong

2008-01-01

Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging

Extracting histones for the specific purpose of label-free MS.

Science.gov (United States)

Govaert, Elisabeth; Van Steendam, Katleen; Scheerlinck, Ellen; Vossaert, Liesbeth; Meert, Paulien; Stella, Martina; Willems, Sander; De Clerck, Laura; Dhaenens, Maarten; Deforce, Dieter

2016-12-01

Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885. © 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of 17α-[(E)-2-[125I]iodoethenyl]androsta-4-6-dien-17β-o l-3-one, an active -site-directed photoaffinity radiolabel for androgen-binding proteins

International Nuclear Information System (INIS)

Diaz Cruz, P.J.; Smith, H.E.; Vanderbilt Univ., Nashville, TN; Danzo, B.J.; Clanton, J.A.; Mason, N.S.

1993-01-01

The active-site-directed photoaffinity radiolabel for androgen-binding proteins, 17α-[(E)-2-[ 125 I]iodoethenyl]androsta-4,6-dien-17β-ol-3-one, was prepared by reaction of 17α-[(E)-2-tributyltin(IV)ethenyl]androsta-4,6-dien-17β-ol-3-one with carrier added sodium iodide-125 in the presence of hydrogen peroxide and acetic acid. Purification by HPLC gave the radiolabeled steroid in 52% radiochemical yield with a specific activity of 27 Ci/mmol and 100% radiochemical purity. (author)

[125I] radioiodinated metaraminol: A new platelet-specific labeling agent

International Nuclear Information System (INIS)

Ohmomo, Y.; Yokoyama, A.; Kawai, K.; Arano, Y.; Horiuchi, K.; Saji, H.; Torizuka, K.

1985-01-01

In our search for a platelet-specific labeling agent, metaraminol (MA), a low-toxic pharmaceutical for the treatment of hypotension and cardiogenic shock, attracted our attention. Its active incorporation and accumulation by platelets have been recognized. At first, the preparation of 125 I radioiodinated metaraminol ( 125 I-MA) was carried out using the chloramine-T method. Then, upon the harvest of platelets as platelet-rich plasma (PRP), their labeling with this new radiopharmaceutical was easily performed by incubation for 10 min at 37 0 C. The cell-labeling efficiency was dependent on cell density, reaching 63.0%+-3.1% at 2.4x10 9 cells/ml. The specific incorporation of 125 I-MA by an active transport system similar to that of 5-hydroxytryptamine (5-HT) as well as by passive diffusion was demonstrated. In vitro studies, the unaltered state of 125 I-MA-labeled platelets with their cellular functions fully retained was estimated. In vivo studies carried out in rabbits with induced thrombi in the femoral artery showed a rather rapid disappearance of the radioactivity from circulating blood, reaching a high thrombus-to-blood activity ratio of 19.8+-4.3 within 30 min of the administration of 125 I-MA-labeled autologous platelets. Thus, with the potential availability of 123 I, 123 I-MA-labeled platelets appear to be a promising agent for thrombus imaging using single-emission computed tomography (CT) studies. (orig.)

Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors

International Nuclear Information System (INIS)

Carlstedt-Duke, J.; Stroemstedt, P.E.; Persson, B.; Cederlund, E.; Gustafsson, J.A.; Joernvall, H.

1988-01-01

Purified rat liver glucocorticoid receptor was covalently charged with [ 3 H]glucocorticoid by photoaffinity labeling (UV irradiation of [ 3 H]triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with [ 3 H]dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with [ 3 H]triamcinolone acetonide and Cys-656 after affinity labeling with [ 3 H]dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A

Synthesis of specifically labelled L-phenylalanines using phenylalanine ammonia lyase activity

International Nuclear Information System (INIS)

Haedener, A.; Tamm, Ch.

1987-01-01

Specifically labelled L-phenylalanines have been prepared using a variety of classical synthetic methods in combination with phenylalanine ammonia lyase (PAL) enzyme activity of the yeast Rhodosporidium toruloides ATCC 10788 or Rhodotorula glutinis IFO 0559, respectively. Thus, L-[2- 2 H]phenyl-[2- 2 H]alanine was formed from (E) -[2,2'- 2 H 2 ]cinnamic acid and ammonia in 46% yield, whereas L-phenyl-[2- 13 C, 15 N]alanine was obtained from (E)-[2- 13 C]cinnamic acid in 45% overall yield. Generally, labelled cinnamic acids were recovered in pure form from the reaction mixture, with a loss of 6-8%. Likewise, unchanged 15 NH 3 was reisolated as 15 NH 4 Cl after steam distillation with overall losses of less than 4%. Labelled cinnamic acids were prepared by Knoevenagel condensations between appropriately labelled benzaldehydes and malonic acids. [2- 2 H]Benzaldehyde was obtained from 2-bromotoluene by decomposition of the corresponding Grignard reagent with 2 H 2 O and subsequent oxidation. Since simple molecules, most of them commercially available in labelled form or otherwise easily accessible, may serve as starting material, and due to its defined stereochemistry, the reaction catalysed by PAL opens a short and attractive route to specifically labelled L-phenylalanines. (author)

Site-Specific Biomolecule Labeling with Gold Clusters

OpenAIRE

Ackerson, Christopher J.; Powell, Richard D.; Hainfeld, James F.

2010-01-01

Site-specific labeling of biomolecules in vitro with gold clusters can enhance the information content of electron cryomicroscopy experiments. This chapter provides a practical overview of well-established techniques for forming biomolecule/gold cluster conjugates. Three bioconjugation chemistries are covered: Linker-mediated bioconjugation, direct gold–biomolecule bonding, and coordination-mediated bonding of nickel(II) nitrilotriacetic acid (NTA)-derivatized gold clusters to polyhistidine (...

Visualization of antigen-specific human cytotoxic T lymphocytes labeled with superparamagnetic iron-oxide particles

Energy Technology Data Exchange (ETDEWEB)

Beer, Ambros J. [Technical University of Munich (TUM), Department of Nuclear Medicine, Klinikum rechts der Isar, Munich (Germany); Holzapfel, Konstantin; Settles, Marcus; Rummeny, Ernst J. [Technical University of Munich, Department of Radiology, Klinikum rechts der Isar, Munich (Germany); Neudorfer, Juliana; Kroenig, Holger; Peschel, Christian; Bernhard, Helga [TUM, Munich, Department of Hematology/Oncology, Klinikum rechts der Isar, Munich (Germany); Piontek, Guido; Schlegel, Juergen [TUM, Munich, Division of Neuropathology, Institute of Pathology, Klinikum rechts der Isar, Munich (Germany)

2008-06-15

New technologies are needed to characterize the migration and survival of antigen-specific T cells in vivo. In this study, we developed a novel technique for the labeling of human cytotoxic T lymphocytes with superparamagnetic iron-oxide particles and the subsequent depiction with a conventional 1.5-T magnetic resonance scanner. Antigen-specific CD8{sup +} T lymphocytes were labeled with ferucarbotran by lipofection. The uptake of ferucarbotran was confirmed by immunofluorescence microscopy using a dextran-specific antibody, and the intracellular enrichment of iron was measured by atomic absorption spectrometry. The imaging of T cells was performed by magnetic resonance on day 0, 2, 7 and 14 after the labeling procedure. On day 0 and 2 post labeling, a pronounced shortening of T2*-relaxation times was observed, which diminished after 7 days and was not detectable anymore after 14 days, probably due to the retained mitotic activity of the labeled T cells. Of importance, the antigen-specific cytolytic activity of the T cells was preserved following ferucarbotran labeling. Efficient ferucarbotran labeling of functionally active T lymphocytes and their detection by magnetic resonance imaging allows the in vivo monitoring of T cells and, subsequently, will impact the further development of T cell-based therapies. (orig.)

Visualization of antigen-specific human cytotoxic T lymphocytes labeled with superparamagnetic iron-oxide particles

International Nuclear Information System (INIS)

Beer, Ambros J.; Holzapfel, Konstantin; Settles, Marcus; Rummeny, Ernst J.; Neudorfer, Juliana; Kroenig, Holger; Peschel, Christian; Bernhard, Helga; Piontek, Guido; Schlegel, Juergen

2008-01-01

New technologies are needed to characterize the migration and survival of antigen-specific T cells in vivo. In this study, we developed a novel technique for the labeling of human cytotoxic T lymphocytes with superparamagnetic iron-oxide particles and the subsequent depiction with a conventional 1.5-T magnetic resonance scanner. Antigen-specific CD8 + T lymphocytes were labeled with ferucarbotran by lipofection. The uptake of ferucarbotran was confirmed by immunofluorescence microscopy using a dextran-specific antibody, and the intracellular enrichment of iron was measured by atomic absorption spectrometry. The imaging of T cells was performed by magnetic resonance on day 0, 2, 7 and 14 after the labeling procedure. On day 0 and 2 post labeling, a pronounced shortening of T2*-relaxation times was observed, which diminished after 7 days and was not detectable anymore after 14 days, probably due to the retained mitotic activity of the labeled T cells. Of importance, the antigen-specific cytolytic activity of the T cells was preserved following ferucarbotran labeling. Efficient ferucarbotran labeling of functionally active T lymphocytes and their detection by magnetic resonance imaging allows the in vivo monitoring of T cells and, subsequently, will impact the further development of T cell-based therapies. (orig.)

Site-specific labeling of proteins with NMR-active unnatural amino acids

International Nuclear Information System (INIS)

Jones, David H.; Cellitti, Susan E.; Hao Xueshi; Zhang Qiong; Jahnz, Michael; Summerer, Daniel; Schultz, Peter G.; Uno, Tetsuo; Geierstanger, Bernhard H.

2010-01-01

A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.

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

Science.gov (United States)

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.

Agonist-induced alterations in lymphocyte β-adrenergic receptor photoaffintiy labelling: effects of phenylarsine oxide

International Nuclear Information System (INIS)

Feldman, R.D.; McArdle, W.; Lai, C.

1986-01-01

In several models, desensitization of the β-adrenergic receptor (βAR) is associated with a decrease in binding of hydrophilic but not hydrophobic βAR ligands. This suggests a sequestration of cell surface βAR. Desensitization of the lymphobyte βAR is also associated with a selective reduction in the photoaffinity labelling of a 55K βAR protein as compared to a 68K βAR protein. In order to examine the relationship between sequestration and reduction in labelling of the 55K peptide, the authors have studied the effect of phenylarsine oxide (PAO; an inhibitor of sequestration) on lymphocyte βAR desensitization. Incubation of cells with PAO prior to desensitization did not block the consequent reduction in isoproterenol-stimulated adenylate cyclase activity. However, the agonist-induced reduction in binding of the hydrophilic βAR ligand CGP-12177 was blocked by PAO (without PAO:57 +/- 4% of control, with PAO: 97 +/- 2% of control). Photolabelling studies with [ 125 I] iodocyanopindolol diazirine revealed that PAO pretreatment also blocked the selective loss in labelling of the 55K βAR protein seen with desensitization. These data suggest that loss of labelling of the 55K protein of the βAR is closely coupled to βAR sequestration

Synthesis of specifically labelled L-phenylalanines using phenylalanine ammonia lyase activity

Energy Technology Data Exchange (ETDEWEB)

Haedener, A.; Tamm, Ch.

1987-11-01

Specifically labelled L-phenylalanines have been prepared using a variety of classical synthetic methods in combination with phenylalanine ammonia lyase (PAL) enzyme activity of the yeast Rhodosporidium toruloides ATCC 10788 or Rhodotorula glutinis IFO 0559, respectively. Thus, L-(2-/sup 2/H)phenyl-(2-/sup 2/H)alanine was formed from (E) -(2,2'-/sup 2/H/sub 2/)cinnamic acid and ammonia in 46% yield, whereas L-phenyl-(2-/sup 13/C, /sup 15/N)alanine was obtained from (E)-(2-/sup 13/C)cinnamic acid in 45% overall yield. Generally, labelled cinnamic acids were recovered in pure form from the reaction mixture, with a loss of 6-8%. Likewise, unchanged /sup 15/NH/sub 3/ was reisolated as /sup 15/NH/sub 4/Cl after steam distillation with overall losses of less than 4%. Labelled cinnamic acids were prepared by Knoevenagel condensations between appropriately labelled benzaldehydes and malonic acids. (2-/sup 2/H)Benzaldehyde was obtained from 2-bromotoluene by decomposition of the corresponding Grignard reagent with /sup 2/H/sub 2/O and subsequent oxidation. Since simple molecules, most of them commercially available in labelled form or otherwise easily accessible, may serve as starting material, and due to its defined stereochemistry, the reaction catalysed by PAL opens a short and attractive route to specifically labelled L-phenylalanines.

Site-Specific Biomolecule Labeling with Gold Clusters

Science.gov (United States)

Ackerson, Christopher J.; Powell, Richard D.; Hainfeld, James F.

2013-01-01

Site-specific labeling of biomolecules in vitro with gold clusters can enhance the information content of electron cryomicroscopy experiments. This chapter provides a practical overview of well-established techniques for forming biomolecule/gold cluster conjugates. Three bioconjugation chemistries are covered: Linker-mediated bioconjugation, direct gold–biomolecule bonding, and coordination-mediated bonding of nickel(II) nitrilotriacetic acid (NTA)-derivatized gold clusters to polyhistidine (His)-tagged proteins. PMID:20887859

Photoaffinity Labeling of the Plasmodium falciparum Chloroquine Resistance Transporter with a Novel Perfluorophenylazido Chloroquine†

Science.gov (United States)

Lekostaj, Jacqueline K.; Natarajan, Jayakumar K.; Paguio, Michelle F.; Wolf, Christian; Roepe, Paul D.

2009-01-01

Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein. PMID:18767816

Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine.

Science.gov (United States)

Lekostaj, Jacqueline K; Natarajan, Jayakumar K; Paguio, Michelle F; Wolf, Christian; Roepe, Paul D

2008-09-30

Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein.

Preparation of [In-111]-labeled-DTPA-bombesin conjugates at high specific activity and stability: Evaluation of labeling parameters and potential stabilizers

Energy Technology Data Exchange (ETDEWEB)

Pujatti, P.B., E-mail: pujatti.pb@gmail.com [Directory of Radiopharmacy, Nuclear and Energy Research Institute (IPEN/CNEN), Av. Prof. Lineu Prestes, 2242 - Cidade Universitaria da USP - Butanta, Sao Paulo - SP - Brazil - CEP: 05508-000 (Brazil); Massicano, A.V.F.; Mengatti, J.; Araujo, E.B. de [Directory of Radiopharmacy, Nuclear and Energy Research Institute (IPEN/CNEN), Av. Prof. Lineu Prestes, 2242 - Cidade Universitaria da USP - Butanta, Sao Paulo - SP - Brazil - CEP: 05508-000 (Brazil)

2012-05-15

The aim of the present work was to obtain stabilized high specific activity (HSA) {sup 111}In-labeled bombesin conjugates for preclinical evaluations. Parameters influencing the kinetics of labeling were investigated and the effect of stabilizers on HSA radiopeptides stability at room temperature were systematically categorized applying chromatography techniques. A SA of 174 GBq/{mu}mol was achieved with high radiochemical purity, but the labeled compounds exhibited low stability. The addition of stabilizers avoided their radiolysis and significantly increased their stability. - Highlights: Black-Right-Pointing-Pointer We aimed to obtain stabilized high specific activity (SA) {sup 111}In-labeled bombesin conjugates. Black-Right-Pointing-Pointer The effect of stabilizers on high SA radiopeptides stability were investigated. Black-Right-Pointing-Pointer A maximum specific activity of 174 GBq/{mu}mol was achieved. Black-Right-Pointing-Pointer The studied stabilizers significantly increased the stability of high SA radiopeptides. Black-Right-Pointing-Pointer These stabilized bombesin conjugates will be applied in preclinical studies.

A new class of photoactivatable and carbene generating reagents with extremely high specific radioactivity. Synthesis, characterization and applications

International Nuclear Information System (INIS)

Weber, T.

1994-01-01

The main objective of this work was the development of new photocrosslinking and labeling reagents which show favourable photochemical properties and can be synthesized in an extremely high specific radioactivity. A key compound in the synthesis of these reagents was 2-tributyltin-4-(3-trifluormethyl-3H-diazirin-3-yl)-benzyl alcohol. Esters of this alcohol can be easily radioiodinated at a specific radioactivity of >2000 Ci/mmol under mild conditions. By experiments with a model compound it was shown that 2-iodo-4-(3-trifluormethyl-3H-diazirin-3-yl)-benzyl esters, upon photolysis, generate highly reactive (singlet) carbenes capable of inserting into CH-bonds. Equally important, there is no indication of loss of iodine under the photolysis conditions applied. Therefore two key requirements for photolabeling reagents are fulfilled. Several photoactivatable radioiodinated phospholipids have been synthesized. Their properties have been evaluated by labeling of the the membranes of intact erythrocytes and influenza viruses. Currently these lipids are used to study the interaction of various proteins (shown are experiments with MARCKS) with membranes. Furthermore a new class of actual label transfer crosslinkers (two thiol specific and one amino specific) have been developed. The range of potentialities of these reagents is currently being investigated. Finally a photoactivatable radioiodinated ceramide analogue suitable as a photoaffinity crosslinker has been developed with the goal of identifying the putative receptor of this second messenger-like lipid. Preliminary studies towards this goal are described. We are convinced that the reagents and methods presented in this work are valuable tools, and that they will find widespread use in future cell-biological and biochemical research. (author) figs., tabs., refs

DNA-specific labelling by deoxyribonucleoside 5'-monophosphates in Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Brendel, M.; Faeth, W.W.; Toper, R.

1975-01-01

Growth of 5'-dTMP low-requiring strains is inhibited by exogenous 5'-dGMP and 5'-GMP at concentrations higher than 5 x 10 -4 M. Synthesis of nucleic acids ceases and cells remain fixed in their respective place in the cell cycle. At concentrations lower than 10 -5 M deoxyribonucleoside 5'-monophosphates may be employed for radioactive labelling, the label being preferentially used for DNA synthesis. Affinity to DNA of the 5'-dNMPs is in the order of 5'-dAMPS > 5'-dGMP > 5'-dCMP > 5'-dUMP. DNA-specific label is achieved with 5'-dAMP when the medium is supplemented with adenine and deoxyadenosine. (orig.) [de

Pharmacological and biochemical properties of the benzodiazepine-GABA receptor in codfish brain in comparison with mammalian brain

International Nuclear Information System (INIS)

Deng, L.

1989-01-01

The GABA receptor of codfish brain is encoded by an ancestral gene of the mammalian GABA receptor based on phylogenetic studies. The mammalian GABA receptor consists of at least two subunits (β and α) which could be photoaffinity labeled by the GABA agonist [ 3 H]muscimol (57 kDa) and the benzodiazepine (BZ) agonist [ 3 H]flunitrazepam (52 kDa), respectively. In contrast, electrophoresis of codfish GABA receptor photoaffinity labeled by the same ligands showed a single radioactive peak on sodium dodecyl surface polyarcylamide gel, giving rise to a relative molecular weight of 56-57 kDa equivalent to the β subunit of 57 kDa in mammals. The homogeneity of purified receptor using benzodiazepine (Ro 7-1986/1) affinity chromatography was further verified by two-dimensional gel electrophoresis based on isoelectric point and molecular weight, in addition to a single band on a silver stained gel and specific activity. The receptor density and affinity constant for [ 3 H]muscimol and [ 3 H]flunitrazepam are comparable to those in bovine, rate, and human brain

Specificity of Facial Expression Labeling Deficits in Childhood Psychopathology

Science.gov (United States)

Guyer, Amanda E.; McClure, Erin B.; Adler, Abby D.; Brotman, Melissa A.; Rich, Brendan A.; Kimes, Alane S.; Pine, Daniel S.; Ernst, Monique; Leibenluft, Ellen

2007-01-01

Background: We examined whether face-emotion labeling deficits are illness-specific or an epiphenomenon of generalized impairment in pediatric psychiatric disorders involving mood and behavioral dysregulation. Method: Two hundred fifty-two youths (7-18 years old) completed child and adult facial expression recognition subtests from the Diagnostic…

The effect of label affinity on the sensitivity and specificity of a hapten radioimmunoassay: A comparison of three [125I]diphenylhydantoin radioligands with the 14C-labelled drug

International Nuclear Information System (INIS)

Rowell, F.J.

1979-01-01

The effects on the sensitivity and specificity of a radioimmunoassay for diphenylhydantoin (DPH) has been investigated using three 125 I-labelled tyrosine ester derivatives of DPH having different bridge lengths between the tyrosine moiety and the DPH moeity and 14 C-labelled DPH. The results demonstrate that for a hapten which does not completely fill the antibody binding sites, greatest sensitivity is achieved when the bridge of the iodine label is most dissimilar to that present in the original immunogen, when the hapten and label affinities are nearly equivalent. Greatest specificity is achieved with the label which most resembles the original immunogen. These results illustrate the difficulty of designing satisfactory labels for assays of both high specificity and sensitivity since minimal changes in label structure may produce greatly amplified changes in the subsequent affinity of the label for the antiserum. (Auth.)

Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

Science.gov (United States)

Pabst, Martin; Benešová, Iva; Fagerer, Stephan R; Jacobsen, Mathias; Eyer, Klaus; Schmidt, Gregor; Steinhoff, Robert; Krismer, Jasmin; Wahl, Fabian; Preisler, Jan; Zenobi, Renato

2016-01-04

We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS.

Identification of a single sinusoidal bile salt uptake system in skate liver

International Nuclear Information System (INIS)

Fricker, G.; Hugentobler, G.; Meier, P.J.; Kurz, G.; Boyer, J.L.

1987-01-01

To identify the sinusoidal bile acid uptake system(s) of skate liver, photoaffinity labeling and kinetic transport studies were performed in isolated plasma membranes as well as intact hepatocytes. In both preparations photoaffinity labeling with the photolabile bile salt derivative revealed the presence of a predominant bile salt binding polypeptide with an apparent molecular weight of 54,000. The [ 3 H]-labeling of this polypeptide was inhibited by taurocholate and cholate in a concentration-dependent manner and was virtually abolished by 1 mM of the anion transport inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Kinetic studies of hepatic uptake with taurocholate, cholate, and the photoreactive bile salt derivative indicated the involvement of a single transport system, and all three substrates mutually competed with the uptake of each other. Finally, irreversible inhibition of the bile salt uptake system of photoaffinity labeling of hepatocytes with high concentrations of photolabile derivative reduced the V max but the K m of taurocholate uptake. These findings strongly indicate that a single polypeptide with an apparent molecular weight of 54,000 is involved in sinusoidal bile salt uptake into skate hepatocytes. These findings contrast with similar studies in rat liver that implicate both a 54,000- and 48,000-K polypeptide in bile salt uptake and are consistent with a single Na + -independent transport mechanism for hepatic bile salt uptake in this primitive vertebrate

Consumer perceptions of specific design characteristics for front-of-package nutrition labels.

Science.gov (United States)

Acton, R B; Vanderlee, L; Roberto, C A; Hammond, D

2018-04-01

An increasing number of countries are developing front-of-package (FOP) labels; however, there is limited evidence examining the impact of specific design characteristics for these labels. The current study investigated consumer perceptions of several FOP label design characteristics, including potential differences among sociodemographic sub-groups. Two hundred and thirty-four participants aged 16 years or older completed nine label rating tasks on a laptop at a local shopping mall in Canada. The rating tasks asked participants to rate five primary design characteristics (border, background presence, background colour, 'caution' symbol and government attribution) on their noticeability, readability, believability and likelihood of changing their beverage choice. FOP labels with a border, solid background and contrasting colours increased noticeability. A solid background increased readability, while a contrasting background colour reduced it. Both a 'caution' symbol and a government attribution increased the believability of the labels and the perceived likelihood of influencing beverage choice. The effect of the design characteristics was generally similar across sociodemographic groups, with modest differences in five of the nine outcomes. Label design characteristics, such as the use of a border, colour and symbols can enhance the salience of FOP nutrition labels and may increase the likelihood that FOP labels are used by consumers.

Biochemical characterization of the pancreatic cholecystokinin receptor using monofunctional photoactivatable probes

International Nuclear Information System (INIS)

Pearson, R.K.; Miller, L.J.; Powers, S.P.; Hadac, E.M.

1987-01-01

Receptor characterization by affinity labeling can be enhanced by taking multiple complementary approaches. To extend our observations on the subunit structure of the rat pancreatic cholecystokinin (CCK) receptor (made using bifunctional cross-linking reagents), we synthesized two monofunctional photoactivatable receptor probes. CCK-8 was acylated with the iodinated aryl azide derivatives, methyl-3-azido-4-hydroxy-5-[ 125 I]iodobenzimidate and N-[4-(4'-azido-3'-[ 125 I]iodophenylazo)benzoyl]-3-aminopropionyl-N- oxy- succinimide. The products were purified by reverse-phase HPLC to a specific radioactivity of 2000 Ci/mmol. Both analogs demonstrated saturable and specific binding to rat pancreatic plasma membranes. Photoaffinity labeling of pancreatic membranes with these monofunctional probes identified an Mr 85,000-95,000 protein that was not part of a larger disulfide-linked complex. High affinity for CCK was demonstrated by the concentration-dependent inhibition of labeling observed with competing CCK-8 (IC50 = 1 nM). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) this protein co-migrates with the major component we identified using a series of cross-linkable, iodinated decapeptide analogs of CCK, and is different from the major protein labeled using 125 I-Bolton Hunter-CCK-33. Thus, these results support the presence of an Mr 85,000-95,000 subunit in the pancreatic CCK receptor, while the small size of these photoaffinity probes and their monovalency suggest that this subunit may contain or be spatially apposed to the active binding site. These probes should be very useful in the further characterization of this and other receptors for this hormone

Site-Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne-Azide Cycloaddition Reactions.

Science.gov (United States)

Perols, Anna; Arcos Famme, Melina; Eriksson Karlström, Amelie

2015-11-01

Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine-reactive probes that target surface-exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site-specifically labeled product. Z32 BPA was synthesized by solid-phase peptide synthesis and further functionalized to give alkyne-Z32 BPA and azide-Z32 BPA for Cu(I) -catalyzed cycloaddition, as well as DBCO-Z32 BPA for Cu-free strain-promoted cycloaddition. The Z32 BPA variants were conjugated to the human IgG1 antibody trastuzumab and site-specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2-expressing cells in immunofluorescence microscopy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

Science.gov (United States)

Keiderling, Timothy A

2017-12-01

Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra. © 2017 Wiley Periodicals, Inc.

Specific Labeling of Zinc Finger Proteins using Non-canonical Amino Acids and Copper-free Click Chemistry

Science.gov (United States)

Kim, Younghoon; Kim, Sung Hoon; Ferracane, Dean; Katzenellenbogen, John A.

2012-01-01

Zinc finger proteins (ZFPs) play a key role in transcriptional regulation and serve as invaluable tools for gene modification and genetic engineering. Development of efficient strategies for labeling metalloproteins such as ZFPs is essential for understanding and controlling biological processes. In this work, we engineered ZFPs containing cysteine-histidine (Cys2-His2) motifs by metabolic incorporation of the unnatural amino acid azidohomoalanine (AHA), followed by specific protein labeling via click chemistry. We show that cyclooctyne promoted [3 + 2] dipolar cycloaddition with azides, known as copper-free click chemistry, provides rapid and specific labeling of ZFPs at high yields as determined by mass spectrometry analysis. We observe that the DNA-binding activity of ZFPs labeled by conventional copper-mediated click chemistry was completely abolished, whereas ZFPs labeled by copper-free click chemistry retain their sequence-specific DNA-binding activity under native conditions, as determined by electrophoretic mobility shift assays, protein microarrays and kinetic binding assays based on Förster resonance energy transfer (FRET). Our work provides a general framework to label metalloproteins such as ZFPs by metabolic incorporation of unnatural amino acids followed by copper-free click chemistry. PMID:22871171

The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

Science.gov (United States)

Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

2016-01-01

MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

Directory of Open Access Journals (Sweden)

Ting Lu

Full Text Available MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye could firmly bind to the surface of adherent cells (Hela and suspended cells (K562 even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

Radiosequence analysis of the human progestin receptor charged with [3H]promegestone. A comparison with the glucocorticoid receptor

International Nuclear Information System (INIS)

Stroemstedt, P.E.B.; Berkenstam, A.; Joernvall, H.G.; Gustafsson, J.A.; Carlstedt-Duke, J.

1990-01-01

Partially purified preparations of the human progestin receptor and the human and rat glucocorticoid receptor proteins were covalently charged with the synthetic progestin, [ 3 H]promegestone, by photoaffinity labeling. After labeling, the denaturated protein was cleaved and the mixture of peptides subjected to radiosequence analysis as previously described for the rat glucocorticoid receptor protein. The radioactivity labels identified, corresponded to Met-759 and Met-909 after photoaffinity labeling of the human progestin receptor, and Met-622 and Cys-754 after labeling of the rat glucocorticoid receptor. The residues labeled in the glucocorticoid receptor are the same as those previously reported to bind triamcinolone actonide. The corresponding residues were also labeled in the human glucocorticoid receptor. Met-759 of the progestin receptor and Met-622 of the rat glucocorticoid receptor are positioned within a segment with an overall high degree of sequence similarity and are equivalent. However, Met-909 (progestin receptor) and Cys-754 (glucocorticoid receptor) do not occur within equivalent segments of the two proteins. Thus, although the two classes of steroid hormone share a common structure within the A-ring, there are subtle differences in their interaction with the two separate receptor proteins

Proteins labelling with 125I and experimental determination of their specific activity

International Nuclear Information System (INIS)

Caro, R.A.; Ciscato, V.A.; Giacomini, S.M.V. de; Quiroga, S.; Radicella, R.

1975-11-01

A standardization of the labelling technique of proteins with 125 I and the control of the obtained products, principally their specific activities was performed, in order to utilize them correctly in radioimmunoassays. The quantities of chloramine-T and sodium metabisulphite were lowered, with regard to the original method, to 3.6 and 9.6 μg respectively. Under these conditions, optimal yields and radioiodinated proteins with good immunological activities were obtained. It was found that the specific activity calculated, as usual, from the yield obtained by electrophoresis, is higher than the real value. For these reasons the yields and the corresponding specific activities were determined from ascending chromatographies performed with 70 per cent methanol as solvent, during two hours in darkness. The radioimmunoassay displacement curves obtained with proteins labelled which the proposed method and the specific activities of which were calculated from their radiochromatographic patterns, were reproducible and gave a percentage of bound radioiodinated protein in the absence of cold protein of 50 +- 4. (author) [es

21 CFR 862.2050 - General purpose laboratory equipment labeled or promoted for a specific medical use.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false General purpose laboratory equipment labeled or... TOXICOLOGY DEVICES Clinical Laboratory Instruments § 862.2050 General purpose laboratory equipment labeled or promoted for a specific medical use. (a) Identification. General purpose laboratory equipment labeled or...

Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

Energy Technology Data Exchange (ETDEWEB)

Monneau, Yoan R. [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Ishida, Yojiro [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Rossi, Paolo; Saio, Tomohide; Tzeng, Shiou-Ru [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Inouye, Masayori, E-mail: inouye@cabm.rutgers.edu [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Kalodimos, Charalampos G., E-mail: ckalodim@umn.edu [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States)

2016-06-15

A simple and cost effective method to independently and stereo-specifically incorporate [{sup 1}H,{sup 13}C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [{sup 1}H,{sup 13}C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [{sup 1}H,{sup 13}C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-{sup 13}C,2,3-{sup 2}H{sub 2},{sup 15}N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[{sup 13}CH{sub 3}]{sup γ2} labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for {sup 2}H-glycine to prevent scrambling.

China Refrigerator Information Label: Specification Development and Potential Impact

Energy Technology Data Exchange (ETDEWEB)

Fridley, David; Fridley, David; Zheng, Nina; Zhou, Nan; Aden, Nathaniel; Lin, Jiang; Jianhong, Cheng; Sakamoto, Tomoyuki

2008-02-01

In the last five years, China's refrigerator market has grown rapidly, and now urban markets are showing signs of saturation, with ownership rates in urban households reaching 92%. Rural markets continue to grow from a much lower base. As a result of this growth, the Chinese government in 2006 decided to revise the refrigerator standards and its associated efficiency grades for the mandatory energy information label. In the Chinese standards process, the efficiency grades for the information label are tied to the minimum standards. Work on the minimum standards revision began in 2006 and continued through the first half of 2007, when the draft standard was completed under the direction of the China National Institute of Standardization (CNIS). Development of the information label grades required consideration of stakeholder input, continuity with the previous grade classification, ease of implementation, and potential impacts on the market. In this process, CLASP, with the support of METI/IEEJ, collaborated with CNIS to develop the efficiency grades, providing technical input to the process, comment and advice on particular technical issues, and evaluation of the results. After three months of effort and three drafts of the final grade specifications, this work was completed. In addition, in order to effectively evaluate the impact of the label on China's market, CLASP further provided assistance to CNIS to collect data on both the efficiency distribution and product volume distribution of refrigerators on the market. The new information label thresholds to be implemented in 2008 maintain the approach first adopted in 2005 of establishing efficiency levels relative to the minimum standard, but increased the related required efficiency levels by 20% over those established in 2003 and implemented in 2005. The focus of improvement was on the standard refrigerator/freezer (class 5), which constitutes the bulk of the Chinese market. Indeed, the new

49 CFR 172.407 - Label specifications.

Science.gov (United States)

2010-10-01

...) Design. (1) Except for size and color, the printing, inner border, and symbol on each label must be as... withstand, without deterioration or a substantial change in color, a 30-day exposure to conditions incident... shown in the appropriate section of this subpart. (d) Color. (1) The background color on each label must...

Synthesis of non-steroidal anti-inflammatory drug analogues for selective studies on the COX-II enzyme

International Nuclear Information System (INIS)

Fleming, S.A.; Ridges, M.D.; Jensen, A.W.

1996-01-01

Synthesis of the azido substituted non-steroidal anti-inflammatory drug 2-(2,6-dichloroanilino)phenylacetic acid and isotope labeling of this compound have been performed and are described. Initial evaluation of the binding ability and photoreactivity indicates that this compound has potential for photoaffinity labeling as well as enzyme selectivity studies. (author)

Synthesis of molecules of biological interest labelled with high specific activity tritium

International Nuclear Information System (INIS)

Petillot, Yves

1975-01-01

Labelled molecules are artificial organic compounds possessing one or several radioactive or steady isotopic atoms. Using tritium to label molecules presents several benefits: a raw material easy to obtain with a high purity and at reasonable cost; synthesised labelled molecules displaying high specific activities very interesting in molecular biology; high resolution of radiographies; relatively simple and quick introduction of tritium atoms in complex molecules. Thus, this report for graduation in organic chemistry addresses the synthesis and study of new labelled molecules which belong to families of organic compounds which have fundamental activities in biology: uridine 3 H-5,6 and thymidine 3 H-methyl which are nucleotides which intervene under the form of phosphates in the synthesis of nucleic acids, oestradiol 3 H-2,4,6,7 which is a powerful estrogenic hormone which naturally secreted by the ovary; and noradrenaline 3 H-1,1' and dopamine 3 H-1,2 which are usually secreted by adrenal medulla and have multiple actions on the nervous system

Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies.

Science.gov (United States)

Brgles, Marija; Mirosavljević, Krunoslav; Noethig-Laslo, Vesna; Frkanec, Ruza; Tomasić, Jelka

2007-03-10

Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG.

40 CFR 170.232 - Knowledge of labeling and site-specific information.

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Knowledge of labeling and site-specific information. 170.232 Section 170.232 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) PESTICIDE PROGRAMS WORKER PROTECTION STANDARD Standard for Pesticide Handlers § 170.232 Knowledge...

Transport of C-13-labelled linoleic and C-13-labelled caprylic acid in rat plasma after administration of specific structured triacylglycerols

DEFF Research Database (Denmark)

Vistisen, Bodil; Høy, Carl-Erik

2004-01-01

the transport of dietary C-13-labelled fatty acids in rat plasma to compare the chylomicron fatty acid metabolism after administration of specific structured, long chain and medium chain triacylglycerols. Rats were fed ML*M, M*LM*, L*L*L* or M*M*M* (L=linoleic acid, 18:2n-6, M=caprylic acid, 8:0, * = C-13......-labelled fatty acid) by gavage. A maximum transport of 0.5% of the administered C-13-labelled 18:2n-6 was observed in 1mL rat plasma both after administration of L*L*L* and ML*M, while approximately 0.04% of the administered C-13-labelled 8:0 was detected in 1mL plasma following administration of M......*M*M* or M*LM*. After L*L*L* administration C-13-labelled 20:4n-6 was observed in plasma, probably formed by elongation and desaturation of 18:2n-6 in the enterocyte or liver cells. Furthermore, C-13-labelled 16:0, 48:0, 18: 1n-9 and 20:4n-6 were observed in plasma of rats fed M*M*M* and M*LM* due...

Tc-99m labeled Sparfloxacin: A specific infection imaging agent

International Nuclear Information System (INIS)

Singh, A.K.; Verma, J.; Bhatnagar, A.; Ali, A.

2003-01-01

Radiolabeled antibiotics are being used for the specific diagnosis of infection by exploiting their specific binding properties to the bacterial component, thereby making it possible to differentiate infection from sterile lesions. A new radiopharmaceutical, Tc-99m Sparfloxacin has been developed for infection imaging. Sparfloxacin is a quinolone based broad-spectrum antibiotic, which is more potent than Ciprofloxacin. Radiolabeling of Sparfloxacin with Tc-99m was standardized using direct labeling protocol. Labeling efficiency, in-vitro and in-vivo stability, blood kinetics and organ distribution studies (in balb/c mice and New Zealand White Rabbits at different time interval up to 24hrs) were carried out. Biological activity of Sparfloxacin after its labeling with Tc-99m was evaluated with S.aureus using Peptone water (DIFCO) as media. Turpentine oil (100 μl) in left thigh and S.aureus (100μl of 3x10 7 cells) in right thigh were injected intramuscularly to create sterile and infective inflammation respectively in six New Zealand white rabbits. The localization kinetics of the radiolabeled complex were studied in the animal model by injecting 70-75MBq of Tc-99m Sparfloxacin intravenously in the ear of rabbit and the images were taken with a Gamma-camera (ECIL) at different post-injection time intervals. Standardized protocol produced >95% labeled complex. About 8% of tracer leached out at 24 hrs when incubated in serum at 37 0 C, confirming high stability of the complex. Blood clearance in rabbit revealed biphasic pattern and 50% of the complex clears from the blood within 5 min. Biodistribution studies in balb/c mice showed hepatobiliary route of excretion. Presence of insignificant amount of tracer at 24 hrs in the stomach confirmed high in vivo stability of the complex. Imaging in rabbits showed significant concentration of tracer in lesions with infection. Typical imaging patterns revealed initial accumulation of radiotracer in both sterile inflammatory

Synthesis of high specific activity tritium labelled [2-3H]-adenosine-5'-triphosphate

International Nuclear Information System (INIS)

Jaiswal, D.K.; Morimoto, H.; Trump, E.L.; Williams, P.G.; Wemmer, D.E.

1996-01-01

A procedure for high level tritium labelling at the C2-H position of adenosine 5'-triphosphate ([2- 3 H]-ATP, 1), based on the tritiodehalogenation reaction of 2-bromoadenosine 5'-triphosphate (2) has been elaborated. This precursor was prepared in a six-step synthesis from guanosine. The tritiodehalogenation of (2) for three hours over palladium oxide in phosphate buffer yielded tritium labelled ATP with high specific activity, in good chemical yield. (author)

Preparation of [35S]sulfobromophthalein of high specific activity

International Nuclear Information System (INIS)

Kurisu, H.; Nilprabhassorn, P.; Wolkoff, A.W.

1989-01-01

Study of the hepatocyte transport mechanism of organic anions such as bilirubin and sulfobromophthalein has been limited by the relatively low specific activities of these ligands. [ 3 H]Bilirubin and [ 35 S]sulfobromophthalein have been available with specific activities of only approximately 100 mCi/mmol. We now report a relatively simple procedure to prepare [ 35 S]sulfobromophthalein at a specific activity of approximately 3000 mCi/mmol. This compound is radiochemically pure and serves as a tracer for authentic sulfobromophthalein as judged by chromatography, hepatocyte uptake, metabolism, and biliary excretion. Use of this material as a photoaffinity probe and as a transported ligand may permit dissection and understanding of its transport mechanism

Localization of 131I-labeled p97-specific Fab fragments in human melanoma as a basis for radiotherapy

International Nuclear Information System (INIS)

Larson, S.M.; Carrasquillo, J.A.; Krohn, K.A.

