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Sample records for 125i-labeled neurotensin binding

  1. Dopaminergic control of 125I-labeled neurotensin binding site density in corticolimbic structures of the rat brain.

    PubMed Central

    Herve, D; Tassin, J P; Studler, J M; Dana, C; Kitabgi, P; Vincent, J P; Glowinski, J; Rostene, W

    1986-01-01

    In the rat brain, destruction of dopaminergic cell groups by injections of 6-hydroxydopamine into the ventral mesencephalic tegmentum results in large decreases in the number of neurotensin binding sites in the mesencephalon and the striatum. In contrast, these lesions produce an increase in the number of 125I-labeled neurotensin binding sites in the lateral part of the prefrontal cortex despite a large decrease in cortical dopamine levels. Increases in the number of 125I-labeled neurotensin binding sites in this cortical area as well as in the entorhinal cortex, the nucleus accumbens, and the central part of the striatum were also obtained after chronic blockade of dopamine neurotransmission by a long-acting neuroleptic pipotiazine palmitic ester. We propose that dopamine inputs regulate the density of postsynaptic neurotensin binding sites through cortical and subcortical dopamine receptors. Therefore, some of the clinical effects of neuroleptics in schizophrenic patients could be partly related to changes in neurotensin neurotransmission. Images PMID:3016745

  2. Increased /sup 125/I-labelled concanavalin A binding to erythrocytes in diabetes mellitus

    SciTech Connect

    Okada, Y.; Arima, T.; Okazaki, S.; Nakata, K.; Nagashima, H.; Yamabuki, T.

    1982-03-01

    Percentage binding of /sup 125/I-labelled concanavalin A to erythrocytes in diabetic patients was significantly higher than that in normal subjects (12.2 +- 2.8 versus 8.1 +- 1.8%, mean +- SD, p < 0.001). Insulin-dependent diabetic patients showed significantly higher concanavalin A binding than non-insulin-dependent diabetic subjects (15.0 +- 1.4 versus 11.4 +- 2.5%, p < 0.01). There was a highly significant correlation between percentage binding of /sup 125/I-labelled concanavalin A and glycosylated haemoglobin.

  3. Formation of complexes between 125I-labelled human or bovine somatotropins and binding proteins in vivo in rat liver and kidney.

    PubMed Central

    Bonifacino, J S; Roguin, L P; Paladini, A C

    1983-01-01

    At 5 min after intravenous injection, both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin were concentrated in rat liver and kidney. When the labelled hormones were administered along with an excess of the corresponding unlabelled hormone, a significant decrease of the uptake was observed in the liver, but not in the kidney. Study of the subcellular distribution of radioiodinated somatotropins in liver revealed that most of the radioactivity was specifically concentrated in the microsomal fraction. In contrast, the kidney fraction that accounted for most of the radioactivity was the 100 000 g supernatant. After solubilization, with 1% (w/v) Triton X-100, of the microsomal fractions obtained from both organs, the radioactive material was analysed by gel filtration on Sepharose CL-6B. By using this approach, it was demonstrated that both 125I-labelled human somatotropin and 125I-labelled bovine somatotropin bind in vivo to proteins present in liver. A small proportion of 125I-labelled human somatotropin was also shown to form complexes with proteins present in kidney. The present results demonstrate that the liver uptake is mainly due to binding of somatotropins to specific proteins, in contrast with the kidney, in which binding to specific sites contributes minimally to the overall uptake. PMID:6615460

  4. Inhibition of /sup 125/I-labeled ristocetin binding to Micrococcus luteus cells by the peptides related to bacterial cell wall mucopeptide precursors: quantitative structure-activity relationships

    SciTech Connect

    Kim, K.H.; Martin, Y.; Otis, E.; Mao, J.

    1989-01-01

    Quantitative structure-activity relationships (QSAR) of N-Ac amino acids, N-Ac dipeptides, and N-Ac tripeptides in inhibition of /sup 125/I-labeled ristocetin binding to Micrococcus luteus cell wall have been developed to probe the details of the binding between ristocetin and N-acetylated peptides. The correlation equations indicate that (1) the binding is stronger for peptides in which the side chain of the C-terminal amino acid has a large molar refractivity (MR) value, (2) the binding is weaker for peptides with polar than for those with nonpolar C-terminal side chains, (3) the N-terminal amino acid in N-Ac dipeptides contributes 12 times that of the C-terminal amino acid to binding affinity, and (4) the interactions between ristocetin and the N-terminal amino acid of N-acetyl tripeptides appear to be much weaker than those with the first two amino acids.

  5. Binding of an ( sup 125 I) labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets

    SciTech Connect

    Dorn, G.W. II; De Jesus, A. )

    1989-12-01

    To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of (125I)BOP was studied. (125I)BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced (125I)BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that (125I)BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.

  6. Binding of /sup 125/I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets

    SciTech Connect

    Meyers, K.M.; Boehme, M.; Inbar, O.

    1982-10-01

    Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using /sup 125/I-labeled E coli O127:B8 endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.

  7. Altered binding of /sup 125/I-labeled calmodulin to a 46. 5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

    SciTech Connect

    Tallant, E.A.; Wallace, R.W.

    1987-02-01

    The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca/sup 2 +//calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of /sup 125/I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis.

  8. Distribution and binding of 18F-labeled and 125I-labeled analogues of ACI-80, a prospective molecular imaging biomarker of disease: a whole hemisphere post mortem autoradiography study in human brains obtained from Alzheimer's disease patients.

    PubMed

    Gulyás, Balázs; Spenger, Christian; Beliczai, Zsuzsa; Gulya, Károly; Kása, Péter; Jahan, Mahabuba; Jia, Zhisheng; Weber, Urs; Pfeifer, Andrea; Muhs, Andreas; Willbold, Dieter; Halldin, Christer

    2012-01-01

    One of the major pathological landmarks of Alzheimer's disease and other neurodegenerative diseases is the presence of amyloid deposits in the brain. The early non-invasive visualization of amyloid is a major objective of recent diagnostic neuroimaging approaches, including positron emission tomography (PET), with an eye on follow-up of disease progression and/or therapy efficacy. The development of molecular imaging biomarkers with binding affinity to amyloid in the brain is therefore in the forefront of imaging biomarker and radiochemistry research. Recently, a dodecamer peptide (amino acid sequence=QSHYRHISPAQV; denominated D1 or ACI-80) was identified as a prospective ligand candidate, binding with high ex vivo affinity to L-Aβ-amyloid (K(d): 0.4 μM). In order to assess the ligand's capacity to visualize amyloid in Alzheimer's disease (AD), two (125)I labeled and three (18)F labeled analogues of the peptide were synthesized and tested in post mortem human autoradiography experiments using whole hemisphere brain slices obtained from deceased AD patients and age matched control subjects. The (18)F-labeled radioligands showed more promising visualization capacity of amyloid that the (125)I-labeled radioligands. In the case of each (18)F radioligands the grey matter uptake in the AD brains was significantly higher than that in control brains. Furthermore, the grey matter: white matter uptake ratio was over ~2, the difference being significant for each (18)F-radioligands. The regional distribution of the uptake of the various radioligands systematically shows a congruent pattern between the high uptake regions and spots in the autoradiographic images and the disease specific signals obtained in adjacent or identical brain slices labeled with histological, immunohistochemical or autoradiographic stains for amyloid deposits or activated astrocytes. The present data, using post mortem human brain autoradiography in whole hemisphere human brains obtained from deceased

  9. Scintillation proximity radioimmunoassay utilizing 125I-labeled ligands

    SciTech Connect

    Udenfriend, S.; Gerber, L.D.; Brink, L.; Spector, S.

    1985-12-01

    A unique type of radioimmunoassay is described that does not require centrifugation or separation. Microbeads containing a fluorophor are covalently linked to antibody. When an /sup 125/I-labeled antigen is added it binds to the beads and, by its proximity, the emitted short-range electrons of the /sup 125/I excite the fluorophor in the beads. The light emitted can be measured in a standard scintillation counter. Addition of unlabeled antigen from tissue extracts displaces the labeled ligand and diminishes the fluorescent signal. Application of scintillation proximity immunoassay to tissue enkephalins, serum thyroxin, and urinary morphine is described. Applications of the principle to study the kinetics of interaction between receptors and ligands are discussed.

  10. Scintillation Proximity Radioimmunoassay Utilizing 125I-Labeled Ligands

    NASA Astrophysics Data System (ADS)

    Udenfriend, Sidney; Diekmann Gerber, Louise; Brink, Larry; Spector, Sydney

    1985-12-01

    A unique type of radioimmunoassay is described that does not require centrifugation or separation. Microbeads containing a fluorophor are covalently linked to antibody. When an 125I-labeled antigen is added it binds to the beads and, by its proximity, the emitted short-range electrons of the 125I excite the fluorophor in the beads. The light emitted can be measured in a standard scintillation counter. Addition of unlabeled antigen from tissue extracts displaces the labeled ligand and diminishes the fluorescent signal. Application of scintillation proximity immunoassay to tissue enkephalins, serum thyroxin, and urinary morphine is described. Applications of the principle to study the kinetics of interaction between receptors and ligands are discussed.

  11. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with 125I-labelled C1q binding and Raji-cell RIA tests

    PubMed Central

    Casali, P.; Bossus, A.; Carpentier, Nicole A.; Lambert, P.-H.

    1977-01-01

    Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample is incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes are detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody. The conglutinin-binding (KgB) test does not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG is 3 μg/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 μg/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the 125I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, 125I-labelled Clq BA and Raji-cell radioimmunoassay (RIA). Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application. PMID:332422

  12. Neurotensin receptor binding levels in basal ganglia are not altered in Huntington's chorea or schizophrenia

    SciTech Connect

    Palacios, J.M.; Chinaglia, G.; Rigo, M.; Ulrich, J.; Probst, A. )

    1991-02-01

    Autoradiographic techniques were used to examine the distribution and levels of neurotensin receptor binding sites in the basal ganglia and related regions of the human brain. Monoiodo ({sup 125}I-Tyr3)neurotensin was used as a ligand. High amounts of neurotensin receptor binding sites were found in the substantia nigra pars compacta. Lower but significant quantities of neurotensin receptor binding sites characterized the caudate, putamen, and nucleus accumbens, while very low quantities were seen in both medial and lateral segments of the globus pallidus. In Huntington's chorea, the levels of neurotensin receptor binding sites were found to be comparable to those of control cases. Only slight but not statistically significant decreases in amounts of receptor binding sites were detected in the dorsal part of the head and in the body of caudate nucleus. No alterations in the levels of neurotensin receptor binding sites were observed in the substantia nigra pars compacta and reticulata. These results suggest that a large proportion of neurotensin receptor binding sites in the basal ganglia are located on intrinsic neurons and on extrinsic afferent fibers that do not degenerate in Huntington's disease.

  13. Derivatives of cyclosporin compatible with antibody-based assays. I. The generation of (/sup 125/I)-labeled cyclosporin

    SciTech Connect

    Mahoney, W.C.; Orf, J.W.

    1985-03-01

    The immunosuppressive drug cyclosporin A, has been successfully iodinated to a specific activity of 300 Ci per gram. /sup 125/I-labeled cyclosporin and (/sup 3/H)cyclosporin are nearly equivalent as tracers in a radioimmunoassay in producing standard lines (suppression by unlabeled cyclosporin) and in assigning values to clinical samples. In addition, the (/sup 125/I)-labeled cyclosporin has greater than twice the sensitivity, and it is stable to long-term storage. Use of a (/sup 125/I)-labeled cyclosporin tracer is more convenient, more reproducible, more precise, and easier than the tritiated-cyclosporin alternative in radioimmunoassay of this compound.

  14. Intracellular modification of /sup 125/I-labeled epidermal growth factor by normal human foreskin fibroblasts

    SciTech Connect

    Schaudies, R.P.; Savage, C.R. Jr.

    1986-02-01

    Intracellular processing of /sup 125/I-labeled epidermal growth factor (EGF) in normal human foreskin fibroblasts was examined after incubation with saturating concentrations of (/sup 125/I)EGF. This report describes the column chromatographic separation of multiple processed forms of EGF generated by human foreskin fibroblasts and their structural characterization. More than 95% of the cell-bound (/sup 125/I)EGF was converted into multiple forms, which were separated into four distinct peaks of radioactivity using columns of Bio-Gel P-150 equilibrated with 0.2% sodium dodecyl sulfate. These were designated peaks 1-4. Cellular generation of these four peaks was dependent on culture conditions. Differences in absolute and relative amounts of peaks 1-4 were observed as a function of time of incubation at 37 C. In addition, chromatographic profiles of cell-associated /sup 125/I varied in relation to cell density. The radioactivity in peak 1 comigrated with /sup 125/I-labeled native EGF on nondenaturing polyacrylamide gels (pH 9.5), whereas peaks 2 and 3 exhibited more rapid electrophoretic mobilities. Electrophoretic mobilities of the radioactivity in peaks 2 and 3 were indistinguishable from those of chemically prepared derivatives of (/sup 125/I)EGF which were lacking either one or six amino acid residues from the carboxyterminus, respectively. The EGF receptor bound the radioactive material in peak 2 with an affinity equal to or greater than that of EGF; however, the radioactivity in peak 3 was bound to a much lesser extent. The radiolabel in both peaks 2 and 3 was greater than 95% precipitable by antiserum to native EGF. The labeled material in peak 4 was composed of (/sup 125/I)monoiodotyrosine, /sup 125/I-, and an unidentified peptide. None of the radiolabeled compounds in peak 4 interacted with the EGF receptor or with antiserum to native EGF.

  15. Direct interaction between the catalytic subunit of the calmodulin-sensitive adenylate cyclase from bovine brain with /sup 125/I-labeled wheat germ agglutinin and /sup 125/I-labeled calmodulin

    SciTech Connect

    Minocherhomjee, A.M.; Selfe, S.; Flowers, N.J.; Storm, D.R.

    1987-07-14

    A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min/sup -1/ and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with /sup 125/I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca/sup 2 +/ concentration dependent. In addition, the catalytic subunit was shown to directly bind /sup 125/I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.

  16. Distribution of sup 125 I-neurotensin binding sites in human forebrain: Comparison with the localization of acetylcholinesterase

    SciTech Connect

    Szigethy, E.; Quirion, R.; Beaudet, A. )

    1990-07-22

    The distribution of 125I-neurotensin binding sites was compared with that of acetylcholinesterase reactivity in the human basal forebrain by using combined light microscopic radioautography/histochemistry. High 125I-neurotensin binding densities were observed in the bed nucleus of the stria terminalis, islands of Calleja, claustrum, olfactory tubercle, and central nucleus of the amygdala; lower levels were seen in the caudate, putamen, medial septum, diagonal band nucleus, and nucleus basalis of Meynert. Adjacent sections processed for cholinesterase histochemistry demonstrated a regional overlap between the distribution of labeled neurotensin binding sites and that of intense acetylcholinesterase staining in all of the above regions, except in the bed nucleus of the stria terminalis, claustrum, and central amygdaloid nucleus, where dense 125I-neurotensin labeling was detected over areas containing only weak to moderate cholinesterase staining. At higher magnification, 125I-neurotensin-labeled binding sites in the islands of Calleja, supraoptic nucleus of the hypothalamus, medial septum, diagonal band nucleus, and nucleus basalis of Meynert were selectively associated with neuronal perikarya found to be cholinesterase-positive in adjacent sections. Moderate 125I-neurotensin binding was also apparent over the cholinesterase-reactive neuropil of these latter three regions. These data suggest that neurotensin (NT) may directly influence the activity of magnocellular cholinergic neurons in the human basal forebrain, and may be involved in the physiopathology of dementing disorders such as Alzheimer's disease, in which these neurons have been shown to be affected.

  17. Absolute quantitative autoradiography of low concentrations of (/sup 125/I)-labeled proteins in arterial tissue

    SciTech Connect

    Schnitzer, J.J.; Morrel, E.M.; Colton, C.K.; Smith, K.A.; Stemerman, M.B.

    1987-12-01

    We developed a method for absolute quantitative autoradiographic measurement of very low concentrations of (/sup 125/I)-labeled proteins in arterial tissue using Kodak NTB-2 nuclear emulsion. A precise linear relationship between measured silver grain density and isotope concentration was obtained with uniformly labeled standard sources composed of epoxy-embedded gelatin containing glutaraldehyde-fixed (/sup 125/I)-albumin. For up to 308-day exposures of 1 micron-thick tissue sections, background grain densities ranged from about two to eight grains/1000 micron 2, and the technique was sensitive to as little as about one grain/1000 micron 2 above background, which correspond to a radioactivity concentration of about 2 x 10(4) cpm/ml. A detailed statistical analysis of variability was performed and the sum of all sources of variation quantified. The half distance for spatial resolution was 1.7 micron. Both visual and automated techniques were employed for quantitative grain density analysis. The method was illustrated by measurement of in vivo transmural (/sup 125/I)-low-density lipoprotein (( /sup 125/I)-LDL) concentration profiles in de-endothelialized rabbit thoracic aortic wall.

  18. Absorption of enzymatically active sup 125 I-labeled bovine milk xanthine oxidase fed to rabbits

    SciTech Connect

    Rzucidlo, S.J. ); Zikakis, J.P. )

    1990-05-01

    Rabbits fed a regular laboratory diet supplemented with a high-fat milk containing xanthine oxidase (XO) were studied to determine the presence of active XO in the blood. A pilot feeding study, where rabbits consumed a high-fat diet containing xanthine oxidase, showed a correlation between dairy food consumption and XO activity in the blood. Antibody to dietary XO was also found. In a second study, rabbits were fed ad libitum the high-fat milk and blood serum samples were tested weekly for XO activity. No elevation in serum XO activity was found. A third study showed that serum XO activity was increased when rabbits were force fed the high-fat milk. The final study consisted of force feeding {sup 125}I-labeled XO to one rabbit to ascertain whether the observed increase in serum XO was due to dietary or endogenous XO. Isoelectric focusing of sera collected from the test rabbit strongly suggested that at least a portion of the serum XO contained the radioactive label. This is the first direct evidence showing the uptake of dietary active XO from the gut.

  19. Radioimmunoassay of salivary cyclosporine with use of /sup 125/I-labeled cyclosporine

    SciTech Connect

    Coates, J.E.; Lam, S.F.; McGaw, W.T.

    1988-08-01

    We prepared /sup 125/I-labeled cyclosporine (/sup 125/I-CS) by modifying the procedure of Mahoney and Orf and characterized it with regards to maximal immunoreactivity (greater than 90%), trichloroacetic acid precipitability (greater than 90%), and stability (90% immunoreactive after five half-lives of /sup 125/I). For a particular preparation of /sup 125/I-CS, we estimated its immunoreaction concentration (50 pmol/L) and the equilibrium constant for its reaction with Sandoz polyclonal antiserum (K = 3.9 X 10(9) L/mol). By substituting /sup 125/I-CS as tracer in the Sandoz radioimmunoassay and by modifying other aspects of the assay, we developed a procedure that is sufficiently sensitive (0.34 micrograms/L) to allow measurement of trough (lowest inter-dose) cyclosporine concentrations in parotid saliva. Of 38 kidney-transplant patients, 35 had measurable concentrations in saliva (mean 8.3, SD 5.2 micrograms/L), and these correlated moderately with paired serum concentrations (r = 0.68, P less than 0.001). We believe that measurement of salivary cyclosporine may offer a simple way of estimating the free fraction of the drug in serum or plasma.

  20. Preliminary Characterization and In Vivo Studies of Structurally Identical (18)F- and (125)I-Labeled Benzyloxybenzenes for PET/SPECT Imaging of β-Amyloid Plaques.

    PubMed

    Yang, Yanping; Zhang, Xiaoyang; Cui, Mengchao; Zhang, Jinming; Guo, Zhide; Li, Yesen; Zhang, Xianzhong; Dai, Jiapei; Liu, Boli

    2015-01-01

    With the assistance of molecular docking and 3D-QSAR models established previously, structurally identical (18)F- and (125)I-labeled benzyloxybenzene derivatives were designed to achieve the early detection of Aβ plaques by PET/SPECT imaging. In competition binding assay, ligands 7a and 12a displayed high binding affinities to Aβ42 aggregates with Ki values of 19.5 nM and 23.9 nM, respectively. Specific plaque labeling was observed on the in vitro autoradiography of brain sections from AD patients and Tg mice. In biodistribution, [(125)I]7a, [(18)F]7a, [(125)I]12a and [(18)F]12a all exhibited high initial brain uptakes (>5% ID/g at 2 min). [(125)I]7a and [(125)I]12a cleared fast from the normal brain regions, while corresponding [(18)F]7a and [(18)F]12a showed slow washout rates. Dynamic microPET/CT and microSPECT/CT imaging data in normal ICR mice were in accordance with in vivo biodistribution results. In vivo metabolism results indicated that the different clearance profiles between the structurally identical (18)F- and (125)I-labeled tracers could be attributed to different biochemical characteristics of the radiometabolites. Radioiodinated benzyloxybenzene derivatives exhibited good in vivo biostability in brain. Ex vivo autoradiography further confirmed the strong in vivo Aβ labeling ability of [(125)I]7a. These new fluorinated and iodinated benzyloxybenzenes can develop into PET/SPECT dual imaging agents targeting Aβ plaques. PMID:26170205

  1. 125I-labeled anti-bFGF monoclonal antibody inhibits growth of hepatocellular carcinoma

    PubMed Central

    Hu, Peng-Hui; Pan, Lan-Hong; Wong, Patrick Ting-Yat; Chen, Wen-Hui; Yang, Yan-Qing; Wang, Hong; Xiang, Jun-Jian; Xu, Meng

    2016-01-01

    AIM: To investigate the inhibitory efficacy of 125I-labeled anti-basic fibroblast growth factor (bFGF) monoclonal antibody (mAb) in hepatocellular carcinoma (HCC). METHODS: bFGF mAb was prepared by using the 1G9B9 hybridoma cell line with hybridization technology and extracted from ascites fluid through a Protein G Sepharose affinity column. After labeling with 125I through the chloramine-T method, bFGF mAb was further purified by a Sephadex G-25 column. Gamma radiation counter GC-1200 detected radioactivity of 125I-bFGF mAb. The murine H22 HCC xenograft model was established and randomized to interventions with control (phosphate-buffered saline), 125I-bFGF mAb, 125I plus bFGF mAb, bFGF mAb, or 125I. The ratios of tumor inhibition were then calculated. Expression of bFGF, fibroblast growth factor receptor (FGFR), platelet-derived growth factor, and vascular endothelial growth factor (VEGF) mRNA was determined by quantitative reverse transcriptase real-time polymerase chain reaction. RESULTS: The purified bFGF mAb solution was 8.145 mg/mL with a titer of 1:2560000 and was stored at -20 °C. After coupling, 125I-bFGF mAb was used at a 1: 1280000 dilution, stored at 4 °C, and its specific radioactivity was 37 MBq/mg. The corresponding tumor weight in the control, 125I, bFGF mAb, 125I plus bFGF mAb, and 125I-bFGF mAb groups was 1.88 ± 0.25, 1.625 ± 0.21, 1.5 ± 0.18, 1.41 ± 0.16, and 0.98 ± 0.11 g, respectively. The tumor inhibition ratio in the 125I, bFGF mAb, 125I plus bFGF mAb, and 125I-bFGF mAb groups was 13.6%, 20.2%, 25.1%, and 47.9%, respectively. Growth of HCC xenografts was inhibited significantly more in the 125I-bFGF mAb group than in the other groups (P < 0.05). Expression of bFGF and FGFR mRNA in the 125I-bFGF mAb group was significantly decreased in comparison with other groups (P < 0.05). Groups under interventions revealed increased expression of VEGF mRNA (except for 125I group) compared with the control group. CONCLUSION: 125I-bFGF m

  2. Direct method for detection and characterization of cell surface receptors for insulin by means of 125I-labeled autoantibodies against the insulin receptor.

    PubMed Central

    Jarrett, D B; Roth, J; Kahn, C R; Flier, J S

    1976-01-01

    Autoantibodies directed against the cell surface receptors for insulin are found in some patients with extreme insulin resistance. These antibodies specifically inhibit the binding of insulin to its receptor. A purified IgG fraction from one patient's plasma was labeled with 125I. The 125I-labeled antireceptor antibody, which initially represented about 0.3% of the total 125I-IgG, was enriched by selective adsorption and subsequent elution from cells rich in insulin receptors. The 125I-antireceptor antibody bound to cells and the binding was inhibited by whole plasma and purified IgG from this patient, as well as whole plasma from another patient with autoantibodies to the insulin receptor. Insulins that differed 300-fold in biological potency and affinity inhibited binding of 125I-antireceptor antibody in direct proportion to their ability to bind to the insulin receptor. The binding of 125I-antireceptor antibody was closely correlated with the binding of 125I-insulin over a wide range of receptor concentrations on different cell types. Experimentally induced reduction of the insulin receptor concentration was associated with parallel decreases in the binding of 125I-antireceptor antibody and 125I-insulin. The preparation of 125I-antireceptor antibody with a high specific activity by cytoadsorption and elution has provided a sensitive method for the detection of receptors and autoantibodies to cell surface components. PMID:1069300

  3. Pharmacokinetics of internally labeled monoclonal antibodies as a gold standard: comparison of biodistribution of /sup 75/Se-, /sup 111/In-, and /sup 125/I-labeled monoclonal antibodies in osteogenic sarcoma xenografts in nude mice

    SciTech Connect

    Koizumi, M.; Endo, K.; Watanabe, Y.; Saga, T.; Sakahara, H.; Konishi, J.; Yamamuro, T.; Toyama, S.

    1989-04-01

    In order to know the true biodistribution of anti-tumor monoclonal antibodies, three monoclonal antibodies (OST6, OST7, and OST15) against human osteosarcoma and control antibody were internally labeled with 75Se by incubating (75Se)methionine and hybridoma cells. 75Se-labeled monoclonal antibodies were evaluated both in vitro and in vivo using the human osteogenic sarcoma cell line KT005, and the results were compared with those of 125I- and 111In-labeled antibodies. 75Se-, 125I- and 111In-labeled monoclonal antibodies had identical binding activities to KT005 cells, and the immunoreactivity was in the decreasing order of OST6, OST7, and OST15. On the contrary, in vivo tumor uptake (% injected dose/g) of 75Se- and 125I-labeled antibodies assessed using nude mice bearing human osteosarcoma KT005 was in the order of OST7, OST6, and OST15. In the case of 111In, the order was OST6, OST7, and OST15. High liver uptake was similarly seen with 75Se- and 111In-labeled antibodies, whereas 125I-labeled antibodies showed the lowest tumor and liver uptake. These data indicate that tumor targeting of antibody conjugates are not always predictable from cell binding studies due to the difference of blood clearance of labeled antibodies. Furthermore, biodistribution of both 111In- and 125I-labeled antibodies are not identical with internally labeled antibody. Admitting that internally labeled antibody is a ''gold standard'' of biodistribution of monoclonal antibody, high liver uptake of 111In-radiolabeled antibodies may be inherent to antibodies. Little, if any, increase in tumor-to-normal tissue ratios of antibody conjugates will be expected compared to those of 111In-labeled antibodies if stably coupled conjugates are administered i.v.

  4. The absorption of 125I-labelled immunoglobulin G by different regions of the gut in young rats

    PubMed Central

    Morris, B.; Morris, R.

    1974-01-01

    1. 125I-labelled homologous IgG was injected into different regions of the small intestine of rats aged 12, 16, 18, 20 and 22 days. At 12 days the proximal and middle regions of the intestine readily absorbed globulin and transmitted it to the circulation. The distal region of the intestine transmitted little to the circulation at all ages tested. 2. The intestine loses its ability to transmit globulin to the circulation in a distal-proximal direction. At 16 and 18 days the ability of the middle region had declined significantly, and this decline continued so that little globulin was transmitted from this region at 20 and 22 days. 3. The proximal intestine retained the ability to transmit globulin to the circulation in significant amounts up to 20 days. 4. There is a close negative correlation between body weight and total radioactivity of the sera of rats which had received doses of labelled globulin into the proximal and middle regions of the intestine. There was no such correlation after injection into the distal intestine — suggesting a restricted throughput of radioactive material by the absorptive cells of this region. 5. These results are discussed in the context of the termination of antibody absorption, and in relation to the results obtained using polyvinyl pyrrolidone. PMID:4436816

  5. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

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

    SciTech Connect

    Denny, J.B.; Blobel, G.

    1984-09-01

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

  7. In Silico Investigation of the Neurotensin Receptor 1 Binding Site: Overlapping Binding Modes for Small Molecule Antagonists and the Endogenous Peptide Agonist.