1983-01-01

33 patients with advanced malignant melanoma were studied after intravenous administration of 131 I-labeled Fab fragments specific for p97, an oncofetal glycoprotein of human melanoma. In all, 47 gamma camera imaging studies were performed for the purpose of localization of metastatic deposits. In addition to tumor, 131 I-Fab uptake was also seen in liver and kidney. 20 of these studies included simultaneous administration of both an 131 I-labeled Fab specific for p97, and an 125 I-labeled Fab not specific for p97. Blood clearance of p97-specific Fab was significantly more rapid than for nonspecific Fab. Eight of these patients had biopsies of subcutaneous nodules at 48 and 72 h postinjection in order to assess whether localization of radioactivity was antigen specific. Antigen-specific localization was observed with average ratios of specific/nonspecific uptake of 3.7 (48 h) and 3.4 (72 h); uptake was strongly correlated with tumor p97 concentration (r . 0.81, P less than 0.01). Also, imaging studies of the bio-distribution of 131 I-labeled anti-p97 Fab in patients selected for high p97 tumor concentration showed avid tumor uptake and more prolonged retention of labeled Fab in tumor than in normal tissues. Based on these studies, we estimated that total 131 I doses of 500 mCi could be safely given to patients before dose-limiting toxicity would be observed

Probing Xylan-Specific Raman Bands for Label-Free Imaging Xylan in Plant Cell Wall

Energy Technology Data Exchange (ETDEWEB)

Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh; Tucker, Melvin P.; Vinzant, Todd; Himmel, Michael E.

2015-06-15

Xylan constitutes a significant portion of biomass (e.g. 22% in corn stover used in this study). Xylan is also an important source of carbohydrates, besides cellulose, for renewable and sustainable energy applications. Currently used method for the localization of xylan in biomass is to use fluorescence confocal microscope to image the fluorescent dye labeled monoclonal antibody that specifically binds to xylan. With the rapid adoption of the Raman-based label-free chemical imaging techniques in biology, identifying Raman bands that are unique to xylan would be critical for the implementation of the above label-free techniques for in situ xylan imaging. Unlike lignin and cellulose that have long be assigned fingerprint Raman bands, specific Raman bands for xylan remain unclear. The major challenge is the cellulose in plant cell wall, which has chemical units highly similar to that of xylan. Here we report using xylanase to specifically remove xylan from feedstock. Under various degree of xylan removal, with minimum impact to other major cell wall components, i.e. lignin and cellulose, we have identified Raman bands that could be further tested for chemical imaging of xylan in biomass in situ.

Carbon-14 labeling of phytoplankton carbon and chlorophyll a carbon: determination of specific growth rates

International Nuclear Information System (INIS)

Welschmeyer, N.A.; Lorenzen, C.J.

1984-01-01

The pattern of photosynthetic 14 C labeling over time is described for phytoplankton. The carbon-specific growth rate (d -1 ) is defined explicitly by changes in the specific activity (dpm μg -1 C) of the algae. For Skeletonema costatum, growing in axenic batch culture, the specific activities of both total cellular carbon and chlorophyll carbon increase at equal rates and the change in specific activity with time follows the predicted pattern. The specific activity of 14 C-labeled chlorophyll a was used to estimate phytoplankton growth rates and C:Chl ratios of field samples in Dabob Bay (Puget Sound), Washington. Growth rates decreased with depth and C:Chl ratios were higher for samples incubated under high light intensity. In several instances the C:Chl ratio increased from the beginning to the end of the incubation; this trend was most conspicuous near surface light intensities and for days of high total incident radiation. On these occasions, Chl a was actively 14 C labeled, yet little (or even negative) change was noted in the concentration of Chl a. These results suggest that some process (or processes) of chlorophyll degradation must be active at the same time that chlorophyll is being synthesized

Increase in the specific radioactivity of tritium-labeled compounds obtained by tritium thermal activation method

International Nuclear Information System (INIS)

Badun, G.A.; Chernysheva, M.G.; Ksenofontov, A.L.

2012-01-01

A method of tritium introduction into different types of organic molecules that is based on the interaction of atomic tritium with solid organic target is described. Tritium atoms are formed on the hot W-wire, which is heated by the electric current. Such an approach is called 'tritium thermal activation method'. Here we summarize the results of labeling globular proteins (lysozyme, human and bovine serum albumins); derivatives of pantothenic acid and amino acids; ionic surfactants (sodium dodecylsulfate and alkyltrimethylammonium bromides) and nonionic high-molecular weight surfactants - pluronics. For the first time it is observed that if the target-compound is fixed and its radicals are stable the specific radioactivity of the labeled product can be drastically increased (up to 400 times) when the target temperature is ca. 295 K compared with the results obtained at 77 K. The influence of labeling parameters as tritium gas pressure, exposure time and W-wire temperature was tested for each target temperature that results in the optimum labeling conditions with high specific radioactivity and chemical yield of the resulting compound. (orig.)

Photoaffinity cross-linking of a radioiodinated probe, 125I-A55453, into alpha 1-adrenergic receptors

International Nuclear Information System (INIS)

Dickinson, K.E.; Leeb-Lundberg, L.M.; Heald, S.L.; Wikberg, J.E.; DeBernardis, J.F.; Caron, M.G.; Lefkowitz, R.J.

1984-01-01

We have synthesized and characterized a high-affinity alpha 1-adrenergic receptor probe, 4-amino-6,7-dimethoxy-2[4'- [5''(3'''- 125 I-iodo-4'''-aminophenyl)pentanoyl]-1'-piperazinyl] quinazoline ( 125 I-A55453). This ligand binds reversibly to rat hepatic plasma membranes with high affinity (KD . 77 +/- 6 pM), and it labels the same number of specific prazosin-competable sites as the alpha 1-adrenergic receptor-selective radioligand [ 125 I] iodo-2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone. Specific binding is stereoselective and competed for by alpha-adrenergic agents with an alpha 1-adrenergic receptor specificity. 125 I-A55453 can be covalently photoincorporated into peptides of rat hepatic and splenic membranes using the bifunctional photoactive cross-linker, N-succinimidyl-6- (4'-azido-2'-nitrophenylamino)hexanoate. Following photolysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of labeled hepatic membranes reveals a major specifically labeled peptide of Mr . 82,000 (+/- 1,000) with minor peptides at Mr . 50,000 (+/- 500), and 40,000 (+/- 300). Covalent incorporation of 125 I-A55453 into the Mr . 82,000 peptide is inhibited by adrenergic drugs with an alpha 1-adrenergic receptor specificity. Labeled splenic membranes demonstrate a broad band of photoincorporated radioactivity centered at Mr . 82,000, and covalent incorporation into this peptide is also attenuated with an alpha 1-adrenergic receptor specificity. This new high-affinity radioiodinated probe has features which should make it useful for the molecular characterization of alpha 1-adrenergic receptors in tissues

Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

Energy Technology Data Exchange (ETDEWEB)

Mas, Guillaume; Crublet, Elodie [Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS) (France); Hamelin, Olivier [CNRS (France); Gans, Pierre; Boisbouvier, Jérôme, E-mail: jerome.boisbouvier@ibs.fr [Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS) (France)

2013-09-28

The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D{sub 2}O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d{sub 10}. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.

Fluorescently labeled dengue viruses as probes to identify antigen-specific memory B cells by multiparametric flow cytometry.

Science.gov (United States)

Woda, Marcia; Mathew, Anuja

2015-01-01

Low frequencies of memory B cells in the peripheral blood make it challenging to measure the functional and phenotypic characteristics of this antigen experienced subset of B cells without in vitro culture. To date, reagents are lacking to measure ex vivo frequencies of dengue virus (DENV)-specific memory B cells. We wanted to explore the possibility of using fluorescently labeled DENV as probes to detect antigen-specific memory B cells in the peripheral blood of DENV immune individuals. Alexa Fluor dye-labeled DENV yielded viable virus that could be stored at -80°C for long periods of time. Using a careful gating strategy and methods to decrease non-specific binding, we were able to identify a small frequency of B cells from dengue immune individuals that bound labeled DENV. Sorted DENV(+) B cells from immune, but not naïve donors secreted antibodies that bound DENV after in vitro stimulation. Overall, Alexa Fluor dye-labeled DENVs are useful reagents to enable the detection and characterization of memory B cells in DENV immune individuals. Copyright © 2014 Elsevier B.V. All rights reserved.

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

Energy Technology Data Exchange (ETDEWEB)

Gaibelet, Gérald [INSERM U563, CHU Purpan, Toulouse (France); CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette (France); Tercé, François [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Bertrand-Michel, Justine [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Lipidomic Platform Metatoul, Toulouse (France); Allart, Sophie [Plateau Technique d’Imagerie Cellulaire, INSERM U1043, Toulouse (France); Azalbert, Vincent [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Lecompte, Marie-France [INSERM U563, Faculté de Médecine de Rangueil, Toulouse (France); Collet, Xavier [Université Toulouse III, UMR 1048, Toulouse (France); INSERM U1048, Toulouse (France); Orlowski, Stéphane, E-mail: stephane.orlowski@cea.fr [INSERM U563, CHU Purpan, Toulouse (France); CEA, SB2SM and UMR8221 CNRS, IBiTec-Saclay, Gif-sur-Yvette (France)

2013-11-01

Highlights: •21-Methylpyrenyl-cholesterol specifically and stably associates to lipoproteins. •It is not esterified by LCAT, and thus reliably labels their peripheral hemi-membrane. •HDL vs. LDL are well distinguishable by various fluorescent labelling characteristics. •LDL peripheral hemi-membrane harbors cholesterol-rich ordered lipid (micro)domains. •Cultured cells can be stained by such labelled lipoproteins-mediated delivery. -- Abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD

21-Methylpyrenyl-cholesterol stably and specifically associates with lipoprotein peripheral hemi-membrane: A new labelling tool

International Nuclear Information System (INIS)

Gaibelet, Gérald; Tercé, François; Bertrand-Michel, Justine; Allart, Sophie; Azalbert, Vincent; Lecompte, Marie-France; Collet, Xavier; Orlowski, Stéphane

2013-01-01

Highlights: •21-Methylpyrenyl-cholesterol specifically and stably associates to lipoproteins. •It is not esterified by LCAT, and thus reliably labels their peripheral hemi-membrane. •HDL vs. LDL are well distinguishable by various fluorescent labelling characteristics. •LDL peripheral hemi-membrane harbors cholesterol-rich ordered lipid (micro)domains. •Cultured cells can be stained by such labelled lipoproteins-mediated delivery. -- Abstract: Lipoproteins are important biological components. However, they have few convenient fluorescent labelling probes currently reported, and their physiological reliability can be questioned. We compared the association of two fluorescent cholesterol derivatives, 22-nitrobenzoxadiazole-cholesterol (NBD-Chol) and 21-methylpyrenyl-cholesterol (Pyr-met-Chol), to serum lipoproteins and to purified HDL and LDL. Both lipoproteins could be stably labelled by Pyr-met-Chol, but virtually not by NBD-Chol. At variance with NBD-Chol, LCAT did not esterify Pyr-met-Chol. The labelling characteristics of lipoproteins by Pyr-met-Chol were well distinguishable between HDL and LDL, regarding dializability, associated probe amount and labelling kinetics. We took benefit of the pyrene labelling to approach the structural organization of LDL peripheral hemi-membrane, since Pyr-met-Chol-labelled LDL, but not HDL, presented a fluorescence emission of pyrene excimers, indicating that the probe was present in an ordered lipid micro-environment. Since the peripheral membrane of LDL contains more sphingomyelin (SM) than HDL, this excimer formation was consistent with the existence of cholesterol- and SM-enriched lipid microdomains in LDL, as already suggested in model membranes of similar composition and reminiscent to the well-described “lipid rafts” in bilayer membranes. Finally, we showed that Pyr-met-Chol could stain cultured PC-3 cells via lipoprotein-mediated delivery, with a staining pattern well different to that observed with NBD

A rapid and convenient method for specific 11C-labelling of synthetic polypeptides containing methionine

International Nuclear Information System (INIS)

Laengstroem, B.; Sjoeberg, S.; Ragnarsson, U.

1981-01-01

11 C-labelling of methionine residues in a synthetic peptide via the preparation of the corresponding protected, pure homocysteine peptide has been investigated. Complete deprotection of the peptide and specific methylation of the homocysteine residue can be performed in one step in liquid ammonia. As a first application of this method the synthesis of the tripeptide, Z-Gly-L-Hcy(Bzl)-Gly-O-Bzl, and its conversion to Gly-Met-Gly and the corresponding labelled Gly-([ 11 C]-methyl)-Met-Gly, is reported. Starting with the protected peptide the labelling was performed in 20 +- 5 min (starting with 11 CO 2 ), yielding the labelled peptide in 92 +- 5 % radiochemical yield. Analyses and preparative LC can be performed within 6 min. (author)

The in vivo fate of a 211At labelled monoclonal antibody with known specificity in a murine system

International Nuclear Information System (INIS)

Vaughan, A.T.M.; Bateman, W.J.; Fisher, D.R.

1982-01-01

A monoclonal antibody reactive against the human transferrin receptor has been labelled with the alpha and X ray emitting isotope Astatine 211. The labelling procedure does not affect the ability of the product to bind to the transferrin receptor on the human leukemic cell line HL60. Using a direct binding assay, 211 At labelled antibody can be specifically inhibited from binding to its target cells by excess unlabelled antibody. Furthermore, the binding inhibition demonstrated in this system correlates to enhanced clonogenic survival of these cells, indicating that very few atoms of 211 At/cell are required for cell death. Data obtained from labelled antibody injected into mice show that the labelled product in serum retains the ability to bind to HL60 cells in vitro, although tissue distributions of the injected activity implies that some of the radiolabel is lost from the protein. Despite this loss of label, preliminary experiments on the localization of labelled antibody to HL60 cells growing s/c in nude mice show that tumor tissue has a higher specific activity than all other tissues, other than blood, after 12 hours. This suggests that further work on the nature of label degradation in vivo is warranted in the context of potential therapeutic and diagnostic studies

Acridinium esters as high-specific-activity labels in immunoassay

International Nuclear Information System (INIS)

Weeks, I.; Beheshti, I.; McCapra, F.; Campbell, A.K.; Woodhead, J.S.

1983-01-01

A chemiluminescent acridinium ester has been synthesized that reacts spontaneously with proteins to yield stable, immunoreactive derivatives of high specific activity. The compound has been used to prepare chemiluminescent monoclonal antibodies to human alpha 1-fetoprotein having average incorporation ratios as great as 2.8 mol of label per mole of antibody, which corresponds to a detection limit of approximately 8 X 10(-19) mol. These antibodies have been used in the preliminary development of a two-site immunochemiluminometric assay for human alpha 1-fetoprotein, which requires only a 30-min incubation and a quantification time of 5 s per sample

Bacterial production of site specific {sup 13}C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

Energy Technology Data Exchange (ETDEWEB)

Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L., E-mail: robert.mcfeeters@uah.edu [University of Alabama in Huntsville, Department of Chemistry (United States)

2017-01-15

Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific {sup 13}C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct {sup 13}C chemical shifts and multiple magnetically equivalent {sup 1}H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with {sup 13}C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated {sup 1}H-{sup 13}C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.

Genetically-directed, cell type-specific sparse labeling for the analysis of neuronal morphology.

Directory of Open Access Journals (Sweden)

Thomas Rotolo

Full Text Available In mammals, genetically-directed cell labeling technologies have not yet been applied to the morphologic analysis of neurons with very large and complex arbors, an application that requires extremely sparse labeling and that is only rendered practical by limiting the labeled population to one or a few predetermined neuronal subtypes.In the present study we have addressed this application by using CreER technology to non-invasively label very small numbers of neurons so that their morphologies can be fully visualized. Four lines of IRES-CreER knock-in mice were constructed to permit labeling selectively in cholinergic or catecholaminergic neurons [choline acetyltransferase (ChAT-IRES-CreER or tyrosine hydroxylase (TH-IRES-CreER], predominantly in projection neurons [neurofilament light chain (NFL-IRES-CreER], or broadly in neurons and some glia [vesicle-associated membrane protein2 (VAMP2-IRES-CreER]. When crossed to the Z/AP reporter and exposed to 4-hydroxytamoxifen in the early postnatal period, the number of neurons expressing the human placental alkaline phosphatase reporter can be reproducibly lowered to fewer than 50 per brain. Sparse Cre-mediated recombination in ChAT-IRES-CreER;Z/AP mice shows the full axonal and dendritic arbors of individual forebrain cholinergic neurons, the first time that the complete morphologies of these very large neurons have been revealed in any species.Sparse genetically-directed, cell type-specific neuronal labeling with IRES-creER lines should prove useful for studying a wide variety of questions in neuronal development and disease.

Detection of magnetic-labeled antibody specific recognition events by combined atomic force and magnetic force microscopy

International Nuclear Information System (INIS)

Hong Xia; Liu Yanmei; Li Jun; Guo Wei; Bai Yubai

2009-01-01

Atomic force (AFM) and magnetic force microscopy (MFM) were developed to detect biomolecular specific interaction. Goat anti-mouse immunoglobulin (anti-IgG) was covalently attached onto gold substrate modified by a self-assembly monolayer of thioctic acid via 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) activation. Magnetic-labeled IgG then specifically adsorbed onto anti-IgG surface. The morphological variation was identified by AFM. MFM was proved to be a fine assistant tool to distinguish the immunorecognized nanocomposites from the impurities by detection of the magnetic signal from magnetic-labeled IgG. It would enhance the understanding of biomolecular recognition process.

Detection of magnetic-labeled antibody specific recognition events by combined atomic force and magnetic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Hong Xia [Center for Advanced Optoelectronic Functional Materials Research, Key Laboratory of UV Light-Emitting Materials and Technology, Ministry of Education, Northeast Normal University, Changchun 130024 (China); College of Chemistry, Jilin University, Changchun 130023 (China)], E-mail: xiahong@nenu.edu.cn; Liu Yanmei; Li Jun; Guo Wei; Bai Yubai [College of Chemistry, Jilin University, Changchun 130023 (China)

2009-09-15

Atomic force (AFM) and magnetic force microscopy (MFM) were developed to detect biomolecular specific interaction. Goat anti-mouse immunoglobulin (anti-IgG) was covalently attached onto gold substrate modified by a self-assembly monolayer of thioctic acid via 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) activation. Magnetic-labeled IgG then specifically adsorbed onto anti-IgG surface. The morphological variation was identified by AFM. MFM was proved to be a fine assistant tool to distinguish the immunorecognized nanocomposites from the impurities by detection of the magnetic signal from magnetic-labeled IgG. It would enhance the understanding of biomolecular recognition process.

Nutrition labelling is a trade policy issue: lessons from an analysis of specific trade concerns at the World Trade Organization.

Science.gov (United States)

Thow, Anne Marie; Jones, Alexandra; Hawkes, Corinna; Ali, Iqra; Labonté, Ronald

2017-01-12

Interpretive nutrition labels provide simplified nutrient-specific text and/or symbols on the front of pre-packaged foods, to encourage and enable consumers to make healthier choices. This type of labelling has been proposed as part of a comprehensive policy response to the global epidemic of non-communicable diseases. However, regulation of nutrition labelling falls under the remit of not just the health sector but also trade. Specific Trade Concerns have been raised at the World Trade Organization's Technical Barriers to Trade Committee regarding interpretive nutrition labelling initiatives in Thailand, Chile, Indonesia, Peru and Ecuador. This paper presents an analysis of the discussions of these concerns. Although nutrition labelling was identified as a legitimate policy objective, queries were raised regarding the justification of the specific labelling measures proposed, and the scientific evidence for effectiveness of such measures. Concerns were also raised regarding the consistency of the measures with international standards. Drawing on policy learning theory, we identified four lessons for public health policy makers, including: strategic framing of nutrition labelling policy objectives; pro-active policy engagement between trade and health to identify potential trade issues; identifying ways to minimize potential 'practical' trade concerns; and engagement with the Codex Alimentarius Commission to develop international guidance on interpretative labelling. This analysis indicates that while there is potential for trade sector concerns to stifle innovation in nutrition labelling policy, care in how interpretive nutrition labelling measures are crafted in light of trade commitments can minimize such a risk and help ensure that trade policy is coherent with nutrition action. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

16 CFR 306.12 - Labels.

Science.gov (United States)

2010-01-01

... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Labels. 306.12 Section 306.12 Commercial..., CERTIFICATION AND POSTING Label Specifications § 306.12 Labels. All labels must meet the following specifications: (a) Layout—(1) For gasoline labels. The label is 3″ (7.62 cm) wide × 21/2″ (6.35 cm) long. The...

A photoaffinity scan maps regions of the p85 SH2 domain involved in phosphoprotein binding.

Science.gov (United States)

Williams, K P; Shoelson, S E

1993-03-15

Src homology 2 (SH2) domains are modular phosphotyrosine binding pockets found within a wide variety of cytoplasmic signaling molecules. Here we develop a new approach to analyzing protein-protein interfaces termed photoaffinity scanning, and apply the method to map regions of the phosphatidylinositol 3-kinase p85 SH2 domain that participate in phospho-protein binding. Each residue except phosphotyrosine (pY) within a tightly binding, IRS-1-derived phosphopeptide (GNGDpYMPMSPKS) was substituted with the photoactive amino acid, benzoylphenylalanine (Bpa). Whereas most substitutions had little effect on binding affinity, Bpa substitution of either Met (+1 and +3 with respect to pY) reduced affinity 50-100-fold to confirm their importance in the pYMXM recognition motif. In three cases photolysis of SH2 domain/Bpa phosphopeptide complexes led to cross-linking of > 50% of the SH2 domain; cross-link positions were identified by microsequence, amino acid composition, and electrospray mass spectrometric analyses. Bpa-1 cross-links within alpha-helix I, whereas Bpa+1 and Bpa+4 cross-link the SH2 domain within the flexible loop C-terminal to alpha-helix II. Moreover, cross-linking at any position prevents SH2 domain cleavage at a trypsin-sensitive site within the flexible loop between beta-strands 1 and 2. Therefore, at least three distinct SH2 regions in addition to the beta-sheet participate in phosphoprotein binding; the loop cross-linked by phosphopeptide residues C-terminal to pY appears to confer specificity to the phosphoprotein/SH2 domain interaction.

Site-Specific Protein Labeling Utilizing Lipoic Acid Ligase (LplA) and Bioorthogonal Inverse Electron Demand Diels-Alder Reaction.

Science.gov (United States)

Baalmann, Mathis; Best, Marcel; Wombacher, Richard

2018-01-01

Here, we describe a two-step protocol for selective protein labeling based on enzyme-mediated peptide labeling utilizing lipoic acid ligase (LplA) and bioorthogonal chemistry. The method can be applied to purified proteins, protein in cell lysates, as well as living cells. In a first step a W37V mutant of the lipoic acid ligase (LplA W37V ) from Escherichia coli is utilized to ligate a synthetic chemical handle site-specifically to a lysine residue in a 13 amino acid peptide motif-a short sequence that can be genetically expressed as a fusion with any protein of interest. In a second step, a molecular probe can be attached to the chemical handle in a bioorthogonal Diels-Alder reaction with inverse electron demand (DA inv ). This method is a complementary approach to protein labeling using genetic code expansion and circumvents larger protein tags while maintaining label specificity, providing experimental flexibility and straightforwardness.

Deuterium labeled cannabinoids

International Nuclear Information System (INIS)

Driessen, R.A.

1979-01-01

Complex reactions involving ring opening, ring closure and rearrangements hamper complete understanding of the fragmentation processes in the mass spectrometric fragmentation patterns of cannabinoids. Specifically labelled compounds are very powerful tools for obtaining more insight into fragmentation mechanisms and ion structures and therefore the synthesis of specifically deuterated cannabinoids was undertaken. For this, it was necessary to investigate the preparation of cannabinoids, appropriately functionalized for specific introduction of deuterium atom labels. The results of mass spectrometry with these labelled cannabinoids are described. (Auth.)

Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI.

Directory of Open Access Journals (Sweden)

Kalle Kipper

Full Text Available The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic

Towards kit formulation of 99mTc labelled somatostatin receptor binding peptides of high specific activity for tumour localization

International Nuclear Information System (INIS)

Behe, M.; Powell, P.; Maecke, H.R.

2001-01-01

The project aimed to develop 99m Tc octreotide analogue for use in nuclear oncology. Several attempts to label SRIF analogues with 99m Tc have used a direct labelling approach but, for this project, HYNIC was chosen as a technetium ligand. A comparison of two different SRIF analogues designed for high specific activity labelling with 99m Tc was done. HYNIC-Octreotide and HYNIC-TOC were prepared and a kit formulation that can be labelled conveniently is currently being studied in a clinical setting. (author)

Clinical applications of cells labelling

International Nuclear Information System (INIS)

Gonzalez, B.M.

1994-01-01

Blood cells labelled with radionuclides are reviewed and main applications are described. Red blood cell labelling by both random and specific principle. A table with most important clinical uses, 99mTc labelling of RBC are described pre tinning and in vivo reduction of Tc, in vitro labelling and administration of labelled RBC and in vivo modified technique. Labelled leucocytes with several 99mTc-complex radiopharmaceuticals by in vitro technique and specific monoclonal s for white cells(neutrofiles). Labelled platelets for clinical use and research by in vitro technique and in vivo labelling

16 CFR 309.17 - Labels.

Science.gov (United States)

2010-01-01

... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Labels. 309.17 Section 309.17 Commercial... ALTERNATIVE FUELS AND ALTERNATIVE FUELED VEHICLES Requirements for Alternative Fuels Label Specifications § 309.17 Labels. All labels must meet the following specifications: (a) Layout: (1) Non-liquid...

Maternal Label and Gesture Use Affects Acquisition of Specific Object Names

Science.gov (United States)

Zammit, Maria; Schafer, Graham

2011-01-01

Ten mothers were observed prospectively, interacting with their infants aged 0 ; 10 in two contexts (picture description and noun description). Maternal communicative behaviours were coded for volubility, gestural production and labelling style. Verbal labelling events were categorized into three exclusive categories: label only; label plus…

Iodoinsulin specifically labelled with 125I and 127I for use as insulin tracer

International Nuclear Information System (INIS)

Bahrami, S.

1981-01-01

In order to describe for the first time iodine-labelled in the B1 position starting with inactive Bolton-Hunter reagents, the essential intermediate steps with secure sup(125,127) iodine positions were synthesized. This labelling position is essential to obtain the structural and biological porperties of insulin on the one hand, and to enable one to trace the metabolism further of the B chain after splitting the insulin in the organism on the other hand. The preparate was made with high specific activity of 420 Ci/mMol. A new high-pressure liquid chromatographic method was developed to separate the products. (RB) [de

Sandwich nucleic acid hybridization: a method with a universally usable labeled probe for various specific tests

International Nuclear Information System (INIS)

Wolf, H.; Leser, U.; Haus, M.; Gu, S.Y.; Pathmanathan, R.

1986-01-01

The use of recombinant m13 phages as hybridization probes offers a considerable advantage over the commonly used recombinant plasmids as the preparation of the DNA probe is very simple and it can easily be labeled directly, e.g. with isotopes with long half-life like 125 I and used for hybridization. However, as the application of nucleic acid hybridization for diagnostic and epidemiological purposes becomes almost unavoidable, the logistic problems of keeping numerous individually labeled hybridization probes increase considerably and may reach prohibitory levels in less well-equipped laboratories. In a new sandwich technique, the first step involves hybridization with an unlabeled recombinant m13 DNA carrying an insert of the desired specificity. In a second step a universally usable labeled probe directed against the m13 part of the recombinant phage DNA is applied. This reduces considerably the problem of preparing and keeping multiple labeled probes in stock. (Auth.)

Synthesis of high specific activity carbon-11 labeled tracers for neuroreceptor studies

International Nuclear Information System (INIS)

Dannals, R.F.; Ravert, H.T.; Wilson, A.A.; Wagner, H.N. Jr; Johns Hopkins Medical Institutions, Baltimore, MD

1989-01-01

The use of short-lived positron-emitting radiotracers together with positron emission tomography (PET) has allowed scientists to acquire previously inaccessible information regarding problems in physiology, biochemistry, and pharmacology in the living human body. In the past five years, successes in the application of PET to the non-invasive determination of the spatial distribution and regional concentration of a variety of neurotransmitter binding sites within the living brain often followed the successful selections and syntheses of appropriately radiolabeled ligands. This presentation concentrates on the synthesis of these high specific activity radiotracers for neuroreceptor PET studies labeled specifically with carbon-11. (author). 15 refs.; 1 fig

The radioactive labeling of monocytes

International Nuclear Information System (INIS)

Ensing, G.J.

1985-01-01

With the aim of studying a possible relationship between circulating monocytes and Sternberg-Reed cells investigations were started on the specific labeling of monocytes. In this thesis the literature on the pertinent data has been reviewed and a series of experiments on the monocyte labeling procedure has been described. The principles of cell labeling with radioactive compounds were discussed. 1. Total separation of the particular cell population to be labeled and subsequent labeling with a non-specific radiopharmaceutical. 2. Specific cell labeling in a mixture of cell types based on a well defined affinity of the cell under study for the radiopharmaceutical used. Next the radionuclides that can be used for cell labeling purposes were discussed with special attention for 111 In and its chelates. The principles of radiodosimetry were also discussed shortly. This section was focussed on the radiation dose the labeled cells receive because of the intracellular localized radioactivity. The radiation burden is high in comparison to amounts of radiation known to affect cell viability. A newly developed method for labeling monocytes specifically by phagocytosis of 111 In-Fe-colloid without apparent loss of cells was described in detail. (Auth.)

Method of preparing tritium-labelled thymidine-5'-monophosphates of high specific activity

International Nuclear Information System (INIS)

Filip, J.; Vesely, J.; Cihak, A.

1976-01-01

A method is described of preparing thymidine-5'-monophosphates labelled with tritium of high specific activity based on enzyme synthesis in vitro. Phosphorylation was carried out using the catalytic effect of an enzyme contained in the supernatant fraction prepared from Yoshida ascites carcinoma in rats. The course of the enzyme reaction can be controlled by the concentration of the individual reaction mixture components. The method described allows obtaining thymidine-5'-monophosphate of radiochemical purity better than 95%. (J.B.)

Protocols for 16S rDNA Array Analyses of Microbial Communities by Sequence-Specific Labeling of DNA Probes

Directory of Open Access Journals (Sweden)

Knut Rudi

2003-01-01

Full Text Available Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for DNA array-based analyses of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Protocols are presented for DNA purification, probe construction, probe labeling, and DNA array hybridizations.

Functional proteomic of Matrix Metallo-proteinases (MMP) dedicated to the detection of active forms of MMP in complex proteome

International Nuclear Information System (INIS)

David, A.

2007-07-01

The Matrix Metallo-proteinases (M.M.P.) represent a family of Zinc dependent extracellular proteinases able to cleave collectively all the proteins constituting the extracellular matrix. Currently, 23 human M.M.P. have been identified and are characterized by their sequence in amino-acids and their highly conserved 3 D structure. These enzymes are expressed constitutively during the tissue remodeling process. Their over-expression in various diseases tightly related to inflammatory processes (arthritis, emphysema, cancer) described M.M.P. as choice therapeutic targets. However, as the tissue remodeling implicates modification of cellular contacts, M.M.P. appear currently as proteins involved in signalling pathways. Recent works demonstrating that M.M.P. are able to cleave substrates, which are different than proteins constituting the extracellular matrix, reinforce this vision. In order to identify the individual role and the protein expression level of M.M.P. in pathological context, we developed a new technique of functional proteomics dedicated to the detection of active forms of M.M.P. in tumour samples. This technique relied on the development of a new photoaffinity probe, based on the structure of a potent phosphinic inhibitor of M.M.P., allowing targeting and isolating active forms of M.M.P. by photoaffinity labelling. Furthermore, as the new developed probe incorporated a radioactive element, photoaffinity labelling permitted to radiolabel the targeted proteins. This probe demonstrated in vitro its remarkable ability to covalently modify the h M.M.P.-12, with a singular cross-linking yield, determined at 42 %, displaying an extremely sensitive detection (2.5 fmoles of h M.M.P.-12). When added to complex proteome, the photoaffinity probe presents the same sensibility of detection for the h M.M.P.-12 (5 fmoles); importantly, in this case, h M.M.P.-12 represents only 0.001 % of the totality of the proteins present in the sample. Moreover, this technique allows

Functional proteomic of Matrix Metallo-proteinases (MMP) dedicated to the detection of active forms of MMP in complex proteome; Proteomique fonctionnelle dediee aux Metalloproteases Matricelles (MMPs): developpement d'une methode extremement sensible permettant la detection des formes actives des MMPs dans des proteomes complexes

Energy Technology Data Exchange (ETDEWEB)

David, A

2007-07-15

The Matrix Metallo-proteinases (M.M.P.) represent a family of Zinc dependent extracellular proteinases able to cleave collectively all the proteins constituting the extracellular matrix. Currently, 23 human M.M.P. have been identified and are characterized by their sequence in amino-acids and their highly conserved 3 D structure. These enzymes are expressed constitutively during the tissue remodeling process. Their over-expression in various diseases tightly related to inflammatory processes (arthritis, emphysema, cancer) described M.M.P. as choice therapeutic targets. However, as the tissue remodeling implicates modification of cellular contacts, M.M.P. appear currently as proteins involved in signalling pathways. Recent works demonstrating that M.M.P. are able to cleave substrates, which are different than proteins constituting the extracellular matrix, reinforce this vision. In order to identify the individual role and the protein expression level of M.M.P. in pathological context, we developed a new technique of functional proteomics dedicated to the detection of active forms of M.M.P. in tumour samples. This technique relied on the development of a new photoaffinity probe, based on the structure of a potent phosphinic inhibitor of M.M.P., allowing targeting and isolating active forms of M.M.P. by photoaffinity labelling. Furthermore, as the new developed probe incorporated a radioactive element, photoaffinity labelling permitted to radiolabel the targeted proteins. This probe demonstrated in vitro its remarkable ability to covalently modify the h M.M.P.-12, with a singular cross-linking yield, determined at 42 %, displaying an extremely sensitive detection (2.5 fmoles of h M.M.P.-12). When added to complex proteome, the photoaffinity probe presents the same sensibility of detection for the h M.M.P.-12 (5 fmoles); importantly, in this case, h M.M.P.-12 represents only 0.001 % of the totality of the proteins present in the sample. Moreover, this technique allows

Functional proteomic of Matrix Metallo-proteinases (MMP) dedicated to the detection of active forms of MMP in complex proteome; Proteomique fonctionnelle dediee aux Metalloproteases Matricelles (MMPs): developpement d'une methode extremement sensible permettant la detection des formes actives des MMPs dans des proteomes complexes

Energy Technology Data Exchange (ETDEWEB)

David, A

2007-07-15

The Matrix Metallo-proteinases (M.M.P.) represent a family of Zinc dependent extracellular proteinases able to cleave collectively all the proteins constituting the extracellular matrix. Currently, 23 human M.M.P. have been identified and are characterized by their sequence in amino-acids and their highly conserved 3 D structure. These enzymes are expressed constitutively during the tissue remodeling process. Their over-expression in various diseases tightly related to inflammatory processes (arthritis, emphysema, cancer) described M.M.P. as choice therapeutic targets. However, as the tissue remodeling implicates modification of cellular contacts, M.M.P. appear currently as proteins involved in signalling pathways. Recent works demonstrating that M.M.P. are able to cleave substrates, which are different than proteins constituting the extracellular matrix, reinforce this vision. In order to identify the individual role and the protein expression level of M.M.P. in pathological context, we developed a new technique of functional proteomics dedicated to the detection of active forms of M.M.P. in tumour samples. This technique relied on the development of a new photoaffinity probe, based on the structure of a potent phosphinic inhibitor of M.M.P., allowing targeting and isolating active forms of M.M.P. by photoaffinity labelling. Furthermore, as the new developed probe incorporated a radioactive element, photoaffinity labelling permitted to radiolabel the targeted proteins. This probe demonstrated in vitro its remarkable ability to covalently modify the h M.M.P.-12, with a singular cross-linking yield, determined at 42 %, displaying an extremely sensitive detection (2.5 fmoles of h M.M.P.-12). When added to complex proteome, the photoaffinity probe presents the same sensibility of detection for the h M.M.P.-12 (5 fmoles); importantly, in this case, h M.M.P.-12 represents only 0.001 % of the totality of the proteins present in the sample. Moreover, this technique allows

Site-Specific Three-Color Labeling of α-Synuclein via Conjugation to Uniquely Reactive Cysteines during Assembly by Native Chemical Ligation.

Science.gov (United States)

Lee, Taehyung C; Moran, Crystal R; Cistrone, Philip A; Dawson, Philip E; Deniz, Ashok A

2018-04-12

Single-molecule fluorescence is widely used to study conformational complexity in proteins, and has proven especially valuable with intrinsically disordered proteins (IDPs). Protein studies using dual-color single-molecule Förster resonance energy transfer (smFRET) are now quite common, but many could benefit from simultaneous measurement of multiple distances through multi-color labeling. Such studies, however, have suffered from limitations in site-specific incorporation of more than two dyes per polypeptide. Here we present a fully site-specific three-color labeling scheme for α-synuclein, an IDP with important putative functions and links to Parkinson disease. The convergent synthesis combines native chemical ligation with regiospecific cysteine protection of expressed protein fragments to permit highly controlled labeling via standard cysteine-maleimide chemistry, enabling more global smFRET studies. Furthermore, this modular approach is generally compatible with recombinant proteins and expandable to accommodate even more complex experiments, such as by labeling with additional colors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fluorescent humanized anti-CEA antibody specifically labels metastatic pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model

Science.gov (United States)

Lwin, Thinzar M.; Miyake, Kentaro; Murakami, Takashi; DeLong, Jonathan C.; Yazaki, Paul J.; Shivley, John E.; Clary, Bryan; Hoffman, Robert M.; Bouvet, Michael

2018-03-01

Specific tumor targeting can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively target and label pancreatic cancer and its metastases in a clinically relevant patient derived xenograft mouse model.

Molecular organization in bacterial cell membranes. Specific labelling and topological distribution of glycoproteins and proteins in Streptomyces albus membranes

Energy Technology Data Exchange (ETDEWEB)

Larraga, V; Munoz, E [Consejo Superior de Investigaciones Cientificas, Madrid (Spain). Instituto de Biologia Celular

1975-05-01

The paper reports about an investigation into the question of the specific labelling and topological distribution of glycoproteins and proteins in Streptomyces albus membranes. The method of sample preparation is described: Tritium labelling of glycoproteins in protoplasts and membranes, iodination of proteins, trypsin treatment and polyacrylamide gel electrophoresis. The findings suggest an asymmetrical distribution of the glycoproteins in membranes and a weak accessibility to iodine label. A structural model of the plasma membranes of Streptomyces albus is proposed similar to the general 'fluid mosaic' model of Singer and Nicholson.

From position-specific isotope labeling towards soil fluxomics: a novel toolbox to assess the microbial impact on biogeochemical cycles

Science.gov (United States)

Apostel, C.; Dippold, M. A.; Kuzyakov, Y.

2015-12-01

Understanding the microbial impact on C and nutrient cycles is one of the most important challenges in terrestrial biogeochemistry. Transformation of low molecular weight organic substances (LMWOS) is a key step in all biogeochemical cycles because 1) all high molecular substances pass the LMWOS pool during their degradation and 2) only LMWOS can be taken up by microorganisms intact. Thus, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the microbial metabolic network and its control mechanism. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools but studies were nearly exclusively based on uniformly labeled substances. However, such tracers do not allow the differentiation of the intact use of the initial substances from its transformation to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of basic metabolites and quantification of isotope incorporation in CO2 and bulk soil enabled following the basic metabolic pathways of microorganisms. However, the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites like phospholipid fatty acids (PLFA) or amino sugars revealed new insights into the soil fluxome: First, it enables tracing specific anabolic pathways in diverse microbial communities in soils e.g. carbon starvation pathways versus pathways reflecting microbial growth. Second, it allows identification of specific pathways of individual functional microbial groups in soils in situ. Tracing metabolic pathways and understanding their regulating factors are crucial for soil C fluxomics i.e. the unravaling of the complex network of C transformations

Synthesis of high specific activity tritium-labelled chloroethylcyclohexylnitrosourea and its application to the study of DNA modification

International Nuclear Information System (INIS)

Siew, E.L.; Habraken, Yvette; Ludlum, D.B.

1991-01-01

A small-scale synthesis of high specific activity, N-(2-chloro-2-[ 3 H-ethyl)-N'-cyclohexyl-N-nitrosourea ([ 3 H]-CCNU) has been accomplished from tritium-labelled ethanolamine. The product is pure by TLC and HPLC analysis and has been used successfully to modify DNA. The overall yield on radioactivity including losses in HPLC purification is approximately 4 percent. The availability of this tritium-labelled compound makes studies of DNA repair and of cellular resistance to N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea possible. (author)

Dynamics of growth/mature-related substances in vegetables using specific triple labeled compound

International Nuclear Information System (INIS)

Yamato, Yoichi; Hamano, Megumi; Yamazaki, Hiroko; Miura, Hiroyuki

2000-01-01

To progress physiological studies of vegetables, development of biosynthetic method for production of triple labeled compounds was attempted in this study and such method for vegetables using specifically labeled sugars was examined. As a sugar compound, 6-C 14 -glucose (14-CG) and 1-H 3 -glucose (3-HG) were given to culture medium for cells derived from tomato embryonic axis and the changes of these compounds were monitored. Tomato embryonic cells were harvested 20 and 44 hours after the addition of 14-CG or 3-CG into the culture medium the cells. The cells were homogenized and the supernatant after centrifugation was applied onto HPLC. Radio analyzer revealed major two peaks in the chromatography of the sugar fraction from the cells after 20 hours from the addition of 14-CG. One was the peak of glucose, itself and the other was estimated to be that of fructose based on the retention time. Whereas in the elution pattern of the sugar fraction after 44 hours from the addition, a peak of sucrose was found along with the peak of glucose. These results indicate that C 14 in 14-CG but not H 3 in 3-HG was transferred into fructose after the metabolism in tomato. Moreover, in both elution patterns, there was a peak positioned at the same retention time, indicating that the compound in this peak was produced from either of 14-CG or 3-HG. Therefore, it is thought that H 3 and C 14 double-labeled compound could be produced from the cell culture added with both labeled compounds; 14-CG and 3-HG. (M.N.)

Synthesis of high specific activity tritium-labelled chloroethylcyclohexylnitrosourea and its application to the study of DNA modification

Energy Technology Data Exchange (ETDEWEB)

Siew, E.L. (State Univ. of New York, Albany, NY (USA). Dept. of Chemistry); Habraken, Yvette; Ludlum, D.B. (Massachusetts Univ., Worcester, MA (USA). Medical School)

1991-02-01

A small-scale synthesis of high specific activity, N-(2-chloro-2-{sup 3}H-ethyl)-N'-cyclohexyl-N-nitrosourea ({sup 3}H-CCNU) has been accomplished from tritium-labelled ethanolamine. The product is pure by TLC and HPLC analysis and has been used successfully to modify DNA. The overall yield on radioactivity including losses in HPLC purification is approximately 4 percent. The availability of this tritium-labelled compound makes studies of DNA repair and of cellular resistance to N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea possible. (author).

Preparation of riboflavin specifically labeled in the 5'-hydroxymethyl terminus using a vitamin B2-aldehyde-forming enzyme from Schizophyllum commune

International Nuclear Information System (INIS)

Kekelidze, T.N.; Edmondson, D.E.; McCormick, D.B.

1995-01-01

A method is described for synthesis of riboflavin selectively labeled in the hydrogens at the 5'-hydroxymethyl position. In this method, a vitamin B 2 -aldehyde-forming enzyme from Schizophyllum commune is used to specifically and completely oxidize the 5'-hydroxymethyl of riboflavin to the 5'-aldehyde. This reaction is monitored spectrophotometrically by the reduction of 2,6-dichlorophenolindophenol at 600 nm. Appearance of aldehyde product was directly quantitated by reverse-phase high-performance liquid chromatography. Product is extracted from the incubation mixture by phenol after saturation with (NH 4 ) 2 SO 4 and then further purified by benzyl alcohol extraction. The 5'-aldehyde is reduced with appropriately labeled sodium borohydride to yield the vitamin specifically labeled in the 5'-hydroxymethyl group. (author)

Application of the quantitative autoradiography for determination of specific activity of labelled non-metallic inclusions

International Nuclear Information System (INIS)

Kowalczyk, J.T.; Wilczynski, A.W.

1983-01-01

The knowledge of specific activity of labelled non-metallic inclusions, i.e. the knowledge of the content of the radiotracer in a single inclusion, allows to obtain new information about the mechanism and the kinetics of steel deoxidation. In order to determine this specific activity quantitative autoradiography was used. Fo; this purpose, various standards of aluminium oxides with different amounts of cerium oxide Ce 2 O 3 and an aluminium-cerium alloy were prepared. The standards and the alloy were activated with thermal neutrons. Then several autoradiographs were made for these standards (ORWO AF-3 films were used). The autoradiographs served as the basis for evaluation of the standardization curves: optical density versus dimension of particles for a constant cerium concentration; optical density versus concentration of cerium for a constant dimension of particle. The samples of liquid steel were deoxidated with Al-Ce alloy. After labelled non-metallic inclusions had been isolated, the autoradiographs were made under the same conditions as for the standards. The standardization curves were used to determine the cerium content in the single inclusions. (author)

Labelling and Self-Esteem: The Impact of Using Specific vs. Generic Labels

Science.gov (United States)

Taylor, Laura Marie; Hume, Ian Robert; Welsh, Nikki

2010-01-01

The aim of this study is to investigate the relationship between being labelled either as having dyslexia or as having general special educational needs (SEN) and a child's self-esteem. Seventy-five children aged between 8 and 15 years categorised as having dyslexia (N = 26), as having general SEN (N = 26) or as having no learning difficulties (N…

The 18F-labelled alkylating agent 2,2,2-trifluoroethyl triflate: synthesis and specific activity

International Nuclear Information System (INIS)

Johnstroem, P.; Stone-Elander, S.

1995-01-01

A method for synthesizing the alkylating agent 2,2,2-trifluoroethyl triflate labelled with [ 18 ]fluoride in the two position is presented. Ethyl [2- 18 )F]-trifluoroacetate was synthesized by the nucleophilic reaction of [ 18 F]F - with ethyl bromodifluoroacetate in DMSO (45-60%, 5 min, 80 o C) and subsequently converted to [2- 18 F]-2,2,2-trifluoroethanol using alane in THF (85-95%, 2 min, 40 o C. Reaction with triflic anhydride in 2,6-lutidine produced [2- 18 F]-2,2,2-trifluoroethyl triflate (70-80%, 1 min, 0 o C. In all three cases the product was removed from the reaction vessel by heating to distil under a stream of nitrogen. [2- 18 F]-2,2,2-Trifluoroethyl triflate was used to label 2-oxoquazepam by N-alkylation in a toulene:DMF mixture (80-85%, 20 min, 120 o C). Although no-carrier-added [ 18 )F]F - was used, considerable unlabelled ethyl trifluoroacetate was produced in the first reaction. Varying the conditions for the fluoro-debromination reaction did not appreciably improve the relative ratio of labelled to unlabelled ester. The specific activity of the labelled 1,4-benzodiazepine-2-one obtained from 1850 MBq [ 18 F]F - was found to be ≅37 MBq/μmol (1mCi/μmol). (Author)

Site-specific fluorescent labeling of nascent proteins on the translating ribosome.

Science.gov (United States)

Saraogi, Ishu; Zhang, Dawei; Chandrasekaran, Sandhya; Shan, Shu-ou

2011-09-28

As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosome-nascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.

Large-scale detection of antigen-specific T cells using peptide-MHC-I multimers labeled with DNA barcodes

DEFF Research Database (Denmark)

Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke Birgitte

2016-01-01

-major histocompatibility complex (MHC) multimers labeled with individual DNA barcodes to screen >1,000 peptide specificities in a single sample, and detect low-frequency CD8 T cells specific for virus- or cancer-restricted antigens. When analyzing T-cell recognition of shared melanoma antigens before and after adoptive...... cell therapy in melanoma patients, we observe a greater number of melanoma-specific T-cell populations compared with cytometry-based approaches. Furthermore, we detect neoepitope-specific T cells in tumor-infiltrating lymphocytes and peripheral blood from patients with non-small cell lung cancer...

Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment

International Nuclear Information System (INIS)

Schedlbauer, Andreas; Auer, Renate; Ledolter, Karin; Tollinger, Martin; Kloiber, Karin; Lichtenecker, Roman; Ruedisser, Simon; Hommel, Ulrich; Schmid, Walther; Konrat, Robert; Kontaxis, Georg

2008-01-01

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when 13 C β and 13 C' shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using 13 C α connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs

Synthesis of high specific activity tritium labelled 1S,2S-(-)-trans-2-isothiocyanato-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)benzene acetamide, a specific irreversible ligand for kappa opioid receptors

Energy Technology Data Exchange (ETDEWEB)

Costa, B.R. de; Thurkauf, A.; Rothman, R.R. (National Inst. of Mental Health, Bethesda, MD (USA)); Jacobson, A.E.; Rice, K.C. (National Inst. of Digestive Diabetes, and Kidney Diseases, Bethesda, MD (USA))

1990-11-01

Optically pure tritium labeled 1S,2S-(-)-trans-2-isothiocyanato-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl )benzeneacetamide, an affinity ligand specific for the kappa opioid receptor was synthesized from optically pure 1S,2S-(-)-trans-2-amino-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide via the sequence of dibromination (57%) followed by catalytic tritiation of the dibromide. The resulting tritium labelled aniline (14% yield, specific activity 31.2 Ci/mmol) was transformed to the title compound in 13.3% yield and 99+% radiochemical purity by treatment with thiophosgene. (author).

Syntheses of 18F-labeled reduced haloperidol and 11C-labeled reduced 3-N-methylspiperone

International Nuclear Information System (INIS)

Ravert, H.T.; Dannals, R.F.; Wilson, A.A.; Wong, D.F.; Wagner, H.N. Jr.

1991-01-01

18 F-Labeled reduced haloperidol and 11 C-labeled reduced 3-N-methylspiperone were synthesized in a convenient and quantitative one step reduction from 18 F-labeled haloperidol and 11 C-labeled N-methylspiperone, respectively. Both products were purified by semipreparative HPLC and were obtained at high specific activity and radiochemical purity. (author)

Preparation of labelled antituberculotics for clarifying specific problems in therapy optimization. 1

International Nuclear Information System (INIS)

Winsel, K.; Iwainsky, H.; Mittag, E.; Kiessling, M.; Koehler, H.

1985-01-01

The preparation of tritium labelled isoniazid according to the Wilzbach method and by catalytic exchange is described. The purified labelled isoniazid is adjusted to an activity of 37 MBq/300 mg isoniazid. It meets the requirements for an injectable pharmaceutical. The tritium labelling is stable under in vitro conditions and in the macroorganism. (author)

Co-Labeling for Multi-View Weakly Labeled Learning.

Science.gov (United States)

Xu, Xinxing; Li, Wen; Xu, Dong; Tsang, Ivor W

2016-06-01

It is often expensive and time consuming to collect labeled training samples in many real-world applications. To reduce human effort on annotating training samples, many machine learning techniques (e.g., semi-supervised learning (SSL), multi-instance learning (MIL), etc.) have been studied to exploit weakly labeled training samples. Meanwhile, when the training data is represented with multiple types of features, many multi-view learning methods have shown that classifiers trained on different views can help each other to better utilize the unlabeled training samples for the SSL task. In this paper, we study a new learning problem called multi-view weakly labeled learning, in which we aim to develop a unified approach to learn robust classifiers by effectively utilizing different types of weakly labeled multi-view data from a broad range of tasks including SSL, MIL and relative outlier detection (ROD). We propose an effective approach called co-labeling to solve the multi-view weakly labeled learning problem. Specifically, we model the learning problem on each view as a weakly labeled learning problem, which aims to learn an optimal classifier from a set of pseudo-label vectors generated by using the classifiers trained from other views. Unlike traditional co-training approaches using a single pseudo-label vector for training each classifier, our co-labeling approach explores different strategies to utilize the predictions from different views, biases and iterations for generating the pseudo-label vectors, making our approach more robust for real-world applications. Moreover, to further improve the weakly labeled learning on each view, we also exploit the inherent group structure in the pseudo-label vectors generated from different strategies, which leads to a new multi-layer multiple kernel learning problem. Promising results for text-based image retrieval on the NUS-WIDE dataset as well as news classification and text categorization on several real-world multi

Scintigraphy with In-111 labeled leukocytes

International Nuclear Information System (INIS)

Itoh, Kazuo; Tsukamoto, Eriko; Furudate, Masayori; Saito, Chihoko.

1987-01-01

With increasing necessity for In-111 labeled leukocyte scintigraphy (ILLS) as a routine examination, a problem of complicated labeling of leukocytes has arisen. In this study, simplified labeling of leukocytes was examined with respect to its ability to detect abscesses. Simplified labeling method yielded significantly satisfactory results for recovery and labeling rates of leukocytes, as compared with conventional recommended method. Therefore, ILLS by simplified technique was clinically applied in 58 patients with suppurative or non-suppurative diseases who gave informed consent. In an analysis of ILLS for detecting suppurative region, the sensitivity, specificity, and corrected specificity were found to be 81 %, 75 %, and 82 %, respectively. (Namekawa, K.)

Photoaffinity Labeling of Developing Jojoba Seed Microsomal Membranes with a Photoreactive Analog of Acyl-Coenzyme A (Acyl-CoA) (Identification of a Putative Acyl-CoA:Fatty Alcohol Acyltransferase.

Science.gov (United States)

Shockey, J. M.; Rajasekharan, R.; Kemp, J. D.

1995-01-01

Jojoba (Simmondsia chinensis, Link) is the only plant known that synthesizes liquid wax. The final step in liquid wax biosynthesis is catalyzed by an integral membrane enzyme, fatty acyl-coenzyme A (CoA):fatty alcohol acyltransferase, which transfers an acyl chain from acyl-CoA to a fatty alcohol to form the wax ester. To purify the acyltransferase, we have labeled the enzyme with a radioiodinated, photoreactive analog of acyl-CoA, 12-[N-(4-azidosalicyl)amino] dodecanoyl-CoA (ASD-CoA). This molecule acts as an inhibitor of acyltransferase activity in the dark and as an irreversible inhibitor upon exposure to ultraviolet light. Oleoyl-CoA protects enzymatic activity in a concentration-dependent manner. Photolysis of microsomal membranes with labeled ASD-CoA resulted in strong labeling of two polypeptides of 57 and 52 kD. Increasing concentrations of oleoyl-CoA reduced the labeling of the 57-kD polypeptide dramatically, whereas the labeling of the 52-kD polypeptide was much less responsive to oleoyl-CoA. Also, unlike the other polypeptide, the labeling of the 57-kD polypeptide was enhanced considerably when photolyzed in the presence of dodecanol. These results suggest that a 57-kD polypeptide from jojoba microsomes may be the acyl-CoA:fatty alcohol acyltransferase.

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

International Nuclear Information System (INIS)

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 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 9 ]-alpha-MSH (II) and [p-azidophenylalanine 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 2 , norvaline 4 ]-alpha-MSH (IV) and [N alpha-(4-azidophenylacetyl)-serine1, leucine 9 ]-alpha-MSH (V) gave much less insertion and [leucine 9 , p-azidophenylalanine 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 13 ]-alpha-MSH was reduced by the weak antagonist H-Phe-Ala-Trp-Gly-Gly-Pro-Val-NH 2 . These experiments prove that hormone receptors can be covalently labelled in tissue with very limited light transparency

Site-specific conjugation and labelling of prostate antibody 7E11C5.3 (CYT-351) with technetium-99m

International Nuclear Information System (INIS)

Stalteri, M.A.; Mather, S.J.; Belinka, B.A.; Coughlin, D.J.; Chengazi, V.U.; Britton, K.E.

1997-01-01

Attachment of chelating agents to the sugar residues of antibodies for subsequent radiolabelling is an attractive approach since it may have less effect on the immunoreactivity than attachment through lysine residues, which are distributed throughout the antibody and may be present near the antigen binding site. We have attached a new hydrazide-linked chelator CYT-395 (Cytogen Corp., Princeton, N.J.) to the sugar residues of the anti-prostate monoclonal antibody 7E11C5.3 and optimised the conditions for labelling the conjugate with technetium-99m in order to compare the conjugate to 7E11C5.3 antibody labelled directly with technetium using a mercaptoethanol reduction technique. Labelling yields of 70%-90% were obtained at specific activities up to 2000 MBq/mg antibody. The stability of the technetium-labelled conjugate in plasma or to a challenge with 0.1 or 1.0 mM cysteine was similar to that of direct-labelled antibody. In nine patients with prostate cancer, the plasma clearance of the labelled conjugate followed a two-compartment model, with an average β-phase half-life of 31.4±3.9 h. The average urinary clearance at 24 h was 15.3±5.0% of the injected dose. In this group of patients there was no significant difference between the blood and urine clearance of the labelled conjugate, and the clearances of the direct-labelled antibody. (orig.). With 5 figs

Human Vitamin B12 Absorption and Metabolism are Measured by Accelerator Mass Spectrometry Using Specifically Labeled 14C-Cobalamin

International Nuclear Information System (INIS)

Carkeet, C; Dueker, S R; Lango, J; Buchholz, B A; Miller, J W; Green, R; Hammock, B D; Roth, J R; Anderson, P J

2006-01-01

There is need for an improved test of human ability to assimilate dietary vitamin B 12 . Assaying and understanding absorption and uptake of B 12 is important because defects can lead to hematological and neurological complications. Accelerator mass spectrometry (AMS) is uniquely suited for assessing absorption and kinetics of 14 C-labeled substances after oral ingestion because it is more sensitive than decay counting and can measure levels of carbon-14 ( 14 C) in microliter volumes of biological samples, with negligible exposure of subjects to radioactivity. The test we describe employs amounts of B 12 in the range of normal dietary intake. The B 12 used was quantitatively labeled with 14 C at one particular atom of the DMB moiety by exploiting idiosyncrasies of Salmonellametabolism. In order to grow aerobically on ethanolamine, S. entericamust be provided with either pre-formed B 12 or two of its precursors: cobinamide and dimethylbenzimidazole (DMB). When provided with 14 C-DMB specifically labeled in the C2 position, cells produced 14 C-B 12 of high specific activity (2.1 GBq/mmol, 58 mCi/mmol) and no detectable dilution of label from endogenous DMB synthesis. In a human kinetic study, a physiological dose (1.5 mg, 2.2 KBq/59 nCi) of purified 14 C-B 12 was administered and showed plasma appearance and clearance curves consistent with the predicted behavior of the pure vitamin. This method opens new avenues for study of B 12 assimilation

Stable isotopes labelled compounds

International Nuclear Information System (INIS)

1982-09-01

The catalogue on stable isotopes labelled compounds offers deuterium, nitrogen-15, and multiply labelled compounds. It includes: (1) conditions of sale and delivery, (2) the application of stable isotopes, (3) technical information, (4) product specifications, and (5) the complete delivery programme

Thrombus imaging with indium-111 and iodine-131-labeled fibrin-specific monoclonal antibody and its F(ab')2 and Fab fragments

International Nuclear Information System (INIS)

Rosebrough, S.F.; Grossman, Z.D.; McAfee, J.G.

1988-01-01

We have previously reported successful imaging of fresh (2-4 hr old) and aged (1-5 days old) canine thrombi with 131 I-labeled intact monoclonal antibody (MAb) specific for fibrin. We now report thrombus imaging with 131 I-labeled F(ab')2 and Fab and 111 In-labeled intact MAb, F(ab')2, and Fab. Indium-111-labeled F(ab')2 proved to be the best imaging agent due to less nonspecific binding in the liver than whole IgG. Image quality was improved by the higher administered dose permissible with 111 In and its better physical characteristics for imaging, compared to 131 I. Immunofluorescence of fresh human histologic sections showed intact MAb and F(ab')2 binding to thrombi, pulmonary emboli, and atherosclerotic plaques, strengthening the feasibility of clinical thrombus imaging

Label-free aptamer-based sensor for specific detection of malathion residues by surface-enhanced Raman scattering

Science.gov (United States)

Nie, Yonghui; Teng, Yuanjie; Li, Pan; Liu, Wenhan; Shi, Qianwei; Zhang, Yuchao

2018-02-01

A novel label-free aptamer surface-enhanced Raman scattering (SERS) sensor for trace malathion residue detection was proposed. In this process, the binding of malathion molecule with aptamer is identified directly. The silver nanoparticles modified with positively charged spermine served as enhancing and capture reagents for the negatively charged aptamer. Then, the silver nanoparticles modified by aptamer were used to specifically capture the malathion. The SERS background spectra of spermine, aptamer, and malathion were recorded and distinguished with the spectrum of malathion-aptamer. To enhance the characteristic peak signal of malathion captured by the aptamer, the aggregate reagents (NaCl, KCl, MgCl2) were compared and selected. The selectivity of this method was verified in the mixed-pesticide standard solution, which included malathion, phosmet, chlorpyrifos-methyl, and fethion. Results show that malathion can be specifically identified when the mixed-pesticide interferences existed. The standard curve was established, presenting a good linear range of 5 × 10- 7 to 1 × 10- 5 mol·L- 1. The spiked experiments for tap water show good recoveries from 87.4% to 110.5% with a relative standard deviation of less than 4.22%. Therefore, the proposed label-free aptamer SERS sensor is convenient, specifically detects trace malathion residues, and can be applied for qualitative and quantitative analysis of other pesticides.

3D label-free prostate specific antigen (PSA) immunosensor based on graphene-gold composites.

Science.gov (United States)

Jang, Hee Dong; Kim, Sun Kyung; Chang, Hankwon; Choi, Jeong-Woo

2015-01-15

Highly sensitive and label-free detection of the prostate specific antigen (PSA) remains a challenge in the diagnosis of prostate cancer. Here, a novel three-dimensional (3D) electrochemical immunosensor capable of sensitive and label-free detection of PSA is reported. This unique immunosensor is equipped with a highly conductive graphene (GR)-based gold (Au) composite modified electrode. The GR-based Au composite is prepared using aerosol spray pyrolysis and the morphology of the composite is the shape of a crumpled GR ball decorated with Au nanoparticles. Unlike the previous research, this novel 3D immunosensor functions very well over a broad linear range of 0-10 ng/mL with a low detection limit of 0.59 ng/mL; furthermore, it exhibits a significantly increased electron transfer and high sensitivity toward PSA. The highest rate of current change with respect to the PSA concentration is 5 μA/(ng/mL). Satisfactory selectivity, reproducibility, and stability of the 3D immunosensor are also exhibited. Copyright © 2014 Elsevier B.V. All rights reserved.

Specific uptake, dissociation, and degradation of 125I-labeled insulin in isolated turtle (Chrysemys dorbigni) thyroid glands

International Nuclear Information System (INIS)

Marques, M.; da Silva, R.S.; Turyn, D.; Dellacha, J.M.

1985-01-01

Thyroid glands from turtles (Chrysemys dorbigni) pretreated with potassium iodide were incubated with 125 I-insulin in the presence or absence of unlabeled insulin, in order to study its specific uptake. At 24 degrees, the specific uptake reached a plateau at 180 min of incubation. The dose of bovine insulin that inhibited 50% of the 125 I-insulin uptake was 2 micrograms/ml of incubation medium. Most of the radioactive material (71%) extracted from the gland, after 30 min incubation with 125 I-insulin, eluted in the same position as labeled insulin on Sephadex G-50. Only 24% eluted in the salt position. After 240 min incubation, increased amount of radioactivity appeared in the Na 125 I position. When bovine insulin was added together with the labeled hormone, a substantial reduction of radioactivity was observed in the insulin and Na 125 I elution positions. Dissociation studies were performed at 6 degrees in glands preincubated with 125 I-insulin either at 24 or 6 degrees. The percentage of trichloroacetic acid (TCA)-soluble radioactive material in the dissociation medium increased with incubation time at both temperatures. However, the degradation activity was lower at 6 than at 24 degrees. The addition of bovine insulin to the incubation buffer containing 125 I-insulin reduced the radioactive degradation products in the dissociated medium. Chloroquine or bacitracin inhibited the degradation activity. Incubation of thyroid glands with 125 I-hGH or 125 I-BSA showed values of uptake, dissociation, and degradation similar to those experiments in which an excess of bovine insulin was added together with the labeled hormone. Thus, by multiple criteria, such as specific uptake, dissociation, and degradation, the presence of insulin-binding sites in the turtle thyroid gland may be suggested

Design and synthesis of tag-free photoprobes for the identification of the molecular target for CCG-1423, a novel inhibitor of the Rho/MKL1/SRF signaling pathway

Directory of Open Access Journals (Sweden)

Jessica L. Bell

2013-05-01

Full Text Available CCG-1423 and related analogues represent a new class of inhibitors of Rho/MKL1/SRF-mediated gene transcription, a pathway that has been implicated in both cancer and fibrosis. The molecular target for these compounds is unknown. To facilitate its identification, a series of tag-free photoaffinity probes was designed and synthesized, each one containing a photoactivatable group and an acetylenic end group for subsequent attachment to a fluorescent tag using click chemistry. All were confirmed to maintain biological activity in a cell-based assay for inhibition of SRE-Luc expression. The functional activity of the most potent probe 24 was further confirmed in an assay for PC-3 cell migration. Photolysis of 24 in intact PC-3 cells followed by cell lysis, click ligation of a fluorescent dye, and gel electrophoresis revealed specific labeling of a single 24 kDa band that could be blocked with an active competitor. Future work will focus on identifying the labeled protein(s.

CFL Labeling Harmonization in the United States, China, Brazil andELI Member Countries: Specifications, Testing, and MutualRecognition

Energy Technology Data Exchange (ETDEWEB)

Fridley, David; Lin, Jiang; Denver, Andrea; Biermayer, Peter; Dillavou, Tyler

2005-07-20

This report examines critical differences among energy-efficient labeling programs for CFLs in Brazil, China, the United States, and the seven members of the international Efficient Lighting Initiative (ELI) in terms of technical specifications and test procedures, and review issues related to international harmonization of these standards.

Syntheses of sup 18 F-labeled reduced haloperidol and sup 11 C-labeled reduced 3-N-methylspiperone

Energy Technology Data Exchange (ETDEWEB)

Ravert, H T; Dannals, R F; Wilson, A A; Wong, D F; Wagner, Jr, H N [Johns Hopkins Medical Institutions, Baltimore, MD (USA)

1991-03-01

{sup 18}F-Labeled reduced haloperidol and {sup 11}C-labeled reduced 3-N-methylspiperone were synthesized in a convenient and quantitative one step reduction from {sup 18}F-labeled haloperidol and {sup 11}C-labeled N-methylspiperone, respectively. Both products were purified by semipreparative HPLC and were obtained at high specific activity and radiochemical purity. (author).

Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet α2-adrenergic receptor

International Nuclear Information System (INIS)

Matsui, Hiroaki; Lefkowitz, R.J.; Caron, M.G.; Regan, J.W.

1989-01-01

The human platelet α 2 -adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, the authors have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [ 3 H]SKF 102229 (an antagonist) or p-azido[ 3 H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [ 3 H]SKF 102229 labeled receptor yielded one peptide of M r 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of M r 4000, which was further digested to the M r 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[ 3 H]clonidine-labeled receptor, a similar M r 2400 peptide was obtained by lysylendopeptidase cleavage. This M r 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet α 2 -adrenergic receptor

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

Energy Technology Data Exchange (ETDEWEB)

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); Schmitt, Eberhard, E-mail: eschmitt@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany); University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen (Germany); Hausmann, Michael, E-mail: hausmann@kip.uni-heidelberg.de [University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany)

2016-07-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei

International Nuclear Information System (INIS)

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

2016-01-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes). Gene targets can be specifically labelled with at least about 20 PNA-probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3D-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.

In vivo tumor angiogenesis imaging with site-specific labeled 99mTc-HYNIC-VEGF

International Nuclear Information System (INIS)

Blankenberg, Francis G.; Backer, Marina V.; Patel, Vimalkumar; Backer, Joseph M.; Levashova, Zoia

2006-01-01

We recently developed a cysteine-containing peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)-maleimide. We then constructed and expressed C-tagged vascular endothelial growth factor (VEGF) and labeled it with HYNIC. We wished to test 99m Tc-HYNIC-C-tagged VEGF ( 99m Tc-HYNIC-VEGF) for the imaging of tumor vasculature before and after antiangiogenic (low continuous dosing, metronomic) and tumoricidal (high-dose) cyclophosphamide treatment. HYNIC-maleimide was reacted with the two thiol groups of C-tagged VEGF without any effect on biologic activity in vitro. 99m Tc-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein (200-300 μCi, 1-2 μg protein) followed by microSPECT imaging 1 h later. Sequencing analysis of HYNIC-containing peptides obtained after digestion confirmed the site-specific labeling of the two accessible thiol groups of C-tagged VEGF. Tumor vascularity was easily visualized with 99m Tc/VEGF in Balb/c mice with 4T1 murine mammary carcinoma 10 days after implantation into the left axillary fat pad in controls (12.3±5.0 tumor/bkg, n=27) along with its decrease following treatment with high (150 mg/kg q.o.d. x 4; 1.14±0.48 tumor/bkg, n=9) or low (25 mg/kg q.d. x 7; 1.03±0.18 tumor/bkg, n=9) dose cyclophosphamide. Binding specificity was confirmed by observing a 75% decrease in tumor uptake of 99m Tc/biotin-inactivated VEGF, as compared with 99m Tc-HYNIC-VEGF. 99m Tc can be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. 99m Tc-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy. (orig.)

The Role of ABC Proteins in Drug Resistant Breast Cancer Cells

Science.gov (United States)

2008-04-01

called the Plasmodium falciparum Chloroquine Transporter (PfCRT). While PfCRT is known to be the main molecular determinant of chloroquine resistance...proteins (such as human P-glycoprotein) and labeled PfCRT with a photoaffinity drug analogue . A manuscript is currently in preparation detailing my results...directly responsible for drug response, the Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) (Fidock et al 2000). While not a member of

A terminal-labelling microcytotoxity assay with 125I-iododeoxyuridine as a label for target cells

International Nuclear Information System (INIS)

Stirrat, G.M.

1976-01-01

The development of a terminal-labelling microcytotoxicity assay is described in which target cells (fetal fibroblasts) were labelled with 125 I-iododeoxyuridine after effector (lymphoid) cells had been incubated with them for 24 h. The time-course for the development of cell-mediated cytotoxicity was assessed following allogeneic skin grafting. 'Non-specific' cytotoxicity detracts from the sensitivity of all microcytotoxicity assays and the terminal-labelling assay using 125 I is no exception. The non-specific effects can be reduced but not eliminated by the removal of adherent cells. The optimum target cell/effector cell ratio would seem to be between 1:100 and 1:250. Residual lymph node cells did not appear to incorporate enough label to affect the test results. In vivo correlates of in vitro findings are still not easy to determine

Preclinical evaluation of melanocortin-1 receptor (MC1-R) specific 68Ga- and 44Sc-labeled DOTA-NAPamide in melanoma imaging.

Science.gov (United States)

Nagy, Gábor; Dénes, Noémi; Kis, Adrienn; Szabó, Judit P; Berényi, Ervin; Garai, Ildikó; Bai, Péter; Hajdu, István; Szikra, Dezső; Trencsényi, György

2017-08-30

Alpha melanocyte stimulating hormone (α-MSH) enhances melanogenesis in melanoma malignum by binding to melanocortin-1 receptors (MC1-R). Earlier studies demonstrated that alpha-MSH analog NAPamide molecule specifically binds to MC1-R receptor. Radiolabeled NAPamide is a promising radiotracer for the non-invasive detection of melanin producing melanoma tumors by Positron Emission Tomography (PET). In this present study the MC1-R selectivity of the newly developed Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using melanoma tumors. DOTA-NAPamide was labeled with Ga-68 and Sc-44 radionuclides. The MC1-R specificity of Ga-68- and Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using MC1-R positive (B16-F10) and negative (A375) melanoma cell lines. For in vivo imaging studies B16-F10 and A375 tumor-bearing mice were injected with 44 Sc/ 68 Ga-DOTA-NAPamide (in blocking studies with α-MSH) and whole body PET/MRI scans were acquired. Radiotracer uptake was expressed in terms of standardized uptake values (SUVs). 44 Sc/ 68 Ga-labeled DOTA-NAPamide were produced with high specific activity (approx. 19 GBq/μmol) and with excellent radiochemical purity (99%DOTA-NAPamide (SUVmean: 0.38±0.02), and Sc-44-DOTA-NAPamide (SUVmean: 0.52±0.13) uptake was observed in subcutaneously growing B16-F10 tumors, than in receptor negative A375 tumors, where the SUVmean values of Ga-68-DOTA-NAPamide and Sc-44-DOTA-NAPamide were 0.04±0.01 and 0.07±0.01, respectively. Tumor-to-muscle (T/M SUVmean) ratios were approximately 15-fold higher in B16-F10 tumor-bearing mice, than that of A375 tumors, and this difference was also significant (p≤0.01) using both radiotracers after 60 min incubation time. Our newly synthesized 44 Sc-labeled DOTA-NAPamide probe showed excellent binding properties to melanocortin-1 receptor (MC1-R) positive melanoma cell and tumors. Due to its high specificity and sensitivity 44 Sc-DOTA-NAPamide is a promising radiotracer in

Stability of rhenium-188 labeled antibody

International Nuclear Information System (INIS)

Lim, B. K.; Jung, J. M.; Jung, J. K.; Lee, D. S.; Lee, M. C.

1999-01-01

For clinical application of beta-emitter labeled antibody, high specific activity is important. Carrier-free Re-188 from W-188/Re-188 generator is an ideal radionuclide for this purpose. However, low stability of Re-188 labeled antibody, especially in high specific activity, due to radiolytic decomposition by high energy (2.1 MeV) beta ray was problem. We studied the stability of Re-188 labeled antibody, and stabilizing effect of several nontoxic radical-quenching agents. Pre-reduced monoclonal antibody (CEA79.4) was labeled with Re-188 by incubating with generator-eluted Re-188-perrhenate in the presence of stannous tartrate for 2 hr at room temperature. Radiochemical purity of each preparation was determined by chromatography (ITLC-SG/acetone, ITLC-SG/Umezawa, Whatman No.1/saline). Human serum albumin was added to the labeled antibodies(2%). Stability of Re-188-CEA79.4 was investigated in the presence of vitamin C, ethanol, or Tween 80 as radical-quenching agents. Specific activities of 4.29∼5.11 MBq/μg were obtained. Labeling efficiencies were 88±4%(n=12). Very low stability after removal of stannous tartrate from the preparation was observed. If stored after purging with N 2 , all the preparations were stable for 10 hr. However, if contacted with air, stability decreased. Perrhenate and Re-188-tartrate was major impurity in declined preparation (12∼47 and 9∼38% each, after 10 hr). Colloid-formation was not a significant problem in all cases. Addition of vitamin C stabilized the labeled antibodies either under N 2 or under air by reducing the formation of perrhenate. High specific activity Re-188 labeled antibody is unstable, especially, in the presence of oxygen. Addition of vitamin C increased the stability

Radioactive labelling of peptidic hormones

International Nuclear Information System (INIS)

Fromageot, P.; Pradelles, P.; Morgat, J.L.; Levine, H.

1976-01-01

The labelling of peptidic hormones requires stability, specificity and sensitivity of the label. Introduction of a radioactive atome is one way to satisfy these criteria. Several processes have been described to prepare radioactive TRF: synthesis of the peptide with labelled aminoacids or introduction of the label into the hormone. In that approach, tritium can be substituted in the imidazole ring, via precursors activating the proper carbon. Monoiodo TRF leads essentially to tritium labelling of the 5 positions whereas monoazo TRF allows the preparation of 3 H TRF labelled in the 2 positions. Di-substituted TRF leads to labelling into the 2 and 5 carbons. Labelled analogs of TRF can be prepared with labelled iodine; further developments of peptide labelling, will be presented. In particular, the homolytic scission of the C-iodine, bond by photochemical activation. The nascent carbon radical can be stabilized by a tritiated scavenger. This approach eliminates the use of heavy metal catalysts

From Position-Specific Labeling to Environmental Fluxomics: Elucidating Biogeochemical Cycles from the Metabolic Perspective (BG Division Outstanding ECS Award Lecture)

Science.gov (United States)

Dippold, Michaela; Apostel, Carolin; Dijkstra, Paul; Kuzyakov, Yakov

2017-04-01

Understanding soil and sedimentary organic matter (SOM) dynamics is one of the most important challenges in biogeoscience. To disentangle the fluxes and transformations of C in soils a detailed knowledge on the biochemical pathways and its controlling factors is required. Biogeochemists' view on the C transformation of microorganisms in soil has rarely exceed a strongly simplified concept assuming that C gets either oxidized to CO2 via the microbial catabolism or incorporated into biomass via the microbial anabolism. Biochemists, however, thoroughly identified in the past decades the individual reactions of glycolysis, pentose-phosphate pathway and citric acid cycle underlying the microbial catabolism. At various points within that metabolic network the anabolic fluxes feeding biomass formation branch off. Recent studies on metabolic flux tracing by position-specific isotope labeling allowed tracing these C transformations in soils in situ, an approach which is qunatitatively complemented by metabolic flux modeling. This approach has reached new impact by the cutting-edge combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites which allows 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. Thus, the combination of position-specific labeling, compound-specific isotope incorporation in biomarkers and quantitative metabolic flux modelling provide the toolbox for quantitative soil fluxomics. Our studies combining position-specific labeled glucose with amino sugar 13C analysis showed that up to 55% of glucose, incorporated into the glucose derivative glucosamine, first passed glycolysis before allocated back via gluconeogenesis. Similarly, glutamate-derived C is allocated via anaplerotic pathways towards fatty acid synthesis and in parallel to its oxidation in citric acid cycle. Thus

Multi-label Learning with Missing Labels Using Mixed Dependency Graphs

KAUST Repository

Wu, Baoyuan

2018-04-06

This work focuses on the problem of multi-label learning with missing labels (MLML), which aims to label each test instance with multiple class labels given training instances that have an incomplete/partial set of these labels (i.e., some of their labels are missing). The key point to handle missing labels is propagating the label information from the provided labels to missing labels, through a dependency graph that each label of each instance is treated as a node. We build this graph by utilizing different types of label dependencies. Specifically, the instance-level similarity is served as undirected edges to connect the label nodes across different instances and the semantic label hierarchy is used as directed edges to connect different classes. This base graph is referred to as the mixed dependency graph, as it includes both undirected and directed edges. Furthermore, we present another two types of label dependencies to connect the label nodes across different classes. One is the class co-occurrence, which is also encoded as undirected edges. Combining with the above base graph, we obtain a new mixed graph, called mixed graph with co-occurrence (MG-CO). The other is the sparse and low rank decomposition of the whole label matrix, to embed high-order dependencies over all labels. Combining with the base graph, the new mixed graph is called as MG-SL (mixed graph with sparse and low rank decomposition). Based on MG-CO and MG-SL, we further propose two convex transductive formulations of the MLML problem, denoted as MLMG-CO and MLMG-SL respectively. In both formulations, the instance-level similarity is embedded through a quadratic smoothness term, while the semantic label hierarchy is used as a linear constraint. In MLMG-CO, the class co-occurrence is also formulated as a quadratic smoothness term, while the sparse and low rank decomposition is incorporated into MLMG-SL, through two additional matrices (one is assumed as sparse, and the other is assumed as low

40 CFR 600.306-86 - Labeling requirements.

Science.gov (United States)

2010-07-01

... general to specific labels or vice versa within a model type, the manufacturer shall, within five calendar... legibility of the fuel economy label is maintained. For this purpose, all fuel economy label information must... cause to be maintained on each automobile: (1) A general fuel economy label (initial, or updated as...