    PubMed

    Lückmann, Michael; Holst, Birgitte; Schwartz, Thue W; Frimurer, Thomas M

    2016-01-01

    The neurotensin receptor 1 (NTSR1) belongs to the family of 7TM, G protein-coupled receptors, and is activated by the 13-amino-acid peptide neurotensin (NTS) that has been shown to play important roles in neurological disorders and the promotion of cancer cells. Recently, a high-resolution x-ray crystal structure of NTSR1 in complex with NTS8-13 has been determined, providing novel insights into peptide ligand recognition by 7TM receptors. SR48692, a potent and selective small molecule antagonist has previously been used extensively as a tool compound to study NTSR1 receptor signaling properties. To investigate the binding mode of SR48692 and other small molecule compounds to NTSR1, we applied an Automated Ligand-guided Backbone Ensemble Receptor Optimization protocol (ALiBERO), taking receptor flexibility and ligand knowledge into account. Structurally overlapping binding poses for SR48692 and NTS8-13 were observed, despite their distinct chemical nature and inverse pharmacological profiles. The optimized models showed significantly improved ligand recognition in a large-scale virtual screening assessment compared to the crystal structure. Our models provide new insights into small molecule ligand binding to NTSR1 and could facilitate the structure-based design of non-peptide ligands for the evaluation of the pharmacological potential of NTSR1 in neurological disorders and cancer. PMID:27491650

  8. Biotin radioligand assay with an /sup 125/I-labeled biotin derivative, avidin, and avidin double-antibody reagents

    SciTech Connect

    Livaniou, E.; Evangelatos, G.P.; Ithakissios, D.S.

    1987-11-01

    We describe a new radioligand assay for determining biotin in biological fluids by using a mixture of N-(beta-(4-OH-3-125I-phenyl)ethyl)- and N-(beta-(4-OH-3,5-di-125I-phenyl)ethyl)biotinamides as radiotracer, avidin as a binding protein, and an avidin double-antibody as a separation reagent. The radiotracer is synthesized by coupling (at pH 8.5, 20-22 degrees C, 90 min) N-hydroxysuccinimidobiotin to radioiodinated tyramine. The assay curve is linear and the assay itself is sensitive (less than 10 ng/L), reproducible (intra- and interassay CVs 4.1% and 7.0%, respectively), and allows the simultaneous handling of more than 100 samples in less than 4 h. Serum samples from apparently normal subjects contained 100-840 ng of biotin per liter (mean 340 ng/L). Pregnant women had low concentrations of biotin (100-300 ng/L) in their serum. Patients undergoing chronic hemodialysis treatment showed high concentrations (0.5-3.0 micrograms/L), which may be ascribable to the inability of avidin, which was used as the assay binding protein, to distinguish biotin from biotinyl derivatives with an intact ureido ring.

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

    SciTech Connect

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

    1985-11-01

    Thyroid glands from turtles (Chrysemys dorbigni) pretreated with potassium iodide were incubated with /sup 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 /sup 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 /sup 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/sup 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/sup 125/I elution positions. Dissociation studies were performed at 6 degrees in glands preincubated with /sup 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 /sup 125/I-insulin reduced the radioactive degradation products in the dissociated medium. Chloroquine or bacitracin inhibited the degradation activity. Incubation of thyroid glands with /sup 125/I-hGH or /sup 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.

  10. Lymphatic flow in humans as indicated by the clearance of /sup 125/I-labeled albumin from the subcutaneous tissue of the leg

    SciTech Connect

    Fernandez, M.J.; Davies, W.T.; Owen, G.M.; Tyler, A.

    1983-08-01

    Since the removal of albumin from the extracellular space and its return to the vascular compartment is the essential function of the lymphatic system, the rate at which it is removed from the interstitial tissue may be regarded as a means of estimating lymphatic efficiency. An objective measure of lymphatic function can be obtained by monitoring the rate of clearance following injection of /sup 125/I-labeled albumin (RIHSA) from the subcutaneous tissue of a limb. The clearance of /sup 125/I-RIHSA from lower limb was monitored in a group of patients with normal limbs, patients with unilateral edema due to deep vein thrombosis, and patients with bilateral edema due to hypoproteinemia. The mean T1/2 in normal legs was 32.7 hr, compared to 23.7 hr in edematous limbs due to deep vein thrombosis and 19.4 in edematous limbs due to hypoproteinemia. There is a clear-cut difference in clearance rate between edematous and nonedematous limbs. This suggests that lymphatic flow is increased in edema due to venous obstruction and hypoproteinemia.

  11. Measurement of cyclosporine concentrations in whole blood: HPLC and radioimmunoassay with a specific monoclonal antibody and /sup 3/H- or /sup 125/I-labeled ligand compared

    SciTech Connect

    Wolf, B.A.; Daft, M.C.; Koenig, J.W.; Flye, M.W.; Turk, J.W.; Scott, M.G.

    1989-01-01

    We compared cyclosporine concentrations in whole blood as measured by HPLC and by RIA with a monoclonal antibody specific for cyclosporine with /sup 3/H- or /sup 125/I-labeled cyclosporine ligand. The /sup 3/H-RIA kit slightly underestimated cyclosporine concentrations (greater than 600 micrograms/L) in comparison with HPLC. Over a wide range of concentrations, cyclosporine measured with the /sup 125/I-RIA kit correlated well with HPLC (slope = 0.99, n = 301, r = 0.98), observed for samples from recipients of kidney, heart, or liver allografts (respective slopes: 1.01, 0.93, and 1.00). The /sup 125/I-RIA standard curve was linear to 1000 micrograms of cyclosporine per liter. Inter- and intra-assay CVs for /sup 125/I-RIA measurements of cyclosporine were less than or equal to 7%. Evidently, the /sup 125/I-RIA kit involving a monoclonal antibody specific for cyclosporine is equivalent to the HPLC assay and can replace it for therapeutic drug monitoring of cyclosporine therapy.

  12. Macrophage function as studied by the clearance of /sup 125/I-labeled polyvinylpyrrolidone in iron-deficient and iron-replete mice

    SciTech Connect

    Kuvibidila, S.; Wade, S.

    1987-01-01

    This study evaluated the effects of iron deficiency and iron repletion on in vivo macrophage function determined by the clearance of /sup 125/I-labeled polyvinylpyrrolidone (PVP). Two experiments were done. There were four groups of C57BL/6 female mice in experiment 1: the iron-deficient (ID), pair-fed (PF), control (C) and the high iron (HI) groups. In experiment 2, there were three ID groups (severe to moderate anemia), three PF, one C and four ID groups that were fed adequate iron for 14 (R-14), 7 (R-7), 3 (R-3) days before or on the day of PVP injection (R-0). The overall rate of PVP clearance from blood was lower in ID than in C or PF groups. This clearance is expressed by a constant, K, calculated from natural log (ln) of the cpm and the time postadministration of PVP that blood was drawn. The theoretical individual macrophages function (alpha PVP), derived from K and the weights of body, spleen and liver, was also lower in ID than in C or PF groups. The impairment was most severe with the most severe iron deficiency. Repletion for 7 to 15 d before PVP administration resulted in a partial correction of the clearance. Moderate undernutrition in the PF group had no effect.

  13. The transmission of -125-I-labelled immunoglobulin G by proximal and distal regions of the small intestine of 16-day-old rats.

    PubMed Central

    Morris, B

    1975-01-01

    1. Standard doses of -125-I-labelled rat IgG were injected into the intestinal lumen of rats aged 16 days, and their sera were sampled 2 and 3 hr later. High concentration quotients were obtained after injection into the proximal small intestime, whereas very little immunoglobulin was transmitted from doses injected into the terminal 20 cm of the small intestine. 2. The villi of the terminal 18--20 cm of the small intestine of 16-day-old rats, the region from which very little transmission of IgG occurred, were lined by tall columnar absorptive cells with very larg supra-nuclear vacuoles. The extent of the terminal intestine, in which this cell type predominated in the absorptive epithelium, varied with age. The importance of defining the precise location of the region of the intestine under examination is stressed. 3. The experimental results and the histological observations are discussed in relation to (a) the results which have been obtained using PVP, which is unsuitable as an indicator of immunoglobulin transport in the rat and (b) the histological composition of the absorptive epithelium and the maturation changes which affect the epithelium between 18 and 21 days. Images A B C D PMID:1127610

  14. Synthesis and evaluation of an (125)I-labeled azide prosthetic group for efficient and bioorthogonal radiolabeling of cyclooctyne-group containing molecules using copper-free click reaction.

    PubMed

    Choi, Mi Hee; Shim, Ha Eun; Nam, You Ree; Kim, Hye Rim; Kang, Jung Ae; Lee, Dong-Eun; Park, Sang Hyun; Choi, Dae Seong; Jang, Beom-Su; Jeon, Jongho

    2016-02-01

    Herein we report the radiosynthesis of a pyridine derived azide prosthetic group for iodine radioisotope labeling of dibenzocyclooctyne (DBCO) conjugated molecules. The radiolabeling of the stannylated precursor 2 was conducted using [(125)I]NaI and chloramine-T to give (125)I-labeled azide ([(125)I]1) with high radiochemical yield (72±8%, n=4) and radiochemical purity (>99%). Using (125)I-labeled azide ([(125)I]1), cyclic RGD peptide and near infrared fluorescent molecule were efficiently labeled with modest to good radiochemical yields. The biodistribution study and SPECT/CT images showed that [(125)I]1 underwent rapid renal clearance. These results clearly demonstrated that [(125)I]1 could be used as an useful radiotracer for in vivo pre-targeted imaging as well as efficient in vitro radiolabeling of DBCO containing molecules. PMID:26748695

  15. Purification of the neurotensin receptor from bovine brain

    SciTech Connect

    Mills, A.; Demoliou-Mason, C.D.; Barnard, E.A.

    1988-01-05

    The neurotensin receptor protein, solubilized with digitonin/asolectin from bovine cerebral cortex membranes, was purified to apparent homogeneity by affinity chromatography using immobilized neurotensin. The product exhibits saturable and specific binding of (3,11-tyrosyl-3,5-/sup 3/H) neurotensin with an apparent affinity (K/sub d/ = 5.5 nM) comparable to that measured in intact membranes and crude soluble extracts. The affinity-purified material, after reduction with 100 mM dithiothreitol, in denaturing gel electrophoresis showed a single polypeptide of M/sub r/ 72,000. Under nonreducing conditions the apparent M/sub r/, however, was 50,000, suggesting the presence of intramolecular disulfide bonds. The purified neurotensin receptor was judged to be homogenous, in that (i) only a single polypeptide was detectable; and (ii) the overall purification was 30,000-50,000-fold, giving a specific neurotensin-binding activity close to the theoretical maximum.

  16. Comparison of /sup 125/I-labeled and /sup 14/C-Labeled peptides of the major outer membrane protein of Chlamydia Trachomatis Strain L2/434 separated by high-performance liquid chromatography

    SciTech Connect

    Judd, R.C.; Caldwell, H.D.

    1985-01-01

    The objective of this study was to determine if in-gel chloramine-T radioiodination adequately labels OM proteins to allow for accurate and precise structural comparison of these molecules. Therefore, intrinsically /sup 14/C-amino acid labeled proteins and /sup 125/I-labeled proteins were cleaved with two endopeptidic reagents and the peptide fragments separated by HPLC. A comparison of retention times of the fragments, as determined by differential radiation counting, thus indicated whether /sup 125/Ilabeling identified of all the peptide peaks seen in the /sup 14/Clabeled proteins. Results demonstrated that radioiodination yields complete and accurate information about the primary structure of outer membrane proteins. In addition, it permits the use of extremely small amounts of protein allowing for method optimization and multiple separations to insure reproducibility.

  17. Existence of B/E and E receptors on Hep-G2 cells: a study using colloidal gold- and /sup 125/I-labeled lipoproteins

    SciTech Connect

    Hesz, A.; Ingolic, E.; Krempler, F.; Kostner, G.M.

    1987-06-01

    The presence of specific receptors for apolipoprotein B (low-density lipoproteins) and apolipoprotein E (HDL-E) on Hep-G2 cells and human skin fibroblasts was studied by chemical methods and by electron microscopy using a differential gold labeling technique. Fibroblasts bound both types of lipoproteins to one and the same receptor (B/E receptor) as deduced from competition experiments with HDL-E and LDL. Labeled HDL-E, on the other hand, was only partially displaced by cold LDL but was completely displaced by unlabeled HDL-E. Scatchard analysis of lipoprotein binding to Hep-G2 cells revealed an approx 10 times higher binding affinity of apoE-containing lipoproteins as compared to apoB-containing ones. No differences between apoE- or apoB-containing lipoproteins with respect to the morphology of cell binding and intracellular processing were observed. The results are compatible with the concept that Hep-G2 cells possess two kinds of receptors, one specific for apoB- and apoE-containing lipoproteins (B/E receptor) and another specific for apoE only. From these studies we conclude that Hep-G2 cells may serve as a suitable model for studying the lipoprotein metabolism in the liver.

  18. Interaction of /sup 125/I-labeled botulinum neurotoxins with nerve terminals. II. Autoradiographic evidence for its uptake into motor nerves by acceptor-mediated endocytosis

    SciTech Connect

    Black, J.D.; Dolly, J.O.

    1986-01-01

    Using pharmacological and autoradiographic techniques it has been shown that botulinum neurotoxin (BoNT) is translocated across the motor nerve terminal membrane to reach a postulated intraterminal target. In the present study, the nature of this uptake process was investigated using electron microscopic autoradiography. It was found that internalization is acceptor-mediated and that binding to specific cell surface acceptors involves the heavier chain of the toxin. In addition, uptake was shown to be energy and temperature-dependent and to be accelerated by nerve stimulation, a treatment which also shortens the time course of the toxin-induced neuroparalysis. These results, together with the observation that silver grains were often associated with endocytic structures within the nerve terminal, suggested that acceptor-mediated endocytosis is responsible for toxin uptake. Possible recycling of BoNT acceptors (an important aspect of acceptor-mediated endocytosis of toxins) at motor nerve terminals was indicated by comparing the extent of labeling in the presence and absence of metabolic inhibitors. On the basis of these collective results, it is concluded that BoNT is internalized by acceptor-mediated endocytosis and, hence, the data support the proposal that this toxin inhibits release of acetylcholine by interaction with an intracellular target.

  19. Reagents for astatination of biomolecules. 5. Evaluation of hydrazone linkers in (211)At- and (125)I-labeled closo-decaborate(2-) conjugates of Fab' as a means of decreasing kidney retention.

    PubMed

    Wilbur, D Scott; Chyan, Ming-Kuan; Hamlin, Donald K; Nguyen, Holly; Vessella, Robert L

    2011-06-15

    benzoate substituent on the hydrazone was chosen for study with (211)At. That reagent was conjugated with 107-1A4 Fab', then labeled (separately) with (125)I and (211)At. The radiolabeled Fab' conjugates were coinjected into nude mice bearing LNCaP human tumor xenografts, and biodistribution data were obtained at 1, 4, and 24 h pi. Tumor targeting was achieved with both (125)I- and (211)At-labeled Fab', but the (211)At-labeled Fab' reached a higher concentration (25.56 ± 11.20 vs 11.97 ± 1.31%ID/g). Surprisingly, while the (125)I-labeled Fab' was cleared from kidney similar to earlier studies, the (211)At-labeled Fab'was not (i.e., kidney conc. for (125)I vs (211)At; 4 h, 13.14 ± 2.03 ID/g vs 42.28 ± 16.38%D/g; 24 h, 4.23 ± 1.57 ID/g vs 39.52 ± 15.87%ID/g). Since the Fab' conjugate is identical in both cases except for the radionuclide, it seems likely that the difference in tissue clearance seen is due to an effect that (211)At has on either the hydrazone cleavage or on the retention of a metabolite. Results from other studies in our laboratory suggest that the latter case is most likely. The hydrazone linkers tested do not provide the tissue clearance sought for (211)At, so additional hydrazones linkers will be evaluated. However, the results support the use of hydrazone linkers when Fab' conjugated with closo-decaborate(2-) reagents are radioiodinated. PMID:21513347

  20. Revisiting the structure of the Vps10 domain of human sortilin and its interaction with neurotensin

    PubMed Central

    Quistgaard, Esben M; Grøftehauge, Morten K; Madsen, Peder; Pallesen, Lone T; Christensen, Brian; Sørensen, Esben S; Nissen, Poul; Petersen, Claus M; Thirup, Søren S

    2014-01-01

    Sortilin is a multifunctional receptor involved in sorting and apoptosis. We have previously reported a 2.0-Å structure of the Vps10 ectodomain in complex with one of its ligands, the tridecapeptide neurotensin. Here we set out to further characterize the structural properties of sortilin and its interaction with neurotensin. To this end, we have determined a new 2.7 Å structure using a crystal grown with a 10-fold increased concentration of neurotensin. Here a second peptide fragment was observed within the Vps10 β-propeller, which may in principle either represent a second molecule of neurotensin or the N-terminal part of the molecule bound at the previously identified binding site. However, in vitro binding experiments strongly favor the latter hypothesis. Neurotensin thus appears to bind with a 1:1 stoichiometry, and whereas the N-terminus does not bind on its own, it enhances the affinity in context of full-length neurotensin. We conclude that the N-terminus of neurotensin probably functions as an affinity enhancer for binding to sortilin by engaging the second binding site. Crystal packing differs partly from the previous structure, which may be due to variations in the degree and pattern of glycosylations. Consequently, a notable hydrophobic loop, not modeled previously, could now be traced. A computational analysis suggests that this and a neighboring loop may insert into the membrane and thus restrain movement of the Vps10 domain. We have, furthermore, mapped all N-linked glycosylations of CHO-expressed human sortilin by mass spectrometry and find that their locations are compatible with membrane insertion of the hydrophobic loops. PMID:24985322

  1. Neurotensin: A role in substance use disorder?

    PubMed

    Ferraro, Luca; Tiozzo Fasiolo, Laura; Beggiato, Sarah; Borelli, Andrea C; Pomierny-Chamiolo, Lucyna; Frankowska, Malgorzata; Antonelli, Tiziana; Tomasini, Maria C; Fuxe, Kjell; Filip, Malgorzata

    2016-02-01

    Neurotensin is a tridecapeptide originally identified in extracts of bovine hypothalamus. This peptide has a close anatomical and functional relationship with the mesocorticolimbic and nigrostriatal dopamine system. Neural circuits containing neurotensin were originally proposed to play a role in the mechanism of action of antipsychotic agents. Additionally, neurotensin-containing pathways were demonstrated to mediate some of the rewarding and/or sensitizing properties of drugs of abuse.This review attempts to contribute to the understanding of the role of neurotensin and its receptors in drug abuse. In particular, we will summarize the potential relevance of neurotensin, its related compounds and neurotensin receptors in substance use disorders, with a focus on the preclinical research. PMID:26755548

  2. The effects of acute exposure to ethanol on neurotensin and guanine nucleotide-stimulation of phospholipase C activity in intact NIE-115 neuroblastoma cells

    SciTech Connect

    Smith, T.L. )

    1990-01-01

    Both ethanol and neurotensin produce sedation and hypothermia. When administered in combination the behavioral effects of these two substances are potentiated. In order to better understand the biochemical nature of this interaction, the direct effects of ethanol on neurotensin receptors and an associated signal transduction process were determined in NIE-115 neuroblastoma cells. Ethanol in physiologically relevant concentrations significantly reduced neurotensin stimulated ({sup 3}H)inositol phosphate production while having no effect on the specific binding of ({sup 3}H)neurotensin. In addition, ethanol up to 200 mM had no effect on GTPYS mediated ({sup 3}H)inositol phosphate production. The results indicate that acute exposure ethanol partially disrupts the normal coupling of activated neurotensin receptors to the guanine nucleotide binding protein associated with phospholipase C.

  3. ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse.

    PubMed

    Barak, Larry S; Bai, Yushi; Peterson, Sean; Evron, Tama; Urs, Nikhil M; Peddibhotla, Satyamaheshwar; Hedrick, Michael P; Hershberger, Paul; Maloney, Patrick R; Chung, Thomas D Y; Rodriguiz, Ramona M; Wetsel, William C; Thomas, James B; Hanson, Glen R; Pinkerton, Anthony B; Caron, Marc G

    2016-07-15

    Pharmacological treatment for methamphetamine addiction will provide important societal benefits. Neurotensin receptor NTR1 and dopamine receptor distributions coincide in brain areas regulating methamphetamine-associated reward, and neurotensin peptides produce behaviors opposing psychostimulants. Therefore, undesirable methamphetamine-associated activities should be treatable with druggable NTR1 agonists, but no such FDA-approved therapeutics exist. We address this limitation with proof-of-concept data for ML314, a small-molecule, brain penetrant, β-arrestin biased, NTR1 agonist. ML314 attenuates amphetamine-like hyperlocomotion in dopamine transporter knockout mice, and in C57BL/6J mice it attenuates methamphetamine-induced hyperlocomotion, potentiates the psychostimulant inhibitory effects of a ghrelin antagonist, and reduces methamphetamine-associated conditioned place preference. In rats, ML314 blocks methamphetamine self-administration. ML314 acts as an allosteric enhancer of endogenous neurotensin, unmasking stoichiometric numbers of hidden NTR1 binding sites in transfected-cell membranes or mouse striatal membranes, while additionally supporting NTR1 endocytosis in cells in the absence of NT peptide. These results indicate ML314 is a viable, preclinical lead for methamphetamine abuse treatment and support an allosteric model of G protein-coupled receptor signaling. PMID:27119457

  4. Estimation of anti-D IgG in red blood cell eluates using the specific radioactivity of 125I-labeled IgG: effect of unlabeled, cytophilic IgG

    SciTech Connect

    Masouredis, S.P.; Mahan, L.C.; Sudora, E.J.; Langley, J.W.; Victoria, E.J.

    1981-01-01

    The specific radioactivity of conventionally prepared 125I IgG anti-D eluates is significantly less (from 1/5 to 1/20) than that of the 125I IgG fraction used to prepare the eluate. This discrepancy is due to the release of unlabeled, cytophilic IgG from normal red blood cells during eluate preparation and does not represent an underestimation of the eluate anti-D IgG content. Cytophilic IgG content of eluates plays an important role in reducing the nonimmunologic binding of labeled antibody IgG. The results justify the assumption used in numerous studies that the specific radioactivity of 125I IgG fractions can be used to provide a valid estimate of the anti-D IgG content of eluates.

  5. Synthesis and bioevaluation of 125I-labeled gold nanorods

    NASA Astrophysics Data System (ADS)

    Shao, Xia; Agarwal, Ashish; Rajian, Justin R.; Kotov, Nicholas A.; Wang, Xueding

    2011-04-01

    A novel technique is described for monitoring the in vivo behavior of gold nanorods (GNRs) using γ-imaging. GNRs were radiolabeled using [125I] sodium iodide in a simple and fast manner with high yield and without disturbing their optical properties. Radiolabeled GNRs were successfully visualized by radioisotope tagging, allowing longitudinal in vivo studies to be performed repeatedly in the same animal. The preliminary biodistribution study showed that PEGylated GNRs have much longer blood circulation times and clear out faster, while bare GNRs accumulate quickly in the liver after systematic administration. The highly efficient method reported here provides an extensively useful tool for guidance of the design and development of new gold nanoparticles as target-specific agents for both diagnostics and photothermal therapy.

  6. Antigen-binding small lymphocytes in the guinea-pig

    PubMed Central

    Donald, D.; Beck, J. Swanson

    1974-01-01

    The time course of the relative distribution of small lymphocytes binding 125I-labelled human thyroglobulin (HTg) in cell suspensions from the peripheral blood and various lymphoid organs was studied in guinea-pigs at progressive intervals up to 28 days after immunization with an emulsion of HTg and BCG in Freund's incomplete adjuvant (FIA). Small lymphocytes binding 125I-labelled HTg were first detected in peripheral blood, popliteal (draining) lymph node, spleen and bone marrow preparations on the 10th day, and in mesenteric (distant) lymph node and thymus preparations on the 14th day after primary immunization. In general, the percentage of these cells increased progressively thereafter until the end of the period of study. Blocking experiments with unlabelled antigens indicated that the binding of 125I-labelled HTg by small lymphocytes was specific. An anti-HTg antibody cytophilic for guinea-pig small lymphocytes was demonstrated by the passive transfer of antigen-binding capacity to lymphocytes of unimmunized animals with hyperimmune guinea-pig serum. It is proposed that, in these experiments, anti-HTg cytophilic antibody was bound first to small lymphocytes in the tissues participating actively in the immune response (popliteal node, spleen and bone marrow) before spilling over into the general circulation to coat lymphocytes at other sites (mesenteric node and thymus). PMID:4604111

  7. Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

    PubMed Central

    Pestonjamasp, K; Amieva, M R; Strassel, C P; Nauseef, W M; Furthmayr, H; Luna, E J

    1995-01-01

    Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein. Images PMID:7612961

  8. Binding of /sup 125/I-hCG to rainbow trout (Salmo gairdneri) testis in vitro. [Human Chorionic Gonadotropin

    SciTech Connect

    Schlaghecke, R.

    1983-02-01

    Homogenates of maturing rainbow trout testes show specific binding sites for /sup 125/I-labeled hCG (. /sup 125/I-labeled hCG). The binding is competitively inhibited by unlabeled hCG and by a hypophyseal extract of rainbow trout. It could be demonstrated that the tissue /sup 125/I-hCG binding specificity is restricted to the gonadal preparation. The trout testis was characterized by determining affinity and capacity from Scatchard plot analysis giving a high constant of dissociation Kd 3.65 x 10(-10)/M and a low binding capacity of 0.88 x 10(-15) M/mg tissue. The test system is markedly dependent on temperature, incubation-time, and pH. The maximum binding was found at 37 degrees during 2 hr of incubation in a buffer of pH 7.5.

  9. Purification of a rat neurotensin receptor expressed in Escherichia coli.

    PubMed Central

    Tucker, J; Grisshammer, R

    1996-01-01

    A truncated rat neurotensin receptor (NTR), expressed in Escherichia coli with the maltose-binding protein fused to its N-terminus and the 13 amino acid Bio tag fused to its C-terminus, was purified to apparent homogeneity in two steps by use of the monomeric avidin system followed by a novel neurotensin column. This purification protocol was developed by engineering a variety of affinity tags on to the C-terminus of NTR. Surprisingly, expression levels varied considerably depending on the C-terminal tag used. Functional expression of NTR was highest (800 receptors/cell) when thioredoxin was placed between the receptor C-terminus and the tag, indicating a stabilizing effect of the thioredoxin moiety. Several affinity chromatography methods were tested for purification. NTR with the in vivo-biotinylated Bio tag was purified with the highest efficiency compared with NTR with the Strep tag or a hexa-histidine tail. Co-expression of biotin ligase improved considerably the in vivo biotinylation of the Bio tag and, therefore, the overall purification yield. Proteolysis of the NTR fusion protein was prevented by removing a protease-sensitive site discovered at the N-terminus of NTR. The ligand binding properties of the purified receptor were similar to those of the membrane-bound protein and the native receptor. The scale-up of this purification scheme, to provide sufficient protein for biophysical studies, is in progress. PMID:8760379

  10. Gonadotropin binding sites in human ovarian follicles and corpora lutea during the menstrual cycle

    SciTech Connect

    Shima, K.; Kitayama, S.; Nakano, R.

    1987-05-01

    Gonadotropin binding sites were localized by autoradiography after incubation of human ovarian sections with /sup 125/I-labeled gonadotropins. The binding sites for /sup 125/I-labeled human follicle-stimulating hormone (/sup 125/I-hFSH) were identified in the granulosa cells and in the newly formed corpora lutea. The /sup 125/I-labeled human luteinizing hormone (/sup 125/I-hLH) binding to the thecal cells increased during follicular maturation, and a dramatic increase was preferentially observed in the granulosa cells of the large preovulatory follicle. In the corpora lutea, the binding of /sup 125/I-hLH increased from the early luteal phase and decreased toward the late luteal phase. The changes in 3 beta-hydroxysteroid dehydrogenase activity in the corpora lutea corresponded to the /sup 125/I-hLH binding. Thus, the changes in gonadotropin binding sites in the follicles and corpora lutea during the menstrual cycle may help in some important way to regulate human ovarian function.

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

    SciTech Connect

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

    1989-09-01

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

  12. (18)F- and (68)Ga-Labeled Neurotensin Peptides for PET Imaging of Neurotensin Receptor 1.