Biochemical study of multiple drug recognition sites on central benzodiazepine receptors

Energy Technology Data Exchange (ETDEWEB)

Trifiletti, R.R.

1986-01-01

The benzodiazepine receptor complex of mammalian brain possesses recognition sites which mediate (at least in part) the pharmacologic actions of the 1,4-benzodiazepines and barbiturates. Evidence is provided suggesting the existence of least seven distinct drug recognition sites on this complex. Interactions between the various recognition sites have been explored using radioligand binding techniques. This information is utilized to provide a comprehensive scheme for characterizing receptor-active drugs on an anxiolytic-anticonvulsant/proconvulsant continuum using radioligand binding techniques, as well as a comprehensive program for identifying potential endogenous receptor-active substances. Further evidence is provided here supporting the notion of benzodiazepine recognition site heterogeneity. Classical 1,4-benzodiazepines do not appear to differentiate two populations of benzodiazepine receptors in an equilibrium sense, but appear to do so in a kinetic sense. An apparent physical separation of the two receptor subtypes can be achieved by differential solubilization. The benzodiazepine binding subunit can be identified by photoaffinity labeling with the benzodiazepine agonist (/sup 3/H)flunitrazepan. Conditions for reproducible partial proteolytic mapping of (/sup 3/H)flunitrazepam photoaffinity labeled receptors are established. From these maps, it is concluded that there are probably no major differences in the primary sequence of the benzodiazepine binding subunit in various regions of the rat central nervous system.

LabelFlow Framework for Annotating Workflow Provenance

Directory of Open Access Journals (Sweden)

Pinar Alper

2018-02-01

Full Text Available Scientists routinely analyse and share data for others to use. Successful data (reuse relies on having metadata describing the context of analysis of data. In many disciplines the creation of contextual metadata is referred to as reporting. One method of implementing analyses is with workflows. A stand-out feature of workflows is their ability to record provenance from executions. Provenance is useful when analyses are executed with changing parameters (changing contexts and results need to be traced to respective parameters. In this paper we investigate whether provenance can be exploited to support reporting. Specifically; we outline a case-study based on a real-world workflow and set of reporting queries. We observe that provenance, as collected from workflow executions, is of limited use for reporting, as it supports queries partially. We identify that this is due to the generic nature of provenance, its lack of domain-specific contextual metadata. We observe that the required information is available in implicit form, embedded in data. We describe LabelFlow, a framework comprised of four Labelling Operators for decorating provenance with domain-specific Labels. LabelFlow can be instantiated for a domain by plugging it with domain-specific metadata extractors. We provide a tool that takes as input a workflow, and produces as output a Labelling Pipeline for that workflow, comprised of Labelling Operators. We revisit the case-study and show how Labels provide a more complete implementation of reporting queries.

Labelling of HBV-DNA probe using reagent made in China

International Nuclear Information System (INIS)

Wang Quanshi

1991-01-01

The labelling hepatitis Bvirus DNA (HBV-DNA) probe was studied by using reagent made in China. The results showed that: (1) The dNTPs with high specific activity was necessary for the labelling of nigh specific activity HBV-DNA probe; (2) reaction of labelling HBV-DNA probe was completed in a few minutes; (3) 0.37 MBq 3 H dTTP (specific activity 1.554TBq/mmol) was enough to label 1 μg HBV-DNA and the specific activity of probe reached 3.4 x 10 cpm/μg; (4) 7 MBqα- 32 P dATP (specific activity > 111 TBq/mmol) can label HBV-DNA probe to specific activity 1.35 x 10 cpm/μg. It was concluded that the reagent made in China can be used for the study in molecular biology

Optimization of the synthesis of a high specific activity 125 I-labelled hapten for radioimmunoassays

International Nuclear Information System (INIS)

Suarez, C.; Simon, M.A.; Paz, D.; Romero del Hombrebueno, B.

1994-01-01

In this first report it is described the synthesis, separation and purification of the 2-radioiodinated histamine ''125 I-labelled histamine by a mixed anhydride reaction. About 75% incorporation of I''1125, from Na''125, I, was achieved with a molecular ratio of 1:1 mixed anhydride:histamine. The radiochemical purity of the conjugate by TLC was >99% and its theoretical specific activity, 3850 mu Ci/mug. Dissolved in ethanol and held at -20 degree centigree under darkness decomposition on storage did not exceed 1% per month

A nanoparticle label/immunochromatographic electrochemical biosensor for rapid and sensitive detection of prostate-specific antigen

Energy Technology Data Exchange (ETDEWEB)

Lin, Ying-Ying; Wang, Jun; Liu, Guodong; Wu, Hong; Wai, Chien M.; Lin, Yuehe

2008-06-15

We present a nanoparticle (NP) label/immunochromatographic electrochemical biosensor (IEB) for rapid and sensitive detection of prostate-specific antigen (PSA) in human serum. This IEB integrates the immunochromatographic strip with the electrochemical detector for transducing quantitative signals. The NP label, made of CdSe@ZnS, serves as a signal-amplifier vehicle. A sandwich immunoreaction was performed on the immunochromatographic strip. The captured NP labels in the test zone were determined by highly sensitive stripping voltammetric measurement of the dissolved metallic component (cadmium) with a disposable-screen-printed electrode, which is embedded underneath the membrane of the test zone. Experimental parameters (e.g., immunoreaction time, the amount of anti-PSA-NP conjugations applied) and electrochemical detection conditions (e.g., preconcentration potential and time) were optimized using this biosensor for PSA detection. The analytical performance of this biosensor was evaluated with serum PSA samples according to the “figure-of-merits” (e.g., dynamic range, reproducibility, and detection limit). The results were validated with enzyme-linked immunosorbent assay (ELISA) and show high consistency. It is found that this biosensor is very sensitive with the detection limit of 0.02 ng/mL PSA and is quite reproducible. This method is rapid, clinically accurate, and less expensive than other diagnosis tools for PSA; therefore, this IEB coupled with a portable electrochemical analyzer shows great promise for simple, sensitive, quantitative point-of-care testing of disease-related protein biomarkers.

In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore

Directory of Open Access Journals (Sweden)

Haibiao Gong

2010-02-01

Full Text Available Overexpression of epidermal growth factor receptor (EGFR is associated with many types of cancers. It is of great interest to noninvasively image the EGFR expression in vivo. In this study, we labeled an EGFR-specific Affibody molecule (Eaff with a near-infrared (NIR dye IRDye800CW maleimide and tested the binding of this labeled molecule (Eaff800 in cell culture and xenograft mouse tumor models. Unlike EGF, Eaff did not activate the EGFR signaling pathway. Results showed that Eaff800 was bound and taken up specifically by EGFR-overexpressing A431 cells. When Eaff800 was intravenously injected into nude mice bearing A431 xenograft tumors, the tumor could be identified 1 hour after injection and it became most prominent after 1 day. Images of dissected tissue sections demonstrated that the accumulation of Eaff800 was highest in the liver, followed by the tumor and kidney. Moreover, in combination with a human EGFR type 2 (HER2-specific probe Haff682, Eaff800 could be used to distinguish between EGFR- and HER2-overexpressing tumors. Interestingly, the organ distribution pattern and the clearance rate of Eaff800 were different from those of Haff682. In conclusion, Eaff molecule labeled with a NIR fluorophore is a promising molecular imaging agent for EGFR-overexpressing tumors.

Applications of site-specific labeling to study HAMLET, a tumoricidal complex of α-lactalbumin and oleic acid.

Science.gov (United States)

Mercer, Natalia; Ramakrishnan, Boopathy; Boeggeman, Elizabeth; Qasba, Pradman K

2011-01-01

Alpha-lactalbumin (α-LA) is a calcium-bound mammary gland-specific protein that is found in milk. This protein is a modulator of β1,4-galactosyltransferase enzyme, changing its acceptor specificity from N-acetyl-glucosamine to glucose, to produce lactose, milk's main carbohydrate. When calcium is removed from α-LA, it adopts a molten globule form, and this form, interestingly, when complexed with oleic acid (OA) acquires tumoricidal activity. Such a complex made from human α-LA (hLA) is known as HAMLET (Human A-lactalbumin Made Lethal to Tumor cells), and its tumoricidal activity has been well established. In the present work, we have used site-specific labeling, a technique previously developed in our laboratory, to label HAMLET with biotin, or a fluoroprobe for confocal microscopy studies. In addition to full length hLA, the α-domain of hLA (αD-hLA) alone is also included in the present study. We have engineered these proteins with a 17-amino acid C-terminal extension (hLA-ext and αD-hLA-ext). A single Thr residue in this extension is glycosylated with 2-acetonyl-galactose (C2-keto-galactose) using polypeptide-α-N-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules. We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity.

Application of amino acid type-specific 1H- and 14N-labeling in a 2H-, 15N-labeled background to a 47 kDa homodimer: Potential for NMR structure determination of large proteins

International Nuclear Information System (INIS)

Kelly, Mark J.S.; Krieger, Cornelia; Ball, Linda J.; Yu Yihua; Richter, Gerald; Schmieder, Peter; Bacher, Adelbert; Oschkinat, Hartmut

1999-01-01

NMR investigations of larger macromolecules (>20 kDa) are severely hindered by rapid 1H and 13C transverse relaxation. Replacement of non-exchangeable protons with deuterium removes many efficient 1H-1H and 1H-13C relaxation pathways. The main disadvantage of deuteration is that many of the protons which would normally be the source of NOE-based distance restraints are removed. We report the development of a novel labeling strategy which is based on specific protonation and 14N-labeling of the residues phenylalanine, tyrosine, threonine, isoleucine and valine in a fully deuterated, 15N-labeled background. This allows the application of heteronuclear half-filters, 15N-editing and 1H-TOCSY experiments to select for particular magnetization transfer pathways. Results from investigations of a 47 kDa dimeric protein labeled in this way demonstrated that the method provides useful information for the structure determination of large proteins

Clinical applications of cells labelling; Aplicaciones clinicas del marcado de celulas

Energy Technology Data Exchange (ETDEWEB)

Gonzalez, B M [Instituto Nacional de Pediatria (Mexico)

1994-12-31

Blood cells labelled with radionuclides are reviewed and main applications are described. Red blood cell labelling by both random and specific principle. A table with most important clinical uses, 99mTc labelling of RBC are described pre tinning and in vivo reduction of Tc, in vitro labelling and administration of labelled RBC and in vivo modified technique. Labelled leucocytes with several 99mTc-complex radiopharmaceuticals by in vitro technique and specific monoclonal s for white cells(neutrofiles). Labelled platelets for clinical use and research by in vitro technique and in vivo labelling.

Optimization of the synthesis of a high specific activity 125I-labelled hapten for radioimmunoassays

International Nuclear Information System (INIS)

Suraez, C.; Paz, D.; Simon, M. A.; Romero del Hombrebueno, B.

1994-01-01

In this first report it is described the synthesis, separation and purification of the 2-radioiodinated histamine- I-labelled histamine by a mixed anhydride reaction. About 75% incorporation of I - 125, from Na 1 25I, was achieved with a molecular ratio of 1:1 mixed anhydride:histamine. The radiochemical purity of the conjugate by TLC was > 99% and its theoretical specific activity, 3850 μCi/μg. Dissolved in ethanol and held at -20 degree centigree under darkness decomposition on storage didn't exceed 1% per month. (Author) 13 refs

Specific localization and imaging of amyloid deposits in vivo using 123I-labeled serum amyloid P component

International Nuclear Information System (INIS)

Hawkins, P.N.; Myers, M.J.; Epenetos, A.A.; Caspi, D.; Pepys, M.B.

1988-01-01

Highly specific, high-resolution scintigraphic images of amyloid-laden organs in mice with experimentally induced amyloid A protein (AA) amyloidosis were obtained after intravenous injection of 123 I-labeled serum amyloid P component (SAP). Interestingly, a much higher proportion (up to 40%) of the injected dose of heterologous human SAP localized to amyloid and was retained there than was the case with isologous mouse SAP, indicating that human SAP binds more avidly to mouse AA fibrils than does mouse SAP. Specificity of SAP localization was established by the failure of the related proteins, human C-reactive protein and Limulus C-reactive protein, to deposit significantly in amyloid and by the absence of human SAP deposition in nonamyloidotic organs. However, only partial correlations were observed between the quantity of SAP localized and two independent estimates, histology and RIA for AA of the amount of amyloid in particular organs. It is not clear which of the three methods used reflects better the extent or clinical significance of the amyloid deposits but in vivo localization of radiolabeled SAP, detectable and quantifiable by gamma camera imaging, is apparently extremely sensitive. These findings establish the use of labeled SAP as a noninvasive in vivo diagnostic probe in experimental amyloidosis, potentially capable of revealing the natural history of the condition, and suggest that it may also be applicable generally as a specific targeting agent for diagnostic and even therapeutic purposes in clinical amyloidosis

Extending Modal Transition Systems with Structured Labels

DEFF Research Database (Denmark)

Bauer, Sebastian S.; Juhl, Line; Larsen, Kim Guldstrand

2012-01-01

We introduce a novel formalism of label-structured modal transition systems that combines the classical may/must modalities on transitions with structured labels that represent quantitative aspects of the model. On the one hand, the specification formalism is general enough to include models like...... weighted modal transition systems and allows the system developers to employ more complex label refinement than in the previously studied theories. On the other hand, the formalism maintains the desirable properties required by any specification theory supporting compositional reasoning. In particular, we...

Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

International Nuclear Information System (INIS)

Stein, J.C.

1985-01-01

Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several 37 P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the 32 P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex

Dual-mode fluorophore-doped nickel nitrilotriacetic acid-modified silica nanoparticles combine histidine-tagged protein purification with site-specific fluorophore labeling.

Science.gov (United States)

Kim, Sung Hoon; Jeyakumar, M; Katzenellenbogen, John A

2007-10-31

We present the first example of a fluorophore-doped nickel chelate surface-modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700-900 TMRs per ca. 23 nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni2+. Silica-embedded TMR retains very high quantum yield, is resistant to quenching by buffer components, and is modestly quenched and only to a certain depth (ca. 2 nm) by surface-attached Ni2+. When exposed to a bacterial lysate containing estrogen receptor alpha ligand binding domain (ERalpha) as a minor component, these beads showed very high specificity binding, enabling protein purification in one step. The capacity and specificity of these beads for binding a his-tagged protein were characterized by electrophoresis, radiometric counting, and MALDI-TOF MS. ERalpha, bound to TMR-SiO2-NTA-Ni++ beads in a site-specific manner, exhibited good activity for ligand binding and for ligand-induced binding to coactivators in solution FRET experiments and protein microarray fluorometric and FRET assays. This dual-mode type TMR-SiO2-NTA-Ni2+ system represents a powerful combination of one-step histidine-tagged protein purification and site-specific labeling with multiple fluorophore species.

The interaction of substituted benzamides with brain benzodiazepine binding sites in vitro.

Science.gov (United States)

Horton, R W; Lowther, S; Chivers, J; Jenner, P; Marsden, C D; Testa, B

1988-08-01

1. The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]-flunitrazepam ([3H]-FNM) from specific binding sites in bovine cortical membranes in vitro. 2. Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration-dependent displacement of [3H]-FNM with IC50 values of 73 nM, 132 nM, 7.7 microM and 5.9 microM, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiapride, sultopride and cisapride were inactive at 10(-5) M. 3. Inhibition by clebopride and Delagrange 2674 of [3H]-FNM binding was apparently competitive and readily reversible. 4. In the presence of gamma-aminobutyric acid (GABA), the ability of diazepam and Delagrange 2674 to displace [3H]-Ro 15-1788 binding was increased 3.6 and 1.6 fold respectively, compared to the absence of GABA, while ethyl beta-carboline-3-carboxylate (beta CCE) and clebopride were less potent in the presence of GABA. 5. Diazepam was 30 fold less potent at displacing [3H]-Ro 15-1788 in membranes that had been photoaffinity labelled with FNM than in control membranes, whereas the potency of beta CCE did not differ. Clebopride and Delagrange 2674 showed a less than two fold loss of potency in photoaffinity labelled membranes. 6. The pattern of binding of clebopride and Delagrange 2674 in these in vitro tests is similar to that found previously with partial agonists or antagonists at BDZ binding sites. 7. Clebopride and Delagrange 2674 inhibited [3H]-FNM binding with similar potency in rat cerebellar and hippocampal membranes, suggesting they have no selectivity for BDZ1 and BDZ2 binding sites. 8. Clebopride and Delagrange 2674 are structurally dissimilar to other BDZ ligands and represent another chemical structure to probe brain BDZ binding sites.

40 CFR 86.1606 - Labeling.

Science.gov (United States)

2010-07-01

... Emission Control Information Update; (2) Full corporate name and trademark of the vehicle manufactuer; (3... tuneup specifications (if changed from the original label specifications) at the applicable altitude. ...

The antibody approach of labeling blood cells

International Nuclear Information System (INIS)

Srivastava, S.C.

1992-01-01

Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated

The antibody approach of labeling blood cells

International Nuclear Information System (INIS)

Srivastava, S.C.

1991-01-01

Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated

The antibody approach of labeling blood cells

Energy Technology Data Exchange (ETDEWEB)

Srivastava, S.C.

1991-12-31

Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

The antibody approach of labeling blood cells

Energy Technology Data Exchange (ETDEWEB)

Srivastava, S.C.

1991-01-01

Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are criticality assessed and evaluated.

The antibody approach of labeling blood cells

Energy Technology Data Exchange (ETDEWEB)

Srivastava, S.C.

1992-12-31

Although the science of blood cell labeling using monoclonal antibodies directed against specific cellular antigens is still in its early stages, considerable progress has recently been accomplished in this area. The monoclonal antibody approach offers the promise of greater selectivity and enhanced convenience since specific cell types can be labeled in vivo, thus eliminating the need for complex and damaging cell separation procedures. This article focuses on these developments with primary emphasis on antibody labeling of platelets and leukocytes. The advantages and the shortcomings of the recently reported techniques are critically assessed and evaluated.

Attitude and Behavior Factors Associated with Front-of-Package Label Use with Label Users Making Accurate Product Nutrition Assessments.

Science.gov (United States)

Roseman, Mary G; Joung, Hyun-Woo; Littlejohn, Emily I

2018-05-01

Front-of-package (FOP) labels are increasing in popularity on retail products. Reductive FOP labels provide nutrient-specific information, whereas evaluative FOP labels summarize nutrient information through icons. Better understanding of consumer behavior regarding FOP labels is beneficial to increasing consumer use of nutrition labeling when making grocery purchasing decisions. We aimed to determine FOP label format effectiveness in aiding consumers at assessing nutrient density of food products. In addition, we sought to determine relationships between FOP label use and attitude toward healthy eating, diet self-assessment, self-reported health and nutrition knowledge, and label and shopping behaviors. A between-subjects experimental design was employed. Participants were randomly assigned to one of four label conditions: Facts Up Front, Facts Up Front Extended, a binary symbol, and no-label control. One hundred sixty-one US primary grocery shoppers, aged 18 to 69 years. Participants were randomly invited to the online study. Participants in one of four label condition groups viewed three product categories (cereal, dairy, and snacks) with corresponding questions. Adults' nutrition assessment of food products based on different FOP label formats, along with label use and attitude toward healthy eating, diet self-assessment, self-reported health and nutrition knowledge, and label and shopping behaviors. Data analyses included descriptive statistics, χ 2 tests, and logistical regression. Significant outcomes were set to α=.05. Participants selected the more nutrient-dense product in the snack food category when it contained an FOP label. Subjective health and nutrition knowledge and frequency of selecting food for healthful reasons were associated with FOP label use (P<0.01 and P<0.05, respectively). Both Facts Up Front (reductive) and binary (evaluative) FOP labels appear effective for nutrition assessment of snack products compared with no label. Specific

Microfluidic Radiometal Labeling Systems for Biomolecules

Energy Technology Data Exchange (ETDEWEB)

Reichert, D E; Kenis, P J. A.

2011-12-29

In a typical labeling procedure with radiometals, such as Cu-64 and Ga-68; a very large (~ 100-fold) excess of the non-radioactive reactant (precursor) is used to promote rapid and efficient incorporation of the radioisotope into the PET imaging agent. In order to achieve high specific activities, careful control of reaction conditions and extensive chromatographic purifications are required in order to separate the labeled compounds from the cold precursors. Here we propose a microfluidic approach to overcome these problems, and achieve high specific activities in a more convenient, semi-automated fashion and faster time frame. Microfluidic reactors, consisting of a network of micron-sized channels (typical dimensions in the range 10 - 300¼m), filters, separation columns, electrodes and reaction loops/chambers etched onto a solid substrate, are now emerging as an extremely useful technology for the intensification and miniaturization of chemical processes. The ability to manipulate, process and analyze reagent concentrations and reaction interfaces in both space and time within the channel network of a microreactor provides the fine level of reaction control that is desirable in PET radiochemistry practice. These factors can bring radiometal labeling, specifically the preparation of radio-labeled biomolecules such as antibodies, much closer to their theoretical maximum specific activities.

Protein Aggregation Inhibitors for ALS Therapy

Science.gov (United States)

2013-07-01

time (2, 5, 10, 15, and 30 min), and disappearance was monitored by HPLC -UV-Vis278nm analysis (Figure 33). Following 10 min irradiation at 312 nm, the...and irradiated with UV light (312 nm, 6W, 4 cm). Photolysis was monitored by HPLC -UV-Vis278nm. Photoaffinity labeling Studies in Whole Cell...well-known compounds resveratrol (57) and curcumin (58), as well as a large family of flavonoids such as quercitin (59) and epicatechin (60), have

Recent developments and applications of clickable photoprobes in medicinal chemistry and chemical biology.

Science.gov (United States)

Lapinsky, David J; Johnson, Douglas S

2015-01-01

Photoaffinity labeling is a well-known biochemical technique that has grown significantly since the turn of the century, principally due to its combination with bioorthogonal/click chemistry reactions. This review highlights new developments and applications of clickable photoprobes in medicinal chemistry and chemical biology. In particular, recent examples of clickable photoprobes for target identification, activity- or affinity-based protein profiling (ABPP or AfBPP), characterization of sterol- or lipid-protein interactions and characterization of ligand-binding sites are presented.

Labeled estrogens as mammary tumor probes

International Nuclear Information System (INIS)

Feenstra, A.

1981-01-01

In this thesis estrogens labeled with a gamma or positron emitting nuclide, called estrogen-receptor binding radiopharmaceuticals are investigated as mammary tumour probes. The requirements for estrogen-receptor binding radiopharmaceuticals are formulated and the literature on estrogens labeled for this purpose is reviewed. The potential of mercury-197/197m and of carbon-11 as label for estrogen-receptor binding radiopharmaceuticals is investigated. The synthesis of 197 Hg-labeled 4-mercury-estradiol and 2-mercury-estradiol and their properties in vitro and in vivo are described. It appears that though basically carbon-11 labeled compounds are very promising as mammary tumour probes, their achievable specific activity has to be increased. (Auth.)

Labelled antibiotics as tumour-localizing agents

International Nuclear Information System (INIS)

Taylor, D.M.; McCready, V.R.

1976-01-01

The published results of clinical and experimental studies of labelled bleomycins and tetracyclines are reviewed. None of the labelled antibiotics yet studied show anything approaching absolute tumour specificity. Clinical trials suggest that 57 Co-bleomycin is superior to either 111 In- or 99 Tcsup(m)-bleomycin and that it may possess some advantages over 67 Ga-citrate in respect of lower uptake in the abdomen and, possibly, lower uptakes in benign and inflammatory lesions. Radioiodine-labelled or 99 Tcsup(m)-labelled tetracyclines appear to be of little value in tumour localization. (author)

Sequence- and structure-dependent DNA base dynamics: Synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA

International Nuclear Information System (INIS)

Spaltenstein, A.; Robinson, B.H.; Hopkins, P.B.

1989-01-01

A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. 1 H NMR, CD, and thermal denaturation studies indicate that 1a (T) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules

Carbon-11 labelled phosgene new synthesis - medical interest

International Nuclear Information System (INIS)

Landais, P.

1985-09-01

This thesis describes a new synthesis of high specific radioactivity carbon-11 labelled phosgene. The latter is an important precursor for the labelling of radiopharmaceuticals used in Positron Emission Tomography. The synthesis is carried out in 10 minutes. First, the carbon-11 labelled methane ( 11 CH 4 ) is chlorinated into carbon tetrachloride on pumice impregnated with copper (II) chloride. A photochemical process had previously been studied but this reaction was strongly inhibited. Then the 11 C-carbon tetrachloride is oxidized into 11 C-phosgene on hot stainless. The 11 C-CGP 12177 has been labelled from this new 11 C-Phosgene synthesis for receptor studies which require high specific radioactivity [fr

Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation

Science.gov (United States)

Pleiner, Tino; Bates, Mark; Trakhanov, Sergei; Lee, Chung-Tien; Schliep, Jan Erik; Chug, Hema; Böhning, Marc; Stark, Holger; Urlaub, Henning; Görlich, Dirk

2015-01-01

Nanobodies are single-domain antibodies of camelid origin. We generated nanobodies against the vertebrate nuclear pore complex (NPC) and used them in STORM imaging to locate individual NPC proteins with nanobody sequence and labeled the resulting proteins with fluorophore-maleimides. As nanobodies are normally stabilized by disulfide-bonded cysteines, this appears counterintuitive. Yet, our analysis showed that this caused no folding problems. Compared to traditional NHS ester-labeling of lysines, the cysteine-maleimide strategy resulted in far less background in fluorescence imaging, it better preserved epitope recognition and it is site-specific. We also devised a rapid epitope-mapping strategy, which relies on crosslinking mass spectrometry and the introduced ectopic cysteines. Finally, we used different anti-nucleoporin nanobodies to purify the major NPC building blocks – each in a single step, with native elution and, as demonstrated, in excellent quality for structural analysis by electron microscopy. The presented strategies are applicable to any nanobody and nanobody-target. DOI: http://dx.doi.org/10.7554/eLife.11349.001 PMID:26633879

Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

Science.gov (United States)

Schmidt, Carla; Grønborg, Mads; Deckert, Jochen; Bessonov, Sergey; Conrad, Thomas; Lührmann, Reinhard; Urlaub, Henning

2014-01-01

The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique. PMID:24448447

Internalization and recycling of receptor-bound gonadotropin-releasing hormone agonist in pituitary gonadotropes

International Nuclear Information System (INIS)

Schvartz, I.; Hazum, E.

1987-01-01

The fate of cell surface gonadotropin-releasing hormone (GnRH) receptors on pituitary cells was studied utilizing lysosomotropic agents and monensin. Labeling of pituitary cells with a photoreactive GnRH derivative, [azidobenzoyl-D-Lys6]GnRH, revealed a specific band of Mr = 60,000. When photoaffinity-labeled cells were exposed to trypsin immediately after completion of the binding, the radioactivity incorporated into the Mr = 60,000 band decreased, with a concomitant appearance of a proteolytic fragment (Mr = 45,000). This fragment reflects cell surface receptors. Following GnRH binding, the hormone-receptor complexes underwent internalization, partial degradation, and recycling. The process of hormone-receptor complex degradation was substantially prevented by lysosomotropic agents, such as chloroquine and methylamine, or the proton ionophore, monensin. Chloroquine and monensin, however, did not affect receptor recycling, since the tryptic fragment of Mr = 45,000 was evident after treatment with these agents. This suggests that recycling of GnRH receptors in gonadotropes occurs whether or not the internal environment is acidic. Based on these findings, we propose a model describing the intracellular pathway of GnRH receptors

Labeling of autologous monocytes with 99mTc-HMPAO at very high specific radioactivity

International Nuclear Information System (INIS)

Hemert, Formijn J. van; Thurlings, Rogier; Dohmen, Serge E.; Voermans, Carlijn; Tak, Paul P.; Eck-Smit, Berthe L.F. van; Bennink, Roelof J.

2007-01-01

Rheumatoid arthritis of joints involves the accumulation of monocyte-derived macrophages in the affected synovial tissue. This process of cell migration can be portrayed scintigraphically in order to monitor noninvasive effects of therapy on the progress of the disease. Scintigraphic detection of inflammation by means of technetium 99m-hexamethylpropylene amine oxime ( 99m Tc-HMPAO)-labeled leukocytes provides a classic example. Present state-of-the-art methods in cell biology allow the isolation of cells like lymphocytes or monocytes, which are less abundant than main blood constituents but, instead, harbor particular functions like specific homing properties. To facilitate scintigraphic imaging of the cell functions involved, the relatively small population of cells must be labeled to radioactive yields as high as possible. We demonstrate that autologous monocytes isolated from 100 ml of peripheral blood can be radiolabeled to a yield of 10 (instead of 1) Bq per cell, allowing scintigraphic analysis of rheumatoid arthritis up to 20 h post injection of patients. The method is based on the instantaneous distribution of lipophilic 99m Tc-HMPAO between the hydrophobic inside of cells and the hydrophilic (aqueous) surrounding of cells, followed by decomposition of the radiopharmaceutical into compounds that are unable to cross the cellular membrane. The procedure provides a method of choice for cell-mediated scintigraphy at low availability of cells with the correct homing properties

Selenium-75-labelled foliate compounds

International Nuclear Information System (INIS)

1974-01-01

A saturation method to analyze a foliate is presented; it uses competitive reaction of the compound to be measured and of a radioactive-labelled version of this compound with a reagent specific to this compound present in insufficient quantity to combine with the whole of the compound and its labelled version, separation of the bound compound from its non-bound homologue and measurement of the radioactivity concentration in the bound compound, the non-bound compound or both. The radioactive isotope used in the labelled foliate is selenium 75 [fr

Proposal for a New Energy Label

DEFF Research Database (Denmark)

Schjær-Jacobsen, Jørgen

2011-01-01

Energy labeling is believed to have great impact on the energy efficiency of household appliances sold. Many different labeling schemes exist around the world. As far as is known, none of these facilitates the easy tightening of specifications to keep up with technological advancements. The present...

Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

International Nuclear Information System (INIS)

Hefke, Frederik; Bagaria, Anurag; Reckel, Sina; Ullrich, Sandra Johanna; Dötsch, Volker; Glaubitz, Clemens; Güntert, Peter

2011-01-01

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with 13 C or/and 15 N such that cross peaks between 13 CO(i – 1) and 15 NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with 13 C and the second with 15 N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B 2 R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.

Preparation of labelled antituberculotics for clarifying specific problems in therapy optimization. 1. Preparation of tritiated isoniazid and injectable solutions, investigation of the stability of labelling

Energy Technology Data Exchange (ETDEWEB)

Winsel, K.; Iwainsky, H. (Forschungsinstitut fuer Lungenkrankheiten und Tuberkulose, Berlin-Buch (German Democratic Republic)); Mittag, E.; Kiessling, M. (Zentralinstitut fuer Kernforschung, Rossendorf bei Dresden (German Democratic Republic)); Koehler, H. (Zentralklinik fuer Herz- und Lungenkrankheiten, Bad Berka (German Democratic Republic))

1985-09-01

The preparation of tritium labelled isoniazid according to the Wilzbach method and by catalytic exchange is described. The purified labelled isoniazid is adjusted to an activity of 37 MBq/300 mg isoniazid. It meets the requirements for an injectable pharmaceutical. The tritium labelling is stable under in vitro conditions and in the macroorganism.

Tritium labeling for bio-med research

International Nuclear Information System (INIS)

Lemmon, R.M.

1980-01-01

A very large fraction of what we know about biochemical pathways in the living cell has resulted from the use of radioactively-labeled tracer compounds; the use of tritium-labeled compounds has been particularly important. As research in biochemistry and biology has progressed the need has arisen to label compounds of higher specific activity and of increasing molecular complexity - for example, oligo-nucleotides, polypeptides, hormones, enzymes. Our laboratory has gradually developed special facilities for handling tritium at the kilocurie level. These facilities have already proven extremely valuable in producing labeled compounds that are not available from commercial sources. The principal ways employed for compound labeling are: (1) microwave discharge labeling, (2) catalytic tritio-hydrogenation, (3) catalytic exchange with T 2 O, and (4) replacement of halogen atoms by T. Studies have also been carried out on tritiation by the replacement of halogen atoms with T atoms. These results indicate that carrier-free tritium-labeled products, including biomacromolecules, can be produced in this way

Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

Science.gov (United States)

Muñoz-Martínez, Francisco; Lu, Peihua; Cortés-Selva, Fernando; Pérez-Victoria, José María; Jiménez, Ignacio A; Ravelo, Angel G; Sharom, Frances J; Gamarro, Francisco; Castanys, Santiago

2004-10-01

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen

DEFF Research Database (Denmark)

Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other...... preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious...

Tritium labeling of simple 7-membered ring compounds

International Nuclear Information System (INIS)

Hiltunen, J.; Peng, C.T.; Yang, Z.C.

1990-01-01

Seven-membered ring compounds, from cycloheptane to complex ring structures containing heteroatoms, substituents and fused phenyl rings, were labeled with tritium, using activated and adsorbed tritium. The 7-membered ring structures are generally stable towards reactions with tritium, which allows compounds like 1-benzosuberone, 1-aza-2-methoxy-1-cycloheptane, iminostilbene and clozapine to be labeled to reasonably high specific activities. The best method varies greatly from compound to compound. By optimizing the labeling conditions and use of efficient support exceptionally good results can be obtained. The Pd-on-alumina support gives consistently higher specific activity and less radioimpurity than other supports. Even molecules containing carbon-halogen bond and hydrogen bound to nitrogen can usually be labeled with tritium at stable positions and without dehalogenation. (author)

Stereoselective synthesis of stable-isotope-labeled amino acids

Energy Technology Data Exchange (ETDEWEB)

Unkefer, C.J.; Martinez, R.A.; Silks, L.A. III [Los Alamos National Laboratory, NM (United States); Lodwig, S.N. [Centralia College, WA (United States)

1994-12-01

For magnetic resonance and vibrational spectroscopies to reach their full potential, they must be used in combination with sophisticated site-specific stable isotope labeling of biological macromolecules. Labeled amino acids are required for the study of the structure and function of enzymes and proteins. Because there are 20 common amino acids, each with its own distinguishing chemistry, they remain a synthetic challenge. The Oppolzer chiral auxiliary provides a general tool with which to approach the synthesis of labeled amino acids. By using the Oppolzer auxiliary, amino acids can be constructed from several small molecules, which is ideal for stable isotope labeling. In addition to directing the stereochemistry at the {alpha}-carbon, the camphorsultam can be used for stereo-specific isotope labeling at prochiral centers in amino acids. By using the camphorsultam auxiliary we have the potential to synthesize virtually any isotopomer of all of the common amino acids.

Stereoselective synthesis of stable-isotope-labeled amino acids

International Nuclear Information System (INIS)

Unkefer, C.J.; Martinez, R.A.; Silks, L.A. III; Lodwig, S.N.

1994-01-01

For magnetic resonance and vibrational spectroscopies to reach their full potential, they must be used in combination with sophisticated site-specific stable isotope labeling of biological macromolecules. Labeled amino acids are required for the study of the structure and function of enzymes and proteins. Because there are 20 common amino acids, each with its own distinguishing chemistry, they remain a synthetic challenge. The Oppolzer chiral auxiliary provides a general tool with which to approach the synthesis of labeled amino acids. By using the Oppolzer auxiliary, amino acids can be constructed from several small molecules, which is ideal for stable isotope labeling. In addition to directing the stereochemistry at the α-carbon, the camphorsultam can be used for stereo-specific isotope labeling at prochiral centers in amino acids. By using the camphorsultam auxiliary we have the potential to synthesize virtually any isotopomer of all of the common amino acids

Somatostatin analogues labelled with 99mTc

International Nuclear Information System (INIS)

Obenaus, E.; Crudo, J.; Edreira, M.; Viaggi, M.; De Castiglia, S.G.

2001-01-01

The aim of the present work was to study the biological and radiochemical behaviour of two somatostatin analogues, the RC-160 and Tyr3Octreotide(TOC) peptides when labelling with 99m Tc by two methods: direct and indirect using S-benzoyl- mercaptoacetyl triglycine (MAG-3) and hydrazinonicotinamide (HYNIC) as chelating agents. RC-160 was labelled with 125I (30% labelling yield) in order to examine its receptor specificity and to study the biodistribution in normal animals. A total binding of 30% and a non specific binding lower than 10% was obtained. On the other hand, the RC-160 was labelled with 99m Tc by a direct method (70% labelling yield), using sodium ascorbate and dithionite in order to reduce the peptide and 99m Tc, respectively. The synthesis of RC-160 with S-benzoyl MAG-3 and TOC with HYNIC, for labelling with 99m Tc are also described. The conjugates were prepared on a small scale and labelled with the radionuclide using tricine as co-ligands for HYNIC conjugates. Chromatographic studies were performed using HPLC system and radiochemical purities higher than 75% and 95% were obtained respectively. Biodistributions studies in normal Wistar rats were performed and results were correlated with chromatographic and protein binding properties. Lower lipophilicity of the labelled conjugates resulted in a higher renal excretion. HYNIC-TOC complex showed promising results when labelling with 99m Tc using tricine as co-ligand although higher stability should be found for ternary co-ligands compared to tricine. (author)

Effect of calorie or exercise labels on menus on calories and macronutrients ordered and calories from specific foods in Hispanic participants: a randomized study.

Science.gov (United States)

Shah, Meena; Bouza, Brooke; Adams-Huet, Beverley; Jaffery, Manall; Esposito, Phil; Dart, Lyn

2016-12-01

The effect of menu labels on food choices is unknown in Hispanics. This study evaluated the impact of menu labels on calories and macronutrients ordered in Hispanics. 372 Hispanics (18-65 years) were randomly assigned to menus with no labels (NL) (n=127), rank-ordered calorie labels plus a statement on energy needs per meal (CL) (n=123), or rank-ordered exercise labels showing minutes of brisk walking necessary to burn the food calories (EL) (n=122). The menus had identical food choices. Participants were instructed to select foods from the assigned menu as if having lunch in a fast food restaurant. One-way analysis of variance found no difference in calories ordered (median (25th and 75th centiles)) by menu condition (NL: 785.0 (465.0, 1010.0) kcal; CL: 790.0 (510.0, 1020.0) kcal; EL: 752.5 (520.0, 1033.8) kcal; p=0.75). Calories from specific foods and macronutrient intake were not different by menu condition. Menu label use was 26.8% in the CL and 25.4% in the EL condition. Calories ordered were not different between those who used and those who did not use the labels. Regression analysis showed that perception of being overweight (p=0.02), selecting foods based on health value (p<0.0001), and meeting exercise guidelines (p<0.0001) were associated with fewer calories ordered. Logistic regression showed that selecting foods based on health value (p=0.01) was associated with higher food label use. Menu labels did not affect food choices in Hispanic participants. Future studies should determine if nutrition, exercise, and weight perception counseling prior to menu labels intervention would result in better food choices. NCT02804503; post-results. Copyright © 2016 American Federation for Medical Research.

Technique of leukocyte harvesting and labeling: problems and perspectives

International Nuclear Information System (INIS)

McAfee, J.G.; Subramanian, G.; Gagne, G.

1984-01-01

Mixed leukocyte suspensions obtained after gravity sedimentation of red cells and labeled with 111 In lipophilic chelates are now widely used clinically for abscess localization at many medical centers. So far, labeling with 111 In-oxine or tropolone has been more successful than any 99 mTc method. More sophisticated approaches are available for isolation and labeling of specific leukocyte cell types, to study their migration in vivo. The most significant advances in cell harvesting include newer density gradients for isopyknic centrifugation, centrifugal elutriation, and flow cytometry. Unlike current radioactive agents which label many cell types indiscriminately, more selective ligands are being developed which bind to specific cell surface receptors. These will label certain leukocyte populations or subtypes while not reacting with others, thereby avoiding laborious separation techniques. Monoclonal antibodies against leukocyte cell-surface antigens appear particularly promising as agents for selective cell labeling

Species specific uptake of radio-labelled phytodetritus by benthic meiofauna from the Baltic Sea

NARCIS (Netherlands)

Ólafsson, E.; Modig, H.; Van de Bund, W.J.