    PubMed

    Maschauer, Simone; Einsiedel, Jürgen; Hübner, Harald; Gmeiner, Peter; Prante, Olaf

    2016-07-14

    The neurotensin (NT) receptor-1 (NTS1) is overexpressed in a variety of carcinomas and is therefore an interesting target for imaging with positron emission tomography (PET). The aim of this study was the development of new NT derivatives based on the metabolically stable peptide sequence NLys-Lys-Pro-Tyr-Tle-Leu suitable for PET imaging. The NT peptides were synthesized by solid-phase supported peptide synthesis and elongated with respective chelators (NODA-GA, DOTA) for (68)Ga-labeling or propargylglycine for (18)F-labeling via copper-catalyzed azide-alkyne cycloaddition. Receptor affinities of the peptides for NTS1 were in the range of 19-110 nM. Biodistribution studies using HT29 tumor-bearing mice showed highest tumor uptake for [(68)Ga]6 and [(68)Ga]8 and specific binding in small-animal PET studies. The tumor uptake of (68)Ga-labeled peptides in vivo significantly correlated with the in vitro Ki values for NTS1. [(68)Ga]8 displayed an excellent tumor-to-background ratio and could therefore be considered as an appropriate molecular probe for NTS1 imaging by PET. PMID:27336295

  13. Neurotensin inversely modulates maternal aggression.

    PubMed

    Gammie, S C; D'Anna, K L; Gerstein, H; Stevenson, S A

    2009-02-18

    Neurotensin (NT) is a versatile neuropeptide involved in analgesia, hypothermia, and schizophrenia. Although NT is released from and acts upon brain regions involved in social behaviors, it has not been linked to a social behavior. We previously selected mice for high maternal aggression (maternal defense), an important social behavior that protects offspring, and found significantly lower NT expression in the CNS of highly protective females. Our current study directly tested NT's role in maternal defense. Intracerebroventricular (i.c.v.) injections of NT significantly impaired defense in terms of time aggressive and number of attacks at all doses tested (0.05, 0.1, 1.0, and 3.0 microg). Other maternal behaviors, including pup retrieval, were unaltered following NT injections (0.05 microg) relative to vehicle, suggesting specificity of NT action on defense. Further, i.c.v. injections of the NT receptor 1 (NT1) antagonist, SR 48692 (30 microg), significantly elevated maternal aggression in terms of time aggressive and attack number. To understand where NT may regulate aggression, we examined Fos following injection of either 0.1 microg NT or vehicle. Thirteen of 26 brain regions examined exhibited significant Fos increases with NT, including regions expressing NT1 and previously implicated in maternal aggression, such as lateral septum, bed nucleus of stria terminalis, paraventricular nucleus, and central amygdala. Together, our results indicate that NT inversely regulates maternal aggression and provide the first direct evidence that lowering of NT signaling can be a mechanism for maternal aggression. To our knowledge, this is the first study to directly link NT to a social behavior. PMID:19118604

  14. "DAKLI": a multipurpose ligand with high affinity and selectivity for dynorphin (kappa opioid) binding sites.

    PubMed Central

    Goldstein, A; Nestor, J J; Naidu, A; Newman, S R

    1988-01-01

    We describe a synthetic ligand, "DAKLI" (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as 125I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin (kappa opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites. PMID:2902630

  15. Interaction of lipids with the neurotensin receptor 1.

    PubMed

    Bolivar, Juan H; Muñoz-García, Juan C; Castro-Dopico, Tomas; Dijkman, Patricia M; Stansfeld, Phillip J; Watts, Anthony

    2016-06-01

    Information about lipid-protein interactions for G protein-coupled receptors (GPCRs) is scarce. Here, we use electron spin resonance (ESR) and spin-labelled lipids to study lipid interactions with the rat neurotensin receptor 1 (NTS1). A fusion protein containing rat NTS1 fully able to bind its ligand neurotensin was reconstituted into phosphatidylcholine (PC) bilayers at specific lipid:protein molar ratios. The fraction of motionally restricted lipids in the range of 40:1 to 80:1 lipids per receptor suggested an oligomeric state of the protein, and the result was unaffected by increasing the hydrophobic thickness of the lipid bilayer from C-18 to C-20 or C-22 chain length PC membranes. Comparison of the ESR spectra of different spin-labelled lipids allowed direct measurement of lipid binding constants relative to PC (Kr), with spin-labelled phosphatidylethanolamine (PESL), phosphatidylserine (PSSL), stearic acid (SASL), and a spin labelled cholesterol analogue (CSL) Kr values of 1.05±0.05, 1.92±0.08, 5.20±0.51 and 0.91±0.19, respectively. The results contrast with those from rhodopsin, the only other GPCR studied this way, which has no selectivity for the lipids analysed here. Molecular dynamics simulations of NTS1 in bilayers are in agreement with the ESR data, and point to sites in the receptor where PS could interact with higher affinity. Lipid selectivity could be necessary for regulation of ligand binding, oligomerisation and/or G protein activation processes. Our results provide insight into the potential modulatory mechanisms that lipids can exert on GPCRs. PMID:26926422

  16. The amide linker in nonpeptide neurotensin receptor ligands plays a key role in calcium signaling at the neurotensin receptor type 2.

    PubMed

    Thomas, James B; Giddings, Angela M; Olepu, Srinivas; Wiethe, Robert W; Warner, Keith R; Sarret, Philippe; Longpre, Jean-Michel; Runyon, Scott P; Gilmour, Brian P

    2015-01-01

    Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesia in relevant preclinical models. The amide bond in nonpeptide NTS1 antagonists plays a central role in receptor recognition and molecular conformation. Using NTS2 FLIPR and binding assays, we found that it is also a key molecular structure for binding and calcium mobilization at NTS2. We found that reversed amides display a shift from agonist to antagonist activity and provided examples of the first competitive nonpeptide antagonists observed in the NTS2 FLIPR assay. These compounds will be valuable tools for determining the role of calcium signaling in vitro to NTS2 mediated analgesia. PMID:25881832

  17. Multipurpose ligand, DAKLI (Dynorphin A-analogue Kappa LIgand), with high affinity and selectivity for dynorphin (. kappa. opioid) binding sites

    SciTech Connect

    Goldstein, A.; Nestor, J.J. Jr.; Naidu, A.; Newman, S.R. )

    1988-10-01

    The authors describe a synthetic ligand, DALKI (Dynorphin A-analogue Kappa LIgand), related to the opioid peptide dynorphin A. A single reactive amino group at the extended carboxyl terminus permits various reporter groups to be attached, such as {sup 125}I-labeled Bolton-Hunter reagent, fluorescein isothiocyanate, or biotin. These derivatives have high affinity and selectivity for the dynorphin ({kappa} opioid) receptor. An incidental finding is that untreated guinea pig brain membranes have saturable avidin binding sites.

  18. Neurotensin-induced miR-133α expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin

    PubMed Central

    Law, Ivy Ka Man; Jensen, Dane; Bunnett, Nigel W.; Pothoulakis, Charalabos

    2016-01-01

    Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133α. Herein, we studied the mechanism of NT-regulated miR-133α expression and examined the role of miR-133α in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133α upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133α or overexpression of aftiphilin (AFTPH), a binding target of miR-133α, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133α regulates NTR1 trafficking through its downstream target AFTPH. PMID:26902265

  19. Neurotensin-induced miR-133α expression regulates neurotensin receptor 1 recycling through its downstream target aftiphilin.

    PubMed

    Law, Ivy Ka Man; Jensen, Dane; Bunnett, Nigel W; Pothoulakis, Charalabos

    2016-01-01

    Neurotensin (NT) triggers signaling in human colonic epithelial cells by activating the G protein-coupled receptor, the neurotensin receptor 1 (NTR1). Activated NTR1 traffics from the plasma membrane to early endosomes, and then recycles. Although sustained NT/NTR1 signaling requires efficient NTR1 recycling, little is known about the regulation of NTR1 recycling. We recently showed that NT/NTR1 signaling increases expression of miR-133α. Herein, we studied the mechanism of NT-regulated miR-133α expression and examined the role of miR-133α in intracellular NTR1 trafficking in human NCM460 colonocytes. We found that NT-induced miR-133α upregulation involves the negative transcription regulator, zinc finger E-box binding homeobox 1. Silencing of miR-133α or overexpression of aftiphilin (AFTPH), a binding target of miR-133α, attenuated NTR1 trafficking to plasma membrane in human colonocytes, without affecting NTR1 internalization. We localized AFTPH to early endosomes and the trans-Golgi network (TGN) in unstimulated human colonic epithelial cells. AFTPH overexpression reduced NTR1 localization in early endosomes and increased expression of proteins related to endosomes and the TGN trafficking pathway. AFTPH overexpression and de-acidification of intracellular vesicles increased NTR1 expression. Our results suggest a novel mechanism of GPCR trafficking in human colonic epithelial cells by which a microRNA, miR-133α regulates NTR1 trafficking through its downstream target AFTPH. PMID:26902265

  20. Specific binding of atrial natriuretic factor in brain microvessels

    SciTech Connect

    Chabrier, P.E.; Roubert, P.; Braquet, P.

    1987-04-01

    Cerebral capillaries constitute the blood-brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. The authors examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using /sup 125/I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity and with a binding capacity of 58 fmol/mg of protein. The binding of /sup 125/I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood-brain barrier function.

  1. Specific Binding of Atrial Natriuretic Factor in Brain Microvessels

    NASA Astrophysics Data System (ADS)

    Chabrier, Pierre E.; Roubert, Pierre; Braquet, Pierre

    1987-04-01

    Cerebral capillaries constitute the blood--brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. We examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using 125I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity (dissociation constant, ≈ 10-10 M) and with a binding capacity of 58 fmol/mg of protein. The binding of 125I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood--brain barrier function.

  2. Characterization of salivary alpha-amylase binding to Streptococcus sanguis

    SciTech Connect

    Scannapieco, F.A.; Bergey, E.J.; Reddy, M.S.; Levine, M.J. )

    1989-09-01

    The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase was the prominent salivary component eluted from S. sanguis. Studies with {sup 125}I-labeled HSMSL or {sup 125}I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of ({sup 125}I)alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.

  3. Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

    SciTech Connect

    Kikuchi, M.; Ishii, S. )

    1989-12-01

    By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

  4. Characterization of salivary alpha-amylase binding to Streptococcus sanguis.

    PubMed Central

    Scannapieco, F A; Bergey, E J; Reddy, M S; Levine, M J

    1989-01-01

    The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase (EC 3.2.1.1) was the prominent salivary component eluted from S. sanguis. Studies with 125I-labeled HSMSL or 125I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of [125I]alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch. Images PMID:2788139

  5. Striatal dopamine receptor plasticity in neurotensin deficient mice

    PubMed Central

    Chastain, Lucy G.; Qu, Hongyan; Bourke, Chase H.; Iuvone, P. Michael; Dobner, Paul R.; Nemeroff, Charles B.; Kinkead, Becky

    2015-01-01

    Schizophrenia is thought to be caused, at least in part, by dysfunction in striatal dopamine neurotransmission. Both clinical studies and animal research have implicated the dopamine neuromodulator neurotensin (NT) in the pathophysiology of schizophrenia. Utilizing male mice lacking the NT gene (NT−/−), these studies examined the consequences of NT deficiency on dopaminergic tone and function, investigating (1) dopamine concentrations and dopamine receptor and transporter expression and binding in dopaminergic terminal regions, and (2) the behavioral effects of selective dopamine receptor agonists on locomotion and sensorimotor gating in adult NT−/− mice compared to wildtype (NT+/+) mice. NT−/− mice did not differ from NT+/+ mice in concentrations of dopamine or its metabolite DOPAC in any brain region examined. However, NT−/− mice showed significantly increased D1 receptor, D2 receptor, and dopamine transporter (DAT) mRNA in the caudate putamen compared to NT+/+ controls. NT−/− mice also showed elevated D2 receptor binding densities in both the caudate putamen and nucleus accumbens shell compared to NT+/+ mice. In addition, some of the behavioral effects of the D1-type receptor agonist SKF-82958 and the D2-type receptor agonist quinpirole on locomotion, startle amplitude, and prepulse inhibition were dose-dependently altered in NT−/− mice, showing altered D1-type and D2-type receptor sensitivity to stimulation by agonists in the absence of NT. The results indicate that NT deficiency alters striatal dopamine receptor expression, binding, and function. This suggests a critical role for the NT system in the maintenance of striatal DA system homeostasis and implicates NT deficiency in the etiology of dopamine-associated disorders such as schizophrenia. PMID:25449842

  6. Brain natriuretic peptide binding sites in rats: In vitro autoradiographic study

    SciTech Connect

    Konrad, E.M.; Thibault, G.; Pelletier, S.; Genest, J.; Cantin, M. )

    1990-08-01

    Brain natriuretic peptide (BNP) is a recently discovered family of natriuretic peptides highly homologous to atrial natriuretic factor (ANF). Quantitative in vitro autoradiography with a computerized microdensitometer demonstrated that the distribution of BNP binding sites is similar to the known distribution pattern of ANF binding sites in rat tissues. Analysis of saturation and competition curves disclosed that the maximal binding capacity for BNP-(Asp-81--Tyr-106) and ANF-(Ser-99--Tyr-126) is similar within the plexiform layer of the olfactory bulb, the choroid plexus, and the adrenal zona glomerulosa. Examination of the competition curves of BNP-(Asp-81--Tyr-106), ANF-(Ser-99--Tyr-126), and des-(Gln-116--Gly-120)ANF-(Asp-102--Cys-121)NH2 (C-ANF, a ligand highly specific for ANF-R2 receptors) for {sup 125}I-labeled BNP-(Asp-81--Tyr-106) and {sup 125}I-labeled ANF-(Ser-99--Tyr-126) binding revealed that ANF fully displaced {sup 125}I-BNP binding and, conversely, BNP completely displaced {sup 125}I-ANF binding in these tissues, whereas C-ANF partially displaced 125-BNP and 125-ANF binding. Angiotensin II, insulin, glucagon, and substance P had no influence on {sup 125}I-BNP binding in the above tissues. These results support the view that BNP and ANF share the same binding sites in rats.

  7. Characteristics of albumin binding to opossum kidney cells and identification of potential receptors.

    PubMed

    Brunskill, N J; Nahorski, S; Walls, J

    1997-02-01

    Albumin re-absorption in the kidney proximal tubule may be pathophysiological in disease. Opossum kidney (OK) cell monolayers were used to investigate the characteristics of [125I]-labelled albumin binding at 4 degrees C. Two binding sites were identified, one with high affinity (KD 154.8 +/-7 mg/l) and low capacity, the other with low affinity (KD 8300 +/- 1000 mg/l) and high capacity. Binding was sensitive to lectins Glycine max and Ulex europaeus I, but not other lectins, indicating involvement of a glycoprotein(s) in the binding process. Binding was also sensitive to a number of agents known to inhibit binding to scavenger receptors. [125I]-Labelled albumin ligand blotting of OK cell membrane proteins identified several albumin-binding proteins with identical lectin affinities to those proteins mediating albumin binding to OK cell monolayers. These results provide initial evidence of the identity of albumin receptors in kidney tubules, and suggest that they may be members of the family of scavenger receptors. PMID:9000429

  8. Modulation of the interaction between neurotensin receptor NTS1 and Gq protein by lipid

    PubMed Central

    Inagaki, Sayaka; Ghirlando, Rodolfo; White, Jim F.; Gvozdenovic-Jeremic, Jelena; Northup, John K.; Grisshammer, Reinhard

    2012-01-01

    Membrane lipids have been implicated to influence the activity of G protein-coupled receptors (GPCRs). Almost all of our knowledge on the role of lipids on GPCR and G protein function comes from work on the visual pigment rhodopsin and its G protein transducin, which reside in a highly specialized membrane environment. Thus insight gained from rhodopsin signaling may not be simply translated to other non-visual GPCRs. Here, we investigated the effect of lipid head group charges on the signal transduction properties of the class A GPCR neurotensin receptor 1 (NTS1) under defined experimental conditions, using self-assembled phospholipid nanodiscs prepared with the zwitter-ionic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), or a POPC/POPG mixture. A combination of dynamic light scattering and sedimentation velocity showed that NTS1 was monomeric in POPC-, POPC/POPG- and POPG-nanodiscs. Binding of the agonist neurotensin to NTS1 occurred with similar affinities and was essentially unaffected by the phospholipid composition. In contrast, Gq protein coupling to NTS1 in various lipid nanodiscs was significantly different and the apparent affinity of Gαq and Gβ1γ1 to activated NTS1 increased with increasing POPG content. NTS1-catalyzed GDP/GTPγS nucleotide exchange at Gαq in the presence of Gβ1γ1 and neurotensin was crucially affected by the lipid type, with exchange rates higher by one or two orders of magnitude in POPC/POPG- and POPG-nanodiscs, respectively, compared to POPC-nanodiscs. Our data demonstrate that negatively charged lipids in the immediate vicinity of a non-visual GPCR modulate the G protein-coupling step. PMID:22306739

  9. Chronic ethanol administration downregulates neurotensin receptors in long- and short-sleep mice.

    PubMed

    Campbell, A D; Erwin, V G

    1993-05-01

    Neurotensin (NT) has been shown to differentially alter many of the physiologic responses to ethanol administration in long-sleep (LS) and short-sleep (SS) mice, which were selectively bred for differences in hypnotic sensitivity to ethanol. These mice have been shown to differ in NT receptor densities in cortical and mesolimbic brain regions and it has been suggested that ethanol actions may be mediated, in part, by neurotensinergic processes. The present study was conducted to further examine this hypothesis by determining the effects of acute and chronic ethanol administration on NT receptor systems in these mice. Scatchard analysis of [3H]NT binding in brain membranes from mice chronically treated with ethanol yielded a one-site model, whereas binding in membranes from control mice were best described by a two-site model. Values for binding capacity (Bmax) were significantly reduced in several brain regions, and binding site density for total, levocabastine-sensitive, and levocabastine-insensitive binding sites were also reduced. The maximum effect was seen after 2 weeks of chronic ethanol consumption. Three weeks after withdrawal from ethanol, Kd and Bmax had returned to control values. Similarly, binding density in all regions for total, levocabastine-sensitive, and levocabastine-insensitive sites had returned to control values within 2 weeks. NT receptor characteristics measured 2 h post-3.0 g/kg ethanol revealed that ethanol caused a rapid downregulation of both subtypes of NT receptors. The finding that both acute and chronic ethanol significantly downregulate the neurotensin receptor systems further supports the hypothesis that ethanol's actions may be mediated in part by neurotensinergic systems. PMID:8100076

  10. Autoradiographic localization of endothelin-1 binding sites in porcine skin

    SciTech Connect

    Zhao, Y.D.; Springall, D.R.; Wharton, J.; Polak, J.M. )

    1991-01-01

    Autoradiographic techniques and {sup 125}I-labeled endothelin-1 were used to study the distribution of endothelin-1 binding sites in porcine skin. Specific endothelin-1 binding sites were localized to blood vessels (capillaries, deep cutaneous vascular plexus, arteries, and arterioles), the deep dermal and connective tissue sheath of hair follicles, sebaceous and sweat glands, and arrector pili muscle. Specific binding was inhibited by endothelin-2 and endothelin-3 as well as endothelin-1. Non-specific binding was found in the epidermis and the medulla of hair follicles. No binding was found in connective tissue or fat. These vascular binding sites may represent endothelin receptors, in keeping with the known cutaneous vasoconstrictor actions of the peptide. If all binding sites are receptors, the results suggest that endothelin could also regulate the function of sweat glands and may have trophic effects in the skin.

  11. Development of cholecystokinin binding sites in rat upper gastrointestinal tract

    SciTech Connect

    Robinson, P.H.; Moran, T.H.; Goldrich, M.; McHugh, P.R.

    1987-04-01

    Autoradiography using /sup 125/I-labeled Bolton Hunter-CCK-33 was used to study the distribution of cholecystokinin binding sites at different stages of development in the rat upper gastrointestinal tract. Cholecystokinin (CCK) binding was present in the distal stomach, esophagus, and gastroduodenal junction in the rat fetus of gestational age of 17 days. In the 20-day fetus, specific binding was found in the gastric mucosa, antral circular muscle, and pyloric sphincter. Mucosal binding declined during postnatal development and had disappeared by day 15. Antral binding declined sharply between day 10 and day 15 and disappeared by day 50. Pyloric muscle binding was present in fetal stomach and persisted in the adult. Pancreatic CCK binding was not observed before day 10. These results suggest that CCK may have a role in the control of gastric emptying and ingestive behavior in the neonatal rat.

  12. Purification of an angiotensin II binding protein by using antibodies to a peptide encoded by angiotensin II complementary RNA

    SciTech Connect

    Elton, T.S.; Dion, L.D.; Bost, K.L.; Oparil, S.; Blalock, J.E.

    1988-04-01

    The authors have generated a monospecific antibody to a synthetic peptide encoded by an RNA complementary to the mRNA for angiotensin II (AII) and determined whether this antibody recognizes the AII receptor. They demonstrate that the antibody competes specifically with /sup 125/I-labeled AII for the same binding site on rat adrenal membranes. Furthermore, they show this antibody inhibits the secretion of aldosterone from cultured rat adrenal cells, suggesting that the antibody recognizes the biologically relevant AII receptor. Finally, they demonstrate that antibody to the complementary peptide can be used to immunoaffinity-purify a protein of M/sub r/ 66,000 that specifically binds radiolabeled AII.

  13. Relationship between in vitro binding activity and in vivo tumor accumulation of radiolabeled monoclonal antibodies

    SciTech Connect

    Sakahara, H.; Endo, K.; Koizumi, M.; Nakashima, T.; Kunimatsu, M.; Watanabe, Y.; Kawamura, Y.; Nakamura, T.; Tanaka, H.; Kotoura, Y.

    1988-02-01

    The relationship between in vitro cell binding and in vivo tumor accumulation of radiolabeled antibodies was studied using /sup 125/I- and /sup 111/In-labeled monoclonal antibodies to human osteosarcoma, and a human osteosarcoma xenograft (KT005) in nude mice. Three monoclonal antibodies--OST6, OST7, and OST15--raised against human osteosarcoma recognize the same antigen molecule. Although the binding of both /sup 125/I- and /sup 111/In-labeled OST6 to KT005 cells was higher than that of radiolabeled OST7 in vitro, /sup 125/I-labeled OST6 showed a faster clearance from the circulation and a lower accumulation in the transplanted tumor than /sup 125/I-labeled OST7. In contrast to the radioiodinated antibodies, the in vivo tumor accumulation of /sup 111/In-labeled OST6 was higher, although not significantly, than that of /sup 111/In-labeled OST7. OST15 showed the lowest binding in vitro, and its in vivo tumor localization was also lower than the others. The discrepancy in tumor uptake between OST6 and OST7 labeled with either /sup 125/I or /sup 111/In may have been a result of differing blood clearance. These results suggest that binding studies can be used to exclude from in vivo use those antibodies which show very poor binding in vitro, while in vivo serum clearance may be a better test for choosing antibodies with similar binding.

  14. Structural prerequisites for G-protein activation by the neurotensin receptor

    DOE PAGESBeta

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-07-24

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist withmore » residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1.« less

  15. Structural prerequisites for G-protein activation by the neurotensin receptor

    SciTech Connect

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-07-24

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1.

  16. Structural prerequisites for G-protein activation by the neurotensin receptor

    PubMed Central

    Krumm, Brian E.; White, Jim F.; Shah, Priyanka; Grisshammer, Reinhard

    2015-01-01

    We previously determined the structure of neurotensin receptor NTSR1 in an active-like conformation with six thermostabilizing mutations bound to the peptide agonist neurotensin. This receptor was unable to activate G proteins, indicating that the mutations restricted NTSR1 to relate agonist binding to G-protein activation. Here we analyse the effect of three of those mutations (E166A3.49, L310A6.37, F358A7.42) and present two structures of NTSR1 able to catalyse nucleotide exchange at Gα. The presence of F3587.42 causes the conserved W3216.48 to adopt a side chain orientation parallel to the lipid bilayer sealing the collapsed Na+ ion pocket and linking the agonist with residues in the lower receptor part implicated in GPCR activation. In the intracellular receptor half, the bulkier L3106.37 side chain dictates the position of R1673.50 of the highly conserved D/ERY motif. These residues, together with the presence of E1663.49 provide determinants for G-protein activation by NTSR1. PMID:26205105

  17. Receptor-like function of heparin in the binding and uptake of neutral lipids

    SciTech Connect

    Bosner, M.S.; Gulick, T.; Riley, D.J.S.; Spilburg, C.A.; Lange, L.G. III )

    1988-10-01

    Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, the authors postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic {sup 125}I-labeled cholesterol esterase and {sup 125}I-labeled triglyceride lipase. This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a K{sub d} of 100 nM and a B{sub max} of approximately 50-60 pmol per mg of vesicle protein. They conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalized pathway for transport of neutral lipids into endothelial and other cells.

  18. Neurotensin enhances estradiol induced DNA synthesis in immature rat uterus

    SciTech Connect

    Mistry, A.; Vijayan, E.

    1985-05-27

    Systemic administration of Neurotensin, a tridecapeptide, in immature rats treated with estradiol benzoate significantly enhances uterine DNA synthesis as reflected by the incorporation of /sup 3/H-thymidine. The peptide may have a direct action on the uterus. Substance P, a related peptide, had no effect on uterine DNA synthesis. 18 references, 4 tables.

  19. Visualization of heparin-binding proteins by ligand blotting with /sup 125/I-heparin

    SciTech Connect

    Cardin, A.D.; Witt, K.R.; Jackson, R.L.

    1984-03-01

    A ligand-blotting procedure which allows detection of heparin-binding proteins is described. Crude commercial heparin was fractionated by chromatography on a column of human plasma low-density lipoproteins immobilized to Sepharose CL-4B. Chromatography yielded an unbound and a bound fraction of heparin, designated URH and HRH, respectively. The HRH fraction was reacted with the N-hydroxysuccinimidyl ester of 3-(p-hydroxyphenyl)propionic acid and then labeled with /sup 125/I. Proteins were separated by 3-20% pore-gradient gel electrophoresis, transferred to nitrocellulose, and then assayed for their ability to bind /sup 125/I-labeled HRH. Human plasma apolipoproteins B-100, B-48, and E of chylomicrons, very low-density lipoproteins, and low-density lipoproteins bound the /sup 125/I-labeled HRH; the radiolabeled haparin did not bind to serum albumin, ferritin, catalase, and lactate dehydrogenase. The ligand-blotting procedure should facilitate the purification of heparin-binding domains from these proteins and, moreover may be applicable to the investigation of heparin-protein interactions in general. 15 references.

  20. Synthesis and Characterization in Vitro and in Vivo of (l)-(Trimethylsilyl)alanine Containing Neurotensin Analogues.

    PubMed

    Fanelli, Roberto; Besserer-Offroy, Élie; René, Adeline; Côté, Jérôme; Tétreault, Pascal; Collerette-Tremblay, Jasmin; Longpré, Jean-Michel; Leduc, Richard; Martinez, Jean; Sarret, Philippe; Cavelier, Florine

    2015-10-01

    The silylated amino acid (l)-(trimethylsilyl)alanine (TMSAla) was incorporated at the C-terminal end of the minimal biologically active neurotensin (NT) fragment, leading to the synthesis of new hexapeptide NT[8-13] analogues. Here, we assessed the ability of these new silylated NT compounds to bind to NTS1 and NTS2 receptors, promote regulation of multiple signaling pathways, induce inhibition of the ileal smooth muscle contractions, and affect distinct physiological variables, including blood pressure and pain sensation. Among the C-terminal modified analogues, compound 6 (JMV2007) carrying a TMSAla residue in position 13 exhibits a higher affinity toward NT receptors than the NT native peptide. We also found that compound 6 is effective in reversing carbachol-induced contraction in the isolated strip preparation assay and at inducing a drop in blood pressure. Finally, compound 6 produces potent analgesia in experimental models of acute and persistent pain. PMID:26348111

  1. Detection and characterization of heparin-binding proteins with a gel overlay procedure

    SciTech Connect

    Mehlman, T.; Burgess, W.H. )

    1990-07-01

    The binding of {sup 125}I-labeled derivatives of heparin has been used by several investigators to identify heparin-binding fragments of different heparin-binding proteins. In this report we utilize the procedure described by J.W. Smith and D.J. Knauer (1987, Anal. Biochem. 160, 105-114) to produce {sup 125}I-fluorescein-heparin. Using this derivative, we compare the use of gel overlay procedures with Western blot procedures for the detection of heparin-binding proteins following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. We show that the gel overlay procedure is a relatively simple and sensitive method for visualizing heparin-binding proteins. In addition, we use the procedure to characterize the heparin-binding properties of heparin-binding growth factor 1 (acidic fibroblast growth factor) with synthetic peptide competitors and site-directed mutants of the growth factor.

  2. Low density lipoprotein receptor-binding activity in human tissues: quantitative importance of hepatic receptors and evidence for regulation of their expression in vivo.

    PubMed Central

    Rudling, M J; Reihnér, E; Einarsson, K; Ewerth, S; Angelin, B

    1990-01-01

    The heparin-sensitive binding of 125I-labeled low-density lipoprotein (LDL) to homogenates from 18 different normal human tissues and some solid tumors was determined. The binding to adrenal and liver homogenates fulfilled criteria established for the binding of LDL to its receptor--namely, (i) saturability, (ii) sensitivity to proteolytic destruction, (iii) inhibition by EDTA, and (iv) heat sensitivity. When the binding of 125I-labeled LDL was assayed at a constant concentration (50 micrograms/ml), the adrenal gland and the ovary had the highest binding of normal tissues. The highest binding per g of tissue overall was obtained in homogenates of a gastric carcinoma and a parotid adenoma. When the weights of the parenchymatous organs were considered, the major amount of LDL receptors was contained in the liver. To study the possible regulation of hepatic LDL-receptor expression, 11 patients were pretreated with cholestyramine (8 g twice a day for 3 weeks). Increased binding activity (+105%, P less than 0.001) was obtained in homogenates from liver biopsies from the cholestyramine-treated patients as compared with 12 untreated controls. It is concluded that the liver is the most important organ for LDL catabolism in humans and that the receptor activity in this organ can be regulated upon pharmacologic intervention. Further studies are needed to confirm the possibility that certain solid tumors can exhibit high numbers of LDL receptors. PMID:2110363

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

    SciTech Connect

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

    1988-11-01

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

  4. Rapid extraction, radioiodination, and in vivo catabolism of 125I-labeled fibrinogen in the horse

    SciTech Connect

    Coyne, C.P.; Hornof, W.J.; Kelly, A.B.; O'Brien, T.R.; DeNardo, S.J.

    1985-12-01

    Two methods were analyzed for the rapid extraction of equine fibrinogen from fresh plasma, using ammonium sulfate-sodium phosphate buffer. Fibrinogen from each of these 2 methods was then radiolabeled with 125I (half-life = 60.2 days, gamma = 35 keV), using monochloroiodine reagent. Mean protein-bound activity was 98.5% and mean clottable radioactivity was 94.1%. Radiolabeled fibrinogen administered IV to 15 horses had an overall mean (+/- SD) plasma half-life of 4.95 +/- 0.44 days.

  5. Optimized method for measuring cyclosporin A with /sup 125/I-labeled cyclosporin

    SciTech Connect

    Felder, R.A.; Mifflin, T.E.; Bastani, B.

    1986-07-01

    We evaluated the use of the new iodinated ligand for the in vitro measurement of cyclosporin A by radioimmunoassay (RIA). Substitution of the iodinated cyclosporin (/sup 125/I-CyA) for the corresponding tritium-labeled analog (/sup 3/H-CyA) considerably simplifies and accelerates the currently available RIA, and improves its precision. Analysis of the respective dose-response curves showed that the 50% B0 value was lower for the /sup 125/I-CyA assay than for the /sup 3/H-CyA assay (37 vs 77 micrograms/L). Use of whole-blood specimens minimized interferences from temperature and hematocrit. We conclude that the use of /sup 125/I-CyA in a commercially available RIA for whole-blood specimens is accessible to most laboratories and provides rapid, reproducible data for management of transplant patients.

  6. Monoclonal antibody OKB7, which identifies the 14OKd complement receptor type 2 (CR/sub 2/), also identifies a 72Kd secreted fragment of CR/sub 2/ that contains the C3d-binding site

    SciTech Connect

    Myones, B.L.; Ross, G.D.

    1986-03-05

    CR/sub 2/ is a 140-145Kd glycoprotein expressed on B lymphocytes which binds both C3d and Epstein-Barr virus (EBV). OKB7, an IgG/sub 2a/ monoclonal antibody to CR/sub 2/, blocks C3d and EBV binding, while HB-5, another monoclonal IgG/sub 2a/ anti-CR/sub 2/, does not. A 72Kd C3d-binding glycoprotein (gp72), isolated from Raji cell media, was previously thought to be CR/sub 2/ because a polyclonal rabbit anti-gp72 inhibited EC3d rosettes. ELISA assay demonstrated that OKB7, but not HB-5, bound to purified gp72 fixed to microtiter wells. Insoluble and soluble gp72 blocked Raji cell uptake of /sup 125/I-labeled OKB7, but not labeled anti-B2 or HB-5. Rabbit anti-gp72 immunoprecipitated bands at 140Kd and 72Kd from /sup 125/I-labelled and solubilized B cell membranes. Culture media from Raji cells grown in the presence /sup 3/H-labeled amino acids was sequentially immunoprecipitated by irrelevant antibody, OKB7, and HB-5. A single 72Kd radiolabeled band was demonstrated only with OKB7, and this was identical to that produced by the immunoprecipitation of /sup 125/I-labeled gp72 with rabbit anti-gp72. Thus, OKB7, which identifies the 140Kd CR/sub 2/ molecule, also identifies a 72Kd shed fragment of CR/sub 2/ isolated from Raji cell media, which contains the C3d-binding site.

  7. Gonadotropin stimulation of cyclic adenosine monophosphate and testosterone production without detectable high-affinity binding sites in purified Leydig cells from rat testis

    SciTech Connect

    Browne, E.S.; Bhalla, V.K. )

    1991-02-01

    Rat testicular interstitial cells were separated by three different gradient-density procedures and, with each, two biochemically and morphologically distinct cell fractions were isolated. The lighter density cells in fraction-I bound iodine 125-labeled human chorionic gonadotropin (hCG) with high-affinity (apparent equilibrium dissociation constant, Kd, approximately 10{sup {minus} 10} M) without producing either cyclic adenosine monophosphate or testosterone in response to hormone action. The heavier-density cells displayed morphologic features typical of Leydig cells and produced cyclic adenosine monophosphate and testosterone in the presence of hCG without detectable {sup 125}I-labeled hCG high-affinity binding. These cell fractions were further characterized by studies using deglycosylated hCG, a known antagonist to hCG action. Cell concentration-dependent studies with purified Leydig cells revealed that maximal testosterone production was achieved when lower cell concentrations (0.5 x 10(6) cells/250 microliters) were used for in vitro hCG stimulation assays. Under these conditions, the {sup 125}I-labeled hCG binding was barely detectable (2.24 fmol; 2,698 sites/cell). Furthermore, these studies revealed that the hCG-specific binding in Leydig cells is overestimated by the classic method for nonspecific binding correction using excess unlabeled hormone. An alternate method is presented.

  8. Neurotensin Changes Propulsive Activity into a Segmental Motor Pattern in the Rat Colon

    PubMed Central

    Li, Hongfei; Chen, Ji-Hong; Yang, Zixian; Huang, Min; Yu, Yuanjie; Tan, Shiyun; Luo, Hesheng; Huizinga, Jan D

    2016-01-01

    Background/Aims Neurotensin is a gut-brain peptide with both inhibitory and excitatory actions on the colonic musculature; our objective was to understand the implications of this for motor patterns occurring in the intact colon of the rat. Methods The effects of neurotensin with concentrations ranging from 0.1–100 nM were studied in the intact rat colon in vitro, by investigating spatio-temporal maps created from video recordings of colonic motility before and after neurotensin. Results Low concentration of neurotensin (0.1–1 nM) inhibited propagating long distance contractions and rhythmic propagating motor complexes; in its place a slow propagating rhythmic segmental motor pattern developed. The neurotensin receptor 1 antagonist SR-48692 prevented the development of the segmental motor pattern. Higher concentrations of neurotensin (10 nM and 100 nM) were capable of restoring long distance contraction activity and inhibiting the segmental activity. The slow propagating segmental contraction showed a rhythmic contraction—relaxation cycle at the slow wave frequency originating from the interstitial cells of Cajal associated with the myenteric plexus pacemaker. High concentrations given without prior additions of low concentrations did not evoke the segmental motor pattern. These actions occurred when neurotensin was given in the bath solution or intraluminally. The segmental motor pattern evoked by neurotensin was inhibited by the neural conduction blocker lidocaine. Conclusions Neurotensin (0.1–1 nM) inhibits the dominant propulsive motor patterns of the colon and a distinct motor pattern of rhythmic slow propagating segmental contractions develops. This motor pattern has the hallmarks of haustral boundary contractions. PMID:26882114

  9. Neurotensin effect on dopamine release and calcium transport in rat striatum: interactions with diphenylalkylamine calcium antagonists.

    PubMed

    Battaini, F; Govoni, S; Di Giovine, S; Trabucchi, M

    1986-03-01

    The release of dopamine was investigated in rat striatal slices exposed in vitro to neurotensin. This peptide increased basal and K+-evoked dopamine release. Moreover neurotensin antagonized the flunarizine-induced inhibition of K+-stimulated dopamine release. The K+-evoked 45Ca2+ accumulation was also inhibited by flunarizine. This effect was antagonized by neurotensin. The results suggest that dopamine release in rat striatum is regulated by different molecular events also of peptidergic nature having as possible mechanism of action an influence on calcium ion movements. PMID:3713871

  10. Isolation of an inhibitory insulin-like growth factor (IGF) binding protein from bone cell-conditioned medium: A potential local regulator of IGF action

    SciTech Connect

    Mohan, S.; Bautista, C.M.; Wergedal, J.; Baylink, D.J. )

    1989-11-01

    Inhibitory insulin-like growth factor binding protein (In-IGF-BP) has been purified to homogeneity from medium conditioned by TE89 human osteosarcoma cells by two different methods using Sephadex G-100 gel filtration, FPLC Mono Q ion-exchange, HPLC C{sub 4} reverse-phase, HPLC CN reverse-phase and affinity chromatographies. In-IGF-BP thus purified appeared to be homogeneous and unique by the following criteria. (i) N-terminal sequence analysis yielded a unique sequence (Asp-Glu-Ala-Ile-His-Cys-Pro-Pro-Glu-Ser-Glu-Ala-Lys-Leu-Ala). (ii) Amino acid composition of In-IGF-BP revealed marked differences with the amino acid compositions of other known PBs. (iii) In-IGF-BP exhibited a single band with molecular mass of 25 kDa under reducing conditions on sodium dodecyl sulfate/polyacrylamide gels. IGF-I and IGF-II but not insulin displaced the binding of {sup 125}I-labeled IGF-I or {sup 125}I-labeled IGF-II binding to In-IGF-BP. In-IGF-BP inhibited basal, IGF-stimulated bone cell proliferation and serum-stimulated bone cell proliferation. Forskolin increases synthesis of In-IGF-BP in TE85 human osteosarcoma cells in a dose-dependent manner. Based on these findings, the authors conclude that In-IGF-BP is a protein that has a unique sequence and significant biological actions on bone cells.

  11. A marine analgesic peptide, Contulakin-G, and neurotensin are distinct agonists for neurotensin receptors: uncovering structural determinants of desensitization properties.

    PubMed

    Lee, Hee-Kyoung; Zhang, Liuyin; Smith, Misty D; Walewska, Aleksandra; Vellore, Nadeem A; Baron, Riccardo; McIntosh, J Michael; White, H Steve; Olivera, Baldomero M; Bulaj, Grzegorz

    2015-01-01

    Neurotensin receptors have been studied as molecular targets for the treatment of pain, schizophrenia, addiction, or cancer. Neurotensin (NT) and Contulakin-G, a glycopeptide isolated from a predatory cone snail Conus geographus, share a sequence similarity at the C-terminus, which is critical for activation of neurotensin receptors. Both peptides are potent analgesics, although affinity and agonist potency of Contulakin-G toward neurotensin receptors are significantly lower, as compared to those for NT. In this work, we show that the weaker agonist properties of Contulakin-G result in inducing significantly less desensitization of neurotensin receptors and preserving their cell-surface density. Structure-activity relationship (SAR) studies suggested that both glycosylation and charged amino acid residues in Contulakin-G or NT played important roles in desensitizing neurotensin receptors. Computational modeling studies of human neurotensin receptor NTS1 and Contulakin-G confirmed the role of glycosylation in weakening interactions with the receptors. Based on available SAR data, we designed, synthesized, and characterized an analog of Contulakin-G in which the glycosylated amino acid residue, Gal-GalNAc-Thr10, was replaced by memantine-Glu10 residue. This analog exhibited comparable agonist potency and weaker desensitization properties as compared to that of Contulakin-G, while producing analgesia in the animal model of acute pain following systemic administration. We discuss our study in the context of feasibility and safety of developing NT therapeutic agents with improved penetration across the blood-brain barrier. Our work supports engineering peptide-based agonists with diverse abilities to desensitize G-protein coupled receptors and further emphasizes opportunities for conotoxins as novel pharmacological tools and drug candidates. PMID:25713532

  12. A marine analgesic peptide, Contulakin-G, and neurotensin are distinct agonists for neurotensin receptors: uncovering structural determinants of desensitization properties

    PubMed Central

    Lee, Hee-Kyoung; Zhang, Liuyin; Smith, Misty D.; Walewska, Aleksandra; Vellore, Nadeem A.; Baron, Riccardo; McIntosh, J. Michael; White, H. Steve; Olivera, Baldomero M.; Bulaj, Grzegorz

    2015-01-01

    Neurotensin receptors have been studied as molecular targets for the treatment of pain, schizophrenia, addiction, or cancer. Neurotensin (NT) and Contulakin-G, a glycopeptide isolated from a predatory cone snail Conus geographus, share a sequence similarity at the C-terminus, which is critical for activation of neurotensin receptors. Both peptides are potent analgesics, although affinity and agonist potency of Contulakin-G toward neurotensin receptors are significantly lower, as compared to those for NT. In this work, we show that the weaker agonist properties of Contulakin-G result in inducing significantly less desensitization of neurotensin receptors and preserving their cell-surface density. Structure-activity relationship (SAR) studies suggested that both glycosylation and charged amino acid residues in Contulakin-G or NT played important roles in desensitizing neurotensin receptors. Computational modeling studies of human neurotensin receptor NTS1 and Contulakin-G confirmed the role of glycosylation in weakening interactions with the receptors. Based on available SAR data, we designed, synthesized, and characterized an analog of Contulakin-G in which the glycosylated amino acid residue, Gal-GalNAc-Thr10, was replaced by memantine-Glu10 residue. This analog exhibited comparable agonist potency and weaker desensitization properties as compared to that of Contulakin-G, while producing analgesia in the animal model of acute pain following systemic administration. We discuss our study in the context of feasibility and safety of developing NT therapeutic agents with improved penetration across the blood-brain barrier. Our work supports engineering peptide-based agonists with diverse abilities to desensitize G-protein coupled receptors and further emphasizes opportunities for conotoxins as novel pharmacological tools and drug candidates. PMID:25713532

  13. Characterization of insulin-like growth factor-binding proteins secreted by isolated sheep thyroid epithelial cells.

    PubMed

    Wang, J F; Becks, G P; Buckingham, K D; Hill, D J

    1990-06-01

    We have characterized the insulin-like growth factor-binding proteins (IGF-BPs) released by isolated sheep thyroid epithelial cells. Thyroid follicles were isolated with collagenase and cultured in Coon's modified F-12 M (0H medium) supplemented with insulin, cortisol, transferrin, glycyl-histidyl-lysine and somatostatin (5H medium) and TSH (6H medium). Conditioned 0H medium specifically bound both 125I-labelled IGF-I and -II, although binding capacity was reduced following acid-gel filtration to separate endogenous IGF-BP complexes, suggesting some destruction of BPs. The binding of 125I-labelled IGF-I or -II to conditioned (0H) medium was progressively displaced by increasing amounts of unlabelled homologous peptides, while fractionation on concanavalin A-Sepharose showed that the IGF-BPs consisted of both glycoprotein and non-glycoprotein components. The molecular sizes of the IGF-BPs were resolved by separation of 0H medium on SDS-PAGE and ligand blot analysis with 125I-labelled IGF-I or -II. Conditioned medium contained four specific binding species for IGF-II of 19, 30, 38 and 46 kDa; all but the smallest also binding radiolabelled IGF-I. Prior fractionation on concanavalin A-Sepharose showed that the 46 kDa binding species was a glycoprotein. Competition studies with increasing concentrations of unlabelled IGF-I or -II during ligand blotting suggested that the 46 and 30 kDa binding species had a greater affinity for IGF-II than IGF-I, while the 38 kDa had a greater relative affinity for IGF-I. Incubation of cells in 5H medium reduced the abundance of the 46 kDa binding protein, while incubation in 6H medium decreased the release of all binding protein species. Results show that isolated thyroid follicles released several forms of IGF-BP with differing relative affinities for IGF-I and -II. Gross changes seen in the presence of BPs between 0H, 5H and 6H media suggest acute hormonal control of release. PMID:1695663

  14. The actions of neurotensin in rat bladder detrusor contractility

    PubMed Central

    Dong, Xingyou; Bai, Xinyu; Zhao, Jiang; Wang, Liang; Wang, Qingqing; Li, Longkun

    2015-01-01

    This study assessed the expression, distribution and function of neurotensin (NTs) and two main neurotensin receptors (NTSR), NTSR1 and NTSR2 in normal rat urinary bladders. NTs is primarily located in the suburothelium and the interstitium of smooth muscle bundles. The NTSR1 and NTSR2 receptor subtypes are found to co-localize with smooth muscle cells (SMCs). NTs not only can directly act on bladder SMCs to induce intracellular calcium mobilization by activating the phospholipase C/inositol triphosphate (PLC/IP3) pathway, promoting extracellular calcium influx through a non-selective cation channels, but may be also involved in the modulation of the cholinergic system. Nowadays, the selective antimuscarinic drugs (solifenacin) and the selective beta 3-adrenergic agonist (mirabegron) are used as the first-line pharmacotherapy for overactive bladder (OAB), but without satisfactory treatment benefits in some patients. This study provided evidence suggesting that bladder NTs may play an important role in the regulation of micturition. Further research is needed to investigate the effects of NTs on bladder contractility and the underlying mechanism, which might reveal that the administration of NTSR antagonists can potentially relieve the symptoms of OAB by coordination with antimuscarinic pharmacotherapy. PMID:26053252

  15. Receptor-Like Function of Heparin in the Binding and Uptake of Neutral Lipids

    NASA Astrophysics Data System (ADS)

    Bosner, Matthew S.; Gulick, Tod; Riley, D. J. S.; Spilburg, Curtis A.; Lange, Louis G.

    1988-10-01

    Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids (e.g., cholesteryl esters, triglycerides, and fatty acids) are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, we postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic 125I-labeled cholesterol esterase (100 kDa) and 125I-labeled triglyceride lipase (52 kDa). This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a Kd of 100 nM and a Bmax of approximately 50-60 pmol per mg of vesicle protein. In contrast, enzymes associated with the hydrolysis of hydrophilic compounds such as amylase, phospholipase A2, and deoxyribonuclease do not bind to intestinal membranes in this manner. Human pancreatic cholesterol esterase also binds specifically and saturably to cultured intestinal epithelial cells (CaCo-2), and soluble heparin significantly diminishes the cellular uptake of the resultant hydrophobic reaction products (cholesterol and free fatty acids). We conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalizable pathway for transport of neutral lipids into endothelial and other cells.

  16. Effect of estradiol-17β on calcitonin receptor bindings in the hen neurohypophysis.

    PubMed

    Nakayama, H; Takahashi, T; Nakagawa-Mizuyachi, K; Kawashima, M

    2011-01-01

    The present study was performed to elucidate whether estradiol-17β (E₂) would affect calcitonin (CT) receptor binding in the hen neurohypophysis. The equilibrium dissociation constant (K(d)) and the maximum binding capacity (B(max)) of the CT receptor in the plasma membrane fraction of the hen neurohypophysis were examined by Scatchard analysis of specific binding of (125)I-labeled chicken CT. A single i.m. injection of E₂ into nonlaying hens caused a decrease in K(d) and B(max) values of the CT receptor. The K(d) and B(max) values of the CT receptor were smaller in laying hens than in nonlaying hens. The present study suggests that E₂ may increase the action of CT on the neurohypophysis in hens. PMID:21177459

  17. Neurotensin Decreases the Proinflammatory Status of Human Skin Fibroblasts and Increases Epidermal Growth Factor Expression

    PubMed Central

    Miguel Neves, Bruno; Cruz, Maria Teresa; Carvalho, Eugénia

    2014-01-01

    Fibroblasts colonization into injured areas during wound healing (WH) is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH. PMID:25180119

  18. Estrophilin immunoreactivity versus estrogen receptor binding activity in meningiomas: evidence for multiple estrogen binding sites

    SciTech Connect

    Lesch, K.P.; Schott, W.; Gross, S.

    1987-09-01

    The existence of estrogen receptors in human meningiomas has long been a controversial issue. This may be explained, in part, by apparent heterogeneity of estrogen binding sites in meningioma tissue. In this study, estrogen receptors were determined in 58 meningiomas with an enzyme immunoassay using monoclonal antibodies against human estrogen receptor protein (estrophilin) and with a sensitive radioligand binding assay using /sup 125/I-labeled estradiol (/sup 125/I-estradiol) as radioligand. Low levels of estrophilin immunoreactivity were found in tumors from 62% of patients, whereas radioligand binding activity was demonstrated in about 46% of the meningiomas examined. In eight (14%) tissue samples multiple binding sites for estradiol were observed. The immunoreactive binding sites correspond to the classical, high affinity estrogen receptors: the Kd for /sup 125/I-estradiol binding to the receptor was approximately 0.2 nM and the binding was specific for estrogens. The second, low affinity class of binding sites considerably influenced measurement of the classical receptor even at low ligand concentrations. The epidemiological and clinical data from patients with meningiomas, and the existence of specific estrogen receptors confirmed by immunochemical detection, may be important factors in a theory of oncogenesis.

  19. Impairement of HT29 Cancer Cells Cohesion by the Soluble Form of Neurotensin Receptor-3

    PubMed Central

    Lacas-Gervais, Sandra; Béraud-Dufour, Sophie; Coppola, Thierry; Mazella, Jean

    2014-01-01

    The neurotensin (NT) receptor-3 (NTSR3), also called sortilin is a multifunctional protein localized at the intracellular and plasma membrane level. The extracellular domain of NTSR3 (sNTSR3) is released by shedding from several cell lines including colonic cancer cells. This soluble protein acts as an active ligand through its ability to bind, to be internalized in the human adenocarcinoma epithelial HT29 cells and to stimulate the PI3 kinase pathway. The aim of this study was to investigate cellular responses induced by sNTSR3 in HT29 cells. The cellular functions of sNTSR3 were monitored by immunofluocytochemistry, electron microscopy and quantitative PCR in order to characterize the cell shape and the expression of adhesion proteins. We evidenced that sNTSR3 significantly regulates the cellular morphology as well as the cell-cell and the cell-matrix adherens properties by decreasing the expession of several integrins and by modifying the structure of desmosomes. Altogether, these properties lead to an increase of cell detachment upon sNTSR3 treatment on HT29, HCT116 and SW620 cancer cells. Our results indicate that sNTSR3 may induce the first phase of a process which weaken HT29 epithelial properties including desmosome architecture, cell spreading, and initiation of cell separation, all events which could be responsible for cancer metastasis. PMID:25221642

  20. Structure-Activity Relationship Studies of Amino Acid Substitutions in Radiolabeled Neurotensin Conjugates.

    PubMed

    Mascarin, Alba; Valverde, Ibai E; Mindt, Thomas L

    2016-01-01

    Radiolabeled derivatives of the peptide neurotensin (NT) and its binding sequence NT(8-13) have been studied as potential imaging probes and therapeutics for NT-1-receptor-positive cancer. However, a direct comparison of reported NT analogues, even if radiolabeled with the same radionuclide, is difficult because different techniques and models have been used for preclinical evaluations. In an effort to identify a suitable derivative of NT(8-13) for radiotracer development, we herein report a side-by-side in vitro comparison of radiometallated NT derivatives bearing some of the most commonly reported amino acid substitutions in their sequence. Performed investigations include cell internalization experiments, determinations of receptor affinity, measurements of the distribution coefficient, and blood serum stability studies. Of the [(177)Lu]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-labeled examples studied, analogues of NT(8-13) containing a short hydrophilic tetraethylene glycol (PEG4 ) spacer between the peptide and the radiometal complex, and a minimum number of substitutions of amino acid residues, exhibited the most promising properties in vitro. PMID:26593062

  1. Profile of the alpha-bungarotoxin-binding regions on the extracellular part of the alpha-chain of Torpedo californica acetylcholine receptor.

    PubMed Central

    Mulac-Jericevic, B; Atassi, M Z

    1987-01-01

    The continuous alpha-neurotoxin-binding regions on the extracellular part (residues 1-210) of the alpha-chain of Torpedo californica acetylcholine receptor were localized by reaction of 125I-labelled alpha-bungarotoxin with synthetic overlapping peptides spanning this entire part of the chain. The specificity of the binding was confirmed by inhibition with unlabelled toxin and, for appropriate peptides, with unlabelled anti-(acetylcholine receptor) antibodies. Five toxin-binding regions were localized within residues 1-10, 32-41, 100-115, 122-150 and 182-198. The third, fourth and fifth (and to a lesser extent the first and second) toxin-binding regions overlapped with regions recognized by anti-(acetylcholine receptor) antibodies. The five toxin-binding regions may be distinct sites or, alternatively, different 'faces' in one (or more) sites. PMID:3435488

  2. Expression of neurotensin messenger RNA in a human carcinoid tumor.

    PubMed Central

    Evers, B M; Ishizuka, J; Townsend, C M; Rajaraman, S; Thompson, J C

    1991-01-01

    Neurotensin (NT), a distal gut peptide, has important regulatory and trophic effects throughout the gut; however the intracellular mechanisms that regulate the gene expression and release of human NT are not known. The purpose of this endeavor was to study a functioning human pancreatic carcinoid cell line (called BON) in vitro that expresses the NT gene, and to study the effect of the cyclic adenosine monophosphate (cAMP) signal-transduction pathway on the expression and release of human NT. RNA was prepared from BON cell line (which has been established in this laboratory); the RNA was analyzed for NT mRNA expression by Northern hybridization with a complementary DNA probe. RNA blot analysis demonstrated that the NT gene is expressed in BON and is transcribed to two mRNAs of 1.0- and 1.5-kb sizes. In the second part of this study, BON cells were treated with either forskolin (FSK), which increases intracellular levels of cAMP, or with serotonin (5-HT), which reduces cAMP in BON cells. Forskolin produced a dose-dependent increase in NT peptide release and, furthermore, FSK (10(-6) mol/L) rapidly increased NT mRNA abundance 1 hour after addition; conversely, 5-HT (10(-5) mol/L) decreased NT mRNA at 1 hour. Neurotensin mRNA levels returned to control values by 3 hours after either FSK or 5-HT, which suggests that the transcript half-life for NT is relatively short. These findings show that the expression and peptide release of human NT is mediated, in part, by the cAMP signal-transduction pathway. Our human carcinoid cell line will provide a useful model to study the in vitro regulation of NT gene expression and peptide release. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. Fig. 6. PMID:1659338

  3. Soybean. beta. -glucan binding sites display maximal affinity for a heptaglucoside phytoalexin-elicitor

    SciTech Connect

    Cosio, E.G.; Waldmueller, T.; Frey, T.; Ebel, J. )

    1990-05-01

    The affinity of soybean {beta}-glucan-binding sites for a synthetic heptaglucan elicitor was tested in a ligand-competition assay against a {sup 125}I-labeled 1,3-1,6-{beta}-glucan preparation (avg. DP=20). Half-maximal displacement of label (IC{sub 50}) was obtained at 9nM heptaglucan, the highest affinity of all fractions tested to date. Displacement followed a uniform sigmoidal pattern and was complete at 1{mu}M indicating access of heptaglucan to all sites available to the labeled elicitor. A mathematical model was used to predict IC{sub 50} values according to the DP of glucan fragments obtained from fungal cell walls. The lowest IC{sub 50} predicted by this model is 3nM. Binding affinity of the glucans was compared with their elicitor activity in a bioassay.

  4. Transient and Stable Expression of the Neurotensin Receptor NTS1: A Comparison of the Baculovirus-Insect Cell and the T-REx-293 Expression Systems

    PubMed Central

    Xiao, Su; White, Jim F.; Betenbaugh, Michael J.; Grisshammer, Reinhard; Shiloach, Joseph

    2013-01-01

    Nowadays, baculovirus-infected insect cells and tetracycline-inducible mammalian cell lines (T-REx-293) are intensively used for G protein-coupled receptor (GPCR) production for crystallography purposes. Here we constructed a suspension T-REx-293 cell line to stably express an engineered neurotensin receptor 1 (NTS1) mutant and we quantitatively compared this cell line with the transient baculovirus-insect cell system throughout a milligram-scale NTS1 expression and purification process. The two systems were comparable with respect to functional NTS1 expression levels and receptor binding affinity for the agonist [3H] neurotensin. However, NTS1 surface display on T-REx-293 cells determined by radio-ligand binding assays was 2.8 fold higher than that on insect cells. This work demonstrates two approaches for preparing milligram quantities of purified NTS1 suitable for structural studies and provides useful input to users in choosing and optimizing an appropriate expression host for other GPCRs. PMID:23696845

  5. Plasma binding proteins for platelet-derived growth factor that inhibit its binding to cell-surface receptors.

    PubMed Central

    Raines, E W; Bowen-Pope, D F; Ross, R

    1984-01-01

    Evidence is presented that the binding of platelet-derived growth factor (PDGF) to plasma constituents inhibits the binding of PDGF to its cell-surface mitogen receptor. Approximately equivalent amounts of PDGF-binding activity were found in plasma from a number of different species known by radioreceptor assay to contain PDGF homologues in their clotted blood. Activation of the coagulation cascade did not significantly alter the PDGF-binding activity of the plasma components. Three molecular weight classes of plasma fractions that inhibit PDGF binding to its cell-surface receptor were defined by gel filtration: approximately equal to 40,000, 150,000, and greater than 500,000. Specific binding of 125I-labeled PDGF to the highest molecular weight plasma fraction could also be demonstrated by gel filtration. The binding of PDGF to these plasma components was reversible under conditions of low pH or with guanidine X HCl, and active PDGF could be recovered from the higher molecular weight fractions. Immunologic and functional evidence is presented that the highest molecular weight plasma fraction may be alpha 2-macroglobulin. A model is proposed in which the activity of PDGF released in vivo may be regulated by association with these plasma binding components and by high-affinity binding to cell-surface PDGF receptors. PMID:6203121

  6. Distribution of Neurotensin and Somatostatin-28 (1-12) in the Minipig Brainstem.

    PubMed

    Sánchez, M L; Vecino, E; Coveñas, R

    2016-08-01

    Using an indirect immunoperoxidase technique, an in depth study has been carried out for the first time on the distribution of fibres and cell bodies containing neurotensin and somatostatin-28 (1-12) (SOM) in the minipig brainstem. The animals used were not treated with colchicine. The distribution of neurotensin- and SOM-immunoreactive fibres was seen to be quite similar and was moderate in the minipig brainstem: a close anatomical relationship between both neuropeptides was observed. The distribution of cell bodies containing neurotensin or SOM was quite different and restricted. Cell bodies containing neurotensin were found in four brainstem nuclei: nucleus centralis raphae, nucleus dorsalis raphae, in the pars centralis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis raphae. Cell bodies containing SOM were found in six nuclei/regions of the brainstem: nucleus ambiguus, nucleus dorsalis motorius nervi vagus, formatio reticularis, nucleus parabrachialis medialis, nucleus reticularis lateralis and nucleus ventralis raphae. According to the observed anatomical distribution of the immunoreactive structures containing neurotensin or SOM, the peptides could be involved in sleep-waking, nociceptive, gustatory, motor, respiratory and autonomic mechanisms. PMID:26250798

  7. The Discovery of Indole Full Agonists of the Neurotensin Receptor 1 (NTSR1)

    PubMed Central

    Di Fruscia, Paolo; He, Yuanjun; Koenig, Marcel; Tabrizifard, Sahba; Nieto, Ainhoa; McDonald, Patricia H.; Kamenecka, Theodore M.