1999-01-01

The diatom Sheletonema costatum is one of the dominant phytoplankton species during spring in the northern Baltic Sea. We followed the uptake of radio-labelled S, costatum by all major meiofauna species in a laboratory experiment. The uptake of labelled diatom carbon varied greatly among major

THE EFFECT OF LABELING INTENSITY, ESTIMATED BY REAL-TIME CONFOCAL LASER SCANNING MICROSCOPY, ON FLOW CYTOMETRIC APPEARANCE AND IDENTIFICATION OF IMMUNOCHEMICALLY LABELED MARINE DINOFLAGELLATES

NARCIS (Netherlands)

VRIELING, EG; DRAAIJER, A; VANZEIJL, WJM; PEPERZAK, L; GIESKES, WWC; VEENHUIS, M; Zeijl, Wilhelmus J.M. van

Two different fluorescein isothiocyanate (FITC) conjugates were used to analyze the effect of labeling intensity on the flow cytometric appearance of marine dinoflagellates labeled with antibodies that specifically recognized the outer cell wall. Location of the labeling was revealed by

Positron emission tomography shows high specific uptake of racemic carbon-11 labelled norepinephrine in the primate heart

International Nuclear Information System (INIS)

Farde, L.; Halldin, C.; Naagren, K.; Suhara, Tetsuya; Karlsson, P.; Schoeps, K.O.; Swahn, C.G.; Bone, D.

1994-01-01

(-)-Norepinephrine is the predominant neurotransmitter of the sympathetic innervation of the heart. Racemic norepinephrine was labelled with carbon-11 and injected i.v. into Cynomolgus monkeys. Five minutes after injection there was a more than tenfold higher radioactivity in the heart than in adjacent tissue. Pretreatment with the norepinephrine reuptake inhibitor desipramine reduced the uptake by more than 80%. The high specific uptake of racemic [ 11 C]norepinephrine indicates that enatiomerically pure(-)-[ 11 C]norepinephrine has promising potential for detailed mapping of the sympathetic innervation of the human myocardium. (orig.)

Positron emission tomography shows high specific uptake of racemic carbon-11 labelled norepinephrine in the primate heart

Energy Technology Data Exchange (ETDEWEB)

Farde, L [Dept. of Clinical Neuroscience, Karolinska Inst., Stockholm (Sweden); Halldin, C [Dept. of Clinical Neuroscience, Karolinska Inst., Stockholm (Sweden); Naagren, K [Turku Univ., Cyclotron/PET Center (Finland); Suhara, Tetsuya [Dept. of Clinical Neuroscience, Karolinska Inst., Stockholm (Sweden); Karlsson, P [Dept. of Clinical Neuroscience, Karolinska Inst., Stockholm (Sweden); Schoeps, K O [Dept. of Clinical Neuroscience, Karolinska Inst., Stockholm (Sweden); Swahn, C G [Dept. of Clinical Neuroscience, Karolinska Inst., Stockholm (Sweden); Bone, D [Dept. of Clinical Neuroscience, Karolinska Inst., Stockholm (Sweden)

1994-04-01

(-)-Norepinephrine is the predominant neurotransmitter of the sympathetic innervation of the heart. Racemic norepinephrine was labelled with carbon-11 and injected i.v. into Cynomolgus monkeys. Five minutes after injection there was a more than tenfold higher radioactivity in the heart than in adjacent tissue. Pretreatment with the norepinephrine reuptake inhibitor desipramine reduced the uptake by more than 80%. The high specific uptake of racemic [[sup 11]C]norepinephrine indicates that enatiomerically pure(-)-[[sup 11]C]norepinephrine has promising potential for detailed mapping of the sympathetic innervation of the human myocardium. (orig.)

Radio-labelling of long-lasting erythropoietin

International Nuclear Information System (INIS)

Liu Guoxia; Zeng Xianyin; Bao Lun; Xu Xiankun; Chen Zhiyu; Liu Xianyi

2004-01-01

The study is designed to investigate the labelling of LL-EPO, purification of labelled compound, and therefore, to prepare the labelled LL-EPO with high purity and biological activity. LL-EPO was labelled with 125 I by the common used chloramine-T and the modified two-phase chloramine-T method, respectively. The labelled compound was purified by both gel filtration and ultrafiltration method, respectively. The purity of the labelled LL-EPO was determined by both trichloroacetic acid (TCA) and SDS-PAGE method, and the biological activity was determined by the reticulocyte counting method. The results demonstrated that the iodine incorporation and specific radioactivities were 89% and 5.82 x 10 5 Bq·μg -1 for LL-EPO labelled by the modified two-phase chloramine-T method and were 20.65% and 3.62 x 10 5 Bq·μg -1 for LL-EPO labelled by the common used chloramine-T method, respectively. The purity of labelled LL-EPO purified by both gel filtration and ultrafiltration were over 96% with TCA method purification. The labelled LL-EPO showed two bands with Rf of 0.28 and 0.49, respectively, which is identical to that of standard LL-EPO through SDS-PAGE. There was no loss of biological activity of LL-EPO after labelling as determined by reticulocyte counting method

Carbon 11 labelled phosgene: a new synthesis - medical interest

International Nuclear Information System (INIS)

Landais, P.

1985-01-01

This thesis describes a new synthesis of high specific radioactivity carbon-11 labelled phosgene. The latter is an important precursor for the labelling of radiopharmaceuticals used in Positron Emission Tomography. The synthesis is carried out in 10 minutes. First, the carbon-11 labelled methane ( 11 CH 4 ) is chlorinated into carbon tetrachloride on pumice impregnated with copper (II) chloride. A photochemical process had previously been studied but this reaction was strongly inhibited. Then the 11 C-carbon tetrachloride is oxidized into 11 C-phosgene on hot stainless. The 11 C-CGP 12177 has been labelled from this new 11 C-Phosgene synthesis for receptor studies which require high specific radioactivity. (author) [fr

Recent developments in blood cell labeling research

Energy Technology Data Exchange (ETDEWEB)

Srivastava, S.C.; Straub, R.F.; Meinken, G.E.

1988-09-07

A number of recent developments in research on blood cell labeling techniques are presented. The discussion relates to three specific areas: (1) a new in vitro method for red blood cell labeling with /sup 99m/Tc; (2) a method for labeling leukocytes and platelets with /sup 99m/Tc; and (3) the use of monoclonal antibody technique for platelet labeling. The advantages and the pitfalls of these techniques are examined in the light of available mechanistic information. Problems that remain to be resolved are reviewed. An assessment is made of the progress as well as prospects in blood cell labeling methodology including that using the monoclonal antibody approach. 37 refs., 4 figs.

Recent developments in blood cell labeling research

International Nuclear Information System (INIS)

Srivastava, S.C.; Straub, R.F.; Meinken, G.E.

1988-01-01

A number of recent developments in research on blood cell labeling techniques are presented. The discussion relates to three specific areas: (1) a new in vitro method for red blood cell labeling with /sup 99m/Tc; (2) a method for labeling leukocytes and platelets with /sup 99m/Tc; and (3) the use of monoclonal antibody technique for platelet labeling. The advantages and the pitfalls of these techniques are examined in the light of available mechanistic information. Problems that remain to be resolved are reviewed. An assessment is made of the progress as well as prospects in blood cell labeling methodology including that using the monoclonal antibody approach. 37 refs., 4 figs

Preparation of radioactive labelled compounds Pt.1. 82Br labelled organic bromine compounds

International Nuclear Information System (INIS)

Otto, R.

1988-05-01

A simple method allowing the preparation of 82 Br labelled organic bromine compounds from olefins with chemical and radiochemical yields between 75 and 95% and the specific activities required, is described [fr

Site-specifically {sup 89}Zr-labeled monoclonal antibodies for ImmunoPET

Energy Technology Data Exchange (ETDEWEB)

Tinianow, Jeff N.; Gill, Herman S.; Ogasawara, Annie; Flores, Judith E.; Vanderbilt, Alexander N.; Luis, Elizabeth; Vandlen, Richard; Darwish, Martine; Junutula, Jagath R.; Williams, Simon-P. [Genentech Research and Early Development, Genentech Inc., South San Francisco, CA 94080 (United States); Marik, Jan [Genentech Research and Early Development, Genentech Inc., South San Francisco, CA 94080 (United States)], E-mail: marik.jan@gene.com

2010-04-15

Three thiol reactive reagents were developed for the chemoselective conjugation of desferrioxamine (Df) to a monoclonal antibody via engineered cysteine residues (thio-trastuzumab). The in vitro stability and in vivo imaging properties of site-specifically radiolabeled {sup 89}Zr-Df-thio-trastuzumab conjugates were investigated. Methods: The amino group of desferrioxamine B was acylated by bromoacetyl bromide, N-hydroxysuccinimidyl iodoacetate, or N-hydroxysuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate to obtain thiol reactive reagents bromoacetyl-desferrioxamine (Df-Bac), iodoacetyl-desferrioxamine (Df-Iac) and maleimidocyclohexyl-desferrioxamine (Df-Chx-Mal), respectively. Df-Bac and Df-Iac alkylated the free thiol groups of thio-trastuzumab by nucleophilic substitution forming Df-Ac-thio-trastuzumab, while the maleimide reagent Df-Chx-Mal reacted via Michael addition to provide Df-Chx-Mal-thio-trastuzumab. The conjugates were radiolabeled with {sup 89}Zr and evaluated for serum stability, and their positron emission tomography (PET) imaging properties were investigated in a BT474M1 (HER2-positive) breast tumor mouse model. Results: The chemoselective reagents were obtained in 14% (Df-Bac), 53% (Df-Iac) and 45% (Df-Chx-Mal) yields. Site-specific conjugation of Df-Chx-Mal to thio-trastuzumab was complete within 1 h at pH 7.5, while Df-Iac and Df-Bac respectively required 2 and 5 h at pH 9. Each Df modified thio-trastuzumab was chelated with {sup 89}Zr in yields exceeding 75%. {sup 89}Zr-Df-Ac-thio-trastuzumab and {sup 89}Zr-Df-Chx-Mal-thio-trastuzumab were stable in mouse serum and exhibited comparable PET imaging capabilities in a BT474M1 (HER2-positive) breast cancer model reaching 20-25 %ID/g of tumor uptake and a tumor to blood ratio of 6.1-7.1. Conclusions: The new reagents demonstrated good reactivity with engineered thiol groups of trastuzumab and very good chelation properties with {sup 89}Zr. The site-specifically {sup 89}Zr-labeled thio

[Academic production on food labeling in Brazil].

Science.gov (United States)

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.

New tools for immunochemistry: internally labelled monoclonal antibodies

International Nuclear Information System (INIS)

Galfre, G.; Cuello, A.C.

1981-01-01

Labelled antibodies are routinely used in a wide variety of immunochemical methods. Over the years several labelling techniques have been developed and the discussion of some of them forms a substantial part of this course. Common to all the procedures is the need to purify the antibodies. The labelling itself consists of coupling the antibodies to a ''label'' molecule by means of a chemical reaction. Preparation in vitro of monoclonal antibodies offers the unique possibility to internally label them. Although this is restricted to radiolabelling, and the specific activity achieved is limited, the procedure is extremely simple, does not require purification prior to labelling and chemical manipulation is not necessary as the antibodies themselves are synthesized from radioactive amino acids. Moreover, different labels can be used ( 14 C, 35 S, 3 H) which have a much longer half-life than 125 I. The choice of labelled amino acid precurors and labelling procedure is discussed. The uses of internally-labelled monoclonal antibodies are indicated. (Auth.)

Synthesis of 13C and 2H labelled retinals: spectroscopic investigations on isotopically labelled rhodopsin and bacteriorhodopsin

International Nuclear Information System (INIS)

Pardoen, J.A.

1986-01-01

In order to develop probes of the structure of chromophores, the author introduces isotopic modifications at specific chromophoric positions as structural probes. To obtain bacteriorhodopsin, rhodopsin and their photoproducts labelled in the chromophore at selected positions, bacterioopsin and opsin were reacted with the appropriate labelled a11-trans and 11-cis retinals. The author describes the synthesis of a11-trans retinal selectively 13 C labelled at different positions. The characterization of these labelled a11-trans retinals by mass spectrometry, 300 MHz 1 H NMR and 75 MHz 13 C NMR spectroscopy is given. The photochemical preparation and isolation of the pure 9-, 11- and 13-cis forms is described in the experimental part. (Auth.)

FDA Online Label Repository

Data.gov (United States)

U.S. Department of Health & Human Services — The drug labels and other drug-specific information on this Web site represent the most recent drug listing information companies have submitted to the Food and Drug...

Pactamycin binding site on archaebacterial and eukaryotic ribosomes

International Nuclear Information System (INIS)

Tejedor, F.; Amils, R.; Ballesta, J.P.G.

1987-01-01

The presence of a photoreactive acetophenone group in the protein synthesis inhibitor pactamycin and the possibility of obtaining active iodinated derivatives that retain full biological activity allow the antibiotic binding site on Saccharomyces cerevisiae and archaebacterium Sulfolobus solfataricus ribosomes to be photoaffinity labeled. Four major labeled proteins have been identified in the yeast ribosomes, i.e., YS10, YS18, YS21/24, and YS30, while proteins AL1a, AS10/L8, AS18/20, and AS21/22 appeared as radioactive spots in S. solfataricus. There seems to be a correlation between some of the proteins labeled in yeast and those previously reported in Escherichia coli indicating that the pactamycin binding sites of both species, which are in the small subunit close to the initiation factors and mRNA binding sites, must have similar characteristics

99mTc-labeling and evaluation of a HYNIC modified small-molecular inhibitor of prostate-specific membrane antigen

International Nuclear Information System (INIS)

Xu, Xiaoping; Zhang, Jianping; Hu, Silong; He, Simin; Bao, Xiao; Ma, Guang; Luo, Jianmin; Cheng, Jingyi; Zhang, Yingjian

2017-01-01

Introduction: Prostate-specific membrane antigen (PSMA) is a well-established target in the development of radiopharmaceuticals for the diagnosis and therapy of prostate cancer (PCa). In this study, we evaluated a novel 99m Tc-labeled small molecular inhibitor of PSMA. Methods: This new small-molecular inhibitor of PSMA, 6-hydrazinonicotinate-Aminocaproic acid-Lysine-Urea-Glutamate (HYNIC-ALUG) was radiolabeled by 99m Tc and was evaluated both in vitro and in vivo using PCa models (PC-3 and LNCaP). Radiation dosimetry was assessed in mice. Results: 99m Tc-HYNIC-ALUG showed excellent stability in different media. A cell assay preliminarily displayed its specificity for PSMA. The inhibitor showed good pharmacokinetics making it suitable for in vivo imaging. PC-3-derived tumors showed no obvious radioactive uptake; however, the LNCaP-derived tumors showed very high radioactive uptake which was significantly decreased by the selective PSMA inhibitor 2-PMPA. Biodistribution in LNCaP xenografts showed an optimum tumor-to-blood ratio of 24.23 ± 3.54 at 2 h. Tumor uptake was also decreased in the inhibition experiment with 2-PMPA (19.45 ± 2.14%ID/g versus 1.42 ± 0.15%ID/g at 2 h). The effective dose of the 99m Tc-HYNIC-ALUG was 8.4E-04 mSv/MBq. Conclusions: A new 99m Tc-labeled PSMA inhibitor with specific accumulation in PSMA-positive tumors and low background in other organs was synthesized. The radiopharmaceutical also showed very low radiation dosimetry. This agent may significantly improve the diagnosis, staging, and subsequent monitoring of therapeutic effects in PCa patients.

Development of radioiodine-labeled 4-hydroxyphenylcysteamine for specific diagnosis of malignant melanoma

International Nuclear Information System (INIS)

Kobayashi, Masato; Nishii, Ryuichi; Shikano, Naoto; Flores, Leo G.; Mizutani, Asuka; Ogai, Kazuhiro; Sugama, Jyunko; Nagamachi, Shigeki; Kawai, Keiichi

2015-01-01

Introduction: A specific diagnosis for melanoma is strongly desired because malignant melanoma has poor prognosis. In a previous study, although radioiodine-125-labeled 4-hydroxyphenyl-L-cysteine ( 125 I-L-PC) was found to have good substrate affinity for tyrosinase enzyme in the melanin metabolic pathway, 123/131 I-L-PC had insufficient substrate affinity for tyrosinase to diagnose melanoma. In this study, we synthesized 4-hydroxyphenylcysteamine (4-PCA) and developed a novel radioiodine-125-labeled 4-hydroxyphenylcysteamine ( 125 I-PCA) to increase affinity for the melanin biosynthesis pathway. Methods: 4-PCA was separated with 2-hydroxyphenylcysteamine (2-PCA), which is an isomer of 4-PCA, and was examined using melting point, proton nuclear magnetic resonance, mass spectrometry and elemental analysis. 125 I-PCA was prepared using the chloramine-T method under no-carrier added conditions. We performed biodistribution experiments using B16 melanoma-bearing mice using 125 I-PCA, 125 I-L-PC, 125 I-α-methyl-L-tyrosine, 123 I-m-iodobenzylguanidine and 67 Ga-citrate. In vitro assay was performed with B16 melanoma cells, and affinity for tyrosinase, DNA polymerase and amino acid transport was evaluated using phenylthiourea, thymidine, ouabine and L-tyrosine inhibitor. In addition, partition coefficients of 125 I-PCA were evaluated. Results: In the synthesis of 4-PCA, analysis values did not differ between calculated and reported values, and 4-PCA was separated from 2-PCA at high purity. In biodistribution experiments, 125 I-PCA was accumulated and retained in B16 melanoma cells when compared with 125 I-L-PC. 125 I-PCA showed the highest values at 60 min after radiotracer injection in melanoma-to-muscle ratios, melanoma-to-blood ratios and melanoma-to-skin ratios. Accumulation of 125 I-PCA was significantly inhibited by phenylthiourea and thymidine. Partition coefficients of 125 I-PCA were lower than those of N-isopropyl-p-[ 123 I]iodoamphetamine and were not

Radiation-induced tritium labelling and product analysis

Energy Technology Data Exchange (ETDEWEB)

Peng, C.T. (California Univ., San Francisco, CA (United States). Dept. of Pharmaceutical Chemistry)

1993-05-01

By-products formed in radiation-induced tritium labelling are identified by co-chromatography with authentic samples or by structure prediction using a quantitative structure-retention index relationship. The by-products, formed from labelling of steroids, polynuclear aromatic hydrocarbons, 7-membered heterocyclic ring structures, 1,4-benzodiazepines, 1-haloalkanes, etc. with activated tritium and adsorbed tritium, are shown to be specifically labelled and anticipated products from known chemical reactions. From analyses of the by-products, one can conclude that the hydrogen abstraction by tritium atoms and the substitution by tritium ions are the mechanisms of labelling. Classification of the tritium labelling methods, on the basis of the type of tritium reagent, clearly shows the active role played by tritium atoms and ions in radiation-induced methods. (author).

Tritium labelled nucleotides: Heterogeneous metal catalyzed exchange labelling of ATP with tritium gas

International Nuclear Information System (INIS)

Jaiswal, D.K.; Morimoto, H.; Williams, P.G.; Wemmer, D.E.

1991-09-01

Adenosine 5' triphosphate (ATP) in aqueous solution has been labeled by exchange with tritium gas in the presence of palladium oxide catalyst. Comparison with our experiments using Pd/BaSO 4 as the catalyst shows that we have obtained product with higher specific activity and improved chemical purity. 3 H NMR spectroscopy of the tritiated ATP shows labelling in both the C-8 and C-2 positions, and the integral ratio of these positions was found to vary from 3:1 to 1:1 under different reaction conditions. 5 refs., 1 fig., 2 tabs

Labelling of histone H5 and its interaction with DNA. 1. Histone H5 labelling with fluorescein isothiocyanate.

Science.gov (United States)

Favazza, M; Lerho, M; Houssier, C

1990-06-01

Histone H5 has been labelled with fluorescein isothiocyanate (FITC) with particular attention to the reaction conditions (pH, reaction time and input FITC/H5 molar ratio) and to the complete elimination of non-covalently bound dye. We preferred to use reaction conditions which yielded non-specific uniform labelling rather than specific alpha-NH2 terminal labelling, in order to obtain higher sensitivity in further studies dealing with the detection of perturbation at the binding sites of H5 on DNA. FITC-labelled H5 was further characterized by absorption and circular dichroism spectroscopy, and the fluorescein probe titrated in the 4-8 pH range. The structural integrity of H5 was found to be preserved after labelling. The positive electrostatic potential of the environment in which the FITC probe is embedded in the arginine/lysine-rich tails of H5 is believed to be responsible for the drop of pK of 1 unit found for H5-FITC as compared to free FITC. For the globular part of H5, the pK of covalently-bound FITC was only slightly lowered; this is a consequence of the much lower content in positively-charged amino-acid side chains in this region.

Chromosome-specific DNA Repeat Probes

Energy Technology Data Exchange (ETDEWEB)

Baumgartner, Adolf; Weier, Jingly Fung; Weier, Heinz-Ulrich G.

2006-03-16

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with {alpha}-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

Manual airway labeling has limited reproducibility

DEFF Research Database (Denmark)

Petersen, Jens; Feragen, Aasa; Thomsen, Laura Hohwü

Purpose: Quantitative airway assessment is often performed in specific branches to enable comparison of measurements between patients and over time. Little is known on the accuracy in locating these branches. We determined inter- and intra-observer agreement of manual labeling of segmental bronch...... disagreement in expert labeling, possibly reflecting large anatomical heterogeneity and changes with inspiration. Consistent airway measurement cannot be guaranteed based on manual localization....

Recognition memory for hue: Prototypical bias and the role of labeling.

Science.gov (United States)

Kelly, Laura Jane; Heit, Evan

2017-06-01

How does the concurrent use of language affect perception and memory for exemplars? Labels cue more general category information than a specific exemplar. Applying labels can affect the resulting memory for an exemplar. Here 3 alternative hypotheses are proposed for the role of labeling an exemplar at encoding: (a) labels distort memory toward the label prototype, (b) labels guide the level of specificity needed in the current context, and (c) labels direct attention to the label's referent among all possible features within a visual scene. University students were shown hues on object silhouettes that they either labeled with basic color categories, made preference judgments about, or indicated the animacy of its category. Experiments 1 and 2 established that there are response shifts toward the category prototype regardless of labeling, showing a pervasive influence of category knowledge on response bias. They also established an effect of labeling whereby labeling decreases the magnitude of shifts. Experiments 3 and 4 investigated the uniqueness and necessity of language in causing the decreased shift-neither of which proved to be the case. Overall, category-relative bias was pervasive and labeling appears to direct attention to the feature resulting in less biased memory. The results highlight that the context at encoding affects how memory is formed. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

Science.gov (United States)

Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.

Tritium labelling of two new analgesic drugs

International Nuclear Information System (INIS)

Santamaria, J.; Rebollo, D.V.; Rivera, P.; Esteban, M.

1986-01-01

The labelling with tritium of two arylpropionic esters was studied. The synthesis between 3 H-Ibuprofen and the two unlabelled alcoholic moieties (Cl-Alkanol and CF 3 -Alkanol) was performed. Assuming that we got ready the acidic moiety, 3 H-Ibuprofen, in our Laboratory, we attempted to label with tritium the alcoholic moiety and then go on to its esterification. Prior to labelling, thermic stability of 2-(4-(3-chlorophenyl)-1-piperazinyl) ethanol (Cl-Alkanol) was studied. As result of this study we had to change the labelling method, so that the Cl-Alkanol was unstable at 70 0 C. Purification was accomplished through thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Concentration, purity and specific activities of the two labelled compounds were determined by ultraviolet, HPLC and liquid scintillation techniques. (author)

High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression.

Directory of Open Access Journals (Sweden)

Sandra J Kuhlman

2008-04-01

Full Text Available We describe a method that combines Cre-recombinase knockin mice and viral-mediated gene transfer to genetically label and functionally manipulate specific neuron types in the mouse brain. We engineered adeno-associated viruses (AAVs that express GFP, dsRedExpress, or channelrhodopsin (ChR2 upon Cre/loxP recombination-mediated removal of a transcription-translation STOP cassette. Fluorescent labeling was sufficient to visualize neuronal structures with synaptic resolution in vivo, and ChR2 expression allowed light activation of neuronal spiking. The structural dynamics of a specific class of neocortical neuron, the parvalbumin-containing (Pv fast-spiking GABAergic interneuron, was monitored over the course of a week. We found that although the majority of Pv axonal boutons were stable in young adults, bouton additions and subtractions on axonal shafts were readily observed at a rate of 10.10% and 9.47%, respectively, over 7 days. Our results indicate that Pv inhibitory circuits maintain the potential for structural re-wiring in post-adolescent cortex. With the generation of an increasing number of Cre knockin mice and because viral transfection can be delivered to defined brain regions at defined developmental stages, this strategy represents a general method to systematically visualize the structure and manipulate the function of different cell types in the mouse brain.

Recognition Memory for Hue: Prototypical Bias and the Role of Labeling

Science.gov (United States)

Kelly, Laura Jane; Heit, Evan

2017-01-01

How does the concurrent use of language affect perception and memory for exemplars? Labels cue more general category information than a specific exemplar. Applying labels can affect the resulting memory for an exemplar. Here 3 alternative hypotheses are proposed for the role of labeling an exemplar at encoding: (a) labels distort memory toward the…

Limited tryptic proteolysis of the benzodiazepine binding proteins in different species reveals structural homologies.

Science.gov (United States)

Friedl, W; Lentes, K U; Schmitz, E; Propping, P; Hebebrand, J

1988-12-01

Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.

Food quality labels from the producers’ perspective

Directory of Open Access Journals (Sweden)

Šárka Velčovská

2016-09-01

Full Text Available The paper deals with analysing the food producer attitudes towards quality labels. The Klasa label, as the most known and the most frequently used food quality label in the Czech Republic, have become the subject of investigation. The aim of the research was to identify the benefits and problems arising from the certification process and the label use. Primary data were collected in online survey based on standardized questionnaire. In census, 86 respondents from the total 218 producers with the Klasa label in the Czech Republic completed the questionnaire. The most of producers (72% have a longer experience with the label, they are using the label for more than four years. The producers’ expectations from the label were fulfilled only partially. A poor state marketing support and missing marketing strategy were identified as general problems of the label. Specific perceived problems are formalities connected with the certification process and certification of poor-quality products. Correlation analysis, t-test and Pearson chi-square test were calculated to discover relations between variables. The results of the study can be beneficial to both, food producers as well as administrator of the label. Identified problems could help them to improve marketing strategy of the label in order to manage the label in effective way and use all benefits arising from the certification. Administrator of the label should make the certification process more effective and transparent, promotion should be focused on the explanation to consumers what the Klasa label guarantees.

Preparation of labelled antituberculotics for clarifying specific problems in therapy optimization. 2

International Nuclear Information System (INIS)

Winsel, K.; Iwainsky, H.; Mittag, E.; Kiessling, M.; Koehler, H.

1985-01-01

Labelling of RMP according to the Wilzbach method results in a fragmentation of the molecule. The synthesis of [ 3 H]-RMP is possible by the reaction of 1-amino-4-[ 3 H]-methylpiperazine with 3-formylrifamycin SV. It consists of the following steps: 1 nitrosopiperazine, 1-aminopiperazine, 1-benzalaminopiperazine, 1-benzalamino-4-[ 3 H]-methylpiperazine, 1-amino-4-[ 3 H]methylpiperazinedihydrochloride. The base was liberated from the hydrochloride directly in the reaction mixture in the presence of 3-formylrifamycin SV. Excluding oxygen and light the reaction is nearly quantitative. The product was purified by preparative thin-layer chromatography. After oral administration measurements of the [ 3 H] activity reflect with good approximation the antimycobacterial activity, although to a small degree the labelled group is eliminated by metabolic processes. (author)

Dynamics of Dengue Virus (DENV)-Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions.

Science.gov (United States)

Woda, Marcia; Friberg, Heather; Currier, Jeffrey R; Srikiatkhachorn, Anon; Macareo, Louis R; Green, Sharone; Jarman, Richard G; Rothman, Alan L; Mathew, Anuja

2016-10-01

The development of reagents to identify and characterize antigen-specific B cells has been challenging. We recently developed Alexa Fluor-labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV(+) class-switched memory B cells (IgD(-)CD27(+) CD19(+) cells) reached up to 8% during acute infection and early convalescence. AF DENV-labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38(-)CD27(+)) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. AF DENVs reveal changes in the phenotype of DENV serotype-specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Efficient preparation of {sup 99m}Tc(III) '4+1' mixed-ligand complexes for peptide labeling with high specific activity

Energy Technology Data Exchange (ETDEWEB)

Kunstler, Jens-Uwe [Biotectid GmbH, Deutscher Platz 5c, 04103 Leipzig (Germany); Seidel, Gesine [Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, P.O. Box 510 119, 01314 Dresden (Germany); Pietzsch, Hans-Jurgen, E-mail: h.j.pietzsch@fzd.d [Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, P.O. Box 510 119, 01314 Dresden (Germany)

2010-09-15

An improved labeling procedure for peptides attached to organometallic {sup 99m}Tc(III) '4+1' mixed-ligand complexes in which the radiometal is coordinated by a tripodal tetradentate chelator 2,2',2''-nitrilotriethanethiol (NS{sub 3}) and a monodentate isocyanide ligand is presented. The labeling procedure was evaluated by the synthesis of [{sup 99m}Tc(NS{sub 3})(L2-RGD)]. The containing radiopharmaceutically interesting RGD-peptide cyclo[Arg-Gly-Asp-D-Tyr-Lys] was modified with 4-isocyanobutanoic acid (L2) as linker conjugated to N{sup 6}-Lys to get the monodentate ligand L2-RGD. The structural identity of the {sup 99m}Tc-conjugate was confirmed by comparison to a Re reference compound. The Tc- and Re-conjugates had matching retention times under identical HPLC conditions. The {sup 99m}Tc-labeling was performed in a novel one-step procedure using the eluate of a {sup 99}Mo/{sup 99m}Tc generator, NS{sub 3}, the isocyanide modified peptide, SnCl{sub 2}, Na{sub 2}EDTA, mannitol and ascorbic acid in the reaction mixture. Using optimized reagents it is possible to label 50 nmol peptide with {sup 99m}Tc within 60 min at room temperature with a radiochemical yield higher than 95% and a specific activity of {approx}20 GBq/{mu}mol.

Radiopharmaceutical potential of I-131 labelled diazepam

International Nuclear Information System (INIS)

Yurt, F.; Unek, P.; Asikoglu, M.; Baggi, S.; Erener, G.; Ozkilic, H.; Uluc, F.; Tuglular, I.

1998-01-01

In this study, diazepam is a derivative of the 1.4 benzodiazepine family that the most widely used drug as anticonvulsant agent has been labeled with I-131, as a new radiopharmaceutical and its radiopharmaceutical potential has been determined. Labeling of diazepam has been performed by iodogen method and optimum labeling conditions have been determined. Optimum reaction conditions are 1 mg for iodogen amount; 1-5 mg for diazepam amount, 15-20 minutes for reaction time and room temperature for reaction temperature. Specific activity of labeled compound was 0,15 Ci/mmol level. N-octanol/water ratio was found 1.9 for 131 IDZ ( 131 I labeled diazepam). In vivo experiments have been carried out to determine radiopharmaceutical potentials of labeled compound. Biodistribution studies on rats showed that 131 IDZ have accumulated in kidneys, liver, lungs and brain tissues. Scintigraphic results taken with gamma camera on rabbits agree with biodistribution results of rats. (author)

Electron paramagnetic resonance spin label titration: a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties

International Nuclear Information System (INIS)

Butterfield, D. Allan; Colvin, Joshua; Liu Jiangling; Wang Jianquan; Bachas, Leonidas; Bhattacharrya, Dibakar

2002-01-01

The immobilization of biological molecules onto polymeric membranes to produce biofunctional membranes is used for selective catalysis, separation, analysis, and artificial organs. Normally, random immobilization of enzymes onto polymeric membranes leads to dramatic reduction in activity due to chemical reactions involved in enzyme immobilization, multiple-point binding, etc., and the extent of activity reduction is a function of membrane hydrophilicity (e.g. activity in cellulosic membrane >> polysulfone membrane). We have used molecular biology to effect site-specific immobilization of enzymes in a manner that orients the active site away from the polymeric membrane surface, thus resulting in higher enzyme activity that approaches that in solution and in increased stability of the enzyme relative to the enzyme in solution. A prediction of this site-specific method of enzyme immobilization, which in this study with subtilisin and organophosphorus hydrolase consists of a fusion tag genetically added to these enzymes and subsequent immobilization via the anti-tag antibody and membrane-bound protein A, is that the active site conformation will more closely resemble that of the enzyme in solution than is the case for random immobilization. This hypothesis was confirmed using a new electron paramagnetic resonance (EPR) spin label active site titration method that determines the amount of spin label bound to the active site of the immobilized enzyme. This value nearly perfectly matched the enzyme activity, and the results suggested: (a) a spectroscopic method for measuring activity and thus the extent of active enzyme immobilization in membrane, which may have advantages in cases where optical methods can not be used due to light scattering interference; (b) higher spin label incorporation (and hence activity) in enzymes that had been site-specifically immobilized versus random immobilization; (c) higher spin label incorporation in enzymes immobilized onto hydrophilic

Dual Mode Fluorophore-Doped Nickel Nitrilotriacetic Acid-Modified Silica Nanoparticles Combine Histidine-Tagged Protein Purification with Site-Specific Fluorophore Labeling

OpenAIRE

Kim, Sung Hoon; Jeyakumar, M.; Katzenellenbogen, John A.

2007-01-01

We present the first example of a fluorophore-doped nickel chelate surface- modified silica nanoparticle that functions in a dual mode, combining histidine-tagged protein purification with site-specific fluorophore labeling. Tetramethylrhodamine (TMR)-doped silica nanoparticles, estimated to contain 700–900 TMRs per ca. 23-nm particle, were surface modified with nitrilotriacetic acid (NTA), producing TMR-SiO2-NTA-Ni+2. Silica-embedded TMR retains very high quantum yield, is resistant to quenc...

Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

Directory of Open Access Journals (Sweden)

Paul Majkut

Full Text Available The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values. In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

18F-Labelling of electron rich iodonium ylides

DEFF Research Database (Denmark)

Petersen, I N; Villadsen, J; Hansen, H D

2017-01-01

in the pursuit of (18)F-labelled 5-HT2A receptor agonist PET-ligands. Subsequent evaluation in pigs showed high brain uptake of the PET ligands but a blocking dose of ketanserin did not significantly reduce the signal in relevant brain regions - indicating that the ligands do not interact specifically with the 5......(18)F-Labelling of aromatic moieties was limited to electron deficient aromatic systems for many years but recent developments have provided access to the direct labelling of electron rich aromatic systems. Herein we report the synthesis and (18)F-labelling of iodonium ylide precursors...

(18)F-Labelling of electron rich iodonium ylides

DEFF Research Database (Denmark)

Petersen, I N; Villadsen, J; Hansen, H D

2017-01-01

in the pursuit of (18)F-labelled 5-HT2A receptor agonist PET-ligands. Subsequent evaluation in pigs showed high brain uptake of the PET ligands but a blocking dose of ketanserin did not significantly reduce the signal in relevant brain regions - indicating that the ligands do not interact specifically with the 5......(18)F-Labelling of aromatic moieties was limited to electron deficient aromatic systems for many years but recent developments have provided access to the direct labelling of electron rich aromatic systems. Herein we report the synthesis and (18)F-labelling of iodonium ylide precursors...

Labeled receptor ligands for spect

International Nuclear Information System (INIS)

Kung, H.F.

1989-01-01

Receptor specific imaging agents for single photon emission computed tomography (SPECT) can potentially be useful in the understanding of basic biochemistry and pharmacology of receptors. SPECT images may also provide tools for evaluation of density and binding kinetics of a specific receptor, information important for diagnosis and patient management. Basic requirements for receptor imaging agents are: (a) they are labeled with short-lived isotopes, (b) they show high selectivity and specific uptake, (c) they exhibit high target/background ratio, and (d) they can be modeled to obtain quantitative information. Several good examples of CNS receptor specific ligands labeled with I-123 have been developed, including iodoQNB, iodoestrogen iodobenzadiazepine, iodobenazepine, iodobenzamides for muscarinic, estrogen benzadiazepine, D-1 and D-2 dopamine receptors. With the advent of newer and faster SPECT imaging devices, it may be feasible to quantitate the receptor density by in vivo imaging techniques. These new brain imaging agents can provide unique diagnostic information, which may not be available through other imaging modalities, such as CT and MRI

Synthesis and evaluation of iodide uptake inhibitors in thyroid gland

International Nuclear Information System (INIS)

Lacotte, Pierre

2012-01-01

This work was intended to discover small organic molecules acting as iodide uptake inhibitors in thyroid cells. These compounds can indeed be derivatized into biochemical probes for further characterization of proteins involved in iodide transport mechanisms. On the long term, these inhibitors also appear as attractive drug candidates for treatment of thyroid pathologies or radioprotection against iodine isotopes. A similar strategy was adopted for both of the two inhibitor families. First, we synthesized a chemical library of around 100 analogues; we measured their IC50 against iodide uptake in FRTL-5 cells to get structure-activity relationships. Absolute configuration of stereo-genic centers was also investigated, and a preferential stereochemistry was found to be responsible for activity. From this basis, around twenty 'second-generation' analogues were synthesized by combining fragments contributing to biological activity. Biological evaluation indicated that nine were very potent inhibitors, with IC50 ≤ 6 nM and satisfying physicochemical properties required for drug candidates. Finally, one photoactivatable biotinylated probe was developed in each family and used for photoaffinity labeling. Several specifically labeled proteins are still under identification and constitute new potential therapeutic targets. (author)

Energy-efficient appliance labeling in China: Lessons for successful labeling programs in varied markets

Energy Technology Data Exchange (ETDEWEB)

Lin, Jiang; Townend, Jeanne; Fridley, David; McNeil, Gary; Silva, Tony; Clark, Robin

2002-08-20

Appliance ownership and production has increased dramatically in China in the past two decades. From extremely low levels in 1980, China's appliance industry has become one of the largest in the world, with sales topping U.S. $14.4 billion in 2000. In 1981, less than 1 percent of urban Chinese households owned a refrigerator; by 1998, that number had increased to over 75 percent. This dramatic increase in sales and ownership leads to an excellent opportunity to impact energy consumption in China by affecting the energy efficiency of appliances being bought and sold. In general, Chinese consumers value energy efficiency and are knowledgeable about the operating costs of major appliances. However, the Chinese marketplace does not provide information that consumers trust about the energy consumption of specific products. Thus, several interdependent organizations have emerged in China to provide information and market supports for energy efficiency. This paper describes the appliance market in China and the evolution of its standards and labeling programs and the agencies that implement them. It discusses the authors' work with these organizations in developing energy efficiency criteria and supporting an energy efficiency endorsement labeling program in China. It describes how the authors have used their experience with ENERGY STAR{reg_sign} and other programs in the U.S. to work with China to develop a successful program specific to Chinese conditions, with a particular emphasis on refrigerators. It then gives the author's market assessment of the Chinese refrigerator market and recommendations for a successful labeling program and transferable lessons for developing energy efficiency labeling programs in varied markets. This paper is based on the authors' market research, their support in setting energy efficiency criteria in China, interviews with Chinese manufacturers, retailers, and sales staff, and the development and implementation of

Dynamics of Dengue Virus (DENV)–Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions

Science.gov (United States)

Woda, Marcia; Friberg, Heather; Currier, Jeffrey R.; Srikiatkhachorn, Anon; Macareo, Louis R.; Green, Sharone; Jarman, Richard G.; Rothman, Alan L.; Mathew, Anuja

2016-01-01

Background. The development of reagents to identify and characterize antigen-specific B cells has been challenging. Methods. We recently developed Alexa Fluor–labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. Results. In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV+ class-switched memory B cells (IgD−CD27+ CD19+ cells) reached up to 8% during acute infection and early convalescence. AF DENV–labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38−CD27+) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. Conclusions. AF DENVs reveal changes in the phenotype of DENV serotype–specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination. PMID:27443614

The tritium labelling of ibuprofen by heterogeneous catalytic exchange

International Nuclear Information System (INIS)

Santamaria, J.; Rebollo, D.V.; Rivera, P.; Estaban, M.

1986-01-01

The tritium labelling of 2-(4-isobutylphenyl) propionic acid (ibuprofen) was performed. The method employed was heterogeneous catalytic exchange between ibuprofen and tritiated water. Prior to labelling, thermic stability of ibuprofen was studied. Purification was accomplished through thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). Concentration, purity and specific activity of the labelled compound were determined by ultraviolet, HPLC and liquid scintillation techniques. (author)

Radioiodine-labelled compounds previously or currently used for tumour localization

International Nuclear Information System (INIS)

Beierwaltes, W.H.

1976-01-01

131 I-labelled human serum albumin, though not used for tumour localization today, is an excellent ''standard'' with which to compare uptake of ''tumour-specific'' radiolabelled compounds. 131 I-labelled fibrinogen and antibodies to fibrinogen have a non-specific uptake in tumours. Nungester, Beierwaltes and Knorpp are credited by Mahaley as first treating a human for cancer with 131 I-labelled antibody globulins (malignant melanoma). Although many theoretical problems remain in obtaining diagnostic localization of 131 I-IgG, Quinones, Mizejewski and Beierwaltes demonstrated the uptake of 131 I-labelled immune antibodies in Syrian hamster cheeck pouch with chorionic gonadotropic hormone as the specific tumour-associated antigen. This model was then used successfully by Goldenberg and Hoffer for demonstrating colon carcinoma by using antibodies to carcinoembryonic antigen. A 131 I-labelled chloroquine analogue, synthesized by Counsell, has been demonstrated by Beierwaltes et al. to concentrate diagnostically and therapeutically in the malignant melanotic melanoma. 131 I-19-iodocholesterol, synthesized by Counsell, has been demonstrated by Beierwaltes et al. to concentrate diagnostically in the human adrenal cortex. It has many unique diagnostic capabilities not available with other routine diagnostic methods available today. (author)

Labeling of monoclonal antibodies with a 67Ga-phenolic aminocarboxylic acid chelate. Part I. Chemistry and labeling technique.

Science.gov (United States)

Schuhmacher, J; Matys, R; Hauser, H; Maier-Borst, W; Matzku, S

1986-01-01

As a chelating agent for labeling antibodies (Abs) with metallic radionuclides, a propionic acid substituted ethylenediamine N,N'-di-[(o-hydroxyphenyl) acetic acid] (P-EDDHA), which tightly complexes 67Ga, was synthesized. The 67Ga-P-EDDHA chelate was coupled in aqueous solution to IgG at a molar ratio of 1:1 via carbodiimide. The average coupling yield was 15%. A specific activity of 4 mCi/mg IgG could be obtained with commercially supplied 67Ga. In vitro stability was evaluated in human serum at 37 degrees C and showed a half-life of about 120 h for the release of 67Ga from the labeled Ab during the initial phase of incubation. This in vitro halflife is similar to that measured for 111In-DTPA labeled Abs. Because of the high stability of the 67Ga-P-EDDHA chelate, the in vivo formation of radioactive labeled transferrin by transchelation, as described for 111In-DTPA labeled Abs, should, however, be reduced by this labeling technique.

Current state of the art of blood cell labeling

International Nuclear Information System (INIS)

Srivastava, S.C.; Straub, R.F.; Meinken, G.E.; Gil, M.C.

1985-01-01

An update on some recent developments in the area of blood cell labeling is provided. Specific topics covered include red cell labeling with /sup 99m/Tc, platelet labeling using an antiplatelet monoclonal antibody, and the labeling of leukocytes with /sup 99m/Tc. Mechanistic information, where available, is discussed. A critical evaluation of current techniques, their pitfalls as well as advantages, and the problems that remain to be resolved, is presented. The promise shown by recent results using the antibody approach for cell labeling is emphasized. An assessment of the progress made in these areas is presented. 38 refs., 10 figs., 6 tabs

Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).

Science.gov (United States)

Meek, Megan E; Van Dolah, Frances M

2016-05-01

Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities. Copyright © 2016 Elsevier B.V. All rights reserved.

Co-isolation of in vivo 32P-labeled specific transcripts and DNA without phenol extraction of nuclease digestion

International Nuclear Information System (INIS)

Hayes, S.; Hayes, C.; Brand, L.

1981-01-01

A method is described for isolation and quantitation of specific intact transcripts, for which a hybridization probe is available, from 32 P-labeled bacterial cells. The RNA is extracted in the absence of R Nase activity by incorporating an inert, physically removable R Nase inhibitor throughout the spheroplasting, cell lysis, and pronase digestion steps. [/sup 32/P]RNA is separated from [ 32 P]DNA, without recourse to phenol extraction of DNase treatment, on a Cs 2 SO/sub 4-/HCONH 2 step gradient in which the precipitated RNA forms a sharp band. Specific transcripts are purified from [ 32 P]RNA by physical separation of the transcript and hybridization probe using gel-exclusion chromatography. The gentleness of this technique enables the co-isolation of DNA and can facilitate the analysis of covalently joined RNA-DNA replication intermediates

21 CFR 201.310 - Phenindione; labeling of drug preparations intended for use by man.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Phenindione; labeling of drug preparations intended for use by man. 201.310 Section 201.310 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL LABELING Specific Labeling Requirements for Specific Drug Products § 201.310 Phenindione;...

Enzyme-labeled Antigen Method: Development and Application of the Novel Approach for Identifying Plasma Cells Locally Producing Disease-specific Antibodies in Inflammatory Lesions

International Nuclear Information System (INIS)

Mizutani, Yasuyoshi; Shiogama, Kazuya; Onouchi, Takanori; Sakurai, Kouhei; Inada, Ken-ichi; Tsutsumi, Yutaka

2016-01-01

In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders

Reductive methods for isotopic labeling of antibiotics

International Nuclear Information System (INIS)

Champney, W.S.

1989-01-01

Methods for the reductive methylation of the amino groups of eight different antibiotics using 3 HCOH or H 14 COH are presented. The reductive labeling of an additional seven antibiotics by NaB 3 H 4 is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented

Disclaimer labels on fashion magazine advertisements: effects on social comparison and body dissatisfaction.

Science.gov (United States)

Tiggemann, Marika; Slater, Amy; Bury, Belinda; Hawkins, Kimberley; Firth, Bonny

2013-01-01

Recent proposals across a number of Western countries have suggested that idealised media images should carry some sort of disclaimer informing readers when these images have been digitally enhanced. The present studies aimed to experimentally investigate the impact on women's body dissatisfaction of the addition of such warning labels to fashion magazine advertisements. Participants were 120 and 114 female undergraduate students in Experiment 1 and Experiment 2 respectively. In both experiments, participants viewed fashion magazine advertisements with either no warning label, a generic warning label, or a specific more detailed warning label. In neither experiment was there a significant effect of type of label. However, state appearance comparison was found to predict change in body dissatisfaction irrespective of condition. Unexpectedly, trait appearance comparison moderated the effect of label on body dissatisfaction, such that for women high on trait appearance comparison, exposure to specific warning labels actually resulted in increased body dissatisfaction. In sum, the present results showed no benefit of warning labels in ameliorating the known negative effect of viewing thin-ideal media images, and even suggested that one form of warning (specific) might be harmful for some individuals. Accordingly, it was concluded that more extensive research is required to guide the most effective use of disclaimer labels. Copyright © 2012 Elsevier Ltd. All rights reserved.

Automatic prostate MR image segmentation with sparse label propagation and domain-specific manifold regularization.

Science.gov (United States)

Liao, Shu; Gao, Yaozong; Shi, Yinghuan; Yousuf, Ambereen; Karademir, Ibrahim; Oto, Aytekin; Shen, Dinggang

2013-01-01

Automatic prostate segmentation in MR images plays an important role in prostate cancer diagnosis. However, there are two main challenges: (1) Large inter-subject prostate shape variations; (2) Inhomogeneous prostate appearance. To address these challenges, we propose a new hierarchical prostate MR segmentation method, with the main contributions lying in the following aspects: First, the most salient features are learnt from atlases based on a subclass discriminant analysis (SDA) method, which aims to find a discriminant feature subspace by simultaneously maximizing the inter-class distance and minimizing the intra-class variations. The projected features, instead of only voxel-wise intensity, will be served as anatomical signature of each voxel. Second, based on the projected features, a new multi-atlases sparse label fusion framework is proposed to estimate the prostate likelihood of each voxel in the target image from the coarse level. Third, a domain-specific semi-supervised manifold regularization method is proposed to incorporate the most reliable patient-specific information identified by the prostate likelihood map to refine the segmentation result from the fine level. Our method is evaluated on a T2 weighted prostate MR image dataset consisting of 66 patients and compared with two state-of-the-art segmentation methods. Experimental results show that our method consistently achieves the highest segmentation accuracies than other methods under comparison.

Imaging small human prostate cancer xenografts after pretargeting with bispecific bombesin-antibody complexes and targeting with high specific radioactivity labeled polymer-drug conjugates

International Nuclear Information System (INIS)

Patil, Vishwesh; Gada, Keyur; Panwar, Rajiv; Ferris, Craig; Khaw, Ban-An; Varvarigou, Alexandra; Majewski, Stan; Weisenberger, Andrew; Tekabe, Yared

2012-01-01

Pretargeting with bispecific monoclonal antibodies (bsMAb) for tumor imaging was developed to enhance target to background activity ratios. Visualization of tumors was achieved by the delivery of mono- and divalent radiolabeled haptens. To improve the ability to image tumors with bsMAb, we have combined the pretargeting approach with targeting of high specific activity radiotracer labeled negatively charged polymers. The tumor antigen-specific antibody was replaced with bombesin (Bom), a ligand that binds specifically to the growth receptors that are overexpressed by many tumors including prostate cancer. Bom-anti-diethylenetriaminepentaacetic acid (DTPA) bispecific antibody complexes were used to demonstrate pretargeting and imaging of very small human prostate cancer xenografts targeted with high specific activity 111 In- or 99m Tc-labeled negatively charged polymers. Bispecific antibody complexes consisting of intact anti-DTPA antibody or Fab' linked to Bom via thioether bonds (Bom-bsCx or Bom-bsFCx, respectively) were used to pretarget PC-3 human prostate cancer xenografts in SCID mice. Negative control mice were pretargeted with Bom or anti-DTPA Ab. 111 In-Labeled DTPA-succinyl polylysine (DSPL) was injected intravenously at 24 h (7.03 ± 1.74 or 6.88 ± 1.89 MBq 111 In-DSPL) after Bom-bsCx or 50 ± 5.34 MBq of 99m Tc-DSPL after Bom-bsFCx pretargeting, respectively. Planar or single photon emission computed tomography (SPECT)/CT gamma images were obtained for up to 3 h and only planar images at 24 h. After imaging, all mice were killed and biodistribution of 111 In or 99m Tc activities were determined by scintillation counting. Both planar and SPECT/CT imaging enabled detection of PC-3 prostate cancer lesions less than 1-2 mm in diameter in 1-3 h post 111 In-DSPL injection. No lesions were visualized in Bom or anti-DTPA Ab pretargeted controls. 111 In-DSPL activity in Bom-bsCx pretargeted tumors (1.21 ± 0.36%ID/g) was 5.4 times that in tumors pretargeted with

Specific and spatial labeling of P0-Cre versus Wnt1-Cre in cranial neural crest in early mouse embryos.

Science.gov (United States)

Chen, Guiqian; Ishan, Mohamed; Yang, Jingwen; Kishigami, Satoshi; Fukuda, Tomokazu; Scott, Greg; Ray, Manas K; Sun, Chenming; Chen, Shi-You; Komatsu, Yoshihiro; Mishina, Yuji; Liu, Hong-Xiang

2017-06-01

P0-Cre and Wnt1-Cre mouse lines have been widely used in combination with loxP-flanked mice to label and genetically modify neural crest (NC) cells and their derivatives. Wnt1-Cre has been regarded as the gold standard and there have been concerns about the specificity of P0-Cre because it is not clear about the timing and spatial distribution of the P0-Cre transgene in labeling NC cells at early embryonic stages. We re-visited P0-Cre and Wnt1-Cre models in the labeling of NC cells in early mouse embryos with a focus on cranial NC. We found that R26-lacZ Cre reporter responded to Cre activity more reliably than CAAG-lacZ Cre reporter during early embryogenesis. Cre immunosignals in P0-Cre and reporter (lacZ and RFP) activity in P0-Cre/R26-lacZ and P0-Cre/R26-RFP embryos was detected in the cranial NC and notochord regions in E8.0-9.5 (4-19 somites) embryos. P0-Cre transgene expression was observed in migrating NC cells and was more extensive in the forebrain and hindbrain but not apparent in the midbrain. Differences in the Cre distribution patterns of P0-Cre and Wnt1-Cre were profound in the midbrain and hindbrain regions, that is, extensive in the midbrain of Wnt1-Cre and in the hindbrain of P0-Cre embryos. The difference between P0-Cre and Wnt1-Cre in labeling cranial NC may provide a better explanation of the differential distributions of their NC derivatives and of the phenotypes caused by Cre-driven genetic modifications. © 2017 Wiley Periodicals, Inc.

Cyclic labellings with constraints at two distances

OpenAIRE

Leese, R; Noble, S D

2004-01-01

Motivated by problems in radio channel assignment, we consider the vertex-labelling of graphs with non-negative integers. The objective is to minimise the span of the labelling, subject to constraints imposed at graph distances one and two. We show that the minimum span is (up to rounding) a piecewise linear function of the constraints, and give a complete specification, together with associated optimal assignments, for trees and cycles.

The preparation of specifically 2H-labeled benz(a)anthracenes and 7,12-dimethylbenz(a)anthracenes

International Nuclear Information System (INIS)

Hallmark, R.K.; Manning, W.B.; Muschik, G.M.

1981-01-01

The 2 + 4 cycloaddition reaction of 1,4-naphthoquinone and substituted styrenes was used to prepare 1-, 2-, 3-, and 4-bromobenz[a]anthracene-7,12-diones (BADs). The corresponding bromobenz[a]anthracenes (BAs) were prepared by aluminum tricyclohexoxide reduction of the diones. Lithiation with t-butyllithium followed by quenching with deuterium oxide gave the specifically 2 H-labeled BAs with deuterium incorporations of > 95%. The 2-, 3-, and 4-bromo-7,12-dimethylbenz[a]anthracenes (DMBAs) were prepared from the bromo BADs in moderate yield by the classical Grignard procedure. Lithium aluminum deuteride reduction gave the 2 H-DMBAs. An alternate synthesis from 3,4-dihydrobenz[a]anthracene 1-(2H)- one was used for the preparation of 1- and 2- 2 H BAs and DMBAs. (author)

Method of preparing thymidine-5'-monophosphate specifically or nonspecifically labelled with 14C or with 3H

International Nuclear Information System (INIS)

Nejedly, Z.; Filip, J.; Ekl, J.; Kolina, J.; Votruba, I.; Skoda, J.

1977-01-01

The invention claims a method for labelled thymidine-5'-monophosphate preparation by cultivating a special thymine-dependent Escherichia coli SPT - strain in the optimum synthetic culture medium containing 0.8 to 1.2 g/ml of labelled thymine. Practically the whole amount of labelled thymine is utilized for cellular deoxyribonucleic acid synthesis. The radioactive biomass obtained is processed using such chemical and enzymatic decomposition procedures as to allow separating the labelled thymidine-5'-monophosphate as the only thymine reaction product. Experiments conducted showed that the radiochemical purity of the thymidine-5'-monophosphate obtained was better than 98%. The absence of other nonactive substances was confirmed by spectrophotometric analysis. The overall product activity was 92.3% of the activity of thymine-2- 14 C introduced in the reaction. (Ha)

Evolution of energy labelling: analysis of international dynamics

International Nuclear Information System (INIS)

Malandrino, Ornella; Roca, Emmanuele

2005-01-01

Currently there are large variety of tools that play a very important role in providing benchmarks for sustainability performance of products and guidance for consumers. The present paper analyses the most important programme related to energy labelling, a market based instrument mainly addressed to consumers, certifying that a specific product is energy efficient. Particular attention has been given to International and European Union Labels of the consumption of energy and other resources by household appliances, to the energy efficient labelling programmes for office equipment and the certification schemes of energy performance of buildings

Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling.

Science.gov (United States)

Schumacher, Dominik; Lemke, Oliver; Helma, Jonas; Gerszonowicz, Lena; Waller, Verena; Stoschek, Tina; Durkin, Patrick M; Budisa, Nediljko; Leonhardt, Heinrich; Keller, Bettina G; Hackenberger, Christian P R

2017-05-01

The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.

Fatty acids labelled in the. omega. -position with iodine isotopes

Energy Technology Data Exchange (ETDEWEB)

Mathieu, J.P.; Busquet, G.; Comet, M. (Universite Scientifique et Medicale de Grenoble, 38 - La Tronche (France)); Riche, F.; Vidal, M. (Laboratoire d' Etudes Dynamiques et Structurales de la Selectivite, 38 - Grenoble (France)); Coornaert, S.; Bardy, A. (CEA, Centre de Saclay, 91 - Gif-sur-Yvette (France)); Godart, J. (Institut des Sciences Nucleaires, 38 - Grenoble (France))

1982-01-01

The synthesis of saturated acetylenic and olefinic (Z or E) ..omega..-iodinated fatty acids has been carried out and their labelling with iodine-131 or 123 by exchange I/sup -/, *I/sup -/ has been studied. The influence of several parameters -water and fatty acid concentrations, specific activity, labelling solution acidity, iodine carrier presence- on this exchange reaction has been noted, enabling experimental conditions to be defined that produce labelling yields of greater than 95%. These results should lead to widespread clinical use of iodine labelled fatty acids.

Development of radiolabeling method for natural juvenile hormones

International Nuclear Information System (INIS)

Okuda, Takashi; Shiotsuki, Takahiro; Kotaki, Toyomi

2000-01-01

This study aimed to develop a new assay method for accurate determination of juvenile hormone (JH) using radioisotope. A new measuring method for JH was designed based on the principle of molecular competition. Namely, JH-binding protein in the insect was used to form a selective binding to JH. At first, corpus allatum was cultured in a medium containing 3 H or 14 C epoxyfarnesyldiazoacetate (EFDA), which is a photoaffinity labeling reagent and JH binding protein (JHBP) was purified using 3 H or 14 C labeled EFDA. Since several kinds of JH homologues different in the molecular structure have been known, it was needed to establish each labeling method appropriate to those homologues. Then, an assay method for micro-quantitative measurement of JH was established utilizing the competition between labeled natural JH and JHBP. Thus, the authors succeeded in the overexpression of the blood JHBA of kind of tabaco moth, H.virescens and also in cloning and overexpression of JHBP in the silk worm. It was confirmed that these JHBPs specifically bind to 3 H labeled JH3, leading to stable supply of blood JHBP. Thus, accurate assay for JH concentration became possible by the use of the labeled JH with natural configuration and the blood JHBP. In the course of this study, several findings were obtained as follows: The JHBP of a sting bug was different from the previously reported ones. The regulation of JH synthesis in the corpus allatum was closely related to the control of diapause in several insects. The JHBP isolated from the cytosol of silk gland was a new protein in respect of amino acid sequence. (M.N.)

Development of radiolabeling method for natural juvenile hormones

Energy Technology Data Exchange (ETDEWEB)

Okuda, Takashi; Shiotsuki, Takahiro; Kotaki, Toyomi [National Inst. of Sericultural and Entomological Science, Tsukuba, Ibaraki (Japan)

2000-02-01

This study aimed to develop a new assay method for accurate determination of juvenile hormone (JH) using radioisotope. A new measuring method for JH was designed based on the principle of molecular competition. Namely, JH-binding protein in the insect was used to form a selective binding to JH. At first, corpus allatum was cultured in a medium containing {sup 3}H or {sup 14}C epoxyfarnesyldiazoacetate (EFDA), which is a photoaffinity labeling reagent and JH binding protein (JHBP) was purified using {sup 3}H or {sup 14}C labeled EFDA. Since several kinds of JH homologues different in the molecular structure have been known, it was needed to establish each labeling method appropriate to those homologues. Then, an assay method for micro-quantitative measurement of JH was established utilizing the competition between labeled natural JH and JHBP. Thus, the authors succeeded in the overexpression of the blood JHBA of kind of tabaco moth, H.virescens and also in cloning and overexpression of JHBP in the silk worm. It was confirmed that these JHBPs specifically bind to {sup 3}H labeled JH3, leading to stable supply of blood JHBP. Thus, accurate assay for JH concentration became possible by the use of the labeled JH with natural configuration and the blood JHBP. In the course of this study, several findings were obtained as follows: The JHBP of a sting bug was different from the previously reported ones. The regulation of JH synthesis in the corpus allatum was closely related to the control of diapause in several insects. The JHBP isolated from the cytosol of silk gland was a new protein in respect of amino acid sequence. (M.N.)

Facial Action Unit Recognition under Incomplete Data Based on Multi-label Learning with Missing Labels

KAUST Repository

Li, Yongqiang

2016-07-07

Facial action unit (AU) recognition has been applied in a wild range of fields, and has attracted great attention in the past two decades. Most existing works on AU recognition assumed that the complete label assignment for each training image is available, which is often not the case in practice. Labeling AU is expensive and time consuming process. Moreover, due to the AU ambiguity and subjective difference, some AUs are difficult to label reliably and confidently. Many AU recognition works try to train the classifier for each AU independently, which is of high computation cost and ignores the dependency among different AUs. In this work, we formulate AU recognition under incomplete data as a multi-label learning with missing labels (MLML) problem. Most existing MLML methods usually employ the same features for all classes. However, we find this setting is unreasonable in AU recognition, as the occurrence of different AUs produce changes of skin surface displacement or face appearance in different face regions. If using the shared features for all AUs, much noise will be involved due to the occurrence of other AUs. Consequently, the changes of the specific AUs cannot be clearly highlighted, leading to the performance degradation. Instead, we propose to extract the most discriminative features for each AU individually, which are learned by the supervised learning method. The learned features are further embedded into the instance-level label smoothness term of our model, which also includes the label consistency and the class-level label smoothness. Both a global solution using st-cut and an approximated solution using conjugate gradient (CG) descent are provided. Experiments on both posed and spontaneous facial expression databases demonstrate the superiority of the proposed method in comparison with several state-of-the-art works.

Facial Action Unit Recognition under Incomplete Data Based on Multi-label Learning with Missing Labels

KAUST Repository

Li, Yongqiang; Wu, Baoyuan; Ghanem, Bernard; Zhao, Yongping; Yao, Hongxun; Ji, Qiang

2016-01-01

Facial action unit (AU) recognition has been applied in a wild range of fields, and has attracted great attention in the past two decades. Most existing works on AU recognition assumed that the complete label assignment for each training image is available, which is often not the case in practice. Labeling AU is expensive and time consuming process. Moreover, due to the AU ambiguity and subjective difference, some AUs are difficult to label reliably and confidently. Many AU recognition works try to train the classifier for each AU independently, which is of high computation cost and ignores the dependency among different AUs. In this work, we formulate AU recognition under incomplete data as a multi-label learning with missing labels (MLML) problem. Most existing MLML methods usually employ the same features for all classes. However, we find this setting is unreasonable in AU recognition, as the occurrence of different AUs produce changes of skin surface displacement or face appearance in different face regions. If using the shared features for all AUs, much noise will be involved due to the occurrence of other AUs. Consequently, the changes of the specific AUs cannot be clearly highlighted, leading to the performance degradation. Instead, we propose to extract the most discriminative features for each AU individually, which are learned by the supervised learning method. The learned features are further embedded into the instance-level label smoothness term of our model, which also includes the label consistency and the class-level label smoothness. Both a global solution using st-cut and an approximated solution using conjugate gradient (CG) descent are provided. Experiments on both posed and spontaneous facial expression databases demonstrate the superiority of the proposed method in comparison with several state-of-the-art works.

Disentangling the Origin of the Kok Effect Using Position Specific Glucose Labeling in Sunflower Leaves

Science.gov (United States)

Gauthier, P. P.; Bender, M. L.; Saenz, N.

2015-12-01

In plants, leaf mitochondrial respiratory CO2 release is inhibited by light. Bessel Kok first demonstrated this inhibition in 1948. Based on curves of CO2 assimilation vs irradiance, it is understood that respiration is maximal in the dark. It then frequently decreases linearly with irradiance until reaching some value around the compensation point, beyond which it is constant. CO2 released by mitochondrial respiration is the result of decarboxylation through pyruvate dehydrogenase (PDH), the tricarboxylic acid pathway (TCAP) and the oxydative pentose phosphate pathway (OPPP). The overall activity of these three reactions is reduced by light. However, their individual contributions to the Kok effect are unknown. We measured the rate of decarboxylation of glucose, position-specifically labeled with 13C, to evaluate the participation of PDH, TCAP and OPPP in the Kok effect of sunflower. Leaves were fed with labeled glucose through their transpiration stream. The δ13C of the CO2 released by the leaf was then measured as a function of irradiance. The results showed that the inhibition of the decarboxylation of carbon positions 3 and 4 in glucose is at the origin of the Kok effect. These are the positions of carbon atoms decarboxylated by PDH. In addition, the rate of decarboxylation of position 1 was not different in the light and in the dark. Thus OPPP plays no role in the Kok effect in sunflower leaves. This work improves our current understanding of leaf mitochondrial respiratory metabolism in the light. Invoking the Kok effect in plant physiology models should improve our ability to simulate carbon fluxes of terrestrial ecosystems.

Glucose transporter of the human brain and blood-brain barrier

International Nuclear Information System (INIS)

Kalaria, R.N.; Gravina, S.A.; Schmidley, J.W.; Perry, G.; Harik, S.I.

1988-01-01

We identified and characterized the glucose transporter in the human cerebral cortex, cerebral microvessels, and choroid plexus by specific D-glucose-displaceable [3H]cytochalasin B binding. The binding was saturable, with a dissociation constant less than 1 microM. Maximal binding capacity was approximately 7 pmol/mg protein in the cerebral cortex, approximately 42 pmol/mg protein in brain microvessels, and approximately 27 pmol/mg protein in the choroid plexus. Several hexoses displaced specific [3H]cytochalasin B binding to microvessels in a rank-order that correlated well with their known ability to cross the blood-brain barrier; the only exception was 2-deoxy-D-glucose, which had much higher affinity for the glucose transporter than the natural substrate, D-glucose. Irreversible photoaffinity labeling of the glucose transporter of microvessels with [3H]cytochalasin B, followed by solubilization and polyacrylamide gel electrophoresis, labeled a protein band with an average molecular weight of approximately 55,000. Monoclonal and polyclonal antibodies specific to the human erythrocyte glucose transporter immunocytochemically stained brain blood vessels and the few trapped erythrocytes in situ, with minimal staining of the neuropil. In the choroid plexus, blood vessels did not stain, but the epithelium reacted positively. We conclude that human brain microvessels are richly endowed with a glucose transport moiety similar in molecular weight and antigenic characteristics to that of human erythrocytes and brain microvessels of other mammalian species

Tritium-labelled abscisic acid

International Nuclear Information System (INIS)

Pluciennik, H.; Michalski, L.

1991-01-01

A simple method for the preparation of biologically active abscisic acid (growth inhibiting plant hormone) labelled with tritium is described. The product obtained has a specific radioactivity of 1.12 GBq mmol -1 : the yield is about 60% as compared to the initial amount of the substance used. (author) 7 refs.; 2 figs

An exploratory study of drinkers views of health information and warning labels on alcohol containers.

Science.gov (United States)

Thomson, Lisa M; Vandenberg, Brian; Fitzgerald, John L

2012-03-01

To identify general and specific features of health information warning labels on alcohol beverage containers that could potentially inform the development and implementation of a new labelling regime in Australia. Mixed methods, including a cross-sectional population survey and a qualitative study of knowledge, attitudes and behaviours regarding alcohol beverage labelling. The population survey used computer-assisted telephone interviews of 1500 persons in Victoria, Australia to gauge the level of support for health information and warning labels. The qualitative study used six focus groups to test the suitability of 12 prototype labels that were placed in situ on a variety of alcohol beverage containers. The telephone survey found 80% to 90% support for a range of information that could potentially be mandated by government authorities for inclusion on labels (nutritional information, alcohol content, health warning, images). Focus group testing of the prototype label designs found that labels should be integrated with other alcohol-related health messages, such as government social advertising campaigns, and specific labels should be matched appropriately to specific consumer groups and beverage types. There are high levels of public support for health information and warning labels on alcohol beverages. This study contributes much needed empirical guidance for developing alcohol beverage labelling strategies in an Australian context. © 2011 Australasian Professional Society on Alcohol and other Drugs.

Studies on quality control of technetium-99m labelling kits

International Nuclear Information System (INIS)

Kim, Jae Rok; Park, Kyung Bae; Awh, Ok Doo

1987-12-01

Various experiments for the quality control of Tc-99m labelled radiopharmaceuticals such as Tc-99m-phytate, Tc-99m-MDP, Tc-99m-Tin Colloid, Tc-99m-DISIDA, Tc-99m-DTPA,Tc-99m-DMSA, Tc-99m-Gulcoheptonate, TC-99m-Pyrophosphate, Tc-99m-HSA, and Tc-99m-HAM were carried out. Labelling yield and radiochemical purity of each of the instant labelling kit of KAERI made were determined by means of radiochromatography. Biodistribution in mice and whole body or specific organ imagings of rabbits were also carried out and discussed the relationship between the data of biodistributions and radiochemical purities. Labelling yeilds were above 98% for almost all of the labelling kits. The radio-pharmaceuticals were accumulated at each target organ with moderate specifities. In case of radiochemical purity of above 98%, the biodistribution and gamma imagings were also better. The kits of MDP and DISIDA were stable at least for four moths while the other kits at least eight months. (Author)

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

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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.

Mobility and height detection of particle labels in an optical evanescent wave biosensor with single-label resolution

Energy Technology Data Exchange (ETDEWEB)

Van Ommering, Kim; Koets, Marjo; Schleipen, Jean J H B; Prins, Menno W J [Philips Research Laboratories, 5656 AE Eindhoven (Netherlands); Somers, Philip A; Van IJzendoorn, Leo J, E-mail: menno.prins@philips.co [Department of Applied Physics, Eindhoven University of Technology, 5600 MB Eindhoven (Netherlands)

2010-04-21

Particle labels are used in biosensors to detect the presence and concentration of analyte molecules. In this paper we demonstrate an optical technique to measure the mobility and height of bound particle labels on a biosensor surface with single-label resolution. The technique is based on the detection of the particle-induced light scattering in an optical evanescent field. We show that the thermal particle motion in the optical evanescent field leads to intensity fluctuations that can accurately be detected. The technique is demonstrated using 290 bp (99 nm) DNA as an analyte and using polystyrene particles and magnetic particles with diameters between 500 and 1000 nm as labels. The particle intensity histograms show that quantitative height measurements are obtained for particles with uniform optical properties, and the intensity versus position plots reflect the analyte-antibody orientation and the analyte flexibility. The novel optical detection technique will lead to biosensors with very high sensitivity and specificity.

Learning from Weak and Noisy Labels for Semantic Segmentation

KAUST Repository

Lu, Zhiwu

2016-04-08

A weakly supervised semantic segmentation (WSSS) method aims to learn a segmentation model from weak (image-level) as opposed to strong (pixel-level) labels. By avoiding the tedious pixel-level annotation process, it can exploit the unlimited supply of user-tagged images from media-sharing sites such as Flickr for large scale applications. However, these ‘free’ tags/labels are often noisy and few existing works address the problem of learning with both weak and noisy labels. In this work, we cast the WSSS problem into a label noise reduction problem. Specifically, after segmenting each image into a set of superpixels, the weak and potentially noisy image-level labels are propagated to the superpixel level resulting in highly noisy labels; the key to semantic segmentation is thus to identify and correct the superpixel noisy labels. To this end, a novel L1-optimisation based sparse learning model is formulated to directly and explicitly detect noisy labels. To solve the L1-optimisation problem, we further develop an efficient learning algorithm by introducing an intermediate labelling variable. Extensive experiments on three benchmark datasets show that our method yields state-of-the-art results given noise-free labels, whilst significantly outperforming the existing methods when the weak labels are also noisy.

Learning from Weak and Noisy Labels for Semantic Segmentation

KAUST Repository

Lu, Zhiwu; Fu, Zhenyong; Xiang, Tao; Han, Peng; Wang, Liwei; Gao, Xin

2016-01-01

A weakly supervised semantic segmentation (WSSS) method aims to learn a segmentation model from weak (image-level) as opposed to strong (pixel-level) labels. By avoiding the tedious pixel-level annotation process, it can exploit the unlimited supply of user-tagged images from media-sharing sites such as Flickr for large scale applications. However, these ‘free’ tags/labels are often noisy and few existing works address the problem of learning with both weak and noisy labels. In this work, we cast the WSSS problem into a label noise reduction problem. Specifically, after segmenting each image into a set of superpixels, the weak and potentially noisy image-level labels are propagated to the superpixel level resulting in highly noisy labels; the key to semantic segmentation is thus to identify and correct the superpixel noisy labels. To this end, a novel L1-optimisation based sparse learning model is formulated to directly and explicitly detect noisy labels. To solve the L1-optimisation problem, we further develop an efficient learning algorithm by introducing an intermediate labelling variable. Extensive experiments on three benchmark datasets show that our method yields state-of-the-art results given noise-free labels, whilst significantly outperforming the existing methods when the weak labels are also noisy.

Labeling of antibodies with a /sup 67/Ga-phenolic aminocarboxylic acid chelate. Pt. 1. Chemistry and labeling technique

Energy Technology Data Exchange (ETDEWEB)

Schuhmacher, J.; Matys, R.; Hauser, H.; Maier-Borst, W.; Matzku, S.

1986-11-01

As a chelating agent for labeling antibodies (Abs) with metallic radionuclides, a propionic acid substituted ethylenediamine N, N'-di-((o-hydroxyphenyl) acetic acid) (P-EDDHA), which tighly complexes /sup 67/Ga, was synthetized. The /sup 67/Ga-P-EDDHA chelate was coupled in aqueous solution to IgG at a molar ratio of 1:1 via carbodiimide. The average coupling yield was 15%. A specific activity of 4 mCi/mg IgG could be obtained with commercially supplied /sup 67/Ga. In vitro stability was evaluated in human serum at 37/sup 0/C and showed a half-life of about 120 h for the release of /sup 67/Ga from the labeled Ab during the initial phase of incubation. This in vitro halflife is similar to that measured for /sup 111/In-DTPA labeled Abs. Because of the high stability of the /sup 67/Ga-P-EDDHA chelate, the in vivo formation of radioactive lebeled transferrin by transchelation, as described for /sup 111/In-DTPA labeled Abs, should, however, be reduced by this labeling technique.

Specific 14C labelling of 3-phosphoglyceric acid by light enhanced dark CO2 fixation in tea leaves

International Nuclear Information System (INIS)

Aoki, Satoshi

1984-01-01

Conditions for light enhanced dark CO 2 fixation (LED), products of LED and distribution pattern of 14 C of 3-phosphoglyceric acid (PGA) were investigated. By LED, 14 C-bicarbonate was abruptly and temporarily incorporated in single cells and discs of tea leaves. Optimal conditions of temperature, preillumination period and light intensity for LED in single cells were 28 deg C, 10 min and 20 klx respectively, and 20 deg C, 20 - 30 min and 40 - 80 klx respectively, in leaf discs. By photosynthesis for 30 sec and 60 sec of leaf discs, although 14 C-bicarbonate was considerably incorporated into PGA and phosphateesters, 14 C was incorporated into malate, serin+glycine and sucrose, too. Malate was predominantly labelled by dark fixation. On the other hand, by LED, 14 C-bicarbonate was incorporated into PGA. PGA was degradated by the modified Sakami's method and their distribution pattern was analyzed. By photosynthesis for 60 sec, 14 C of C-1 carbon (carboxylic carbon), C-2 carbon and C-3 carbon of PGA were 70, 17 and 13 %, respectively. By LED of 60 sec, however, 97 % of 14 C was at C-1. From these results, it is clear that carboxylic carbon of PGA was specifically labelled from 14 C-bicarbonate by LED. (Kubozono, M.)

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

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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.

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

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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.

Specific binding assay technique; standardization of reagent

International Nuclear Information System (INIS)

Huggins, K.G.; Roitt, I.M.

1979-01-01

The standardization of a labelled constituent, such as anti-IgE, for use in a specific binding assay method is disclosed. A labelled ligand, such as IgE, is standardized against a ligand reference substance, such as WHO standard IgE, to determine the weight of IgE protein represented by the labelled ligand. Anti-light chain antibodies are contacted with varying concentrations of the labelled ligand. The ligand is then contacted with the labelled constituent which is then quantitated in relation to the amount of ligand protein present. The preparation of 131 I-labelled IgE is described. Also disclosed is an improved specific binding assay test method for determining the potency of an allergen extract in serum from an allergic individual. The improvement involved using a parallel model system of a second complex which consisted of anti-light chain antibodies, labelled ligand and the standardized labelled constituent (anti-IgE). The amount of standardized labelled constituent bound to the ligand in the first complex was determined, as described above, and the weight of ligand inhibited by addition of soluble allergen was then used as a measure of the potency of the allergen extract. (author)

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

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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.

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

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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.

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

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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.

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

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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.

Radioactive labelling of alkaloids with morphine skeleton

International Nuclear Information System (INIS)

Toth, Geza; Sirokman, Ferenc

1985-01-01

Results achieved by the sup(14)C, sup(125)I and sup(3)H labelling of alkaloids with morphine skeleton for kinetic, receptor, metabolims and pharmacological investigations are summarized and evaluated. The methods for the preparation of sup(3)H labelled dihydromorphine, dihydroethylmorphine, dihydrocodeine, naloxone and naloxazone are described. The compounds have higher specific molar activity than those referred to in literature which makes them suitable for a number of investigations. (author)

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

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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.

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

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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.

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

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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.

Monoclonal antibodies and coupling reagents to cell membrane proteins for leukocyte labeling

International Nuclear Information System (INIS)

McAfee, J.G.; Gagne, G.; Subramanian, G.; Schneider, R.F.

1984-01-01

Current gamma-emitting agents for tagging leukocytes, In-111 oxine or tropolone, label all cell types indiscriminantly, and nuclear localization in lymphocytes results in radiation damage. Coupling reagents and murine monoclonal antibodies (Mab) specific for cell surface antigens of human leukocytes were tried as cell labeling agents to avoid nuclear localization. 10/sup 8/ mixed human leukocytes in Hepes buffer were added to tubes coated with 5 mg of dry cyclic dianhydride of DTPA for 15 minutes at room temperature. After washing, 0.1 ml of In-111 Cl in ACD (pH 6.8) was added. After 30 minutes, a cell labeling yield of 23% was obtained. Washing the cells in an elutriation centrifuge showed that this label was irreversible. Mab for cell surface antigens of human granulocytes were labeled with 300 μCi of I-125 using the Iodobead technic and unbound activity was removed by gel column chromatography. 1-10 μg were added to 10/sup 8/ mixed leukocytes in 0.5 ml plasma or saline for 1 hr. With Mab anti-leu M4 (clone G7 E11), an IgM, the cell labeling yield was 21%, irreversible, and specific for granulocytes. With anti-human leukocyte Mab NEI-042 (clone 9.4), and IgG2a, and anti-granulocyte Mab MAS-065 (clone FMCl1) an IgG1, the cell labeling was relatively unstable. Labeling of leukocyte subpopulations with Mab is feasible, and the binding of multivalent IgM is stronger than that of other immunoglobulins. DTPA cyclic anhydride is firmly bound to cell membranes, but the labeling is non-specific

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

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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.

5'Azido-N-1-napthylphthalamic acid, a photolabile analog of N-1-naphthylphthalamic acid

International Nuclear Information System (INIS)

Voet, J.G.; Howley, K.S.; Shumsky, J.S.

1987-01-01

A photolabile analog of N-1-naphthylphthalamic acid (NPA), 5'-azido-N-1-naphthylphthalamic acid (Az-NPA), has been synthesized and characterized. This potential photoaffinity label for the plasma membrane NPA binding protein competes with [ 3 H]NPA for binding sites on Curcurbita pepo L. (zucchini) hypocotyl cell membranes with K/sub 0.5/ = 2.8 x 10 -7 molar. The K/sub 0.5/ for NPA under these conditions is 2 x 10 -8 molar, indicating that the affinity of Az-NPA for the membranes is only 14-fold lower than NPA. While the binding of Az-NPA to NPA binding sites is reversible in the dark, exposure of the Az-NPA treated membranes to light results in a 30% loss in [ 3 H]NPA binding ability. Pretreatment of the membranes with NPA protects the membranes against photodestruction of [ 3 H]NPA binding sites by Az-NPA supporting the conclusion that Az-NPA destroys these sites by specific covalent attachment

Labelled antibody techniques in glycoprotein estimation

International Nuclear Information System (INIS)

Hazra, D.K.; Ekins, R.P.; Edwards, R.; Williams, E.S.