    2014-01-01

    Neurotensin (NT) is an endogenous tridecapeptide found in the central nervous system (CNS) and in peripheral tissues. Neurotensin exerts a wide range of physiological effects and it has been found to play a critical role in a number of human diseases, such as schizophrenia, Parkinson’s disease and drug addiction. The discovery of small-molecule non-peptide neurotensin receptor (NTSR) modulators would represent an important breakthrough as such compounds could be used as pharmacological tools, to further decipher the cellular functions of neurotensin, and potentially as therapeutic agents to treat human disease. Herein, we report the identification of non-peptide low-micromolar neurotensin receptor 1 (NTSR1) full agonists, discovered through structural optimization of the known NTSR1 partial agonist 1. In vitro cellular screenings, based on an intracellular Ca2+ mobilization assay, revealed our best hit molecule 8 (SR-12062) to have an EC50 of 2 μM at NTSR1 with full agonist behaviour (Emax = 100%), showing a higher efficacy and ~ 90-fold potency improvement compared to parent compound 1 (EC50 = 178 μM; Emax = 17%). PMID:24997685

  8. Multiple toxic doses of methamphetamine alter neurotensin concentrations in various region of the rat brain

    SciTech Connect

    Hanson, G.R.; Merchant, K.; Gibb, J.W.; Letter, A.A.

    1986-03-05

    The authors have previously reported that multiple high doses of methamphetamine (METH) alter neuronal monoamine metabolism and release. Recently, Hokfelt et al. showed that neurotensin, a tridecapeptide, has neurotransmitter properties which may be involved with DA neuronal activity. In the present study they investigated the possible effects of METH on the CNS neurotensin system. Five doses of METH (15 mg/kg) were administered every 6 h; control and treated rats were sacrificed 18 h after the last dose and concentrations of neurotensin-like immuno-reactivity (NTLI) were measured by radioimmunoassay. NTLI was elevated 200-300% in the nucleus accumbens, neostriatum, and substantia nigra; 30-40% increases in NTLI were measured in the hippocampus and hypothalamus. No change was observed in amygdala, A-10 or periaqueductal gray. In contrast to the above measured areas, the frontal lobe and olfactory bulb showed decreases of 25-35%. These findings demonstrate that METH treatment alters the activities of several CNS neurotensin systems, possibly due to the influence of this drug on DA pathways. The variability in the type and magnitude of these responses suggests that DA and neurotensin systems interact by more than one mechanism.

  9. Immunological properties of prolactin and studies on a gonadotropin binding inhibitor

    SciTech Connect

    Chang, Y.S.

    1985-01-01

    The physiological role of prolactin in horses has not yet been well defined. With the availability of highly purified ePRL for inducing antibody formation in rabbits and for radiolabeling with Na/sup 125/I, a very sensitive (0.4-0.6 ng/ml) and highly specific homologous RIA for ePRL was developed. A heterologous RIA using /sup 125/I-labeled ovine PRL and anti-ePRL antiserum was also developed and compared to the homologous RIA for ePRL. Of the two systems, it is concluded that this homologous RIA system is more suitable and more reliable for measuring prolactin concentration in horse serum samples. Until now, biochemical information on PRL has not been available for reptilian species. Sea turtle (Chelonia mydas) prolactin was purified from pituitary extracts by selective precipitation, DEAE-cellulose chromatography and gel filtration. Similar to other species of PRL, sea turtle PRL is a 22,000-24,000 daltons protein and contains a high content of glutamic acid, aspartic acid, serine and leucine, the N-terminal amino acid residue. Gonadotropin (FSH) binding inhibitor was partially purified from sheep testes by ammonium sulfate fractionation and ion exchange chromatography. The FSH-BI (molecular weight: 50,000 daltons, estimated by gel filtration) contains a protein moiety necessary for binding inhibitory activity. The inhibition of the binding of /sup 125/I-labeled ovine FSH to its receptor by the FSH-BI is not competitive. Both in vivo and in vitro biological studies of FSH-BI preparations in rats indicated various effects on FSH and LH activities at the gonadal level. These findings suggest a physiological role for FSH-BI in the regulation of reproduction.

  10. Deletion in the first cysteine-rich repeat of low density lipoprotein receptor impairs its transport but not lipoprotein binding in fibroblasts from a subject with familial hypercholesterolemia

    SciTech Connect

    Leitersdorf, E.; Hobbs, H.H.; Fourie, A.M.; Jacobs, M.; Van Der Westhuyzen, D.R.; Coetzee, G.A. )

    1988-11-01

    The ligand-binding domain of the low density lipoprotein (LDL) receptor is composed of seven cysteine-rich repeats, each {approx} 40 amino acids long. Previous studies showed that if the first repeat of the ligand-binding domain (encoded by exon 2) is deleted, the receptor fails to bind an anti-LDL receptor monoclonal antibody (IgG-C7) but continues to bind LDL with high affinity. Cultured fibroblasts from a Black South African Xhosa patient (TT) with the clinical syndrome of homozygous familial hypercholesterolemia demonstrated high-affinity cell-surface binding of {sup 125}I-labeled LDL but not {sup 125}I-labeled IgG-C7. previous haplotype analysis, using 10 restriction fragment length polymorphic sites, suggested that the patient inherited two identical LDL receptor alleles. The polymerase chain reaction technique was used to selectively amplify exon 2 of the LDL receptor gene from this patient. Sequence analysis of the amplified fragment disclosed a deletion of six base pairs that removes two amino acids, aspartic acid and glycine, from the first cysteine-rich ligand binding repeat. The mutation creates a new Pst I restriction site that can be used to detect the deletion. The existence of this mutant allele confirms that the epitope of IgG-C7 is located in the first cysteine-rich repeat and that this repeat is not necessary for LDL binding. The mutant gene produced a normally sized 120-kilodalton LDL receptor precursor protein that matured to the 160-kilodalton form at less than one-fourth the normal rate.

  11. Plasma levels of human neurotensin: methodological and physiological considerations.

    PubMed

    Ferris, C F; George, J K; Eastwood, G; Potegal, M; Carraway, R E

    1991-01-01

    The ingestion of a meal high in fat content is known to increase circulating levels of neurotensin (NT) in humans. However, the magnitude of the postprandial rise of NT in the general circulation and its physiological significance have been subject of much debate. The present study examines circulating levels of NT in male volunteers prior to and following each of their three daily meals (ca. 31 g fat/meal). The response observed are also compared to that elicited by the direct instillation of intralipid (ca. 44 g fat) into the duodenum. NT levels were determined by radioimmunoassay of acid/acetone extracted plasma fractionated by high pressure liquid chromatography. Meals caused a significant but modest increase in NT levels, with the largest increment (ca. 4 fmol/ml) occurring after breakfast. In contrast, NT levels increased ca. 20 fmol/ml with intraduodenal instillation of lipid. The meal-stimulated increases in circulating NT measured here are 4- to 5-fold less than those reported by others, the difference most likely reflecting the lesser amount of lipid ingested. Previous studies provided subjects with single meals containing in excess of 120 g of fat; the 30 g of fat ingested by our subjects, ca. 33% of total caloric intake, is near that recommended by the U.S. Senate, Select Committee on Nutritional and Human Needs. These data show that diets with a reasonable fat content have only a modest effect on circulating levels of NT. PMID:2067972

  12. Mephedrone alters basal ganglia and limbic neurotensin systems

    PubMed Central

    German, Christopher L.; Hoonakker, Amanda H.; Fleckenstein, Annette E.; Hanson, Glen R.

    2014-01-01

    Mephedrone (4-methylmethcathinone) is a synthetic cathinone designer drug that alters presynaptic dopamine (DA) activity like many psychostimulants. However, little is known about the postsynaptic dopaminergic impacts of mephedrone. The neuropeptide neurotensin (NT) provides inhibitory feedback for basal ganglia and limbic DA pathways, and postsynaptic D1-like and D2-like receptor activity affects NT tissue levels. This study evaluated how mephedrone alters basal ganglia and limbic system NT content and the role of NT receptor activation in drug consumption behavior. Four 25 mg/kg injections of mephedrone increased NT content in basal ganglia (striatum, substantia nigra and globus pallidus) and the limbic regions (nucleus accumbens core), while a lower dosage (5 mg/kg/injection) only increased striatal NT content. Mephedrone-induced increases in basal ganglia NT levels were mediated by D1-like receptors in the striatum and the substantia nigra by both D1-like and D2-like receptors in the globus pallidus. Mephedrone increased substance P content, another neuropeptide, in the globus pallidus, but not in the dorsal striatum or substantia nigra. Finally, the NT receptor agonist PD149163 blocked mephedrone self-administration, suggesting reduced NT release, as indicated by increased tissue levels, likely contributing to patterns of mephedrone consumption. PMID:24678634

  13. Lectin-binding properties of Aeromonas caviae strains

    PubMed Central

    Rocha-de-Souza, Cláudio M.; Hirata-Jr, Raphael; Mattos-Guaraldi, Ana L.; Freitas-Almeida, Angela C.; Andrade, Arnaldo F. B.

    2008-01-01

    The cell surface carbohydrates of four strains of Aeromonas caviae were analyzed by agglutination and lectin-binding assays employing twenty highly purified lectins encompassing all sugar specificities. With the exception of L-fucose and sialic acid, the sugar residues were detected in A. caviae strains. A marked difference, however, in the pattern of cell surface carbohydrates in different A. caviae isolates was observed. Specific receptors for Tritricum vulgaris (WGA), Lycopersicon esculentum (LEL) and Solanum tuberosum (STA) (D-GlcNAc-binding lectins) were found only in ATCC 15468 strain, whereas Euonymus europaeus (EEL, D-Gal-binding lectin) sites were present exclusively in AeQ32 strain, those for Helix pomatia (HPA, D-GalNAc-binding lectin) in AeC398 and AeV11 strains, and for Canavalia ensiformes (Con A, D-Man-binding lectin) in ATCC 15468, AeC398, AeQ32 and AeV11 strains, after bacterial growing at 37°C. On the other hand, specific receptors for WGA and EEL were completely abrogated growing the bacteria at 22°C. Binding studies with 125I- labeled lectins from WGA, EEL and Con A were performed. These assays essentially confirmed the selectivity, demonstrated in the agglutination assays of these lectins for the A. caviae strains. PMID:24031204

  14. Influence of pertussis toxin on thermic responses to morphine and neurotensin in rats.

    PubMed

    Basilico, L; Abbondi, M; Fumagalli, A; Parolaro, D; Gori, E; Giagnoni, G

    1992-11-10

    The influence of pertussis toxin (PTX) on thermic responses elicited by morphine and neurotensin was evaluated in unrestrained rats kept at 22 degrees C. High doses of morphine (9-36 micrograms/rat i.c.v.) lowered body temperature and low doses (1.25, 2.5 micrograms/rat i.c.v.) produced hyperthermia. The hyperthermic effect was more resistant than the hypothermic effect to naloxone antagonism. Neurotensin (50, 100 micrograms/rat i.c.v.) induced marked hypothermia followed by hyperthermia. I.c.v. injection of PTX (1 microgram), six days before morphine (18 micrograms/rat i.c.v.), replaced the opiate hypothermia by consistent hyperthermia and reduced by 60% the hyperthermia elicited by morphine (2.5 micrograms/rat i.c.v.). The toxin also affected the thermic responses induced by neurotensin (50 micrograms/rat i.c.v.) administered six days after PTX (1 microgram/rat i.c.v.). The initial hypothermia was enhanced by 173% and the late hyperthermia was fully antagonized. It thus appears that PTX-sensitive G-proteins play different roles in the molecular events underlying the thermoregulatory responses to morphine and neurotensin. PMID:1451736

  15. Reversible calcitonin binding to solubilized sheep brain binding sites.

    PubMed Central

    Sexton, P M; Schneider, H G; D'Santos, C S; Mendelsohn, F A; Kemp, B E; Moseley, J M; Martin, T J; Findlay, D M

    1991-01-01

    In this study we have solubilized and characterized binding sites for calcitonin (CT) from sheep brainstem. Autoradiography of 125I-labelled salmon CT (125I-sCT) binding to sheep diencephalon revealed a similar pattern of binding to that seen in other species, although the extent of distribution was greater in the sheep. CT binding activity could be extracted from membranes with either CHAPS or digitonin, but not with beta-octyl glucoside, 125I-sCT binding was saturable, with a dissociation constant for CHAPS-solubilized membranes of 2.8 +/- 0.5 nM and a maximum binding site concentration of 6.2 +/- 1.6 pmol/mg of protein. In competition binding studies, various CTs and their analogues demonstrated a similar rank order of potency to that seen in other CT receptor systems, Optimal binding occurred in the pH range 6.5-7.5, and was decreased in the presence of NaCl concentrations greater than 200 mM. In contrast with most other CT receptor binding systems, in which binding is poorly reversible, the binding of 125I-sCT to sheep brain binding sites underwent substantial dissociation upon addition of excess unlabelled sCT, with 40% and 46% dissociation after 2 h at 4 degree C in particulate and solubilized membranes respectively. Photoaffinity labelling of the binding site with the biologically active analogue 125I-[Arg11,18,4-azidobenzoyl-Lys14]sCT and analysis on SDS/PAGE under reducing conditions revealed a specific protein band of Mr approximately solubilized and particulate brain membranes. This is in accordance with the molecular size of CT receptors in other tissues where two species of receptor have been identified. one of Mr approximately 71,000 and another of Mr approximately 88,000. These results demonstrate the presence of high concentrations of CT binding sites in sheep brain which display different kinetic properties to those of CT receptors found in other tissues. Images Fig. 1. Fig. 6. PMID:1846527

  16. Antidiuretic effects of neurotensin in chloralose anaesthetized dogs.

    PubMed

    Kivlighn, S D; Jandhyala, B S

    1990-06-01

    1. Effects of cerebroventricular and/or intravenous infusions of neurotensin (NT), an endogenous tridecapeptide, on haemodynamics and renal function were investigated in chloralose anaesthetized dogs. 2. Cerebroventricular infusions (i.c.v.) of NT (10-6 mol/L and 10-5 mol/L, 0.1 mL/min) for 30 min did not produce any significant alterations in the measured variables. In the vagotomized dogs, intravenous (i.v.) infusion of NT (10(-5) mol/L) at a rate of 0.1 mL/min for 30 min significantly lowered the arterial blood pressure and glomerular filtration rate; these effects were accompanied by pronounced reductions in the urine flow and urinary sodium excretion and marked increases in urine osmolality. 3. In the dogs with vagi intact, i.v. infusions of NT failed to produce any alterations in the blood pressure; however, renal effects of NT were essentially identical to those observed in the vagotomized dogs. 4. Infusions of NT (10(-6) mol/L) and/or NT-metabolites NT1-8 and NT8-13 (10(-5) mol/L) directly into the renal artery failed to produce any significant alterations in the urine flow. Antidiuretic effects of i.v. NT were not prevented by acute renal denervation, adrenalectomy, or pretreatment of the animals with naloxone. However, morphine pretreatment completely abolished the hypotensive and anti-diuretic effects of NT. 5. It is proposed that i.v. infusion of NT rapidly facilitates the secretion of an endogenous substance possessing potent antidiuretic properties and opiate mechanisms are involved in mediating such an effect. Although it appears unlikely, a role for vasopressin cannot be ruled out. PMID:2390805

  17. Exogenous neurotensin modulates sperm function in Japanese Black cattle.

    PubMed

    Umezu, Kohei; Hiradate, Yuuki; Oikawa, Toshinori; Ishiguro, Hirotoshi; Numabe, Takashi; Hara, Kenshiro; Tanemura, Kentaro

    2016-08-25

    Recently, the conception rates after artificial insemination have been pointed out to decline continuously. To overcome this problem, the control of frozen and thawed sperm quality is required. However, the mechanism of bovine sperm functional regulation is still largely unknown. In mammals, the ejaculated sperm are capable of showing fertilizing ability during migration in the female reproductive organs. It is well known that these female organs secrete several factors contributing to sperm capacitation. We previously reported that neurotensin (NT) secreted from the oviduct and cumulus cells enhanced sperm capacitation and acrosome reaction in mice. In this study, we confirmed the expression of the NT receptor (NTR1) in the bovine sperm neck region and the secretion of NT in the bovine uterus and oviduct. The similar expression patterns of NT and NTR1 suggests a conserved mechanism of sperm functional regulation between mouse and cattle. Thus, we examined the effects of exogenous NT on the bovine sperm functions. First, we showed that NT induced sperm protein tyrosine phosphorylation in a dose-dependent manner, suggesting that NT enhances sperm capacitation. Second, we showed that NT induced acrosome reactions of capacitated sperm in a dose-dependent manner, suggesting that NT facilitates acrosome reaction. Finally, we used a computer-aided sperm analysis system to show that NT did not have a great effect on sperm motility. These results suggest that NT acts as a facilitator of sperm capacitation and acrosome reaction in the female reproductive tracts in cattle, highlighting the importance of NT-mediated signaling to regulate sperm functions. PMID:27210588

  18. Exogenous neurotensin modulates sperm function in Japanese Black cattle

    PubMed Central

    UMEZU, Kohei; HIRADATE, Yuuki; OIKAWA, Toshinori; ISHIGURO, Hirotoshi; NUMABE, Takashi; HARA, Kenshiro; TANEMURA, Kentaro

    2016-01-01

    Recently, the conception rates after artificial insemination have been pointed out to decline continuously. To overcome this problem, the control of frozen and thawed sperm quality is required. However, the mechanism of bovine sperm functional regulation is still largely unknown. In mammals, the ejaculated sperm are capable of showing fertilizing ability during migration in the female reproductive organs. It is well known that these female organs secrete several factors contributing to sperm capacitation. We previously reported that neurotensin (NT) secreted from the oviduct and cumulus cells enhanced sperm capacitation and acrosome reaction in mice. In this study, we confirmed the expression of the NT receptor (NTR1) in the bovine sperm neck region and the secretion of NT in the bovine uterus and oviduct. The similar expression patterns of NT and NTR1 suggests a conserved mechanism of sperm functional regulation between mouse and cattle. Thus, we examined the effects of exogenous NT on the bovine sperm functions. First, we showed that NT induced sperm protein tyrosine phosphorylation in a dose-dependent manner, suggesting that NT enhances sperm capacitation. Second, we showed that NT induced acrosome reactions of capacitated sperm in a dose-dependent manner, suggesting that NT facilitates acrosome reaction. Finally, we used a computer-aided sperm analysis system to show that NT did not have a great effect on sperm motility. These results suggest that NT acts as a facilitator of sperm capacitation and acrosome reaction in the female reproductive tracts in cattle, highlighting the importance of NT-mediated signaling to regulate sperm functions. PMID:27210588

  19. Activation of neurotensin receptor type 1 attenuates locomotor activity.

    PubMed

    Vadnie, Chelsea A; Hinton, David J; Choi, Sun; Choi, YuBin; Ruby, Christina L; Oliveros, Alfredo; Prieto, Miguel L; Park, Jun Hyun; Choi, Doo-Sup

    2014-10-01

    Intracerebroventricular administration of neurotensin (NT) suppresses locomotor activity. However, the brain regions that mediate the locomotor depressant effect of NT and receptor subtype-specific mechanisms involved are unclear. Using a brain-penetrating, selective NT receptor type 1 (NTS1) agonist PD149163, we investigated the effect of systemic and brain region-specific NTS1 activation on locomotor activity. Systemic administration of PD149163 attenuated the locomotor activity of C57BL/6J mice both in a novel environment and in their homecage. However, mice developed tolerance to the hypolocomotor effect of PD149163 (0.1 mg/kg, i.p.). Since NTS1 is known to modulate dopaminergic signaling, we examined whether PD149163 blocks dopamine receptor-mediated hyperactivity. Pretreatment with PD149163 (0.1 or 0.05 mg/kg, i.p.) inhibited D2R agonist bromocriptine (8 mg/kg, i.p.)-mediated hyperactivity. D1R agonist SKF-81297 (8 mg/kg, i.p.)-induced hyperlocomotion was only inhibited by 0.1 mg/kg of PD149163. Since the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in the behavioral effects of NT, we examined whether microinjection of PD149163 into these regions reduces locomotion. Microinjection of PD149163 (2 pmol) into the NAc, but not the mPFC suppressed locomotor activity. In summary, our results indicate that systemic and intra-NAc activation of NTS1 is sufficient to reduce locomotion and NTS1 activation inhibits D2R-mediated hyperactivity. Our study will be helpful to identify pharmacological factors and a possible therapeutic window for NTS1-targeted therapies for movement disorders. PMID:24929110

  20. Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase in embryonic mouse brain cell cultures

    SciTech Connect

    Shanker, G.; Pieringer, R.A.

    1986-05-01

    Previously, the authors demonstrated that ornithine decarboxylase was stimulated by insulin in cultures of embryonic mouse brain cells. In the present work, they have investigated the presence and specificity of insulin receptors in these cultures. A time study showed that maximum binding of /sup 125/(I) labelled insulin was around 75 min. Other studies measured the influence of concentration and age on insulin binding. A displacement study using increasing concentrations of cold insulin, glucagon or growth hormone demonstrated that the specificity of the receptors for insulin was rather high. It was also found that insulin displayed a clear dose-dependent stimulation of thymidine incorporation into the brain cells. Insulin also stimulated the glial enzyme 2':3'-cyclic nucleotide phosphohydrolase (CNP-ase). The results suggest a dual role for insulin; it regulates both cell proliferation as well as differentiation.

  1. Binding of complement regulators factor H and C4b binding protein to group A streptococcal strains isolated from tonsillar tissue and blood.

    PubMed

    Suvilehto, Jari; Jarva, Hanna; Seppänen, Mikko; Siljander, Tuula; Vuopio-Varkila, Jaana; Meri, Seppo

    2008-06-01

    Group A streptococcus (GAS) is the most common pathogen causing bacterial pharyngitis. We isolated streptococcal strains from tonsils removed from patients with tonsillar disease (n=202) and studied their ability to bind the complement regulators factor H (FH) and C4b binding protein (C4BP) using 125 I-labeled proteins. Blood isolates of GAS (n=10) were obtained from patients with bacteraemia. Streptococci were isolated from 21% of the tonsillitis patients. The emm and T types of the GAS strains were determined. Of the 26 GAS strains studied, only six could bind FH and/or C4BP above the threshold levels. The fraction of the offered radioactive protein bound ranged between 6-12% for FH and 19-56% for C4BP. The clinical course of the tonsillar disease was not related to the binding of FH or C4BP by GAS. The binding strains were mostly of the T4M4 or T28M28 type. From the invasive strains (n=10), three bound FH (binding level: 8-11%) and two C4BP (36-39%). The binding correlated only partially to M-protein (emm) type suggesting that the binding was not exclusively due to M-protein. The results indicate that complement regulator binding by GAS is only partially related to pathogenicity and not a universal property of all group A streptococci. PMID:18538613

  2. Radioiodinated rat parathyroid hormone-(1-34) binds to its receptor on rat osteosarcoma cells in a manner consistent with two classes of binding sites

    SciTech Connect

    Seitz, P.K.; Nickols, G.A.; Nickols, M.A.; McPherson, M.B.; Cooper, C.W. )

    1990-04-01

    Binding of 125I-labeled rat (r) PTH-(1-34) to ROS 17/2.8 osteoblastic bone cells and to membranes from these cells was examined. Competitive binding inhibition experiments were performed using unlabeled rPTH-(1-34) with particular emphasis on concentrations of peptide below 1 nM. In intact cells, binding of labeled rPTH-(1-34) was highly specific, and inhibition of binding by unlabeled ligand suggested the presence of two classes of binding sites, one with high affinity and low capacity (KD = 40 pM, approximately 20% of total binding sites) and the other with lower affinity and high capacity (KD = 2 nM, approximately 80% of the sites). Membranes prepared from ROS cells also exhibited a pattern of binding from competitive inhibition curves consistent with two distinct binding sites (KD = 30 pM and 6 nM). In intact ROS cells, cellular cAMP levels increased over the range of 10(-11)-10(-9) M rPTH-(1-34) with an ED50 intermediate between the two KD values (0.25 nM). These data suggest that osteoblastic bone cells possess two distinct classes of membrane receptors for PTH. Since the KD of the higher affinity site more closely approximates circulating concentrations of PTH, binding to this site may have physiologic relevance.

  3. Localization and synthesis of the hormone-binding regions of the human thyrotropin receptor

    SciTech Connect

    Atassi, M.Z.; Manshouri, T. ); Sakata, Shigeki )

    1991-05-01

    Two regions of human thyrotropin (thyroid-stimulating hormone, TSH) receptor (TSHR) were selected on the basis that they exhibit no sequence resemblance to luteinizing hormone/chorionic gonadotropin receptor. Five synthetic overlapping peptides (12-30, 24-44, 308-328, 324-344, and 339-364) were studied for their ability to bind {sup 125}I-labeled human TSH (hTSH), its isolated {alpha} and {beta} subunits, bovine TSH, ovine TSH, human luteinizing hormone, and human follicle-stimulating hormone. The human TSHR peptides 12-30 and 324-344 exhibited remarkable binding activity to human, bovine, and ovine TSH and to the {beta} chain of hTSH. Lower binding activity resided in the adjacent overlapping peptides, probably due to the contribution of the shared overlap to the binding. The specificity of TSH binding to these peptides was confirmed by their inability to bind human luteinizing hormone, human follicle-stimulating hormone, and the {alpha} chain of hTSH. Thyrotropins did not bind to bovine serum albumin or to peptide controls unrelated to the TSHR system. It is concluded that the binding of TSH to its receptor involves extensive contacts and that the TSHR peptides 12-30 and 324-344 contain specific binding regions for TSH that might be either independent sites or two faces (subsites) within a large binding site.

  4. Novel Drosophila receptor that binds multiple growth factors

    SciTech Connect

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-05-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10/sup -6/ to 10/sup -8/ M. The 100 kDa protein can be affinity-labeled with these /sup 125/I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by /sup 125/I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors.

  5. Ventral Midbrain NTS1 Receptors Mediate Conditioned Reward Induced by the Neurotensin Analog, D-Tyr[11]neurotensin

    PubMed Central

    Rouibi, Khalil; Bose, Poulomee; Rompré, Pierre-Paul; Warren, Richard A.

    2015-01-01

    The present study was aimed at characterizing the mechanisms by which neurotensin (NT) is acting within the ventral midbrain to induce a psychostimulant-like effect. In a first experiment, we determine which subtype(s) of NT receptors is/are involved in the reward-inducing effect of ventral midbrain microinjection of NT using the conditioned place-preference (CPP) paradigm. In a second study, we used in vitro patch clamp recording technique to characterize the NT receptor subtype(s) involved in the modulation of glutamatergic neurotransmission (excitatory post-synaptic current, EPSC) in ventral tegmental neurons that expressed (Ih+), or do not express (Ih-), a hyperpolarization-activated cationic current. Behavioral studies were performed with adult male Long-Evans rats while electrophysiological recordings were obtained from brain slices of rat pups aged between 14 and 21 days. Results show that bilateral ventral midbrain microinjections of 1.5 and 3 nmol of D-Tyr[11]NT induced a CPP that was respectively attenuated or blocked by co-injection with 1.2 nmol of the NTS1/NTS2 antagonist, SR142948, and the preferred NTS1 antagonist, SR48692. In electrophysiological experiments, D-Tyr[11]NT (0.01-0.5 μM) attenuated glutamatergic EPSC in Ih+ but enhanced it in Ih- neurons. The attenuation effect (Ih+ neurons) was blocked by SR142948 (0.1 μM) while the enhancement effect (Ih- neurons) was blocked by both antagonists (0.1 μM). These findings suggest that (i) NT is acting on ventral midbrain NTS1 receptors to induce a rewarding effect and (ii) that this psychostimulant-like effect could be due to a direct action of NT on dopamine neurons and/or an enhancement of glutamatergic inputs to non-dopamine (Ih-) neurons. PMID:26733785

  6. Design and synthesis of [(125)I]Pyricoxib: A novel (125)I-labeled cyclooxygenase-2 (COX-2) inhibitors.

    PubMed

    Tietz, Ole; Dzandzi, James; Bhardwaj, Atul; Valliant, John F; Wuest, Frank

    2016-03-15

    Cyclooxygenase-2 (COX-2) is the key enzyme in the prostaglandin synthesis pathway which is involved in various pathophysiological conditions. The enzyme is membrane bound and located inside of the endoplasmic reticulum and nuclear membrane. Effective perfusion of inhibitors to the active site requires lipophilic drugs, which consequently display high unspecific background accumulation, for example, in fatty tissues. The objective of this work was the development of a small molecule radiolabeled with a long-lived iodine radioisotope to enable longer imaging times and better target-to-background ratios. A group of iodinated compounds (8-10) was synthesized and identified as selective COX-2 inhibitors (COX-2 IC50=0.85-13 μM). Molecular docking results provided the theoretical support for the experimental COX-2 inhibition data. Furthermore, a novel (125)I-containing trifluoro-pyrimidine compound ([(125)I]Pyricoxib) was prepared via radioiododestannylation reaction as potent and selective COX-2 inhibitor. Radiosynthesis of [(125)I]Pyricoxib was accomplished with innovative fluorous chemistry using fluorous chloroamine-T (F-CAT) as novel oxidizing agent in high radiochemical yields of 91 ± 4%. PMID:26898334

  7. Identification of cross-reactive promastigote cell surface antigens of some leishmanial stocks by 125I labeling and immunoprecipitation.

    PubMed Central

    Gardiner, P R; Jaffe, C L; Dwyer, D M

    1984-01-01

    Externally oriented surface membrane constituents of promastigotes from several Leishmania species were radiolabeled with 125I. Autoradiographs of cell surface-labeled and sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated proteins of the stocks revealed distinctive patterns of bands in the molecular weight range of 6,000 to 240,000. Immunoprecipitation of detergent extracts of the labeled promastigote stocks with anti-Leishmania donovani membrane serum demonstrated that each of the stocks contained some antigenically cross-reactive determinants. The electrophoretic patterns of these determinants serve both to distinguish the parasite stocks (by unique, species-specific patterns) and to indicate antigenic similarities in stocks thought to be different by other biochemical criteria. At least 12 cross-reactive cell surface antigens in two New World leishmanias are recognized by polyvalent anti-L. donovani serum, suggesting that these common leishmanial antigens may account for the documented serological cross-reactivities among various Leishmania species. In all stocks tested, an iodinated protein was identified which had a relative molecular weight of 65,000 under reducing conditions but which demonstrated an increase in relative mobility in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Distinctive patterns of the antigens common to the several stocks were also demonstrated with the use of monoclonal antibodies. Images PMID:6363295

  8. /sup 125/I-labeled radioimmunoassay kits for progesterone evaluated for use in an in vitro fertilization program

    SciTech Connect

    Blight, L.F.; White, G.H.

    1983-06-01

    We have evaluated two commercially available /sup 125/I radioimmunoassay kits (Diagnostic Products Corp., DPC; and Radioassay Systems Laboratories, RSL) for measurement of serum or plasma progesterone, to determine their suitability for use in in vitro fertilization programs. Both kits were suitably rapid for program requirements. Results by both were linear with concentration up to 60 nmol/L, and both had acceptable lower detection limits of 0.3 nmol/L. Kit-determined progesterone concentrations (y) for 100 patients' samples correlated well with results by our existing 3H radioimmunoassay method (y . 1.11x + 0.2, r . 0.965 for the DPC kit; y . 1.01x + 1.4, r . 0.974 for the RSL kit). Mean analytical recovery for the RSL kit was 116%, that for the DPC kit, 202%. Within-batch precision, expressed as the mean CV for three concentrations of progesterone, was 6.5% for the RSL kit, and 16.4% for the DPC kit; between-day CV was 8.1% for the RSL kit, 17.7% for the DPC kit. We conclude that the RSL kit provides a rapid, precise, and accurate assay for serum progesterone, suitable for use in a fertilization program, but do not recommend the DPC kit for either this purpose or the more general purpose of tracking menstrual cycles.

  9. The effects of cortisone acetate on stomach evacuation and the absorption of 125I-labelled globulins in young rats

    PubMed Central

    Morris, B.; Morris, R.

    1974-01-01

    1. After short exposure (3-5 hr) to I.P. cortisone acetate (5 mg), the reduced transmission of labelled globulin to the circulation in 14-day-old rats is due to the slow release of the oral dose from the stomach. The ability of the small intestine to absorb and transmit globulin to the circulation is comparable in control and experimental animals. 2. About 26 hr after cortisone acetate treatment (5 mg), the greatly reduced absorption of labelled globulin from oral doses administered to rats aged 15 days is due to the combined effects of the slower release of the dose from the stomach and to changes which have occurred in the small intestine. 3. About 50 hr after the administration of 5 mg cortisone acetate the effect on the rate of stomach evacuation is minimal in rats aged 16 days. When labelled globulin is introduced directly into the duodenum of these animals virtually no absorption occurs. 4. The results obtained from the experiments in which labelled globulin was injected into the duodenum support the contention that the proximal half of the small intestine is an important site for macromolecular transport. PMID:4854797

  10. (125)I labeling of clomiphene and biodistribution studies for possible use as a model in breast cancer imaging.

    PubMed

    Ibrahim, I T; El-Kolaly, M T; Aboumanei, M H; Abdelbary, A

    2016-09-01

    Clomiphene has growth-inhibitory effects of breast cancer cells, clomiphene was successfully labeled with (125)I via direct electrophilic substitution reaction with labeling yield 97%. It was obtained at optimum substrate amount of 0.5mg, Chloramine-T was used as an oxidizing agent at optimum amount of 25µg. Labeling reactions was done at pH 5 at ambient temperature. This study showed good in vitro and in vivo stability of the (125)I-clomiphene. The radiolabeled compound showed high ascetic fluid uptake of 18.12±0.27% at 30min post-injection. Solid tumor uptake of (125)I-clomiphene was 12.48±0.32% at 30min post-injection. This data revealed the localization of tracer in tumor tissue with high percent sufficient to use (125)I-clomiphene as a promising tool for the diagnosis of breast cancer. PMID:27337647

  11. Distribution of neurotensin/neuromedin N mRNA in rat forebrain: Unexpected abundance in hippocampus and subiculum

    SciTech Connect

    Alexander, M.J.; Miller, M.A.; Dorsa, D.M.; Bullock, B.P.; Helloni, R.H. Jr.; Dobner, P.R.; Leeman, S.E. )

    1989-07-01

    The authors have used in situ hybridization to determine the regional distribution of mRNA encoding the neurotensin/neuromedin N (NT/N) precursor in the forebrain of the adult male rat. Cells containing NT/N mRNA are widely distributed in the forebrain. These areas include the septum, bed nucleus of the stria terminalis, preoptic area, hypothalamus, amygdala, accumbens nucleus, caudate-putamen, and piriform and retrosplenial cortex. In general, the regional distribution of NT/N mRNA corresponds to the previously determined distribution of neurotensin-immunoreactive cell bodies; however, several notable exceptions were observed. The most striking difference occurs specifically in the CA1 region of the hippocampus, where intense labeling is associated with the pyramidal cell layer despite the reported absence of neurotensin-immunoreactive cells in this region. A second major discrepancy between NT/N mRNA abundance and neurotensin-immunoreactivity occurs in the intensely labeled subiculum, a region that contains only scattered neurotensin-immunoreactive cells in the adult. These results suggest that, in specific regions of the forebrain, NT/N precursor is processed to yield products other than neurotensin. In addition, these results provide an anatomical basis for studying the physiological regulation of NT/N mRNA levels in the forebrain.

  12. Saccharin and Cyclamate Inhibit Binding of Epidermal Growth Factor

    NASA Astrophysics Data System (ADS)

    Lee, L. S.

    1981-02-01

    The binding of 125I-labeled mouse epidermal growth factor (EGF) to 18 cell lines, including HeLa (human carcinoma), MDCK (dog kidney cells), HTC (rat hepatoma), K22 (rat liver), HF (human foreskin), GM17 (human skin fibroblasts), XP (human xeroderma pigmentosum fibroblasts), and 3T3-L1 (mouse fibroblasts), was inhibited by saccharin and cyclamate. The human cells were more sensitive to inhibition by these sweeteners than mouse or rat cells. EGF at doses far above the physiological levels reversed the inhibition in rodent cells but not in HeLa cells. In HeLa cells, the doses of saccharin and cyclamate needed for 50% inhibition were 3.5 and 9.3 mg/ml, respectively. Glucose, 2-deoxyglucose, sucrose, and xylitol did not inhibit EGF binding. Previous studies have shown that phorbol esters, strongly potent tumor promoters, also inhibit EGF binding to tissue culture cells. To explain the EGF binding inhibition by such greatly dissimilar molecules as phorbol esters, saccharin, and cyclamate, it is suggested that they operate through the activation of a hormone response control unit.

  13. Neurotensin analog NT77 induces regulated hypothermia in the rat.

    PubMed

    Gordon, Christopher J; McMahon, Beth; Richelson, Elliott; Padnos, Beth; Katz, Laurence

    2003-10-01

    The potential use of hypothermia as a therapeutic treatment for stroke and other pathological insults has prompted the search for drugs that can lower core temperature. Ideally, a drug is needed that reduces the set-point for control of core temperature (T(c)) and thereby induces a regulated reduction in T(c). To this end, a neurotensin analog (NT77) that crosses the blood brain barrier and induces hypothermia was assessed for its effects on the set-point for temperature regulation in the Sprague-Dawley rat by measuring behavioral and autonomic thermoregulatory responses. Following surgical implanation of radiotransmitters to monitor T(c), rats were placed in a temperature gradient and allowed to select from a range of ambient temperatures (T(a)) while T(c) was monitored by radiotelemetry. There was an abrupt decrease in selected T(a) from 29 to 16 degrees C and a concomitant reduction in T(c) from 37.4 to 34.0 degrees C 1 hr after IP injection of 5.0 mg/kg NT77. Selected T(a) and T(c) then recovered to control levels by 1.5 hr and 4 hr, respectively. Oxygen consumption (M) and heat loss (H) were measured in telemetered rats housed in a direct calorimeter maintained at a T(a) of 23.5 degrees C. Injection of NT77 initially led to a reduction in M, little change in H, and marked decrease in T(c). H initially rose but decreased around the time of the maximal decrease in T(c). Overall, NT77 appears to induce a regulated hypothermic response because the decrease in T(c) was preceded by a reduction in heat production, no change in heat loss, and preference for cold T(a)'s. Inducing a regulated hypothermic response with drugs such as NT77 may be an important therapy for ischemic disease and other insults. PMID:12967685

  14. Active sites of salivary proline-rich protein for binding to Porphyromonas gingivalis fimbriae.

    PubMed Central

    Kataoka, K; Amano, A; Kuboniwa, M; Horie, H; Nagata, H; Shizukuishi, S

    1997-01-01

    Porphyromonas gingivalis fimbriae specifically bind salivary acidic proline-rich protein 1 (PRP1) through protein-protein interactions. The binding domains of fimbrillin (a subunit of fimbriae) for PRP1 were analyzed previously (A. Amano, A. Sharma, J.-Y. Lee, H. T. Sojar, P. A. Raj, and R. J. Genco, Infect. Immun. 64:1631-1637, 1996). In this study, we investigated the sites of binding of the PRP1 molecules to the fimbriae. PRP1 (amino acid residues 1 to 150) was proteolysed to three fragments (residues 1 to 74 [fragment 1-74], 75 to 129, and 130 to 150). 125I-labeled fimbriae clearly bound fragments 75-129 and 130-150, immobilized on a polyvinylidene difluoride membrane; both fragments also inhibited whole-cell binding to PRP1-coated hydroxyapatite (HAP) beads by 50 and 83%, respectively. However, the N-terminal fragment failed to show any effect. Analogous peptides corresponding to residues 75 to 89, 90 to 106, 107 to 120, 121 to 129, and 130 to 150 of PRP1 were synthesized. The fimbriae significantly bound peptide 130-150, immobilized on 96-well plates, and the peptide also inhibited binding of 125I-labeled fimbriae to PRP1-coated HAP beads by almost 100%. Peptides 75-89, 90-106, and 121-129, immobilized on plates, showed considerable ability to bind fimbriae. For further analysis of active sites in residues 130 to 150, synthetic peptides corresponding to residues 130 to 137, 138 to 145, and 146 to 150 were prepared. Peptide 138-145 (GRPQGPPQ) inhibited fimbrial binding to PRP1-coated HAP beads by 97%. This amino acid sequence was shared in the alignment of residues 75 to 89, 90 to 106, and 107 to 120. Six synthetic peptides were prepared by serial deletions of individual residues from the N and C termini of peptide GRPQGPPQ. Peptide PQGPPQ was as inhibitory as peptide GRPQGPPQ. Further deletions of the dipeptide Pro-Gln from the N and C termini of peptide PQGPPQ resulted in significant loss of the inhibitory effect. These results strongly suggest that PQGPPQ

  15. Light-chain binding sites on renal brush-border membranes

    SciTech Connect

    Batuman, V.; Dreisbach, A.W.; Cyran, J.

    1990-05-01

    Immunoglobulin light chains are low-molecular-weight proteins filtered at the renal glomerulus and catabolized within the proximal tubular epithelium. Excessive production and urinary excretion of light chains are associated with renal dysfunction. They also interfere with proximal renal tubule epithelial functions in vitro. We studied the binding of 125I-labeled kappa- and lambda-light chains, obtained from the urine of multiple myeloma patients, to rat and human renal proximal tubular brush-border membranes. Light-chain binding to brush borders was also demonstrated immunologically by flow cytometry. Computer analysis of binding data was consistent with presence of a single class of low-affinity, high-capacity, non-cooperative binding sites with relative selectivity for light chains on both rat and human kidney brush-border membranes. The dissociation constants of light chains ranged from 1.6 X 10(-5) to 1.2 X 10(-4) M, and maximum binding capacity ranged from 4.7 +/- 1.3 X 10(-8) to 8.0 +/- 0.9 X 10(-8) (SD) mol/mg protein at 25 degrees C. Kappa- and lambda-light chains competed with each other for binding with comparable affinity constants. Competition by albumin and beta-lactoglobulin, however, was much weaker, suggesting relative site selectivity for light chains. These binding sites probably function as endocytotic receptors for light chains and possibly other low-molecular-weight proteins.

  16. Effects of heparin on insulin binding and biological activity

    SciTech Connect

    Kriauciunas, K.M.; Grigorescu, F.; Kahn, C.R.

    1987-02-01

    The effect of heparin, a polyanionic glycosaminoglycan known to alter the function of many proteins, on insulin binding and bioactivity was studied. Cultured human lymphocytes (IM-9) were incubated with varying concentrations of heparin, then extensively washed, and /sup 125/I-labeled insulin binding was measured. Heparin at concentrations used clinically for anticoagulation (1-50 U/ml) inhibited binding in a dose-dependent manner; 50% inhibition of binding occurred with 5-10 U/ml. Scatchard analysis indicated that the decrease in binding was due to a decrease in both the affinity and the apparent number of available insulin receptors. The effect occurred within 10 min at 22 degrees C and persisted even after the cells were extensively washed. Inhibition of insulin binding also occurred when cells were preincubated with heparinized plasma or heparinized serum but not when cells were incubated with normal serum or plasma from blood anticoagulated with EDTA. By contrast, other polyanions and polycations, e.g., poly-L-glutamic acid, poly-L-lysine, succinylated poly-L-lysine, and histone, did not inhibit binding. Heparin also inhibited insulin binding in Epstein-Barr (EB) virus-transformed lymphocytes but had no effect on insulin binding to isolated adipocytes, human erythrocytes, or intact hepatoma cells. When isolated adipocytes were incubated with heparin, there was a dose-dependent inhibition of insulin-stimulated glucose oxidation and, to a lesser extent, of basal glucose oxidation. Although heparin has no effect on insulin binding to intact hepatoma cells, heparin inhibited both insulin binding and insulin-stimulated autophosphorylation in receptors solubilized from these cells.

  17. Sex differences in neurotensin and substance P following nicotine self-administration in rats.

    PubMed

    Pittenger, Steven T; Swalve, Natashia; Chou, Shinnyi; Smith, Misty D; Hoonakker, Amanda J; Pudiak, Cindy M; Fleckenstein, Annette E; Hanson, Glen R; Bevins, Rick A

    2016-08-01

    Investigator-administered nicotine alters neurotensin and substance P levels in Sprague-Dawley rats. This finding suggested a role of the dopamine-related endogenous neuropeptides in nicotine addiction. We sought to extend this observation by determining the responses of neurotensin and substance P systems (assessed using radioimmunoassay) in male and female rats following nicotine self-administration (SA). Male and female Sprague-Dawley were trained to self-administer nicotine, or receive saline infusions yoked to a nicotine-administering rat during daily sessions (1-h; 21 days). Brains were extracted 3 h after the last SA session. Nicotine SA increased tissue levels of neurotensin in the males in the anterior and posterior caudate, globus pallidus, frontal cortex, nucleus accumbens core and shell, and ventral tegmental area. Nicotine SA also increased tissue levels of neurotensin in the females in the anterior caudate, globus pallidus, nucleus accumbens core and shell, but not in the posterior caudate, frontal cortex, or ventral tegmental area. There were fewer sex differences observed in the substance P systems. Nicotine SA increased tissue levels of substance P in both the males and females in the posterior caudate, globus pallidus, frontal cortex, nucleus accumbens shell, and ventral tegmental area. A sex difference was observed in the nucleus accumbens core, where nicotine SA increased tissue levels of substance P in the males, yet decreased levels in the females. The regulation of neuropeptides following nicotine SA may play a role in the susceptibility to nicotine dependence in females and males. Synapse 70:336-346, 2016. © 2016 Wiley Periodicals, Inc. PMID:27074301

  18. Pleiotropic effects of bombesin and neurotensin on intestinal mucosa: not just trefoil peptides.

    PubMed

    Assimakopoulos, Stelios-F; Scopa, Chrisoula-D; Nikolopoulou, Vassiliki-N; Vagianos, Constantine-E

    2008-06-14

    Bombesin and neurotensin are neuropeptides which exert a wide spectrum of biological actions on gastrointestinal tissues influencing intestinal growth and adaptation, intestinal motility, blood flow, secretion, nutrient absorption and immune response. Based mainly on their well-established potent enterotrophic effect, numerous experimental studies investigated their potential positive effect on the atrophic or injured intestinal mucosa. These peptides proved to be effective mucosa-healing factors, but the potential molecular and cellular mechanisms for this action remained unresolved. In a recently published study (World J Gastroenterol 2008; 14(8): 1222-1230), it was shown that their protective effect on the intestine in experimentally induced inflammatory bowel disease was related to anti-inflammatory, antioxidant and antiapoptotic actions. These results are in close agreement with our previous studies on jaundiced and hepatectomized rats that showed a regulatory effect of bombesin and neurotensin on critical cellular processes such as enterocyte' proliferation and death, oxidative stress and redox equilibrium, tight junctions' formation and function, and inflammatory response. The pleiotropic effects of bombesin and neurotensin on diverse types of intestinal injury may justify their consideration for clinical trials. PMID:18567096

  19. Association of Neurotensin receptor 1 gene polymorphisms with processing speed in healthy Chinese-Han subjects.

    PubMed

    Wang, Man; Ma, Hui; Huang, Ying-lin; Zhu, Gang; Zhao, Jing-ping

    2014-12-01

    Neurotensin modulates dopamine and serotonin transmission in the brain. The study investigated whether genetic polymorphisms in the Neurotensin receptor 1 gene were associated with performance on processing speed and executive function. A total of 129 healthy Chinese-Han volunteers were recruited. Genotyping for three SNPs, including rs6090453, rs6011914, and rs2427422, was analyzed by using a PCR and a restriction fragment length polymorphism analysis. Performances of processing speed and executive function were assessed by using Trail Making Test-A (TMT-A), Wisconsin Card Sorting Test, and Stroop Color-Word Test. We found significant differences in the outcomes of TMT-A score among rs6090453C/G (F(2,126)=4.405, P=0.014) and rs2427422A/G (F(2,126)=7.498, P=0.001) genotypes. Neurotensin receptor 1 SNP polymorphisms were significantly associated with the variance in processing speed performance in a sample of Chinese college students. PMID:25159184

  20. Autoradiographic demonstration of oxytocin-binding sites in the macula densa

    SciTech Connect

    Stoeckel, M.E.; Freund-Mercier, M.J. )

    1989-08-01

    Specific oxytocin (OT)-binding sites were localized in the rat kidney with use of a selective {sup 125}I-labeled OT antagonist ({sup 125}I-OTA). High concentrations of OT binding sites were detected on the juxtaglomerular apparatus with use of the conventional film autoradiographic technique. No labeling occurred on other renal structures. The cellular localization of the OT binding sites within the juxtaglomerular apparatus was studied in light microscope autoradiography, on semithin sections from paraformaldehyde-fixed kidney slices incubated in the presence of {sup 125}I-OTA. These preparations revealed selective labeling of the macula densa, mainly concentrated at the basal pole of the cells. Control experiments showed first that {sup 125}I-OTA binding characteristics were not noticeably altered by prior paraformaldehyde fixation of the kidneys and second that autoradiographic detection of the binding sites was not impaired by histological treatments following binding procedures. In view of the role of the macula densa in the tubuloglomerular feedback, the putative OT receptors of this structure might mediate the stimulatory effect of OT on glomerular filtration.

  1. Binding proteins for growth hormone and prolactin in rabbit kidney cytosol

    SciTech Connect

    Herington, A.C.; Stevenson, J.L.; Ymer, S.I. )

    1988-09-01

    Two soluble, receptor-like binding proteins with apparent somatotrophic (growth hormone (GH)) and lactogenic (prolactin (PRL)) specificities, respectively, and that are present in rabbit kidney cytosol have now been examined in more detail using specific GH receptor and PRL receptor monoclonal antibodies (MAb). Gel chromatography of {sup 125}I-labeled human GH ({sup 125}I-hGH) kidney cytosol complexes in the absence of these MAbs revealed two specifically bound regions of radioactivity at molecular weights (MW) of {approximately}120,000 and {approximately}60,000, which are similar in size to complexes formed by the native GH receptor of rabbit liver cytosol and the PRL receptor of mammary gland. Co-incubation with GH-receptor MAb inhibited {sup 125}I-hGH binding only to the higher MW (120,000) species, whereas the PRL-receptor MAb inhibited only the lower MW (60,000) species, thus establishing definitively the hormonal specificities of the two binding proteins. The presence of both GH- and PRL-specific binding subunits in cytosol was confirmed using covalent cross-linking techniques. No GH binding protein was detected in kidney membranes. The presence of naturally soluble, receptor-like binding proteins for GH and PRL in kidney cytosol preparations raises the possibility of their playing a role in the intracellular regulation of kidney function and/or metabolism.

  2. Temperature dependence of high-affinity CCK receptor binding and CCK internalization in rat pancreatic acini

    SciTech Connect

    Williams, J.A.; Bailey, A.C.; Roach, E. Univ. of California, San Francisco )

    1988-04-01

    {sup 125}I-labeled cholecystokinin (CCK) binding and internalization were studied as a function of temperatures in isolated rat pancreatic acini. At 37{degree}C, acini readily bound and degraded {sup 125}I-CCK. When labeled hormone binding was inhibited by increasing amounts of unlabeled CCK, competition-inhibition curves were biphasic, consistent with both high- (K{sub d}, 18 pM) and low-affinity (K{sub d}, 13 nM) binding sites. At 4{degree}C, acini bound only one-third as much {sup 125}I-CCK and degradation was essentially abolished. At 4{degree}C, CCK competition curves were consistent with a single class of low-affinity binding sites (K{sub d}, 19 nM). Internalization of {sup 125}I-CCK was evaluated by three washing procedures utilizing acid, base, and trypsin. All were shown to remove membrane-bound {sup 125}I-CCK, and this finding was validated for trypsin by electron microscope autotradiography. When internalization of {sup 125}I-CCK was evaluated as a function of the medium concentration of CCK, both high- and low-affinity components were observed. These results suggest that high-affinity CCK binding and CCK internalization are separate temperature-sensitive processes. Moreover, internalization is not uniquely associated with high-affinity binding.

  3. Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin

    SciTech Connect

    Fuchs, G.; Mobassaleh, M.; Donohue-Rolfe, A.; Montgomery, R.K.; Grand, R.J.; Keusch, G.T.

    1986-08-01

    This study examined the binding of purified /sup 125/I-labeled shigella toxin to rabbit jejunal microvillus membranes (MVMs). Toxin binding was concentration dependent, saturable, reversible, and specifically inhibited by unlabeled toxin. The calculated number of toxin molecules bound at 4/sup 0/C was 7.9 X 10(10) (3 X 10(10) to 2 X 10(11))/micrograms of MVM protein or 1.2 X 10(6) per enterocyte. Scatchard analysis showed the binding site to be of a single class with an equilibrium association constant, K, of 4.7 X 10(9) M-1 at 4/sup 0/C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 4/sup 0/C dissociated from MVM when the temperature was raised to 37/sup 0/C, but reassociated when the temperature was again brought to 4/sup 0/C. There was no structural or functional change of MVM due to toxin as monitored by electron microscopy or assay of MVM sucrase activity. These studies demonstrate a specific binding site for shigella toxin on rabbit MVMs. The physiological relevance of this receptor remains to be determined.

  4. Atrial natriuretic factor binding sites in experimental congestive heart failure

    SciTech Connect

    Bianchi, C.; Thibault, G.; Wrobel-Konrad, E.; De Lean, A.; Genest, J.; Cantin, M. )

    1989-10-01

    A quantitative in vitro autoradiographic study was performed on the aorta, renal glomeruli, and adrenal cortex of cardiomyopathic hamsters in various stages of heart failure and correlated, in some instances, with in vivo autoradiography. The results indicate virtually no correlation between the degree of congestive heart failure and the density of 125I-labeled atrial natriuretic factor ((Ser99, Tyr126)ANF) binding sites (Bmax) in the tissues examined. Whereas the Bmax was increased in the thoracic aorta in moderate and severe heart failure, there were no significant changes in the zona glomerulosa. The renal glomeruli Bmax was lower in mild and moderate heart failure compared with control and severe heart failure. The proportion of ANF B- and C-receptors was also evaluated in sections of the aorta, adrenal, and kidney of control and cardiomyopathic hamsters with severe heart failure. (Arg102, Cys121)ANF (des-(Gln113, Ser114, Gly115, Leu116, Gly117) NH2) (C-ANF) at 10(-6) M displaced approximately 505 of (Ser99, Tyr126)125I-ANF bound in the aorta and renal glomeruli and approximately 20% in the adrenal zona glomerulosa in both series of animals. These results suggest that ANF may exert a buffering effect on the vasoconstriction of heart failure and to a certain extent may inhibit aldosterone secretion. The impairment of renal sodium excretion does not appear to be related to glomerular ANF binding sites at any stage of the disease.

  5. The Internalization of Neurotensin by the Low-Affinity Neurotensin Receptors (NTSR2 and vNTSR2) Activates ERK 1/2 in Glioma Cells and Allows Neurotensin-Polyplex Transfection of tGAS1.