1977-01-01

The problems in the radioimmunoassay of the glycoprotein hormones (pituitary LH, FSH and TSH and human chlorionic gonadotrophin HGG) are reviewed viz: limited specificity and sensitivity in the clinical context, interpretation of disparity between bioassay and radioimmunoassay, and interlaboratory variability. The advantages and limitations of the labelled antibody techniques - classical immonoradiometric methods and 2-site or 125 I-anti-IgG indirect labelling modifications are reviewed in general, and their theoretical potential in glycoprotein assays examined in the light of previous work. Preliminary experiments in the development of coated tube 2-site assay for glycoproteins using 125 I anti-IgG labelling are described, including conditions for maximizing solid phase extraction of the antigen, iodination of anti-IgG, and assay conditions such as effects of temperature of incubation with antigen 'hormonefree serum', heterologous serum and detergent washing. Experiments with extraction and antigen-specific antisera raised in the same or different species are described as exemplified by LH and TSH assay systems, the latter apparently promising greater sensitivity than radioimmunoassay. Proposed experimental and mathematical optimisation and validation of the method as an assay system is outlined, and the areas for further work delineated. (orig.) [de

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

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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.

Efficient Multi-Label Feature Selection Using Entropy-Based Label Selection

Directory of Open Access Journals (Sweden)

Jaesung Lee

2016-11-01

Full Text Available Multi-label feature selection is designed to select a subset of features according to their importance to multiple labels. This task can be achieved by ranking the dependencies of features and selecting the features with the highest rankings. In a multi-label feature selection problem, the algorithm may be faced with a dataset containing a large number of labels. Because the computational cost of multi-label feature selection increases according to the number of labels, the algorithm may suffer from a degradation in performance when processing very large datasets. In this study, we propose an efficient multi-label feature selection method based on an information-theoretic label selection strategy. By identifying a subset of labels that significantly influence the importance of features, the proposed method efficiently outputs a feature subset. Experimental results demonstrate that the proposed method can identify a feature subset much faster than conventional multi-label feature selection methods for large multi-label datasets.

Labeling proteins on live mammalian cells using click chemistry.

Science.gov (United States)

Nikić, Ivana; Kang, Jun Hee; Girona, Gemma Estrada; Aramburu, Iker Valle; Lemke, Edward A

2015-05-01

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

Rapid and specific biotin labelling of the erythrocyte surface antigens of both cultured and ex-vivo Plasmodium parasites

Directory of Open Access Journals (Sweden)

Thompson Joanne

2007-05-01

Full Text Available Abstract Background Sensitive detection of parasite surface antigens expressed on erythrocyte membranes is necessary to further analyse the molecular pathology of malaria. This study describes a modified biotin labelling/osmotic lysis method which rapidly produces membrane extracts enriched for labelled surface antigens and also improves the efficiency of antigen recovery compared with traditional detergent extraction and surface radio-iodination. The method can also be used with ex-vivo parasites. Methods After surface labelling with biotin in the presence of the inhibitor furosemide, detergent extraction and osmotic lysis methods of enriching for the membrane fractions were compared to determine the efficiency of purification and recovery. Biotin-labelled proteins were identified on silver-stained SDS-polyacrylamide gels. Results Detergent extraction and osmotic lysis were compared for their capacity to purify biotin-labelled Plasmodium falciparum and Plasmodium chabaudi erythrocyte surface antigens. The pellet fraction formed after osmotic lysis of P. falciparum-infected erythrocytes is notably enriched in suface antigens, including PfEMP1, when compared to detergent extraction. There is also reduced co-extraction of host proteins such as spectrin and Band 3. Conclusion Biotinylation and osmotic lysis provides an improved method to label and purify parasitised erythrocyte surface antigen extracts from both in vitro and ex vivo Plasmodium parasite preparations.

Multi-label Learning with Missing Labels Using Mixed Dependency Graphs

KAUST Repository

Wu, Baoyuan; Jia, Fan; Liu, Wei; Ghanem, Bernard; Lyu, Siwei

2018-01-01

This work focuses on the problem of multi-label learning with missing labels (MLML), which aims to label each test instance with multiple class labels given training instances that have an incomplete/partial set of these labels (i.e., some

Recent progress in fluorine-18 labelled peptide radiopharmaceuticals

Energy Technology Data Exchange (ETDEWEB)

Okarvi, S.M. [Cyclotron and Radiopharmaceuticals Department, King Faisal Specialist Hospital and Research Centre, Riyadh (Saudi Arabia)

2001-07-01

The application of biologically active peptides labelled with positron-emitting nuclides has emerged as a useful and interesting field in nuclear medicine. Small synthetic receptor-binding peptides are currently the preferred agents over proteins and antibodies for diagnostic imaging of various tumours. Due to the smaller size of peptides, both higher target-to-background ratios and rapid blood clearance can often be achieved with radiolabelled peptides. Hence, short-lived positron emission tomography (PET) isotopes are potential candidates for labelling peptides. Among a number of positron-emitting nuclides, fluorine-18 appears to be the best candidate for labelling bioactive peptides by virtue of its favourable physical and nuclear characteristics. The major disadvantage of labelling peptides with {sup 18}F is the laborious and time-consuming preparation of the {sup 18}F labelling agents. In recent years, various techniques have been developed which allow efficient labelling of peptides with {sup 18}F without affecting their receptor-binding properties. Moreover, the development of a variety of prosthetic groups has facilitated the efficient and site-specific labelling of peptides with {sup 18}F. The {sup 18}F-labelled peptides hold enormous clinical potential owing to their ability to quantitatively detect and characterise a wide variety of human diseases when using PET. Recently, a number of {sup 18}F-labelled bioactive peptides have shown great promise as diagnostic imaging agents. This review presents the recent developments in {sup 18}F-labelled biologically active peptides used in PET. (orig.)

Recent progress in fluorine-18 labelled peptide radiopharmaceuticals

International Nuclear Information System (INIS)

Okarvi, S.M.

2001-01-01

The application of biologically active peptides labelled with positron-emitting nuclides has emerged as a useful and interesting field in nuclear medicine. Small synthetic receptor-binding peptides are currently the preferred agents over proteins and antibodies for diagnostic imaging of various tumours. Due to the smaller size of peptides, both higher target-to-background ratios and rapid blood clearance can often be achieved with radiolabelled peptides. Hence, short-lived positron emission tomography (PET) isotopes are potential candidates for labelling peptides. Among a number of positron-emitting nuclides, fluorine-18 appears to be the best candidate for labelling bioactive peptides by virtue of its favourable physical and nuclear characteristics. The major disadvantage of labelling peptides with 18 F is the laborious and time-consuming preparation of the 18 F labelling agents. In recent years, various techniques have been developed which allow efficient labelling of peptides with 18 F without affecting their receptor-binding properties. Moreover, the development of a variety of prosthetic groups has facilitated the efficient and site-specific labelling of peptides with 18 F. The 18 F-labelled peptides hold enormous clinical potential owing to their ability to quantitatively detect and characterise a wide variety of human diseases when using PET. Recently, a number of 18 F-labelled bioactive peptides have shown great promise as diagnostic imaging agents. This review presents the recent developments in 18 F-labelled biologically active peptides used in PET. (orig.)

Private label brand equity: a conceptual framework

OpenAIRE

Xara-Brasil, Duarte; Marreiros, Cristina; Dionísio, Andreia

2012-01-01

Trabalho apresentado na AMA/ACRA First Triennial Conference, 18-21 de abril de 2012, Seattle, USA This paper presents a conceptual framework to analyze private label brand equity in a retail context. Several authors proposed brand equity models as Aaker (1996), Keller (1993) and Yoo and Donthu (2001), and specific research has been done in retail industry (Jara & Cliquet, 2009), (Pappu & Quester, 2006). To study private label brand equity, we suggest a framework based on the Yo...

Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samples

DEFF Research Database (Denmark)

Truhlar, Stephanie M E; Cervantes, Carla F; Torpey, Justin W

2008-01-01

. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" (15)N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot...

Simultaneous neuron- and astrocyte-specific fluorescent marking

Energy Technology Data Exchange (ETDEWEB)

Schulze, Wiebke [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Hayata-Takano, Atsuko [Molecular Research Center for Children' s Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, 2-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kamo, Toshihiko [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nakazawa, Takanobu, E-mail: takanobunakazawa-tky@umin.ac.jp [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Nagayasu, Kazuki [iPS Cell-based Research Project on Brain Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kasai, Atsushi; Seiriki, Kaoru [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Interdisciplinary Program for Biomedical Sciences, Institute for Academic Initiatives, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Shintani, Norihito [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ago, Yukio [Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Farfan, Camille [Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); and others

2015-03-27

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein.

Simultaneous neuron- and astrocyte-specific fluorescent marking

International Nuclear Information System (INIS)

Schulze, Wiebke; Hayata-Takano, Atsuko; Kamo, Toshihiko; Nakazawa, Takanobu; Nagayasu, Kazuki; Kasai, Atsushi; Seiriki, Kaoru; Shintani, Norihito; Ago, Yukio; Farfan, Camille

2015-01-01

Systematic and simultaneous analysis of multiple cell types in the brain is becoming important, but such tools have not yet been adequately developed. Here, we aimed to generate a method for the specific fluorescent labeling of neurons and astrocytes, two major cell types in the brain, and we have developed lentiviral vectors to express the red fluorescent protein tdTomato in neurons and the enhanced green fluorescent protein (EGFP) in astrocytes. Importantly, both fluorescent proteins are fused to histone 2B protein (H2B) to confer nuclear localization to distinguish between single cells. We also constructed several expression constructs, including a tandem alignment of the neuron- and astrocyte-expression cassettes for simultaneous labeling. Introducing these vectors and constructs in vitro and in vivo resulted in cell type-specific and nuclear-localized fluorescence signals enabling easy detection and distinguishability of neurons and astrocytes. This tool is expected to be utilized for the simultaneous analysis of changes in neurons and astrocytes in healthy and diseased brains. - Highlights: • We develop a method for the specific fluorescent labeling of neurons and astrocytes. • Neuron-specific labeling is achieved using Scg10 and synapsin promoters. • Astrocyte-specific labeling is generated using the minimal GFAP promoter. • Nuclear localization of fluorescent proteins is achieved with histone 2B protein

Progress of new label-free techniques for biosensors: a review.

Science.gov (United States)

Sang, Shengbo; Wang, Yajun; Feng, Qiliang; Wei, Ye; Ji, Jianlong; Zhang, Wendong

2016-01-01

The detection techniques used in biosensors can be broadly classified into label-based and label-free. Label-based detection relies on the specific properties of labels for detecting a particular target. In contrast, label-free detection is suitable for the target molecules that are not labeled or the screening of analytes which are not easy to tag. Also, more types of label-free biosensors have emerged with developments in biotechnology. The latest developed techniques in label-free biosensors, such as field-effect transistors-based biosensors including carbon nanotube field-effect transistor biosensors, graphene field-effect transistor biosensors and silicon nanowire field-effect transistor biosensors, magnetoelastic biosensors, optical-based biosensors, surface stress-based biosensors and other type of biosensors based on the nanotechnology are discussed. The sensing principles, configurations, sensing performance, applications, advantages and restriction of different label-free based biosensors are considered and discussed in this review. Most concepts included in this survey could certainly be applied to the development of this kind of biosensor in the future.

Searching for flavor labels in food products: the influence of color-flavor congruence and association strength.

Science.gov (United States)

Velasco, Carlos; Wan, Xiaoang; Knoeferle, Klemens; Zhou, Xi; Salgado-Montejo, Alejandro; Spence, Charles

2015-01-01

Prior research provides robust support for the existence of a number of associations between colors and flavors. In the present study, we examined whether congruent (vs. incongruent) combinations of product packaging colors and flavor labels would facilitate visual search for products labeled with specific flavors. The two experiments reported here document a Stroop-like effect between flavor words and packaging colors. The participants were able to search for packaging flavor labels more rapidly when the color of the packaging was congruent with the flavor label (e.g., red/tomato) than when it was incongruent (e.g., yellow/tomato). In addition, when the packaging color was incongruent, those flavor labels that were more strongly associated with a specific color yielded slower reaction times and more errors (Stroop interference) than those that were less strongly tied to a specific color. Importantly, search efficiency was affected both by color/flavor congruence and association strength. Taken together, these results therefore highlight the role of color congruence and color-word association strength when it comes to searching for specific flavor labels.

Improved tumor localization with (strept)avidin and labeled biotin as a substitute for antibody

International Nuclear Information System (INIS)

Hnatowich, D.J.; Fritz, B.; Virzi, F.; Mardirossian, G.; Rusckowski, M.

1993-01-01

We have investigated tumor localization with labeled biotin administered subsequent to unlabeled and unconjugated streptavidin. Nude mice bearing anti-CEA tumors (LS174T) received 10 μg of 111 In-labeled anti-CEA antibody (C110) or 111 In-labeled streptavidin with sacrifice 5 h later. In an examination of pretargeting, other animals received 50 μg of unlabeled streptavidin followed 3 h later with 1 μg of 111 In-labeled biotin (EB 1 ) and sacrifice 2 h later. The biodistribution of labeled streptavidin was similar to that of labeled specific antibody except for lower blood and higher kidney levels. Tumor levels were also lower with labeled streptavidin but, because of still lower levels in liver and blood, the tumor/normal tissue ratios were improved. When unlabeled streptavidin was administered and followed by labeled biotin (pretargeting), tumor levels were further reduced modestly; however, normal tissue levels were greatly reduced such that the tumor/blood and tumor/liver ratios were 10.6 and 2.2 vs 1.5 and 0.5 for the specific antibody. Improvements were seen in all tissues sampled with the exception of kidney and muscle. A further control of labeled biotin alone (without the preinjection of streptavidin) showed minimal accumulations in all tissues with the exception of kidneys. In conclusion, the accumulation of (strept)avidin by passive diffusion in tumor may be comparable, at early times, to the accumulation of specific antibody. (author)

Improved tumor localization with (strept)avidin and labeled biotin as a substitute for antibody

International Nuclear Information System (INIS)

Hnatowich, D.J.; Fritz, B.; Virzi, F.; Mardirossian, G.; Rusckowski, M.

1993-01-01

We have investigated tumor localization with labeled biotin administered subsequent to unlabeled and unconjugated streptavidin. Nude mice bearing anti-CEA tumors (LS174T) received 10 μg of 111 In-labeled anti-CEA antibody (C110) or 111 In-labeled streptavidin with sacrifice 5 h later. In an examination of pretargeting, other animals received 50 μg of unlabeled streptavidin followed 3 h later with 1 μg of 111 In-labeled biotin (EB 1 ) and sacrifice 2 h later. The biodistribution of labeled streptavidin was similar to that of labeled specific antibody except for lower blood and higher kidney levels. Tumor levels were also lower with labeled streptavidin but, because of still lower levels in liver and blood, the tumor/normal tissue ratios were improved. When unlabeled streptavidin was administered and followed by labeled biotin (pretargeting), tumor levels were further reduced modestly; however, normal tissue levels were greatly reduced such that the tumor/blood and tumor/liver ratios were 10.6 and 2.2 vs 1.5 and 0.5 for the specific antibody. Improvements were seen in all tissues sampled with the exception of kidney and muscle. A further control of labeled biotin alone showed minimal accumulation in all tissues with the exception of kidneys. In conclusion, the accumulation of (strept)avidin by passive diffusion in tumor may be comparable, at early times, to the accumulation of specific antibody. By combining the administration of unlabeled (strept)avidin with labeled biotin, tumor targeting may potentially be improved. (author)

The tritium labeling of Butibufen by heterogeneous catalytic exchange

International Nuclear Information System (INIS)

Santamaria, J.; Rebollo, D.

1986-01-01

The labeling of a new non-steroidal antiinflammatory agent, Butibufen (2-(4-isobutylphenyl) butyric acid) was studied. The method used was heterogeneous catalytic exchange between Butibufen and tritiated water, obtained in situ. Purification was accomplished through thin layer chromatography. Concentration, purity and specific activity of the labeled drug were determined by ultraviolet and liquid scintillation techniques. (Author) 7 refs

Front of Pack Labels Enhance Attention to Nutrition Information in Novel & Commercial Brands.

Science.gov (United States)

Becker, Mark W; Bello, Nora M; Sundar, Raghav P; Peltier, Chad; Bix, Laura

2015-10-01

1) To assess whether Front-of-Pack (FOP) nutrition labels garner attention more readily than more complete, mandated nutrition information (the Nutrition Facts Panel (NFP), required in the US), and 2) To determine whether label design characteristics, specifically, color coding and/or coding with facial icons, increase attention to the FOP label. In two experiments, we tracked the allocation of attention while participants (n=125) viewed novel and commercial packages with varied FOP designs using a change detection methodology. We found empirical evidence that FOP labels are attended more often, and earlier, than the currently mandated NFP, and that this benefit is due both to its placement on the front of the package and to the design characteristics of the FOP. Specifically, the use of color in FOPs increased attention to the label, but there was no evidence that coding information via facial icons impacted attention. Our work supports a growing body of evidence supporting the use of FOP labels to attract attention to nutritional information. Findings may be relevant to inform policy decisions on labeling standards.

Off-Label Uses of Omalizumab.

Science.gov (United States)

El-Qutob, David

2016-02-01

The off-label use of medicines is a common and extensive clinical practice. Omalizumab has been licensed for use in severe allergic asthma and chronic urticaria. Omalizumab dosing was based on body weight and baseline serum IgE concentration. All patients are required to have a baseline IgE between 30 and 700 IU/ml and body weight not more than 150 kg. The use of off-label drugs may lead to several problems including adverse effects and an increased risk/benefit balance. In this article, there are summarized off-label uses of omalizumab in the last recent years in diseases in which IgE maybe or certainly has a corner role such as allergic rhinitis, allergic bronchopulmonary aspergillosis, anaphylaxis, keratoconjunctivitis, food allergy, drug allergy, urticaria, angioedema, non-atopic asthma, atopic dermatitis, nasal polyps, Churg-Strauss syndrome, eosinophilic otitis media, chronic rhinosinusitis, bullous pemphigoid, contact dermatitis, and others. Use in pregnancy asthmatic women and pre-co-administration with specific immunotherapy will also be revised.

Labeling of lectin receptors during the cell cycle.

Science.gov (United States)

Garrido, J

1976-12-01

Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling.

Substrate coated with receptor and labelled ligand for assays

International Nuclear Information System (INIS)

1980-01-01

Improvements in the procedures for assaying ligands are described. The assay consists of a polystyrene tube on which receptors are present for both the ligand to be assayed and a radioactively labelled form of the ligand. The receptors on the bottom portion of the tube are also coated with labelled ligands, thus eliminating the necessity for separate addition of the labelled ligand and sample during an assay. Examples of ligands to which this method is applicable include polypeptides, nucleotides, nucleosides and proteins. Specific examples are given in which the ligand to be assayed is digoxin, the labelled form of the ligand is 3-0-succinyl digoxyigenin tyrosine ( 125 I) and the receptor is digoxin antibody. (U.K.)

Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons.

Science.gov (United States)

Juárez-Morales, José L; Martinez-De Luna, Reyna I; Zuber, Michael E; Roberts, Alan; Lewis, Katharine E

2017-09-01

A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry. However, the X. laevis tetraploid genome and long generation time make it difficult to investigate how neurons are specified. The opacity of X. laevis embryos also makes it hard to connect functional classes of neurons and the genes that they express. We demonstrate here that Tol2 transgenic constructs using zebrafish enhancers that drive expression in specific zebrafish spinal neurons label equivalent neurons in X. laevis and that the incorporation of a Gal4:UAS amplification cassette enables cells to be observed in live X. laevis tadpoles. This technique should enable the molecular phenotypes, morphologies and physiologies of distinct X. laevis spinal neurons to be examined together in vivo. We have used an islet1 enhancer to label Rohon-Beard sensory neurons and evx enhancers to identify V0v neurons, for the first time, in X. laevis spinal cord. Our work demonstrates the homology of spinal cord circuitry in zebrafish and X. laevis, suggesting that future work could combine their relative strengths to elucidate a more complete picture of how vertebrate spinal cord neurons are specified, and function to generate behavior. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1007-1020, 2017. © 2017 Wiley Periodicals, Inc.

Tub-Tag Labeling; Chemoenzymatic Incorporation of Unnatural Amino Acids.

Science.gov (United States)

Helma, Jonas; Leonhardt, Heinrich; Hackenberger, Christian P R; Schumacher, Dominik

2018-01-01

Tub-tag labeling is a chemoenzymatic method that enables the site-specific labeling of proteins. Here, the natural enzyme tubulin tyrosine ligase incorporates noncanonical tyrosine derivatives to the terminal carboxylic acid of proteins containing a 14-amino acid recognition sequence called Tub-tag. The tyrosine derivative carries a unique chemical reporter allowing for a subsequent bioorthogonal modification of proteins with a great variety of probes. Here, we describe the Tub-tag protein modification protocol in detail and explain its utilization to generate labeled proteins for advanced applications in cell biology, imaging, and diagnostics.

Synthesis of specifically 2H-labeled reserpines, 3,4,5-trimethoxybenzoic acids, and syringic acid

International Nuclear Information System (INIS)

Roth, R.W.; Fischer, D.L.; Pachta, J.M.; Althaus, J.F.

1982-01-01

3,4,5-Trimethoxy- 2 H 9 -, 4-methoxy-3,5-dimethoxy- 2 H 6 , and 4-hydroxyl-3,5-dimethoxy- 2 H 6 -benzoic acids were prepared from n-propyl 3,4,5-trihydroxybenzoate (n-propyl gallate) by means of appropriate alkylation-hydrolysis sequences employing iodomethane- 2 H 3 or dimethyl- 2 H 6 -sulfate as the deuterium source. 4-Methoxy- 2 H 3 -3,5-dimethoxybenzoic acid was similarly prepared from ethyl 4-hydroxy-3,5-dimethoxybenzoate. The labeled trimethoxybenzoic acids were converted to the corresponding 2 H-labeled reserpines by condensation of the acid chlorides with methyl reserpate in pyridine according to the classical procedure. The labeled reserpine analog methyl 18-0-(4-hydroxy-3,5-dimethoxy- 2 H 6 )benzoyl reserpate was likewise prepared from 4-hydroxy-3,5-dimethoxy- 2 H 6 -benzoic acid via the intermediate methyl 18-0-(4-ethoxycarbonyloxy-3,5-dimethoxy- 2 H 6 -benzoyl)reserpate (syrosingopine- 2 H 6 ). The isotopic purity of each compound exceeded 99 atom percent 2 H. (author)

Chemical aspects of labeling sucralfate with 99mTcO4

International Nuclear Information System (INIS)

Billinghurst, M.W.; Abrams, D.N.; Lawson, M.S.

1989-01-01

Two formulations of [ 99m Tc]sucralfate have been used to image gastric and duodenal ulcers and inflammatory bowel disease. One formulation is a complexation of [ 99m Tc]HSA with sucralfate. The second is prepared by directly labeling sucralfate with [ 99m Tc]pertechnetate in the presence of stannous ion. An in vitro study of the factors affecting the production and stability of these labeled sucralfate preparations was conducted. Both formulations were stable at the acidic pH likely encountered in the stomach. However, at pH greater than 6 the albumin-sucralfate complex began to dissociate while directly labeled sucralfate was stable to a pH of 9. Conversely it was shown that directly labeled sucralfate was more susceptible to loss of 99m Tc to other chelating species. Sucralfate complexed with [ 99m Tc]HSA was radiochemically stable up to a specific activity of 26 GBq (700 mCi) per gram while directly labeled sucralfate showed decreased 24-hr stability at specific activities greater than 837 mCi (31 GBq) per gram

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

International Nuclear Information System (INIS)

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

1991-01-01

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

Site-specific Orientation of an α-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy

Science.gov (United States)

Ding, Bei; Laaser, Jennifer E.; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T.; Chen, Zhan

2013-01-01

Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single isotope labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138 degrees from the surface normal, and the transition dipole of the isotope labeled C=O group is tilted at 23 degrees from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrated that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

Site-specific orientation of an α-helical peptide ovispirin-1 from isotope-labeled SFG spectroscopy.

Science.gov (United States)

Ding, Bei; Laaser, Jennifer E; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T; Chen, Zhan

2013-11-27

Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C═O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution.

Gold Nanoparticle Labels Amplify Ellipsometric Signals

Science.gov (United States)

Venkatasubbarao, Srivatsa

2008-01-01

The ellipsometric method reported in the immediately preceding article was developed in conjunction with a method of using gold nanoparticles as labels on biomolecules that one seeks to detect. The purpose of the labeling is to exploit the optical properties of the gold nanoparticles in order to amplify the measurable ellipsometric effects and thereby to enable ultrasensitive detection of the labeled biomolecules without need to develop more-complex ellipsometric instrumentation. The colorimetric, polarization, light-scattering, and other optical properties of nanoparticles depend on their sizes and shapes. In the present method, these size-and-shape-dependent properties are used to magnify the polarization of scattered light and the diattenuation and retardance of signals derived from ellipsometry. The size-and-shape-dependent optical properties of the nanoparticles make it possible to interrogate the nanoparticles by use of light of various wavelengths, as appropriate, to optimally detect particles of a specific type at high sensitivity. Hence, by incorporating gold nanoparticles bound to biomolecules as primary or secondary labels, the performance of ellipsometry as a means of detecting the biomolecules can be improved. The use of gold nanoparticles as labels in ellipsometry has been found to afford sensitivity that equals or exceeds the sensitivity achieved by use of fluorescence-based methods. Potential applications for ellipsometric detection of gold nanoparticle-labeled biomolecules include monitoring molecules of interest in biological samples, in-vitro diagnostics, process monitoring, general environmental monitoring, and detection of biohazards.

Synthesis of positron labeled photoactive compounds: 18F labeled aryl azides for positron labeling of biochemical molecules

International Nuclear Information System (INIS)

Hashizume, Kazunari; Hashimoto, Naota; Miyake, Yoshihiro

1995-01-01

The authors have prepared various [ 18 F] fluorine labeled aryl azides as a novel photoactive compounds suitable for positron labeling of biochemical molecules. The introduction of fluorine substituents to aryl azides can be expected to have dramatic effects on their nature and reactivity toward photolysis. Positron labeled reagents for labeling proteins or peptides have recently attracted considerable attention due to their wide applicability in biochemistry and positron emission tomography (PET). Various labeled azide compounds are often used in biochemistry for radiolabeling biological molecules by photolysis, but there have been no reports on the preparation or use of fluorine-18 labeled azides. The authors now report a novel synthesis of 18 F-labeled aryl azides which will have wide application in the biochemistry and nuclear medicine as a means for 18 F-fluorine labeling for proteins, peptides, and nucleic acids. 2 tabs

Lectin Ulex europaeus agglutinin I specifically labels a subset of primary afferent fibers which project selectively to the superficial dorsal horn of the spinal cord.

Science.gov (United States)

Mori, K

1986-02-19

To examine differential carbohydrate expression among different subsets of primary afferent fibers, several fluorescein-isothiocyanate conjugated lectins were used in a histochemical study of the dorsal root ganglion (DRG) and spinal cord of the rabbit. The lectin Ulex europaeus agglutinin I specifically labeled a subset of DRG cells and primary afferent fibers which projected to the superficial laminae of the dorsal horn. These results suggest that specific carbohydrates containing L-fucosyl residue is expressed selectively in small diameter primary afferent fibers which subserve nociception or thermoception.

Radioisotope labelling of several major insect pest

International Nuclear Information System (INIS)

Sutrisno, Singgih

1981-01-01

Radioisotope uptake by insects could take place through various parts i.e. mouth, cuticula, intersegmental, secretion and excretion organs. Usually insects are labelled internally by feeding them on an artificial diet containing radioisotope solution. Labelling of several insect pests of cabbage (Crocidolomia binotalis) Zell and Plutella maculipennis Curt and rice (Chilo suppressalis Walker) by dipping of the pupae in 32 P solution showed a promising result. Pupae of Crocidolomia binotalis Zell dipped in 3 ml solution of 32 P with specific activities of 1, 3, 5 and 7 μCi/ml had developed labelled adults of sufficiently high radioactivity levels for ecological studies. Similar results were also obtained with Plutella maculipennis Curt and Chilo suppressalis Walker with doses of 1, 3, 5, 7 and 9 μCi/ml 32 P solution. The best doses for radioisotope labelling by dipping of the insects Crocidolomia binotalis Zell, Plutella maculipennis Curt, and Chilo suppressalis Walker were 1, 9, and 7 μCi/ml respectivelly. (author)

Synthesis of labelled ecdysone precursors

International Nuclear Information System (INIS)

Haag, T.; Hetru, C.; Nakatani, Y.; Luu, B.; Meister, M.; Pichat, L.; Audinot, M.

1985-01-01

High specific activity tritiated 3β,14α-dihydroxy-5β-cholest-7-en-6-one, has been prepared using a precursor which permits rapid and easy labelling. This compound is converted to ecdysone under in vitro conditions by insect prothoracic glands, a well known site of ecdysone biosynthesis. (author)

Labeling Lanreotide with 125I and 188Re

International Nuclear Information System (INIS)

Bai Hongsheng

2000-01-01

Lanreotide is a new somatostatin analogue. It can bind to human somatostatin receptor (hSSTR) subtype 2 through 5 with high affinity and to hSSTR subtype I with low affinity. We investigate labeling condition, quality control and stability in vitro of 125 I-Lanreotide and 188 Re-lanreotide respectively. (A) Lanreotide is labeled with 125 I using Chloramine T. The effect of reaction condition (such as reaction time, pH value, Lanreotide amount, quantity of Chloramine T and reaction volume) on labeling yield is investigated in detail. (B) The labeling yield and radiochemical purity (RP) is measured with paper chromatography (PC) and Sep-Pak C 18 Cartridge. (C) The stability of 125 I-Lanreotide in vitro is investigated by labeling compound incubating for 48 hours at 37 deg C in the 0.9% sodium chloride solution and RP is tested by PC at specific time intervals. (D) Lanreotide is labeled directly with 188 Re via the mixture of citrate and tartate using stannous chloride as reduced agent. The influence of reaction conditions such as pH, temperature, amount of stannous chloride, amount of Lanreotide and reaction time on labeling yield is investigated in detail. At the time, the stability in vitro quality control and animal test are evaluated

Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

Energy Technology Data Exchange (ETDEWEB)

Lentes, K.U.; Venter, J.C.

1986-05-01

Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 ..mu..M clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 ..mu..g trypsin/mg membrane protein yielded H/sub 2/O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain.

Probabilistic atlas based labeling of the cerebral vessel tree

Science.gov (United States)

Van de Giessen, Martijn; Janssen, Jasper P.; Brouwer, Patrick A.; Reiber, Johan H. C.; Lelieveldt, Boudewijn P. F.; Dijkstra, Jouke

2015-03-01

Preoperative imaging of the cerebral vessel tree is essential for planning therapy on intracranial stenoses and aneurysms. Usually, a magnetic resonance angiography (MRA) or computed tomography angiography (CTA) is acquired from which the cerebral vessel tree is segmented. Accurate analysis is helped by the labeling of the cerebral vessels, but labeling is non-trivial due to anatomical topological variability and missing branches due to acquisition issues. In recent literature, labeling the cerebral vasculature around the Circle of Willis has mainly been approached as a graph-based problem. The most successful method, however, requires the definition of all possible permutations of missing vessels, which limits application to subsets of the tree and ignores spatial information about the vessel locations. This research aims to perform labeling using probabilistic atlases that model spatial vessel and label likelihoods. A cerebral vessel tree is aligned to a probabilistic atlas and subsequently each vessel is labeled by computing the maximum label likelihood per segment from label-specific atlases. The proposed method was validated on 25 segmented cerebral vessel trees. Labeling accuracies were close to 100% for large vessels, but dropped to 50-60% for small vessels that were only present in less than 50% of the set. With this work we showed that using solely spatial information of the vessel labels, vessel segments from stable vessels (>50% presence) were reliably classified. This spatial information will form the basis for a future labeling strategy with a very loose topological model.

Mixed Map Labeling

Directory of Open Access Journals (Sweden)

Maarten Löffler

2016-12-01

Full Text Available Point feature map labeling is a geometric visualization problem, in which a set of input points must be labeled with a set of disjoint rectangles (the bounding boxes of the label texts. It is predominantly motivated by label placement in maps but it also has other visualization applications. Typically, labeling models either use internal labels, which must touch their feature point, or external (boundary labels, which are placed outside the input image and which are connected to their feature points by crossing-free leader lines. In this paper we study polynomial-time algorithms for maximizing the number of internal labels in a mixed labeling model that combines internal and external labels. The model requires that all leaders are parallel to a given orientation θ ∈ [0, 2π, the value of which influences the geometric properties and hence the running times of our algorithms.

Determination of gluconeogenesis in vivo with 14C-labeled substrates

International Nuclear Information System (INIS)

Katz, J.

1985-01-01

A mitochondrial model of gluconeogenesis and the tricarboxylic acid cycle, where pyruvate is metabolized via pyruvate carboxylase and pyruvate dehydrogenase, and pyruvate kinase is examined. The effect of the rate of tricarboxylic acid flux and the rates of the three reactions of pyruvate metabolism on the labeling patterns from [ 14 C]pyruvate and [ 14 C]acetate are analyzed. Expressions describing the specific radioactivities and 14 C distribution in glucose as a function of these rates are derived. Specific radioactivities and isotopic patterns depend markedly on the ratio of the rates of pyruvate carboxylation and decarboxylation to the rate of citrate synthesis, but the effect of phosphoenolpyruvate hydrolysis is minor. The effects of these rates on 1) specific radioactivity of phosphoenolpyruvate, 2) labeling pattern in glucose, and 3) contribution of pyruvate, acetyl-coenzyme A, and CO 2 to glucose carbon are illustrated. To determine the contribution of lactate or alanine to gluconeogenesis, experiments with two compounds labeled in different carbons are required. Methods in current use to correct for the dilution of 14 C in gluconeogenesis from [ 14 C]pyruvate are shown to be erroneous. The experimental design and techniques to determine gluconeogenesis from 14 C-labeled precursors are presented and illustrated with numerical examples

Study on Chinese herbal medicine active ingredients labelled with tritium

International Nuclear Information System (INIS)

Dong Mo; Bao Guangliang

2008-01-01

Chinese medicinal herb active ingredients was labeled with triteium by using exchange of new synthesized tritiated water or exchange of low-pressure gas-liquid. The active ingredients was Genipin, acetylalkannin and chlorogenic acid .The radiochemical purity of the three labeled compounds were more than 95% after TLC and HPLC purification. The specific activities of tritium labeled-genipin, acetylalkannin and chlorogenic acid were 5.97, 3.24 and 470 GBq/g, respectively. The results indicated that the unstable Chinese medicinal herb active ingredients could be labeled with tritium by the methods of exchange of new synthesized tritiated water and exchange of low-pressure gas-liquid. (authors)

Selective retrograde labeling of cholinergic neurons with [3H]choline

International Nuclear Information System (INIS)

Bagnoli, P.; Beaudet, A.; Stella, M.; Cuenod, M.

1981-01-01

Evidence is presented which is consistent with a specific retrograde labeling of cholinergic neurons following [ 3 H]choline application in their zone of termination. [ 3 H]Choline injection in the rat hippocampus leads to perikaryal retrograde labeling in the ipsilateral medial septal nuclease and nucleus of the diagonal band, thus delineating an established cholinergic pathway, while only diffuse presumably anterograde labeling was observed in the lateral septum, the entorhinal cortex, and the opposite hippocampus. After [ 3 H]choline injection in the pigeon visual Wulst, only the ipsilateral thalamic relay, of all inputs, showed similar perikaryal retrograde labeling, an observation supporting the suggestion that at least some thalamo-Wulst neurons are cholinergic

Read the Label First! Protect Your Household

Science.gov (United States)

... and pesticides could harm children, pets, or the environment if not used and stored correctly. Choose a product that is labeled for your specific pest. EPA encourages consumers to consider using EPA-registered ...

Re-purification of labelled ferritin antigen with HPLC

International Nuclear Information System (INIS)

Zhang Haoyi; Jin Lichun

2002-01-01

Objective: To improve the quality of long-term stored labelled ferritin antigen with HPLC. Methods: The antigen was analyzed and purified with HPLC and again analyzed with RIA afterwards. Results: Ferritin antigen underwent significant polymerization after long-term (aggregation) storage. After re-purification with HPLC, its immuno-activity and labelled specific radioactivity were both significantly improved. Conclusion: Quality of stored ferritin RIA kit could be greatly improved after re-purification with HPLC

Functional interaction between the two halves of the photoreceptor-specific ATP binding cassette protein ABCR (ABCA4). Evidence for a non-exchangeable ADP in the first nucleotide binding domain.

Science.gov (United States)

Ahn, Jinhi; Beharry, Seelochan; Molday, Laurie L; Molday, Robert S

2003-10-10

ABCR, also known as ABCA4, is a member of the superfamily of ATP binding cassette transporters that is believed to transport retinal or retinylidene-phosphatidylethanolamine across photoreceptor disk membranes. Mutations in the ABCR gene are responsible for Stargardt macular dystrophy and related retinal dystrophies that cause severe loss in vision. ABCR consists of two tandemly arranged halves each containing a membrane spanning segment followed by a large extracellular/lumen domain, a multi-spanning membrane domain, and a nucleotide binding domain (NBD). To define the role of each NBD, we examined the nucleotide binding and ATPase activities of the N and C halves of ABCR individually and co-expressed in COS-1 cells and derived from trypsin-cleaved ABCR in disk membranes. When disk membranes or membranes from co-transfected cells were photoaffinity labeled with 8-azido-ATP and 8-azido-ADP, only the NBD2 in the C-half bound and trapped the nucleotide. Co-expressed half-molecules displayed basal and retinal-stimulated ATPase activity similar to full-length ABCR. The individually expressed N-half displayed weak 8-azido-ATP labeling and low basal ATPase activity that was not stimulated by retinal, whereas the C-half did not bind ATP and exhibited little if any ATPase activity. Purified ABCR contained one tightly bound ADP, presumably in NBD1. Our results indicate that only NBD2 of ABCR binds and hydrolyzes ATP in the presence or absence of retinal. NBD1, containing a bound ADP, associates with NBD2 to play a crucial, non-catalytic role in ABCR function.

Searching for flavor labels in food products: The influence of color-flavor congruence and association strength

Directory of Open Access Journals (Sweden)

Carlos eVelasco

2015-03-01

Full Text Available Prior research provides robust support for the existence of a number of associations between colors and flavors. In the present study, we examined whether congruent (vs. incongruent combinations of product packaging colors and flavor labels would facilitate visual search for products labelled with specific flavors in a Stroop-like manner. Across two experiments, a Stroop-like effect between flavor words and packaging colors is documented and we demonstrate that people are able to search for packaging flavor labels more rapidly when the color of the packaging is congruent with the flavor label (e.g., red/tomato than when it is incongruent (e.g., yellow/tomato. In addition, when the packaging color was incongruent, those flavor labels that were more strongly associated with a specific color yielded slower reaction times and more errors (Stroop interference than those that were less strongly tied to a specific color. Importantly, search efficiency was affected both by color/flavor congruence and association strength. Taken together, these results therefore highlight the role of color congruence and color-word association strength when it comes to searching for specific flavor labels.

Synthesis of tritium-labeled vitamin A and its analogs

International Nuclear Information System (INIS)

Rhee, S.W.; Bubb, J.E.