    PubMed

    Ayala-Sarmiento, Alberto E; Martinez-Fong, Daniel; Segovia, José

    2015-08-01

    Glioblastoma is the most malignant primary brain tumor and is very resistant to treatment; hence, it has a poor prognosis. Neurotensin receptor type 1 (NTSR1) plays a key role in cancer malignancy and has potential therapeutic applications. However, the presence and function of neurotensin (NTS) receptors in glioblastoma is not clearly established. RT-PCR assays showed that healthy (non-tumor) astroglial cells and C6 glioma cells express NTSR2 and its isoform (vNTSR2) rather than NTSR1. In glioma cells, NTS promotes the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK 1/2), an effect that was completely abolished by blocking the internalization of the NTS/NTSR complex. We demonstrated pharmacologically that the internalization is dependent on the activation of NTSR2 receptors and it was prevented by levocabastine, a NTSR2 receptor antagonist. The internalization of NTSR2 and vNTSR2 was further demonstrated by its ability to mediate gene transfer (transfection) via the NTS-polyplex system. Expression of reporter transgenes and of the pro-apoptotic soluble form of growth arrest specific 1 (tGAS1) was observed in glioma cells. A significant reduction on the viability of C6 cells was determined when tGAS1 was transfected into glioma cells. Conversely, astroglial cells could neither internalize NTS nor activate ERK 1/2 and could not be transfected by the NTS-polyplex. These results demonstrate that the internalization process of NTSR2 receptors is a key regulator necessary to trigger the activation of the ERK 1/2. Our data support a new internalization pathway in glioma C6 cells that involve NTSR2/vNTSR2, which can be used to selectively transfer therapeutic genes using the NTS-polyplex system. PMID:25772140

  6. Platelet-collagen adhesion enhances platelet aggregation induced by binding of VWF to platelets

    SciTech Connect

    Laduca, F.M.; Bell, W.R.; Bettigole, R.E. State Univ. of New York, Buffalo )

    1987-11-01

    Ristocetin-induced platelet aggregation (RIPA) was evaluated in the presence of platelet-collagen adhesion. RIPA of normal donor platelet-rich plasma (PRP) demonstrated a primary wave of aggregation mediated by the binding of von Willebrand factor (VWF) to platelets and a secondary aggregation wave, due to a platelet-release reaction, initiated by VWF-platelet binding and inhibitable by acetylsalicylic acid (ASA). An enhanced RIPA was observed in PRP samples to which collagen had been previously added. These subthreshold concentrations of collagen, which by themselves were insufficient to induce aggregation, caused measurable platelet-collagen adhesion. Subthreshold collagen did not cause microplatelet aggregation, platelet release of ({sup 3}H)serotonin, or alter the dose-responsive binding of {sup 125}I-labeled VWF to platelets, which occurred with increasing ristocetin concentrations. However, ASA inhibition of the platelet release reaction prevented collagen-enhanced RIPA. These results demonstrate that platelet-collagen adhesion altered the platelet-release reaction induced by the binding of VWF to platelets causing a platelet-release reaction at a level of VWF-platelet binding not normally initiating a secondary aggregation. These findings suggest that platelet-collagen adhesion enhances platelet function mediated by VWF.

  7. Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding sites on the beta-chain of human haemoglobin by synthetic overlapping peptides encompassing the entire chain.

    PubMed

    Yoshioka, N; Atassi, M Z

    1986-03-01

    A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146. PMID:3718478

  8. Mono(125I)iodo-Tyr10,MetO17)-vasoactive intestinal polypeptide. Preparation, characterization, and use for radioimmunoassay and receptor binding

    SciTech Connect

    Martin, J.L.; Rose, K.; Hughes, G.J.; Magistretti, P.J.

    1986-04-25

    Vasoactive intestinal polypeptide (VIP) was labeled with sodium (125I)iodide using the chloramine-T method and subsequently purified by reverse-phase high performance liquid chromatography.Three main 125I-labeled peaks designated A, B, and C resulted from the radioiodination and purification procedures. They were characterized by electrophoresis of tryptic fragments; Edman degradation (for Peaks A and C); enzymatic digestion to amino acids by leucine aminopeptidase, carboxypeptidase Y and Pronase; and treatment with cyanogen bromide. Peak A corresponds to VIP monoiodinated on Tyr10 and with the Met17 residue oxidized to methionine sulfoxide. This (mono(125I)iodo-Tyr10,MetO17)VIP displays the following characteristics. 1) It constitutes quantitatively the major product of the iodination procedure (62.5%); 2) it is well resolved from other labeled and unlabeled products; 3) it is stable (2 months at -20 degrees C); 4) it possesses a high specific activity (2050 Ci/mmol); 5) it maintains the biological activity of native VIP; and 6) it binds to antibody and membrane recognition sites in a specific, saturable, and reversible manner. Reduction of (mono(125I)iodo-Tyr10, Met-O17)VIP to (mono(125I)iodo-Tyr10)VIP does not improve the performance of the tracer in a radioimmunoassay. The method described in this article is simple and rapid and yields a molecular form of 125I-labeled VIP that has been fully characterized and is suitable for use in biological studies.

  9. Identification of the endothelial cell binding site for factor IX.

    PubMed Central

    Cheung, W F; van den Born, J; Kühn, K; Kjellén, L; Hudson, B G; Stafford, D W

    1996-01-01

    We previously demonstrated that the primary region of factor IX and IXa responsible for saturable specific binding to bovine aortic endothelial cells resides in residues 3-11 at the amino terminus of factor IX. We also demonstrated that mutations of lysine to alanine at residue 5, factor IX K5A, or valine to lysine at residue 10, factor IX V10K, resulted in a molecule unable to bind to endothelial cells. Moreover, a mutation with lysine to arginine at residue 5, factor IX K5R, resulted in a factor IX molecule with increased affinity for the endothelial cell binding site. In this paper we report that collagen IV is a strong candidate for the factor IX binding site on endothelial cells. Factor IX and factor IX K5R compete with 125I-labeled factor IX for binding to tetrameric collagen IV immobilized on microtiter plates, while factor X, factor VII, and factor IX K5A or V10K fail to compete. The Kd for wild-type factor IX binding to collagen IV in the presence of heparin was 6.8 +/- 2 nM, and the Kd for factor IX K5R was 1.1 +/- 0.2 nM, which agrees well with our previously published Kd values of 7.4 and 2.4 nM for binding of the same proteins to endothelial cells. Our working assumption is that we have identified the endothelial cell binding site and that it is collagen IV. Its physiological relevance remains to be determined. PMID:8855310

  10. Neurotensin Is Coexpressed, Coreleased, and Acts Together With GLP-1 and PYY in Enteroendocrine Control of Metabolism.

    PubMed

    Grunddal, Kaare V; Ratner, Cecilia F; Svendsen, Berit; Sommer, Felix; Engelstoft, Maja S; Madsen, Andreas N; Pedersen, Jens; Nøhr, Mark K; Egerod, Kristoffer L; Nawrocki, Andrea R; Kowalski, Timothy; Howard, Andrew D; Poulsen, Steen Seier; Offermanns, Stefan; Bäckhed, Fredrik; Holst, Jens J; Holst, Birgitte; Schwartz, Thue W

    2016-01-01

    The 2 gut hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are well known to be coexpressed, costored, and released together to coact in the control of key metabolic target organs. However, recently, it became clear that several other gut hormones can be coexpressed in the intestinal-specific lineage of enteroendocrine cells. Here, we focus on the anatomical and functional consequences of the coexpression of neurotensin with GLP-1 and PYY in the distal small intestine. Fluorescence-activated cell sorting analysis, laser capture, and triple staining demonstrated that GLP-1 cells in the crypts become increasingly multihormonal, ie, coexpressing PYY and neurotensin as they move up the villus. Proglucagon promoter and pertussis toxin receptor-driven cell ablation and reappearance studies indicated that although all the cells die, the GLP-1 cells reappear more quickly than PYY- and neurotensin-positive cells. High-resolution confocal fluorescence microscopy demonstrated that neurotensin is stored in secretory granules distinct from GLP-1 and PYY storing granules. Nevertheless, the 3 peptides were cosecreted from both perfused small intestines and colonic crypt cultures in response to a series of metabolite, neuropeptide, and hormonal stimuli. Importantly, neurotensin acts synergistically, ie, more than additively together with GLP-1 and PYY to decrease palatable food intake and inhibit gastric emptying, but affects glucose homeostasis in a more complex manner. Thus, neurotensin is a major gut hormone deeply integrated with GLP-1 and PYY, which should be taken into account when exploiting the enteroendocrine regulation of metabolism pharmacologically. PMID:26469136

  11. Selective uptake of cholesteryl esters of low-density lipoproteins is mediated by the lipoprotein-binding site in HepG2 cells and is followed by the hydrolysis of cholesteryl esters.

    PubMed Central

    Brissette, L; Charest, M C; Falstrault, L

    1996-01-01

    The study described in this paper shows that 125I-labelled low-density lipoproteins (LDL) interact with high- and low-affinity binding sites on human hepatoma (HepG2) cells. The former site is the LDL receptor and the latter is the lipoprotein-binding site (LBS). The association of 125I-labelled LDL and [3H]cholesteryl ethers-LDL with HepG2 cells revealed a 4-fold selective uptake of cholesteryl esters (CE) in a 4 h incubation period, which correlated with the depletion of CE mass in LDL. This selective uptake was not observed when the cells were incubated in the presence of a 100-fold excess of high-density lipoprotein 3, conditions where only the LDL receptor is being monitored. Also, no reduction in uptake was observed in the presence of IgG-C7, an anti-(LDL receptor) monoclonal antibody. Both findings indicate that the selective uptake occurs through the LBS and that the LBS contributes more to the entry of CE from LDL into the cell than does the LDL receptor. The fates of CE entering the cell via the LDL receptor and the LBS were also followed. To achieve this, LDL were labelled with [3H]cholesteryl oleate and the hydrolysis of [3H]cholesteryl oleate was monitored. The results indicated that 45% of the CE were hydrolysed after a 4 h incubation period, irrespective of the site of entry. Chloroquine (100 microM) was shown to inhibit hydrolysis, indicating that lysosomal enzymes were responsible for the hydrolysis of LDL-CE, whichever pathway was used. Thus our results reveal, for the first time, that the mass of CE entering the cell via the LBS is substantial and that hydrolysis of CE is by lysosomal enzyme activity. Overall, this suggests that the LBS has significant physiological importance. PMID:8836127

  12. Study of follitropin receptors in testis using a homologous system. Binding of porcine follitropin to plasma membranes from immature porcine testis and correlation with adenylate cyclase stimulation.

    PubMed

    Maghuin-Rogister, G; Closset, J; Combarnous, Y; Hennen, G; Dechenne, C; Ketelslegers, J M

    1978-05-01

    The properties of follitropin receptors in immature porcine testis were determined using highly purified porcine follitropin. 1. The characteristics of follitropin binding to a subcellular fraction rich in plasma membranes were studied using a 125I-labelled follitropin with high specific activity (75-100 Ci/g) and high binding activity. The binding is dependent on time, temperature and pH. It is specific to follitropin as demonstrated by the very low binding activity of the follitropin alpha and beta subunits and of the other glycoprotein hormones. Scatchard analysis of binding data indicated an equilibrium association constant of 2 x 10(10) M-1 and a concentration of high affinity binding sites of 500 fmol/mg membrane proteins. 2. A sensitive radio-ligand receptor assay was developed. Fifty percent inhibition of binding was obtained with as little as 2 ng of porcine follitropin. Ovine and bovine follitropins and pregnant mare serum gonadotropin gave binding inhibition curves parallel to that given by porcine follitropin. With equine and human follitropin, significantly different slopes were recorded. 3. Kinetics of dissociation of labelled follitropin from its testis receptors showed the presence of at least two compartments with fast and slow dissociation rate constants. The ratio between the sizes of the slow and fast compartments appeared dependent upon preincubation time. 4. A temporal correlation was observed between binding of follitropin to testis receptors and activation of membrane bound adenylate cyclase. PMID:207514

  13. Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding

    NASA Technical Reports Server (NTRS)

    Zhang, J. Z.; Wu, Y.; Williams, B. Y.; Rodney, G.; Mandel, F.; Strasburg, G. M.; Hamilton, S. L.

    1999-01-01

    This study presents evidence for a close relationship between the oxidation state of the skeletal muscle Ca2+ release channel (RyR1) and its ability to bind calmodulin (CaM). CaM enhances the activity of RyR1 in low Ca2+ and inhibits its activity in high Ca2+. Oxidation, which activates the channel, blocks the binding of 125I-labeled CaM at both micromolar and nanomolar Ca2+ concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation of hyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1 with N-ethylmaleimide completely blocks oxidant-induced intersubunit cross-linking and inhibits Ca2+-free 125I-CaM but not Ca2+/125I-CaM binding. These studies suggest that 1) the sites on RyR1 for binding apocalmodulin have features distinct from those of the Ca2+/CaM site, 2) oxidation may alter the activity of RyR1 in part by altering its interaction with CaM, and 3) CaM may protect RyR1 from oxidative modifications during periods of oxidative stress.

  14. Staphylococcal enterotoxin induced mitogenesis: toxin binding and cell-cell interactions.

    PubMed

    Buxser, E S; Bonventre, P F; Archer, D L

    1983-07-01

    The binding characteristics of 125I-labelled staphylococcal enterotoxin A (125I-SEA), a T-cell mitogen, to murine lymphoid cell subpopulations were analyzed. Both T- and B-lymphocytes from murine spleens possess specific binding sites for SEA, as do T-lymphocytes from thymus. B-lymphocytes appear to have a greater capacity for binding of 125-SEA than do T-lymphocytes from either thymus or spleen. Enterotoxin did not specifically bind to thioglycollate-induced peritoneal exudate cells (PECs), used as a source of macrophages. Adherent PECs however, incorporated 125-ISEA by fluid phase endocytosis. When exposed to SEA and thoroughly washed, macrophages stimulate lymphocyte mitogenesis in spleen or thymus cell cultures not directly exposed to toxin. Maximum mitogenic stimulation took place only when both PECs and lymphocytes were exposed to SEA. The presence of splenic B-lymphocytes enhanced the mitogenic response of thymus derived T-cells to SEA. Thus, B-lymphocytes appear to contribute to SEA mitogenesis. These data suggest that mitogenic stimulation and possibly other immunological phenomena associated with SEA occur as a result of complex interactions between cellular components of the immune system. PMID:6605472

  15. Identification by cross-linking of a beta-bungarotoxin binding polypeptide in chick brain membranes.

    PubMed Central

    Rehm, H; Betz, H

    1983-01-01

    beta-Bungarotoxin (beta-BTX) is a snake venom neurotoxin which inhibits neurotransmitter release from different types of nerve terminals. To identify presynaptic membrane components potentially important in neurosecretion, 125I-labeled beta-BTX (mol. wt. 21 000) was cross-linked to a high-affinity binding site in synaptic membrane fractions of chick brain using the photoactivable cross-linker N-succinimidyl-6(4'-azido-2'-nitrophenylamino)-hexanoate. Electrophoretic analysis of the cross-linked membrane proteins under both reducing and non-reducing conditions revealed a single [125I]beta-BTX-polypeptide adduct of apparent mol. wt. 116 000 (+/- 2000). The labeling of this band was prevented under conditions previously shown to inhibit the binding of [125I]beta-BTX to its high-affinity binding site. It is concluded that the cross-linking procedure identified a polypeptide of the presynaptic binding site for beta-BTX, and that this polypeptide has a mol. wt. of 95 000. Images Fig. 1. Fig. 2. Fig. 3. PMID:6628364

  16. Specific binding of acrosome-reaction-inducing substance to the head of starfish spermatozoa.

    PubMed

    Ushiyama, A; Araki, T; Chiba, K; Hoshi, M

    1993-05-01

    In the starfish, spermatozoa undergo the acrosome reaction upon encountering the jelly coat of eggs. A highly sulphated glycoprotein in the jelly coat is called acrosome-reaction-inducing substance (ARIS) because it is the key signal molecule to trigger the acrosome reaction. The activity of ARIS is mainly attributed to its sulphate and saccharide residues. The extremely large molecular size and species-specific action of ARIS suggest the presence of a specific ARIS receptor on the sperm surface, but no experimental evidence for the receptor has been presented. We therefore measured specific binding of ARIS and its pronase digest (P-ARIS), which retains the full activity of ARIS, to homologous spermatozoa by using fluorescein-isothiocyanate-labelled ARIS and 125I-labelled P-ARIS, respectively. The spermatozoa had the ability to bind ARIS, as well as P-ARIS, specifically. The binding was species-specific and mostly localised to the head region of spermatozoa. Scatchard plot analysis indicated the presence of one class of ARIS receptor on the surface of acrosome-intact spermatozoa. Furthermore, the specific binding of P-ARIS to the anterior region of sperm heads was microscopically confirmed by using P-ARIS conjugated to polystyrene latex beads with intense fluorescence. It is concluded that starfish spermatozoa have a specific receptor for ARIS on the surface of the anterior region of heads. PMID:8081808

  17. Enhancement of in vivo binding of [123I]beta-CIT by MK-801 in rat brain.

    PubMed

    Nakano, T; Takatoku, K; Matoba, Y; Iwamoto, B; Nishiura, M; Inoue, O; Nishimura, T

    1998-12-01

    The effects of MK-801, a noncompetitive NMDA receptor antagonist, on in vivo and in vitro binding of radioactive iodine ([123I] or [125I]) labeled beta-CIT [RTI-55, 3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester] were investigated in rat brain. In the in vitro binding study, 10 pM of [125I]beta-CIT was incubated with either 0.03 microM or 3 microM of MK-801 at 24 degrees C for 60 min. In vitro, no alterations in [125I]beta-CIT binding in any region of rat brain slices were detected after addition of MK-801. In the in vivo binding study, [123I]beta-CIT was intravenously injected into rats 30 min after intraperitoneal injection of 0.03-1 mg/kg of MK-801. The in vivo [123I]beta-CIT binding in the striatum, frontal cortex, occipital cortex, hypothalamus, and thalamus was significantly increased by pretreatment with 1 mg/kg of MK-801. Kinetic analysis using the cerebellum as a reference region revealed that the increases in in vivo [123I]beta-CIT binding induced by MK-801 were mainly due to increases in both input rate constant k3 and output rate constant k4. The results of this study indicate that the glutamatergic system, including NMDA receptor, plays an important role in regulating neurotransmission in the dopaminergic or serotonergic systems in intact brain. PMID:9826232

  18. Interaction of the two components of leukocidin from Staphylococcus aureus with human polymorphonuclear leukocyte membranes: sequential binding and subsequent activation.

    PubMed Central

    Colin, D A; Mazurier, I; Sire, S; Finck-Barbançon, V

    1994-01-01

    The sequential interaction between the two components S and F of leukocidin from Staphylococcus aureus and the membrane of human polymorphonuclear neutrophils has been investigated in the presence of 1 mM Ca2+. With 125I-labeled components, it has been shown that binding of the F component occurred only after binding of the S component. The kinetic constants of binding of both components were not statistically different (Kd, approximately 5 nM; Bm, approximately 35,000 molecules per cell), and both Hill coefficients were 1. The application of increasing concentrations of leukocidin provoked a dose-dependent secretion of the granule content, as determined by hexosaminidase and lysozyme activity measurements. Furthermore, the separate perfusion of S and F components on human polymorphonuclear neutrophils deposited on a filter induced secretion of the granules content only when the perfusion of the S component preceded that of the F component. We conclude, therefore, that (i) S-component binding is a prerequisite for F-component binding and for subsequent activation of polymorphonuclear neutrophils and (ii) there is a specific binding site for the S component in the plasma membrane. PMID:8039887

  19. The integrin alpha IIb beta 3 contains distinct and interacting binding sites for snake-venom RGD (Arg-Gly-Asp) proteins. Evidence that the receptor-binding characteristics of snake-venom RGD proteins are related to the amino acid environment flanking the sequence RGD.

    PubMed Central

    Rahman, S; Lu, X; Kakkar, V V; Authi, K S

    1995-01-01

    We have previously demonstrated [Lu, Williams, Deadman, Salmon, Kakkar, Wilkinson, Baruch, Authi and Rahman (1994) Biochem. J. 304, 929-936] the preferential antagonism of the interactions of the integrin alpha IIb beta 3 on activated platelets with three immobilized glycoprotein ligands (fibrinogen, fibronectin and von Willebrand factor) by a selected panel of snake-venom RGD (Arg-Gly-Asp)-containing proteins including the disintegrins kistrin and elegantin, and the neurotoxin variant dendroaspin. Kistrin and dendroaspin, although structurally unrelated, contain similar amino acids flanking the tripeptide RGD and behaved as identical antagonists preferentially inhibiting platelet adhesion to immobilized fibrinogen as opposed to fibronectin. In contrast, elegantin, which shares extensive sequence similarity with kistrin but has different amino acids around the tripeptide RGD, preferentially inhibited platelet adhesion to immobilized fibronectin as opposed to fibrinogen. To develop further insights into the mechanisms underlying the preferential antagonism shown by the venom proteins in the adhesion studies, we, in the present study, sought to determine the binding properties of kistrin, elegantin and dendroaspin to the alpha IIb beta 3 complex by radioligand kinetic and competition studies. In direct binding experiments, both kistrin and dendroaspin were observed to bind to a single class of binding site on ADP-activated platelets with apparent equilibrium dissociation constant (Kdapp) values of 42 +/- 2 nM and 21 +/- 6 nM respectively. In competition studies, dendroaspin blocked the binding of 125I-labelled kistrin to ADP-activated platelets in a simple competitive manner, with an apparent equilibrium inhibition constant (Kiapp) of 143 +/- 14 nM, from which an indirect Kdapp = 22 nM for dendroaspin was determined. This result suggests that kistrin and dendroaspin bind to the same site on the integrin alpha IIb beta 3 consistent with their similar inhibitory

  20. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    SciTech Connect

    Daughaday, W.H.; Trivedi, B.

    1987-07-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of /sup 125/I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound /sup 125/I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.

  1. Effects of the neurotensin NTS1 receptor agonist PD149163 on visual signal detection in rats

    PubMed Central

    Hillhouse, Todd M.; Prus, Adam J.

    2013-01-01

    Antipsychotic drugs provide limited efficacy for cognitive impairment in schizophrenia. Recent studies have found that the neurotensin NTS1 receptor agonist and putative atypical antipsychotic drug PD149163 reverses deficits in sensory-gating and novel object recognition, suggesting that this compound may have the potential to improve cognitive functioning in schizophrenia. The present study sought to extend these investigations by evaluating the effects of PD149163 on sustained attention using a visual signal detection operant task in rats. PD149163, the atypical antipsychotic drug clozapine, and the dopamine D2/3 receptor antagonist raclopride all significantly decreased percent “hit” accuracy, while none of these compounds altered “correct rejections” (compared to vehicle control). Clozapine and raclopride significantly increased response latency, while high doses of PD149163 and raclopride significantly increased trial omissions. Nicotine, which was tested as a positive control, significantly improved overall performance in this task and did not affect response latency or trial omissions. The present findings suggest that neurotensin NTS1 receptor agonists, like antipsychotic drugs, may inhibit sustained attention in this task despite having different pharmacological mechanisms of action. PMID:24076181

  2. Neurotensin-produced antinociception in the rostral ventromedial medulla is partially mediated by spinal cord norepinephrine

    PubMed Central

    Buhler, A. V.; Proudfit, H. K.; Gebhart, G. F.

    2008-01-01

    Microinjection of neurotensin (NT) into the rostral ventromedial medulla (RVM) produces dose-dependent antinociception. Here we show that antinociception produced by intra RVM microinjection of neurotensin (NT) or the selective NT receptor subtype 1 (NTR1) agonist PD149163 can be partially blocked by intrathecal (i.t.) yohimbine, an α2-adrenoceptor antagonist and by methysergide, a serotonin receptor antagonist. Antinociception produced by the NTR2 agonist beta-lactotensin (β-LT) is blocked by intrathecal (i.t.) yohimbine, but not by methysergide i.t.. It is not known which noradrenergic cell group is involved in this newly identified noradrenergic component of NTR-mediated antinociception. These experiments provide the first evidence that selective activation of NTR2 in the RVM produces antinociception. These results also provide evidence that activation of NTR1 in the RVM produces antinociception through spinal release of norepinephrine (NE) and serotonin, and that activation of NTR2 in the RVM produces antinociception mediated by spinal release of NE. PMID:17664042

  3. AB007. Neurotensin derived from cancer stroma contributes to castration resistance via promoting neuroendocrine transdifferentiation

    PubMed Central

    Zhu, Shimiao; Shang, Zhiqun; Tian, Hao; Flores-Morales, Amilcar; Niu, Yuanjie

    2016-01-01

    The mechanism of neuroendocrine transdifferentiation (NED) during the development of castration-resistant prostate cancer (CRPC) remains undefined. Although androgen-deprivation therapy (ADT) can impair tumor cell growth, ADT can also triggers a parallel reaction, leading to increased neurotensin (NTS) production in cancer associated stromal cells which drives NED. Here, we systematically explore the NTS network in tumor microenvironment that drives NED following ADT. The CK8+/CK14+ intermediate cells, as opposed to other epithelial cells, can be transdifferentiated to neuroendocrine (NE) status by excessive NTS through simultaneous activation of neurotensin receptor 1 (NTSR1)-PRKACB and 3 (NTSR3)-AHNAK axes. The importance of PRKACB and AHNAK in NED development was then confirmed in human prostate tumor tissues. More importantly, we demonstrated SR48692 (an inhibitor of NTSR1) could inhibit NED and prevent castration resistance in prostate tumor from xenografts and TRAMP models. We propose that targeting this pathway could provide benefit for patients with tumors expressing high levels of NTS following ADT.

  4. The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

    PubMed Central

    Young, S P; Bomford, A; Williams, R

    1984-01-01

    Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin. PMID:6743230

  5. Solid-phase receptor binding assay for /sup 125/I-hCG

    SciTech Connect

    Bortolussi, M.; Selmin, O.; Colombatti, A.

    1987-01-01

    A solid-phase radioligand-receptor assay (RRA) to measure the binding of /sup 125/I-labelled human chorionic gonadotropin (/sup 125/I-hCG) to target cell membranes has been developed. The binding of /sup 125/I-hCG to membranes immobilized on the wells of microtitration plates reached a maximum at about 3 hours at 37 degrees C, was saturable, displayed a high affinity (Ka = 2.4 X 10(9) M-1) and was specifically inhibited by unlabelled hCG. In comparison with RRAs carried out with membranes in suspension, the solid-phase RRA is significantly simpler and much faster to perform as it avoids centrifugation or filtration procedures. The solid-phase RRA was adapted profitably to process large numbers of samples at the same time. It proved particularly useful as a screening assay to detect anti-hCG monoclonal antibodies with high inhibitory activity for binding of /sup 125/I-hCG to its receptors.

  6. Removal of 14 N-terminal amino acids of lactoferrin enhances its affinity for parenchymal liver cells and potentiates the inhibition of beta- very low density lipoprotein binding.