1985-01-01

Metabolic and pharmacologic studies of Vitamin A and its analogs related to the prevention of lung cancer and other epithelial cancers required tritium-labeled Vitamin A analogs and β-carotene at high specific activity. Syntheses of some of the isomers were therefore developed in the laboratory, as described in the paper. The advantages of the scheme shown are that : 1. Tritiums are introduced into the molecule by catalytic hydrogenation, thus affording high specific activity. 2. It uses an allylic rearrangement of tritiated vinyl-β-ionol to C 15 -phosphonium salt, which is condensed with C 5 -nitrile to give C 20 -skeleton of retinonitrile. 3. It permits the development of milder methods to convert tritium-labeled retinaldehyde, as a common intermediate, to the other retinoids (i.e., retinoic acid, retinol, and retinyl acetate). Furthermore, tritium-labeled all-trans-β-carotene, an important carotenoid, has been obtained from the retinaldehyde

Inflammation Scan Using {sup 99m}Tc-HMPAO Labelled Leukocytes

Energy Technology Data Exchange (ETDEWEB)

Yang, Woo Jin; Chung, Soo Kyo; Shinn, Kyung Sub; Bahk, Yong Whee; Kim, Hoon Kyo [Catholic University College of Medicine, Seoul (Korea, Republic of)

1989-07-15

Inflammation scan using radiolabelled leukocytes has high sensitivity and specificity. Several methods for labelling leukocytes have been evaluated using P-32 diisopropyl fluorophosphate (DFP -32), H-3 thymidine, Cr-51 chromate, Ga-67 citrate and {sup 99m}Tc-sulfur colloid. In-111-oxine has proved so far to be the most reliable agent for labelling leukocytes. In-111-oxine is, however, expensive, not easily available when needed, and its radiation dose to leukocytes is relatively high. Moreover, resolution of the resultant image is relatively poor. {sup 99m}Tc is still the agent of choice because of, as compared with the indium, its favorable physical characteristics, lower cost and availability. Now the technique for labelling the leukocytes with technetium is successfully obtained using the lipophilic HMPAO with higher efficiency for granulocytes than for other cells. With this technique it is possible to label leukocytes in plans to improve the viability of the leukocytes. Inflammation scan using {sup 99m}Tc-HMPAO has been evaluated in several laboratories, and difference in methods for separation and labelling accounts for difference in efficiency, viability and biodistribution of the labelled leukocytes. We performed inflammation scan using leukocytes labelled with {sup 99m}Tc-HMPAO in three dogs 24 hours after inoculation of live E. Coli and S. Aureus in their right abdominal wall. We separated mixed leukocytes by simple sedimentation using 6% hetastarch (HES) and labelled the leukocytes with {sup 99m}Tc-HMPAO in 20% cell free plasma diluted with phosphate buffer solution. Uptake was high in the liver and spleen but is was minimal in the lungs on whole body scan. Kidneys and intestine showed minimal activity although it was high in the urinary bladder. Uptake of labelled leukocytes in the inflammation site was definite on 2 hour-postinjection scan and abscess was clearly delineated on 24 hour-delayed scan with high target-to-nontarget ratio. 4). Inflammation

Food Labels

Science.gov (United States)

... Staying Safe Videos for Educators Search English Español Food Labels KidsHealth / For Teens / Food Labels What's in ... to have at least 95% organic ingredients. Making Food Labels Work for You The first step in ...

Use of Take-set system for radiopharmaceutical labelling: example of indium-111 labeled pentetreotide (Octreoscan) preparation

International Nuclear Information System (INIS)

Ben Reguiga, M.; Sinegre, M.; Besse, H.; Stievenart, J.L.; Le Guludec, D.

2009-01-01

The quality of indium 111 radiolabelled pentetreotide preparation (Octreoscan, Covidien) depends on several factors among which the use of a special transfer needle (Sterican) especially conceived to avoid the metal impurities introduction into the reactional medium during labelling. This device, usually provided by the supplier, can exceptionally present defects (twisted needle, folded bevel...) preventing its use for the preparation. In order to manage this risk, we propose in the present technical note an alternative labelling method, based on an adaptation of the original one and using another transfer device, the Take-setSWAN system, which permits to obtain high quality Octreoscan preparations that meet the product approval specifications. (authors)

Dual-process theory and consumer response to front-of-package nutrition label formats.

Science.gov (United States)

Sanjari, S Setareh; Jahn, Steffen; Boztug, Yasemin

2017-11-01

Nutrition labeling literature yields fragmented results about the effect of front-of-package (FOP) nutrition label formats on healthy food choice. Specifically, it is unclear which type of nutrition label format is effective across different shopping situations. To address this gap, the present review investigates the available nutrition labeling literature through the prism of dual-process theory, which posits that decisions are made either quickly and automatically (system 1) or slowly and deliberately (system 2). A systematically performed review of nutrition labeling literature returned 59 papers that provide findings that can be explained according to dual-process theory. The findings of these studies suggest that the effectiveness of nutrition label formats is influenced by the consumer's dominant processing system, which is a function of specific contexts and personal variables (eg, motivation, nutrition knowledge, time pressure, and depletion). Examination of reported findings through a situational processing perspective reveals that consumers might prefer different FOP nutrition label formats in different situations and can exhibit varying responses to the same label format across situations. This review offers several suggestions for policy makers and researchers to help improve current FOP nutrition label formats. © The Author(s) 2017. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Direct isotope determination of isotopically labelled lipids by field desorption mass spectrometry

International Nuclear Information System (INIS)

Lehmann, W.D.; Kessler, M.

1982-01-01

Lipids labelled with deuterium or carbon-14 have been investigated by field desorption mass spectrometry for determination of their degree of labelling. This application is demonstrated for free fatty acids, cholesterol, cholesteryl esters, triglycerides, and L-α-phosphatidylcholines. Comparison of the molecular ion groups of the non-labelled and of the labelled compounds enables a fast and reliable determination of the degree of labelling. For multiply labelled compounds the label distribution is also obtained from the molecular ion group. In addition, for cholesteryl esters and for phosphatidylcholines structurally significant fragment ions provide information about the position of the label. Several hundred nanograms of the compound are typically required for a single analysis with a relative standard error of 0.5-2% in the value calculated for atom% hydrogen-2 or for the specific carbon-14 activity. (orig.) [de

Governance of private label as a strategic asset: developing a brand valuation model

Directory of Open Access Journals (Sweden)

Renato Giovannini

2017-12-01

Full Text Available This paper aims at identifying which factors should be considered in the building of an economic evaluation model for the private label brand. In fact, some specific characteristics of private label, with respect to industrial brand, make unusable the consolidated models available. The results of the paper are the definition of some specific factors of private label, the assumptions about how these features impact on the traditional economic evaluation models and how these could be included in a model. Because of the complexity of the topic, the hypothesis is to build a model of synthesis, made of two parts: one part for a Financial-Based evaluation of Brand Equity, with the addition of some specific factors and indicators to the traditional formulas, while the other part is for a Consumer-based evaluation of Brand Equity, thanks to an index that summarizes the strength of private label brands from the consumer perspective. The private label economic evaluation has some relevant managerial implications on the retail system, on the vertical supply chain relationships and on the understanding of the strategic nature of this asset.

Nutrition Labeling

DEFF Research Database (Denmark)

Grunert, Klaus G

2013-01-01

because consumers will avoid products that the label shows to be nutritionally deficient, but also because food producers will try to avoid marketing products that appear, according to the label, as nutritionally problematic, for example, because of a high content of saturated fat or salt. Nutrition......Nutrition labeling refers to the provision of information on a food product’s nutritional content on the package label. It can serve both public health and commercial purposes. From a public health perspective, the aim of nutrition labeling is to provide information that can enable consumers...... to make healthier choices when choosing food products. Nutrition labeling is thus closely linked to the notion of the informed consumer, that chooses products according to their aims, on the basis of the information at their disposal. Because many consumers are assumed to be interested in making healthy...

Surface-Enhanced Raman Scattering Nanoparticles as Optical Labels for Imaging Cell Surface Proteins

Science.gov (United States)

MacLaughlin, Christina M.

Assaying the expression of cell surface proteins has widespread application for characterizing cell type, developmental stage, and monitoring disease transformation. Immunophenotyping is conducted by treating cells with labelled targeting moieties that have high affinity for relevant surface protein(s). The sensitivity and specificity of immunophenotyping is defined by the choice of contrast agent and therefore, the number of resolvable signals that can be used to simultaneously label cells. Narrow band width surface-enhanced Raman scattering (SERS) nanoparticles are proposed as optical labels for multiplexed immunophenotying. Two types of surface coatings were investigated to passivate the gold nanoparticles, incorporate SERS functionality, and to facilitate attachment of targeting antibodies. Thiolated poly(ethylene glycol) forms dative bonds with the gold surface and is compatible with multiple physisorbed Raman-active reporter molecules. Ternary lipid bilayers are used to encapsulate the gold nanoparticles particles, and incorporate three different classes of Raman reporters. TEM, UV-Visible absorbance spectroscopy, DLS, and electrophoretic light scattering were used characterize the particle coating. Colourimetric protein assay, and secondary antibody labelling were used to quantify the antibody conjugation. Three different in vitromodels were used to investigate the binding efficacy and specificity of SERS labels for their biomarker targets. Primary human CLL cells, LY10 B lymphoma, and A549 adenocarcinoma lines were targeted. Dark field imaging was used to visualize the colocalization of SERS labels with cells, and evidence of receptor clustering was obtained based on colour shifts of the particles' Rayleigh scattering. Widefield, and spatially-resolved Raman spectra were used to detect labels singly, and in combination from labelled cells. Fluorescence flow cytometry was used to test the particles' binding specificity, and SERS from labelled cells was also

Immunoassay of 5-methyltetrahydrofolate: use of 125I-labeled protein A as the tracer molecule for specific antibody

International Nuclear Information System (INIS)

Langone, J.J.

1980-01-01

A sensitive and specific solid-phase radioimmunoassay for 5-methyltetrahydrofolate (5-MTHFA) has been developed. 125 I-Labeled staphylococcal Protein A ( 125 I-PA) was used as the tracer molecule for rabbit IgG antibodies bound to 5-MTHFA immobilized on polyacrylamide beads. The dose-dependent inhibition of antibody binding by fluid-phase drug was reflected in decreased binding of 125 I-PA. This inhibition, determined in the presence of known amounts of 5-MTHFA, served as the basis for quantification of 5-MTHFA in test samples. An early bleeding was relatively specific; 4.5 ng 5-MTHFA inhibited immune binding by 50% compared to 7700 ng folinic acid or 1200 ng tetrahydrofolate. Other folic acid analogs, including methotrexate, failed to inhibit significantly. The assay using a later bleeding was more sensitive since 1.6 ng 5-MTHFA gave 50% inhibition (detection limit 0.2 ng), but folinic acid cross-reacted significantly. Absorption with immobilized folinic acid markedly enhanced the specificity of this antiserum and resulted in a 15 to 20% increase in maximum inhibition by 5-MTHFA. The assay could be carried out in the presence of 0.025 ml human serum or urine without affecting the standard curve, and was used to determine levels of 5-MTHFA in serum of drug-treated rabbits

Rhenium 188 labelling of peptide conjugates

International Nuclear Information System (INIS)

Melendez-Alafort, Laura

2001-01-01

Many human tumours express high levels, of somatostatin receptors. In order to make possible a radiotherapeutic treatment of this kind for tumour a series of somatostatin analogues that can tightly chelate beta emitting isotopes have been developed in recent years. The work carried out for this thesis has been aimed towards development of a new therapeutic radiopharmaceutical for treatment of somatostatin receptor positive tumours. The first chapters describe work with technetium-99m to establish the labelling and analytical conditions for a somatostatin analogue, [Tyr 3 ]-octreotide (TOC), as a precursor to undertaking labelling studies with the beta emitter rhenium-188. 6-Hydrazinopyridine-3-carboxylic acid (HYNIC) was conjugated to TOC and labelled with 99m using different coligands. Then the stability, receptor binding and biodistribution of each complex were assessed. 99m Tc-HYNIC-TOC using EDDA as coligand showed the best characteristics, and was superior for tumour imaging in humans than the commercially available 111 In-DTPA-octreotide. The conditions for labelling the HYNIC-TOC conjugate with 188 Re were then optimised using tricine as a co-ligand. A labelling yield of ∼80% was achieved. After purification however, the stability of the complex was low. The use of other coligand systems which had proved useful for 99m Tc labelling was explored, but yields were very poor. Other chelators such as diethylenetriamine pentaacetic acid (DTPA), dimercaptosuccinic acid (DMSA) and mercaptoacetyltriglycine (MAG 3 ) were studied as potential co-ligand agents to label the HYNIC-TOC conjugate with 188 Re but, again low yields of the labelled peptide complexes were achieved. A novel 188 Re-HYNIC complex was prepared in high yields using N-N-disubstituted dithiocarbamates as coligands. However to date, the specific activities achieved with this system are relatively low. The use of the [ 99m Tc(CO) 3 (H 2 O) 3 ] complex to label the HYNIC-TOC conjugate was investigated

Comparative Analysis of Baby Food Labelling in Hungary and in Romania: Consumers’ Perspective

OpenAIRE

Noémi Hajdú; Katalin Lipták; Zsuzsa Săplăcan

2018-01-01

Baby food represents a growing segment of the food industry; the baby food labelling issues affect more and more mothers who want to make better and safer nutritional choices. In a continuously improving food labelling regulation environment consumer studies regarding the baby food labelling are very limited. Present article has an exploratory nature and aims to find specific patterns of baby food buying behaviour and labelling preference in Romania and Hungary, and also to rev...

Isotopically labelled vitamin D derivatives and processes for preparing same

International Nuclear Information System (INIS)

Deluca, H.R.; Schnoes, H.K.; Napoli, J.L.; Fivizzani, M.A.

1981-01-01

This invention relates to 26,27-isotopically labelled vitamin D 3 compounds, including radiolabelled vitamin D 3 compounds of high specific activity, methods for their preparation, and intermediates obtained in their synthesis. The method involves reacting an ester of a 26,27-dinor-vitamin D-25-carboxylic acid with an isotopically labelled methyl Grignard reagent or methyl lithium reagent to obtain a 26,27-isotopically labelled compound in which at least some of the H atoms and/or C atoms are heavy isotopes. (author)

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

Science.gov (United States)

2013-11-07

... raising of animals, such as ``no antibiotics administered'' or ``vegetarian fed''; (4) instructional or... Standards and Labeling Policy Book includes animal production claims; omega fatty acid guidance; allergen... inclusion of Country of Origin Labeling on all labels; the production and sale of labels by USDA; developing...

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

International Nuclear Information System (INIS)

Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

2015-01-01

Highlights: • An efficient signal amplification strategy for label-free EIA is proposed. • Divalent biotinylated AP and monovalent biotinylated ZZ were prepared via Avitag–BirA system. • The above site-specific biotinylated fusion proteins form complex via SA–biotin interaction. • The mechanism relies on the ZZ–Avi-B/SA/AP–(Avi-B) 2 complex. • The analytical signals are enhanced (32-fold) by the proposed strategy. - Abstract: Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

Energy Technology Data Exchange (ETDEWEB)

Tang, Jin-Bao [School of Pharmacy, Weifang Medical University, Weifang 261053 (China); Tang, Ying [Affiliated Hospital of Weifang Medical University, Weifang 261041 (China); Yang, Hong-Ming, E-mail: yanghongming2006@sohu.com [School of Pharmacy, Weifang Medical University, Weifang 261053 (China)

2015-02-15

Highlights: • An efficient signal amplification strategy for label-free EIA is proposed. • Divalent biotinylated AP and monovalent biotinylated ZZ were prepared via Avitag–BirA system. • The above site-specific biotinylated fusion proteins form complex via SA–biotin interaction. • The mechanism relies on the ZZ–Avi-B/SA/AP–(Avi-B){sub 2} complex. • The analytical signals are enhanced (32-fold) by the proposed strategy. - Abstract: Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable

Mothers' labeling responses to infants' gestures predict vocabulary outcomes.

Science.gov (United States)

Olson, Janet; Masur, Elise Frank

2015-11-01

Twenty-nine infants aged 1;1 and their mothers were videotaped while interacting with toys for 18 minutes. Six experimental stimuli were presented to elicit infant communicative bids in two communicative intent contexts - proto-declarative and proto-imperative. Mothers' verbal responses to infants' gestural and non-gestural communicative bids were coded for object and action labels. Relations between maternal labeling responses and infants' vocabularies at 1;1 and 1;5 were examined. Mothers' labeling responses to infants' gestural communicative bids were concurrently and predictively related to infants' vocabularies, whereas responses to non-gestural communicative bids were not. Mothers' object labeling following gestures in the proto-declarative context mediated the association from infants' gesturing in the proto-declarative context to concurrent noun lexicons and was the strongest predictor of subsequent noun lexicons. Mothers' action labeling after infants' gestural bids in the proto-imperative context predicted infants' acquisition of action words at 1;5. Findings show that mothers' responsive labeling explain specific relations between infants' gestures and their vocabulary development.

Carbon "Quantum" Dots for Fluorescence Labeling of Cells.

Science.gov (United States)

Liu, Jia-Hui; Cao, Li; LeCroy, Gregory E; Wang, Ping; Meziani, Mohammed J; Dong, Yiyang; Liu, Yuanfang; Luo, Pengju G; Sun, Ya-Ping

2015-09-02

The specifically synthesized and selected carbon dots of relatively high fluorescence quantum yields were evaluated in their fluorescence labeling of cells. For the cancer cell lines, the cellular uptake of the carbon dots was generally efficient, resulting in the labeling of the cells with bright fluorescence emissions for both one- and two-photon excitations from predominantly the cell membrane and cytoplasm. In the exploration on labeling the live stem cells, the cellular uptake of the carbon dots was relatively less efficient, though fluorescence emissions could still be adequately detected in the labeled cells, with the emissions again predominantly from the cell membrane and cytoplasm. This combined with the observed more efficient internalization of the same carbon dots by the fixed stem cells might suggest some significant selectivity of the stem cells toward surface functionalities of the carbon dots. The needs and possible strategies for more systematic and comparative studies on the fluorescence labeling of different cells, including especially live stem cells, by carbon dots as a new class of brightly fluorescent probes are discussed.

Labelling of biological structures with technetium 99 m

International Nuclear Information System (INIS)

Bernardo Filho, M.

1988-01-01

The labelling of red blood cells (RBC) with technetium 99m ( 99m Tc) depends on several factors, as the stannous ion (Sn ++ ) concentration, the time and temperature of incubation, the anticoagulant utilized, the presence of plasma proteins (PP) and others. Although the blinding of 99m Tc with hemoglobin and PP are similar, they appear to have specific characteristics as demonstrated by precipitation with alcohol, acetone, trichloroacetic acid, hydrochloric acid and mercury chloride. The bacterial cultures labeled with Technetium- 99m , at optimal Sn ++ ion concentration, presents a large stability and their viability is not altered by this treatment. The electrophoretic mobility, the hydrophobicity, the cationized ferritin distribution and the adherence to human buccal epithelial cells are not modified either. The possibility of labelling with 99m Tc of planaria and cercariae of Schistossoma mansoni evaluative cycle increases the utilization of this radionuclide to an experimental level. The results described with the labelling of these biological structures with 99m Tc demonstrated that stable labeled and viable operations are obtained. (author)

Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay.

Science.gov (United States)

Dolati, Somayeh; Ramezani, Mohammad; Nabavinia, Maryam Sadat; Soheili, Vahid; Abnous, Khalil; Taghdisi, Seyed Mohammad

2018-05-15

Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (K d  = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk. Copyright © 2018 Elsevier Inc. All rights reserved.

'Snack' versus 'meal': The impact of label and place on food intake.

Science.gov (United States)

Ogden, Jane; Wood, Chloe; Payne, Elise; Fouracre, Hollie; Lammyman, Frances

2018-01-01

Eating behaviour is influenced by both cognitions and triggers in the environment. The potential difference between a 'snack' and a 'meal' illustrates these factors and the way in which they interact, particularly in terms of the label used to describe food and the way it is presented. To date no research has specifically explored the independent and combined impact of label and presentation on eating behaviour. Using a preload/taste test design this experimental study evaluated the impact of label ('snack' vs. 'meal') and place ('snack' vs. 'meal') of a preload on changes in desire to eat and subsequent food intake. Eighty female participants consumed a pasta preload which labelled as either a 'snack' or a 'meal' and presented as either a 'snack' (standing and eating from a container) or a 'meal' (eating at a table from a plate), generating four conditions. The results showed main effects of label and place with participants consuming significantly more sweet mass (specifically chocolate) at the taste test when the preload had been labelled a 'snack' and more total mass and calories when the preload had been presented as a 'snack'. No label by place interactions were found. The results also showed a combined effect of both label and place with those who had eaten the preload both labelled and presented as a 'snack' consuming significantly more in terms of nearly all measures of food intake than those in the other conditions. To conclude, label and presentation influence subsequent food intake both independently and combined which is pertinent given the increase in 'snacking' in contemporary culture. Copyright © 2017 Elsevier Ltd. All rights reserved.

Synthesizing labeled compounds

International Nuclear Information System (INIS)

London, R.E.; Matwiyoff, N.A.; Unkefer, C.J.; Walker, T.E.

1983-01-01

A metabolic study is presented of the chemical reactions provided by isotopic labeling and NMR spectroscopy. Synthesis of 13 C-labeled D-glucose, a 6-carbon sugar, involves adding a labeled nitrile group to the 5-carbon sugar D-arabinose by reaction with labeled hydrogen cyanide. The product of this reaction is then reduced and hydrolyzed to a mixture of the labeled sugars. The two sugars are separated by absorption chromotography. The synthesis of 13 C-labeled L-tyrosine, an amino acid, is also presented

A revised method of labeling mouse IgG with yttrium-90

International Nuclear Information System (INIS)

Arbab, A.S.; Koizumi, Kiyoshi; Araki, Tsutomu

1996-01-01

We report the successful labeling of mouse IgG with yttrium-90 (Y-90) using isothiocyanatobenzyl-ethylenediaminetetraacetic acid (SCN-Bz-EDTA) as a chelating agent and compared the result with labeling by indium-111 (In-111). After conjugating IgG with SCN-Bz-EDTA, a predetermined volume of conjugated IgG was mixed with different volumes of either Y-90 or In-111 acetate and incubated at 37degC. Labeling efficiency was assessed at specific intervals upto 3 hr. After 3 hr, the mixtures were challenged with Na 2 EDTA to evaluate the transchelation of labeled Y-90 or In-111. All mixtures showed labeling efficiency of around 50% with Y-90 and the leveling was fairly preserved even after Na 2 EDTA challenge. However, labeling with In-111 was unsuccessful when conjugated IgG was not separated from the unconjugated form. When separated, however, In-111 showed more than 80% labeling efficiency though labeling with In-111 could not tolerate Na 2 EDTA challenge. In conclusion IgG was efficiently labeled by Y-90 using SCN-Bz-EDTA though labeling with In-111 showed some problems associated with this method. (author)

Tritiated bovine fibrinogen labelled in fibrinopeptide A region

International Nuclear Information System (INIS)

Wegrzynowicz, Z.; Kloczewiak, M.; Kopec, M.

1974-01-01

The method is described for labelling of bovine fibrinogen with 3 H-AcOAc. Preparations labelled at pH 7.8 with 10 to 40 molar excess of 3 H-AcOAc were found to contain 8 to 13 moles of acetyl residues per mole of fibrinogen. The content of clottable protein and UV spectra were unchanged as compared with control unlabelled preprations. The rate of clotting with thrombin was only slightly affected. The investigations on distribution of 3 H in products of proteolysis of 3 H-fibrinogen by thrombin and plasmin demonstrated a preferential labelling of fibrinopeptide A, absence of radioactive tracer in fibrinopeptide B, significantly higher specific radioactivity of fragment E than that of fragment D. Incorporation of the label into fibrinopeptide A opens the possibility for application of 3 H-fibrinogen as a convenient substrate for selective investigations on the enzymatic phase of clotting. (author)

Examination of the evidence for off-label use of gabapentin.

Science.gov (United States)

Mack, Alicia

2003-01-01

(1) Describe the relevance of off-label use of gabapentin to managed care pharmacy; (2) summarize recent FDA warnings and media reports related to off-label gabapentin use; (3) review medical information pertaining to the off-label use of gabapentin; (4) outline alternatives to off-label use of gabapentin in an evidence-based fashion, where literature exists to support such alternatives; and (5) encourage key clinicians and decision makers in managed care pharmacy to develop and support programs that restrict the use of gabapentin to specific evidence-based situations. Gabapentin is approved by the U.S. Food and Drug Administration (FDA) for adjunctive therapy in treatment of partial seizures and postherpetic neuralgia. Various off-label (unapproved) uses have been reported, and the use of gabapentin for off-label purposes has reportedly exceeded use for FDAapproved indications. Pharmaceutical marketing practices and physician dissatisfaction with currently available pharmacological treatment options may be key factors that contribute to this prescribing trend. Recently, the media has focused on these issues, noting that many cases of reported safety and effectiveness of gabapentin for off-label use may have been fabricated. A thorough review of the medical and pharmacy literature related to off-label use of gabapentin was performed, and a summary of the literature for the following conditions is presented: bipolar disorder, peripheral neuropathy, diabetic neuropathy, complex regional pain syndrome, attention deficit disorder, restless legs syndrome, trigeminal neuralgia, periodic limb movement disorder of sleep, migraine headaches, and alcohol withdrawal syndrome. A common theme in the medical literature for gabapentin is the prevalence of open-label studies and a lack of randomized controlled clinical trials for all but a small number of indications. In the majority of circumstances where it has reported potential for.off-label. use, gabapentin is not the optimal

Preclinical evaluation of somatostatin analogs bearing two macrocyclic chelators for high specific activity labeling with radiometals

International Nuclear Information System (INIS)

Storch, D.; Schmitt, J.S.; Waldherr, C.; Maecke, H.R.; Waser, B.; Reubi, J.C.

2007-01-01

Radiometallated analogues of the regulatory peptide somatostatin are of interest in the in vivo localization and targeted radiotherapy of somatostatin receptor-overexpressing tumors. An important aspect of their use in vivo is a fast and efficient labeling (complexation) protocol for radiometals along with a high specific activity. We describe in this manuscript synthetic methods for the coupling of two chelators (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid = DOTA) to the bioactive peptide [Tyr 3 ,Thr 8 ]-octreotide (TATE) in order to increase the specific activity (radioactivity in Bq per mole peptide). The full chelator-linker-peptide conjugate was assembled on solid support using standard Fmoc chemistry. Two DOTA-chelators were linked to the peptide using lysine or N,N'-bis(3-aminopropyl)-glycine (Apg); in addition, pentasarcosine (Sar 5 ) was used as a spacer between the chelators and the peptide to probe its influence on biology and pharmacology. Complexation rates with In 3+ and Y 3+ salts and the corresponding radiometals were high, the bis-DOTA-derivatives showed higher complexation rates and gave higher specific activity than DOTA-TATE. Pharmacological and biological data of the complexed molecules did not show significant differences if compared to the parent peptide [ 111/nat In-DOTA]-TATE except for [( 111/nat In-DOTA) 2 -Apg]-TATE which showed a lower binding affinity and rate of internalization into tumor cells. The biodistribution of [( 111/nat In-DOTA)-Lys( 111/nat In-DOTA)]-TATE in the rat tumor model (AR4-2J) showed a high and specific (as shown by a blocking experiment) tracer uptake in somatostatin receptor-positive tissue but a lower tumor uptake compared to [ 111/nat In-DOTA]-TATE. (orig.)

Application of product life cycle concept to private label management

Directory of Open Access Journals (Sweden)

Sandra Horvat

2013-06-01

Full Text Available Private labels have recorded significant growth rates worldwide, becoming a serious threat to manufacturer brands. Development of private labels in many different product categories increased the complexity of their management. Therefore, this paper examines the possibility of using the product life cycle concept in private label management. Given that private labels are a specific brand type, it is necessary to adjust certain elements of the product life cycle concept, as it was developed on the basis of manufacturer brands. For instance, in the growth stage of the product life cycle, retailers expand private labels to a number of product categories and use the push strategy while manufacturers tend to expand their distribution network in the expansion of their brands and predominantly use the pull strategy in doing so. Furthermore, there is a focus shift from low-price strategy, predominantly used in the introduction phase, to increasing the quality and private label value in the later stages of the product life cycle.

Study on labelling conditions of 125I-synkavit by the iodogen method

International Nuclear Information System (INIS)

Oezdemir, D.; Uenak, P.

1994-01-01

Labeling conditions of synkavit (2-methyl-1,4-naphtoquinol disodium phosphate) with iodine-125 have been studied. Labeling temperature, reaction time, successive using of iodogen coated tubes, iodogen amount and synkavit concentrations have been determined to get optimum conditions for maximum labeling. Final results showed that when the labeling temperature, reaction time, synkavit concentration and iodogen amount were, at room temperature, 15 min (in the case of successive using of three iodogen coated tubes), 2 mg ml -1 and 5 mg, respectively; labeling yield was 90% and specific activities of the order of 555 GBq mmol -1 (15 Ci mmol -1 have been obtained. (author) 14 refs.; 4 figs

ML-MG: Multi-label Learning with Missing Labels Using a Mixed Graph

KAUST Repository

Wu, Baoyuan

2015-12-07

This work focuses on the problem of multi-label learning with missing labels (MLML), which aims to label each test instance with multiple class labels given training instances that have an incomplete/partial set of these labels (i.e. some of their labels are missing). To handle missing labels, we propose a unified model of label dependencies by constructing a mixed graph, which jointly incorporates (i) instance-level similarity and class co-occurrence as undirected edges and (ii) semantic label hierarchy as directed edges. Unlike most MLML methods, We formulate this learning problem transductively as a convex quadratic matrix optimization problem that encourages training label consistency and encodes both types of label dependencies (i.e. undirected and directed edges) using quadratic terms and hard linear constraints. The alternating direction method of multipliers (ADMM) can be used to exactly and efficiently solve this problem. To evaluate our proposed method, we consider two popular applications (image and video annotation), where the label hierarchy can be derived from Wordnet. Experimental results show that our method achieves a significant improvement over state-of-the-art methods in performance and robustness to missing labels.

Assay for vitamin B12 absorption and method of making labeled vitamin B12

Science.gov (United States)

Anderson, Peter J [Davis, CA; Dueker, Stephen [Davis, CA; Miller, Joshua [Davis, CA; Green, Ralph [Elmacero, CA; Roth, John [Davis, CA; Carkeet, Colleen [Silver Spring, MD; Buchholz,; Bruce, A [Orinda, CA

2012-06-19

The invention provides methods for labeling vitamin B12 with .sup.14C, .sup.13C, tritium, and deuterium. When radioisotopes are used, the invention provides for methods of labeling B12 with high specific activity. The invention also provides labeled vitamin B12 compositions made in accordance with the invention.

125Iodine labeling of β-hexosaminidase A without modifying its properties

International Nuclear Information System (INIS)

Kusiak, J.W.; Barranger, J.A.

1979-01-01

Human placental β-hexosaminidase A was labeled with 125 iodine to high specific activity with the retention of conformational integrity as judged by the retention of enzymatic activity. The oligosaccharide structure also appeared to be intact since the labeled enzyme was cleared from the circulation of the rat with a half-life identical to that of the unlabeled enzyme and an excess of unlabeled enzyme effectively blocked the clearance of the labelled form. Furthermore, the pattern of inhibition of clearance of the native and labeled enzymes by asialofetuin and mannans was identical. The useful and mild procedure for labeling enzymes may be of general importance in the preparation of enzymes for metabolic studies in normal animals and animal models of genetic lysosomal storage disorders. (Auth.)

Photolabeling and radioligand binding of human erythrocyte NaK-ATPase with 125I-derivatives of cymarin and digitoxigenin

International Nuclear Information System (INIS)

Lowndes, J.M.

1988-01-01

NaK-ATPase is an enzyme which maintains Na + and K + gradients across the plasma membrane of eukaryotic cells, and is specifically inhibited by cardiac glycosides. The cardiac glycoside binding site is located primarily on the catalytic α subunit but the glycoprotein β and proteolipid-γ subunits may also contribute to the structure of the site. In order to label the cardiac glycoside binding site of human erythrocytes, four photoaffinity ligands with very high specific radioactivity were synthesized. The compounds, which are abbreviated [ 125 I]AISC, [ 125 I]AIPP-GluD, [ 125 I]AIPP-GalD and [ 125 I]IA-GalD, were all effective photolabels for NaK-ATPase as shown by ouabain-protectable, covalent labeling of the α, β, and proteolipid-γ subunits. In order to study the possible existence of a very high affinity binding site in erythrocyte NaK-ATPase, a carrier-free radioligand, [ 125 I]I-TASC, was synthesized; this compound had the same structure as [ 125 I]AISC except that a light-sensitive azide group was replaced with a hydroxyl group. Competitive binding assays with cymarin against 0.2 nM [ 125 I]I-TASC suggested two classes of erythrocyte binding sites. Scatchard analysis of direct [ 125 I]I-TASC binding indicated that the very high affinity, low capacity class of erythrocyte bindings sites had a K D of 54 pM and a B max of 23 fmol/mg protein

Women's Relationship to Feminism: Effects of Generation and Feminist Self-Labeling

Science.gov (United States)

Duncan, Lauren E.

2010-01-01

The relative importance to feminism of generation and feminist self-labeling was explored in a sample of 667 women riding buses to a 1992 March on Washington for Reproductive Rights. Specifically, generational (Generation X vs. Baby Boomers) and feminist self-labeling (strong feminists vs. weak feminists vs. nonfeminists) similarities and…

Preparation of radioactively labeled dehydroxy methylepoxy quinomicin, an NF-kB function inhibitor

International Nuclear Information System (INIS)

Chaicharoenpong, Chanya; Umezawa, Kazuo

2003-10-01

Dehydroxymethylepoxyquinomicin (DHMEQ), a synthetic derivative of epoxyquinomicin C, is a potent and specific inhibitor of NF-kB in cultured cells. Tritium-labeled DHMEQ was synthesized by reduction reaction between epoxyquinone 5 and sodium borotritium. Specific radioactivity of the synthesized tritium-labeled DHMEQ was 15.45 mCi/mmol. It should be useful to study the mechanism of action and the stability of DHMEQ in cultured cells

Technetium-99m labelled hydrazinonicotinamido human non-specific polyclonal immunoglobulin G for detection of infectious foci: a comparison with two other technetium-labelled immunoglobulin preparations

Energy Technology Data Exchange (ETDEWEB)

Claessens, R.A.M.J. [University Hospital Nijmegen, Department of Nuclear Medicine, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Boerman, O.C. [University Hospital Nijmegen, Department of Nuclear Medicine, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Koenders, E.B. [University Hospital Nijmegen, Department of Nuclear Medicine, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Oyen, W.J.G. [University Hospital Nijmegen, Department of Nuclear Medicine, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Meer, J.W.M. van der [University Hospital Nijmegen, Department of Nuclear Medicine, P.O. Box 9101, 6500 HB Nijmegen (Netherlands); Corstens, F.H.M. [University Hospital Nijmegen, Department of Nuclear Medicine, P.O. Box 9101, 6500 HB Nijmegen (Netherlands)

1996-04-01

In this study we compared the tissue distribution of three different {sup 99m}Tc-hIgG preparations in groups of five Wistar rats with a focal intramuscular infection with Staphylococcus aureus. We compared {sup 99m}Tc-HYNIC-hIgG with {sup 99m}Tc-hIgG labelled via the so-called Schwarz method (reduction of disulphide bonds) and with the {sup 99m}Tc-labelled commercially available Technescan-HIG. Unlike the HYNIC linker, in the two other labelling methods free sulph-hydryl groups are involved in the binding of {sup 99m}Tc. High-performance liquid chromatography analysis of the labelled preparations and of plasma samples revealed aggregate or polymer formation in all three agents; this was least pronounced in the product labelled by means of the Schwarz method. The tested preparations did not show signs of degradation in vitro. The difference in linker chemistry was reflected in the tissue distribution. Thus the biodistribution of {sup 99m}Tc-HYNIC-hIgG was significantly different from the distribution of the two other preparations: abscess (1.4%{+-}0.2%ID/g), muscle, liver, spleen, plasma, lung, bone marrow, and small intestine concentrations were higher at 24 h p.i.; kidney uptake (1.19%{+-}0.08%ID/g) was significantly lower. The abscess-to-plasma and the abscess-to-muscle ratios (0.5 and 11, respectively), however, were in the same range for the three preparations tested. Quantitative analysis of the scintigraphs revealed that the total body clearance of {sup 99m}Tc-HYNIC-hIgG was significantly slower than for the other agents. The abscess uptake of {sup 99m}Tc-HYNIC-hIgG as a percentage of the remaining body activity was significantly higher. Based on its high abscess uptake, its low uptake in the kidneys and the high percentage of its abscess uptake in relation to the remaining body activity, we conclude that {sup 99m}Tc-HYNIC-hIgG seems superior to the two other preparations tested for the detection of infections. (orig.). With 7 figs., 2 tabs.

A new technique of ion beam tritium labelling

International Nuclear Information System (INIS)

Zhang Nianbao; Sheng Shugang; Yao Fuzeng

1990-01-01

In this paper a new technique is reported for tritium labelling of proteins, peptides and other nonvolatile organic compounds. A tritium ion beam is accelerated to bombard solid sample target for producing tritium exchange with hydrogen. The tritium labelling method has been applied to tritiated soybean trypsin inhibitor, ribonuclease A, elastin, pachyman and others totalled 11. After purifying by dialysis, ion exchange chromatography and gel filtration, the tritiated proteins and polysaccharide were obtained with specific activity over 37 GBq/mmol, without decomposition and with biological activity well preserved. By amino acid analysis of tritiated protein it was shown that the relative specific radioactivities for His., Tyr. and Phe. residues were higher while those for Val., Ile. and Ser. residues were lower

Information on nutritional supplement labels: time for legislation ...

African Journals Online (AJOL)

To assist this process, it is important to have specific knowledge and understanding of the claims made on labels of nutritional supplement products used for general, and more specifically, for sports consumers. The industry is not regulated, and therefore the claims that are made may not always be accurate. Method: The ...

101 labeled brain images and a consistent human cortical labeling protocol

Directory of Open Access Journals (Sweden)

Arno eKlein

2012-12-01

Full Text Available We introduce the Mindboggle-101 dataset, the largest and most complete set of free, publicly accessible, manually labeled human brain images. To manually label the macroscopic anatomy in magnetic resonance images of 101 healthy participants, we created a new cortical labeling protocol that relies on robust anatomical landmarks and minimal manual edits after initialization with automated labels. The Desikan-Killiany-Tourville (DKT protocol is intended to improve the ease, consistency, and accuracy of labeling human cortical areas. Given how difficult it is to label brains, the Mindboggle-101 dataset is intended to serve as brain atlases for use in labeling other brains, as a normative dataset to establish morphometric variation in a healthy population for comparison against clinical populations, and contribute to the development, training, testing, and evaluation of automated registration and labeling algorithms. To this end, we also introduce benchmarks for the evaluation of such algorithms by comparing our manual labels with labels automatically generated by probabilistic and multi-atlas registration-based approaches. All data and related software and updated information are available on the http://www.mindboggle.info/data/ website.

101 Labeled Brain Images and a Consistent Human Cortical Labeling Protocol

Science.gov (United States)

Klein, Arno; Tourville, Jason

2012-01-01

We introduce the Mindboggle-101 dataset, the largest and most complete set of free, publicly accessible, manually labeled human brain images. To manually label the macroscopic anatomy in magnetic resonance images of 101 healthy participants, we created a new cortical labeling protocol that relies on robust anatomical landmarks and minimal manual edits after initialization with automated labels. The “Desikan–Killiany–Tourville” (DKT) protocol is intended to improve the ease, consistency, and accuracy of labeling human cortical areas. Given how difficult it is to label brains, the Mindboggle-101 dataset is intended to serve as brain atlases for use in labeling other brains, as a normative dataset to establish morphometric variation in a healthy population for comparison against clinical populations, and contribute to the development, training, testing, and evaluation of automated registration and labeling algorithms. To this end, we also introduce benchmarks for the evaluation of such algorithms by comparing our manual labels with labels automatically generated by probabilistic and multi-atlas registration-based approaches. All data and related software and updated information are available on the http://mindboggle.info/data website. PMID:23227001

ML-MG: Multi-label Learning with Missing Labels Using a Mixed Graph

KAUST Repository

Wu, Baoyuan; Lyu, Siwei; Ghanem, Bernard

2015-01-01

This work focuses on the problem of multi-label learning with missing labels (MLML), which aims to label each test instance with multiple class labels given training instances that have an incomplete/partial set of these labels (i.e. some