    PubMed

    Ziere, G J; Bijsterbosch, M K; van Berkel, T J

    1993-12-25

    Lactoferrin inhibits the hepatic uptake of lipoprotein remnants, and we showed earlier that arginine residues of lactoferrin are involved. In this study, lactoferrin was treated with aminopeptidase M (APM), which resulted in removal of 14 N-terminal amino acids, including 4 clustered arginine residues at positions 2-5 (APM-lactoferrin). After intravenous injection into rats, 125I-labeled APM-lactoferrin was cleared within 10 min by the liver parenchymal cells (74.7% of the dose). In contrast to native lactoferrin, APM-lactoferrin was rapidly internalized after liver association (> 80% of the liver-associated radioactivity was internalized within 10 min). Binding of APM-lactoferrin to isolated parenchymal liver cells was saturable with a Kd of 186 nM (750,000 sites/cell). This is in striking contrast to the binding of native lactoferrin (Kd 10 microM; 20 x 10(6) sites/cell). Preinjection of rats with 20 mg of APM-lactoferrin/kg of body weight reduced the liver association of beta-very low density lipoprotein (beta-VLDL) by 50%, whereas lactoferrin had no effect at this dose. With isolated parenchymal liver cells, APM-lactoferrin was a more effective competitor for beta-VLDL binding than native lactoferrin (50% inhibition at 0.5 mg/ml versus 8.0 mg/ml). Selective modification of the arginines of APM-lactoferrin with 1,2-cyclohexanedione reduced the liver association by approximately 60% and abolished the capacity of APM-lactoferrin to inhibit the binding of 125I-labeled beta-VLDL in vitro. In conclusion, our data indicate that the four-arginine cluster of lactoferrin at positions 2-5 is involved in its massive, low affinity association of lactoferrin with the liver, possibly to proteoglycans, but is not essential for the inhibition of lipoprotein remnant uptake. The Arg-Lys sequence at positions 25-31, which resembles the binding site of apolipoprotein E, may mediate the high affinity binding of lactoferrin and block the binding of beta-VLDL to the remnant receptor

  7. Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species

    PubMed Central

    Caccia, Silvia; Hernández-Rodríguez, Carmen Sara; Mahon, Rod J.; Downes, Sharon; James, William; Bautsoens, Nadine; Van Rie, Jeroen; Ferré, Juan

    2010-01-01

    Background Evolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac. Methodology/Principal Findings Bioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with 125I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in 125I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins. Conclusion/Significance This is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same

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

    SciTech Connect

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

    1987-07-28

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

  9. Monoclonal antibody (H107) inhibiting IgE binding to Fc epsilon R(+) human lymphocytes.

    PubMed

    Noro, N; Yoshioka, A; Adachi, M; Yasuda, K; Masuda, T; Yodoi, J

    1986-08-15

    A hybridoma-producing monoclonal antibody blocking the binding of human IgE to lymphocytes Fc receptor (Fc epsilon R) was established by the fusion of murine myeloma cells. P3X63.653.Ag8, with BALB/c spleen cells immunized with Fc epsilon R(+) human B lymphoblastoid cell line cells, RPMI1788. A clone of the hybridoma (H107) produced a monoclonal IgG2b antibody that inhibited the rosette formation of Fc epsilon R(+) human B lymphoblastoid cell line cells (RPMI1788, RPMI8866, CESS, Dakiki, and IM9) with fixed ox red blood cells (ORBC) conjugated with human IgE (IgE-ORBC). In contrast, the rosette formation with IgG-conjugated ORBC (IgG-ORBC) on Fc gamma R(+), Fc epsilon R(-) Daudi cells were not affected by the H107 antibodies. A close association of Fc epsilon R and the antigenic determinant recognized by H107 antibody was suggested by the following results. First, the bindings of 125I-labeled IgE (125I-IgE) or 125I-labeled H107 IgG2b antibody (125I-H107) to RPMI8866 cells were inhibited by cold human IgE and H107 IgG2b but not by other classes of human Ig (IgA and IgG), MPC11 IgG2b, or unrelated monoclonal antibodies. Second, H107 antibody reacted with Fc epsilon R(+) B cell lines but not with Fc epsilon R(-) B cell lines as determined by an indirect immunofluorescence. Third, Fc epsilon R(+) cells were depleted by the incubation in the dish coated with H107 antibody or IgE but not in the dish coated with unrelated antibodies. Finally, there was a correlation between the increase of Fc epsilon R(+) cells and that of H107(+) cells in the peripheral blood lymphocytes of the patients with atopic dermatitis. The surface antigens on Fc epsilon R(+) RPMI8866 cells recognized by H107 antibodies had the molecular size of 45,000 as determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PMID:2942602

  10. Bacillus thuringiensis delta-endotoxin binding to brush border membrane vesicles of rice stem borers.

    PubMed

    Alcantara, Edwin P; Aguda, Remedios M; Curtiss, April; Dean, Donald H; Cohen, Michael B

    2004-04-01

    The receptor binding step in the molecular mode of action of five delta-endotoxins (Cry1Ab, Cry1Ac, Cry1C, Cry2A, and Cry9C) from Bacillus thuringiensis was examined to find toxins with different receptor sites in the midgut of the striped stem borer (SSB) Chilo suppressalis (Walker) and yellow stem borer (YSB) Scirpophaga incertulas (Walker) (Lepidoptera: Pyralidae). Homologous competition assays were used to estimate binding affinities (K(com)) of (125)I-labelled toxins to brush border membrane vesicles (BBMV). The SSB BBMV affinities in decreasing order was: Cry1Ab = Cry1Ac > Cry9C > Cry2A > Cry1C. In YSB, the order of decreasing affinities was: Cry1Ac > Cry1Ab > Cry9C = Cry2A > Cry1C. The number of binding sites (B(max)) estimated by homologous competition binding among the Cry toxins did not affect toxin binding affinity (K(com)) to both insect midgut BBMVs. Results of the heterologous competition binding assays suggest that Cry1Ab and Cry1Ac compete for the same binding sites in SSB and YSB. Other toxins bind with weak (Cry1C, Cry2A) or no affinity (Cry9C) to Cry1Ab and Cry1Ac binding sites in both species. Cry2A had the lowest toxicity to 10-day-old SSB and Cry1Ab and Cry1Ac were the most toxic. Taken together, the results of this study show that Cry1Ab or Cry1Ac could be combined with either Cry1C, Cry2A, or Cry9C for more durable resistance in transgenic rice. Cry1Ab should not be used together with Cry1Ac because a mutation in one receptor site could diminish binding of both toxins. PMID:15027071

  11. Toxin a from Clostridium difficile binds to rabbit erythrocyte glycolipids with therminal Gal. cap alpha. 1-3Gal. beta. 1-4GlcNaC sequences

    SciTech Connect

    Clark, G.F.; Krivan, H.; Wilkins, T.; Smith, D.F.

    1987-05-01

    Toxin A is one of two clostridial toxins implicated as the causative agent of pseudomembranous colitis in patients undergoing postoperative antibiotic therapy. Evidence that the carbohydrate binding determinant for this toxin is a glycoconjugate(s) with non-reducing Gal..cap alpha..1-3Gal..beta..1-4GlcNAc has recently been reported. Specific agglutination of rabbit erythrocytes by Toxin A is inhibited by bovine thyroglobulin and prevented by pretreatment of cells with ..cap alpha..-galactosidase. Total lipid extracts from rabbit erythrocytes were subjected to thin layer chromatography and the chromatogram overlaid with purified /sup 125/I-labeled Toxin A. Two major and several minor toxin-binding glycolipids were detected following autoradiography. The major toxin-binding glycolipids were identified as pentasaccharide- and decasaccharide-ceramides expressing terminal Gal..cap alpha..1-3Gal..beta..1-4GlcNAc sequences. Treatment of the toxin-binding glycolipids with ..cap alpha..-galactosidase abolished binding. Forsmann glycolipid, globoside, Gal..cap alpha..1-4 Gal..beta..1-4Glc-cer, and Gal..cap alpha..1-3Gal..beta..1-4Glc-cer did not bind the toxin. These observations are consistent with the proposed carbohydrate specificity of the toxin for the non-reducing terminal sequence, Gal..cap alpha..1-3Gal..beta..1-4GlcNAc.

  12. Receptor binding characterization of the benzodiazepine radioligand sup 125 I-Ro16-0154: Potential probe for SPECT (Single Photon Emission Computed Tomography) brain imaging

    SciTech Connect

    Johnson, E.W.; Woods, S.W.; Zoghbi, S.; Baldwin, R.M.; Innis, R.B. ); McBride, B.J. )

    1990-01-01

    The binding of an iodinated benzodiazepine (BZ) radioligand has been characterized, particularly in regard to its potential use as a neuroreceptor brain imaging agent with SPECT (Single Photon Emission Computed Tomography). Ro16-0154 is an iodine-containing BZ antagonist and a close analog of Ro15-1788. In tissue homogenates prepared from human and monkey brain, the binding of {sup 125}I-labeled Ro16-0154 was saturable, of high affinity, and had high ratios of specific to non-specific binding. Physiological concentrations of NaCl enhanced specific binding approximately 15% compared to buffer without this salt. Kinetic studies of association and dissociation demonstrated a temperature dependent decrease in affinity with increasing temperature. Drug displacement studies confirmed that {sup 125}I-Ro16-0154 binds to the central type BZ receptor: binding is virtually identical to that of {sup 3}H-Ro15-1788 except that {sup 125}I-Ro16-0154 shows an almost 10 fold higher affinity at 37{degree}C. These in vitro results suggest that {sup 123}I-labeled Ro16-0154 shows promise as a selective, high affinity SPECT probe of the brain's BZ receptor.

  13. Synthesis and binding affinity of an iodinated juvenile hormone

    SciTech Connect

    Prestwich, G.D.; Eng, W.S.; Robles, S.; Vogt, R.G.; Wisniewski, J.R.; Wawrzenczyk, C.

    1988-01-25

    The synthesis of the first iodinated juvenile hormone (JH) in enantiomerically enriched form is reported. This chiral compound, 12-iodo-JH I, has an iodine atom replacing a methyl group of the natural insect juvenile hormone, JH I, which is important in regulating morphogenesis and reproduction in the Lepidoptera. The unlabeled compound shows approximately 10% of the relative binding affinity for the larval hemolymph JH binding protein (JHBP) of Manduca sexta, which specifically binds natural /sup 3/H-10R,11S-JH I (labeled at 58 Ci/mmol) with a KD of 8 X 10(-8) M. It is also approximately one-tenth as biologically active as JH I in the black Manduca and epidermal commitment assays. The 12-hydroxy and 12-oxo compounds are poor competitors and are also biologically inactive. The radioiodinated (/sup 125/I)12-iodo-JH I can be prepared in low yield at greater than 2500 Ci/mmol by nucleophilic displacement using no-carrier-added /sup 125/I-labeled sodium iodide in acetone; however, synthesis using sodium iodide carrier to give the approximately 50 Ci/mmol radioiodinated ligand proceeds in higher radiochemical yield with fewer by-products and provides a radioligand which is more readily handled in binding assays. The KD of (/sup 125/I)12-iodo-JH I was determined for hemolymph JHBP of three insects: M. sexta, 795 nM; Galleria mellonella, 47 nM; Locusta migratoria, 77 nM. The selectivity of 12-iodo-JH I for the 32-kDa JHBP of M. sexta was demonstrated by direct autoradiography of a native polyacrylamide gel electrophoresis gel of larval hemolymph incubated with the radioiodinated ligand. Thus, the in vitro and in vivo activity of 12-iodo-JH I indicate that it can serve as an important new gamma-emitting probe in the search for JH receptor proteins in target tissues.

  14. Neurotensin Induces Presynaptic Depression of D2 Dopamine Autoreceptor-Mediated Neurotransmission in Midbrain Dopaminergic Neurons

    PubMed Central

    Piccart, Elisabeth; Courtney, Nicholas A.; Branch, Sarah Y.; Ford, Christopher P.

    2015-01-01

    Increased dopaminergic signaling is a hallmark of severe mesencephalic pathologies such as schizophrenia and psychostimulant abuse. Activity of midbrain dopaminergic neurons is under strict control of inhibitory D2 autoreceptors. Application of the modulatory peptide neurotensin (NT) to midbrain dopaminergic neurons transiently increases activity by decreasing D2 dopamine autoreceptor function, yet little is known about the mechanisms that underlie long-lasting effects. Here, we performed patch-clamp electrophysiology and fast-scan cyclic voltammetry in mouse brain slices to determine the effects of NT on dopamine autoreceptor-mediated neurotransmission. Application of the active peptide fragment NT8–13 produced synaptic depression that exhibited short- and long-term components. Sustained depression of D2 autoreceptor signaling required activation of the type 2 NT receptor and the protein phosphatase calcineurin. NT application increased paired-pulse ratios and decreased extracellular levels of somatodendritic dopamine, consistent with a decrease in presynaptic dopamine release. Surprisingly, we observed that electrically induced long-term depression of dopaminergic neurotransmission that we reported previously was also dependent on type 2 NT receptors and calcineurin. Because electrically induced depression, but not NT-induced depression, was blocked by postsynaptic calcium chelation, our findings suggest that endogenous NT may act through a local circuit to decrease presynaptic dopamine release. The current research provides a mechanism through which augmented NT release can produce a long-lasting increase in membrane excitability of midbrain dopamine neurons. SIGNIFICANCE STATEMENT Whereas plasticity of glutamate synapses in the brain has been studied extensively, demonstrations of plasticity at dopaminergic synapses have been more elusive. By quantifying inhibitory neurotransmission between midbrain dopaminergic neurons in brain slices from mice we have

  15. NHERF-1 regulation of EGF and neurotensin signalling in HT-29 epithelial cells

    SciTech Connect

    Kruger, Wade A.; Monteith, Gregory R.; Poronnik, Philip

    2013-03-22

    Highlights: ► NHERF-1 expression was abundant throughout HT-29 cells consistent with a cancerous phenotype. ► Knockdown of NHERF-1 lead to a significant reduction in cell proliferation. ► EGF and neurotensin-mediated proliferation was inhibited by knockdown of NHERF-1. ► Neurotensin-mediated Ca{sup 2+} response was abolished by knockdown of NHERF-1. -- Abstract: Neurotensin receptors (NT-R) and the epidermal growth factor receptors (EGF-R) are commonly overexpressed in many epithelial origin tumours. In addition to their role as mitogenic mediators through specific cell signalling, recent studies indicate that the activity/expression of scaffold proteins responsible for the assembly and coordination of the signalling complexes may also have central roles in epithelial transformation. In particular, the “epithelial” PSD-95/Dlg/Zo-1 (PDZ) scaffold/adapter protein, Na{sup +}/H{sup +} exchanger regulatory factor isoform one (NHERF-1), has been identified as a potential regulator of cellular transformation. NHERF-1 is a known regulator of EGF-R function and plays numerous roles in G-protein-coupled receptor signalling. Because of the synergistic signalling between these two potent mitogens, we investigated a potential role for NHERF-1 in the molecular mechanism linking the aberrant proliferative phenotype initiated by some G-Protein-coupled receptor activators in the colon adenocarcinoma HT-29 cell line. Knockdown (80%) of endogenous NHERF-1 leads to significant reduction in proliferation rate; an effect that could not be recovered by exogenous application of either NT or EGF. Inhibition of the EGF-R with AG1487 also inhibited proliferation and this effect could not be recovered with NT. Knockdown of NHERF-1 significantly altered the expression of the EGF-R, and almost completely abolished the NT-mediated increases in intracellular free Ca{sup 2+}. Knockdown of NHERF-1 also attenuated UTP-mediated purinergic Ca{sup 2+} signalling. Taken together, these data

  16. OB protein binds specifically to the choroid plexus of mice and rats.

    PubMed

    Devos, R; Richards, J G; Campfield, L A; Tartaglia, L A; Guisez, Y; van der Heyden, J; Travernier, J; Plaetinck, G; Burn, P

    1996-05-28

    Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor. PMID:8643634

  17. OB protein binds specifically to the choroid plexus of mice and rats.

    PubMed Central

    Devos, R; Richards, J G; Campfield, L A; Tartaglia, L A; Guisez, Y; van der Heyden, J; Travernier, J; Plaetinck, G; Burn, P

    1996-01-01

    Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8643634

  18. Calcitonin receptor binding in the hen anterior pituitary during an oviposition cycle.

    PubMed

    Nakayama, Hiroyuki; Takahashi, Tetsuya; Nakagawa-Mizuyachi, Kaori; Kawashima, Mitsuo

    2011-10-01

    The equilibrium dissociation constant (K(d) ) and the maximum binding capacity (B(max) ) of calcitonin (CT) receptor in the plasma membrane of the anterior pituitary in hens were examined by Scatchard analysis of specific binding of (125) I-labeled chicken CT. Values of K(d) and B(max) of CT receptor were smaller in laying hens than in non-laying hens. A decrease in the K(d) and B(max) value of CT receptor was observed in the anterior pituitary after the injection of estradiol-17β and progesterone into nonlaying hens, but not changed after the injection of 5α-dihydrotestosterone. During an oviposition cycle, the K(d) and the B(max) value decreased 3 h before oviposition. In non-laying hens, neither the K(d) nor the B(max) value changed during a full day period. The present study suggests that the CT action on the anterior pituitary may increase 3 h before oviposition by the effect of estradiol-17β and progesterone in laying hens. PMID:21951904

  19. Low doses of neurotensin in the preoptic area produce hyperthermia. Comparison with other brain sites and with neurotensin-induced analgesia.

    PubMed

    Benmoussa, M; Chait, A; Loric, G; de Beaurepaire, R

    1996-01-01

    High amounts of neurotensin (NT) are found in the preoptic area of the hypothalamus, an area known to be involved in the regulation of body temperature. It is generally believed that NT is a peptide that produces hypothermia, and several sites in the brain have been proposed to mediate NT-induced hypothermia, including the preoptic area. However, the doses of NT used in these experiments were always very high (microgram order) whereas, according to Goedert, the total brain content of NT in the rat does not exceed 10 ng. We therefore reinvestigated the effects of microinjections of NT in the brain, using high (5 micrograms) and low (50 and 5 ng) doses, into the preoptic area and other brain sites (cerebral ventricles, posterior hypothalamus, and nucleus accumbens), and we also studied, as a comparison, the effects of high and low doses of NT on pain sensitivity in the same sites. The results show that the preoptic area has unique properties in the regulation of body temperature: low doses of NT in the preoptic area produce a hyperthermic response, whereas high doses produce hypothermia. In comparison, NT produces hypothermia in the posterior hypothalamus whatever the dose, and NT has analgesic effects in the preoptic area only at high doses. Besides, NT has no thermic effect, but does have an analgesic effect, in the nucleus accumbens. The selectivity of the actions of high doses of NT, as well as the mechanism of action of NT (possibly an endogenous neuroleptic), are discussed. PMID:8705314

  20. Construction of recombinant HEK293 cell lines for the expression of the neurotensin receptor NTSR1.

    PubMed

    Xiao, Su; Shiloach, Joseph; Grisshammer, Reinhard

    2015-01-01

    G protein-coupled receptors (GPCRs) are associated with a wide array of diseases and are targets of most of the medicines sold worldwide. Despite their clinical importance, only 25 unique GPCR structures have been determined as of April 2014. The first step for structural studies is to establish the expression of correctly folded, functional receptors in recombinant host cells at quantities to allow subsequent purification and crystallization trials. Here we describe the T-REx™-inducible expression system to construct and select a stable HEK293 cell line for high-level expression of functional neurotensin receptor type I (NTSR1). We also present the protocols used for the adaptation of the cells into suspension culture, as well as the optimization of the induction parameters for NTSR1 expression, which led to 1 mg of purified NTSR1 per liter suspension culture in bioreactors. PMID:25563176

  1. An insulin receptor mutant (Asp707 --> Ala), involved in leprechaunism, is processed and transported to the cell surface but unable to bind insulin.

    PubMed

    Hart, L M; Lindhout, D; Van der Zon, G C; Kayserilli, H; Apak, M Y; Kleijer, W J; Van der Vorm, E R; Maassen, J A

    1996-08-01

    We have identified a homozygous mutation near the carboxyl terminus of the insulin receptor (IR) alpha subunit from a leprechaun patient, changing Asp707 into Ala. Fibroblasts from this patient had no high affinity insulin binding sites. To examine the effect of the mutation on IR properties, the mutant IR was stably expressed in Chinese hamster ovary cells. Western blot analysis and metabolic labeling showed a normal processing of the mutant receptor to alpha and beta subunits. No increase in high affinity insulin binding sites was observed on Chinese hamster ovary cells expressing the mutant receptor, and also, affinity cross-linking of 125I-labeled insulin by disuccinimidyl suberate to these cells failed to label the mutant alpha subunit. Biotinylation of cell surface proteins by biotin succinimidyl ester resulted in efficient biotinylation of the mutant IR alpha and beta subunits, showing its presence on the cell surface. On solubilization of the mutant insulin receptor in Triton X-100-containing buffers, 125I-insulin was efficiently cross-linked to the receptor alpha subunit by disuccinimidyl suberate. These studies demonstrate that Ala707 IR is normally processed and transported to the cell surface and that the mutation distorts the insulin binding site. Detergent restores this site. This is an example of a naturally occurring mutation in the insulin receptor that affects insulin binding without affecting receptor transport and processing. This mutation points to a major contribution of the alpha subunit carboxyl terminus to insulin binding. PMID:8702527

  2. Role of apolipoprotein A-I in HDL binding to a rat hepatoma cell in culture

    SciTech Connect

    Gottlieb, B.A.

    1985-01-01

    The binding of HDL to rat Fu5AH hepatoma cells at 4/sup 0/, and uptake and degradation at 37/sup 0/, was investigated in monolayer cultures. HDL, free of apo E and apo A-IV, was obtained from the plasma of nephrotic rats (HDLne). /sup 125/I-labeled HDLne bound to the cells in a specific, saturable manner. By Scatchard analysis, two classes of binding sites were obtained: a high affinity binding site (Kd = 1.25 +/- 0.023 ..mu..g/ml, or 5 x 10/sup -9/ M), and a lower affinity site (Kd = 45 +/- 15 ..mu..g/ml, or 1.8 x 10/sup -7/ M). In competitive binding experiments, normal rat HDL was nearly as effective as HDLne, but rat VLDL and human lipoproteins were ineffective. Rat apo A-I/phospholipid complexes also did not complete effectively for HDLne binding, although they were capable of binding to the cells. However, LDL (1.02 < d < 1.063) from nephrotic rat plasma, containing 20% of apo A-I, was as effective as rat HDL in competing for HDLne binding when the competition was expressed as a function of apo A-I content. Control experiments indicated that labeled apo A-I from HDLne did not exchange appreciably with unlabeled apo A-I on the LDLne. When the hepatoma cells were allowed to internalize and degrade HDLne at 37/sup 0/, the acid-soluble products (iodotyrosine and iodide) were derived almost entirely from the breakdown of apo A-I. We conclude that the rat hepatoma cell (Fu5AH) has high affinity HDL binding sites which recognize apo A-I-lipid complexes in which apo A-I an appropriate conformation.

  3. Receptor binding sites for substance P in surgical specimens obtained from patients with ulcerative colitis and Crohn disease

    SciTech Connect

    Mantyh, C.R.; Gates, T.S.; Zimmerman, R.P.; Welton, M.L.; Passaro, E.P. Jr.; Vigna, S.R.; Maggio, J.E.; Kruger, L.; Mantyh, P.W.

    1988-05-01

    Several lines of evidence indicate that tachykinin neuropeptides (substance P (SP), substance K (SK), and neuromedin K (NK)) play a role in regulating the inflammatory and immune responses. To test this hypothesis in a human inflammatory disease, quantitative receptor autoradiography was used to examine possible abnormalities in tachykinin binding sites in surgical specimens from patients with inflammatory bowel disease. In all cases, specimens were processed for quantitative receptor autoradiography by using /sup 125/I-labeled Bolton-Hunter conjugates of NK, SK, and SP. In colon tissue obtained from ulcerative colitis and Crohn disease patients, very high concentrations of SP receptor binding sites are expressed by arterioles and venules located in the submucosa, muscalairs mucosa, external circular muscle, external longitudinal muscle, and serosa, in contrast to control patients. These results demonstrate that receptor binding sites for SP, but not SK or NK, are ectopically expressed in high concentrations by cells involved in mediating inflammatory and immune responses. These data suggest that SP may be involved in the pathophysiology of inflammatory bowel disease and might provide some insight into the interaction between the nervous system and the regulation of inflammation and the immune response in human inflammatory disease.

  4. Evidence for a role of endogenous neurotensin in the development of sensitization to the locomotor stimulant effect of morphine.

    PubMed

    Lévesque, Karine; Lamarche, Caroline; Rompré, Pierre-Paul

    2008-10-10

    This experiment was aimed at exploring the role of endogenous neurotensin in the development of sensitization to the locomotor stimulant effect of morphine. During the induction phase (Days 1, 3, 5 and 7), male Long-Evans adult rats were treated with the neurotensin antagonist SR-48692 (160, 320 or 640 microg/kg, i.p.) or its vehicle, followed by morphine (5.0 mg/kg, i.p.) or its vehicle, and their locomotor activity (ambulatory, non-ambulatory and vertical activity) was measured for 2 h. One week after the last injection, each group received a single injection of morphine (2.5 mg/kg, i.p.) and their locomotor activity was again measured for 2 h (sensitization test, day 14). Results show that SR-48692 alone did not change locomotion. Morphine stimulated locomotor activity, an effect that was stronger on day 7 than on day 1. The two higher doses of SR-48692 attenuated the acute stimulant effect of morphine and prevented the observed increase from day 1 to day 7. The sensitization test on day 14 showed that rats pre-treated with morphine alone displayed significantly stronger ambulatory and vertical activity than vehicle pre-treated rats, a sensitization effect that was attenuated by SR-48692. The present results suggest that endogenous neurotensin contributes to the acute locomotor stimulant effect of morphine and to the induction of its sensitization. PMID:18706409

  5. Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes.

    PubMed

    Boldt, D H; Banwell, J G

    1985-12-13

    Ingestion of red kidney bean phytohemagglutinin causes impaired growth and intestinal malabsorption, and facilitates bacterial colonization in the small intestine of weanling rats. We have studied interactions of the highly purified phytohemagglutinin erythroagglutinating (E4) and mitogenic (L4) isolectins with microvillous membrane vesicles prepared from rat small intestines. E4 and L4 were radioiodinated with 125I by the chloramine-T technique. E4 and L4 isolectins both bound to microvillous membrane vesicles. Binding was saturable and reversible. Each mg of membrane protein bound 744 +/- 86 micrograms E4 and 213 +/- 21 micrograms L4. The apparent Ka for E4 and L4 binding was 2.5 x 10(-6) and 13.0 x 10(-6) M-1, respectively. Binding of each 125I-labelled isolectin was abolished by 100-fold excess of unlabelled isolectin. In each case binding also was inhibited by appropriate oligosaccharide inhibitors, indicating that isolectin-microvillous membrane interactions were mediated by carbohydrate recognition. Patterns of saccharide inhibition of isolectin binding were different for E4 and L4. Competitive binding experiments demonstrated mutual noncompetitive inhibition of E4 and L4 binding consistent with steric hindrance. Therefore, E4 and L4 each bound to its own set of receptors. Based on the known saccharide specificities of E4 and L4, these data indicate that there are differences in expression of complex asparagine-linked biantennary and tri- or tetraantennary oligosaccharides at the microvillous surface. The data also provide the possibility that direct interactions of one or more phytohemagglutinin isolectins with intestinal mucosa in vivo may contribute to the antinutritional effects associated with ingestion of crude red kidney beans. PMID:4063394

  6. Characterization of the resistance to Vip3Aa in Helicoverpa armigera from Australia and the role of midgut processing and receptor binding

    PubMed Central

    Chakroun, Maissa; Banyuls, Núria; Walsh, Tom; Downes, Sharon; James, Bill; Ferré, Juan

    2016-01-01

    Crops expressing genes from Bacillus thuringiensis (Bt crops) are among the most successful technologies developed for the control of pests but the evolution of resistance to them remains a challenge. Insect resistant cotton and maize expressing the Bt Vip3Aa protein were recently commercialized, though not yet in Australia. We found that, although relatively high, the frequency of alleles for resistance to Vip3Aa in field populations of H. armigera in Australia did not increase over the past four seasons until 2014/15. Three new isofemale lines were determined to be allelic with previously isolated lines, suggesting that they belong to one common gene and this mechanism is relatively frequent. Vip3Aa-resistance does not confer cross-resistance to Cry1Ac or Cry2Ab. Vip3Aa was labeled with 125I and used to show specific binding to H. armigera brush-border membrane vesicles (BBMV). Binding was of high affinity (Kd = 25 and 19 nM for susceptible and resistant insects, respectively) and the concentration of binding sites was high (Rt = 140 pmol/mg for both). Despite the narrow-spectrum resistance, binding of 125I-labeled Vip3Aa to BBMV of resistant and susceptible insects was not significantly different. Proteolytic conversion of Vip3Aa protoxin into the activated toxin rendered the same products, though it was significantly slower in resistant insects. PMID:27095284

  7. Characterization of the resistance to Vip3Aa in Helicoverpa armigera from Australia and the role of midgut processing and receptor binding.

    PubMed

    Chakroun, Maissa; Banyuls, Núria; Walsh, Tom; Downes, Sharon; James, Bill; Ferré, Juan

    2016-01-01

    Crops expressing genes from Bacillus thuringiensis (Bt crops) are among the most successful technologies developed for the control of pests but the evolution of resistance to them remains a challenge. Insect resistant cotton and maize expressing the Bt Vip3Aa protein were recently commercialized, though not yet in Australia. We found that, although relatively high, the frequency of alleles for resistance to Vip3Aa in field populations of H. armigera in Australia did not increase over the past four seasons until 2014/15. Three new isofemale lines were determined to be allelic with previously isolated lines, suggesting that they belong to one common gene and this mechanism is relatively frequent. Vip3Aa-resistance does not confer cross-resistance to Cry1Ac or Cry2Ab. Vip3Aa was labeled with (125)I and used to show specific binding to H. armigera brush-border membrane vesicles (BBMV). Binding was of high affinity (Kd = 25 and 19 nM for susceptible and resistant insects, respectively) and the concentration of binding sites was high (Rt = 140 pmol/mg for both). Despite the narrow-spectrum resistance, binding of (125)I-labeled Vip3Aa to BBMV of resistant and susceptible insects was not significantly different. Proteolytic conversion of Vip3Aa protoxin into the activated toxin rendered the same products, though it was significantly slower in resistant insects. PMID:27095284

  8. Relative abilities of distinct isotypes of human major histocompatibility complex class II molecules to bind streptococcal pyrogenic exotoxin types A and B.

    PubMed Central

    Imanishi, K; Igarashi, H; Uchiyama, T

    1992-01-01

    The relative ability of distinct isotypes of human leukocyte antigen class II molecules to bind streptococcal pyrogenic exotoxins A and B (SPE A and SPE B, respectively) was investigated by a direct-binding assay with 125I-labeled toxin for SPE A and by a functional assay system measuring the accessory cell activity of human leukocyte antigen class II transfectants in toxin-induced T-cell activation for SPE A and SPE B. SPE A binding was observed in L cells transfected with DQw1 genes. By contrast, it was not detected in L cells transfected with DR2, DR4, DPw4 or DP(Cp63) genes. All the transfectants supported SPE-induced interleukin-2 production by human T cells except the DP transfectants for SPE B. Levels of accessory cell activity were low in the DP transfectants induced by stimulation with SPE A and in the DR and DP transfectants induced by SPE B. The results indicate that SPE A and SPE B bind well to DQ molecules, less well to DR molecules, and very weakly to DP molecules. PMID:1452333

  9. Translocation of protein kinase C to membranes induced by TNF does not cause the inhibition of EGF binding to human wish cells.

    PubMed

    Katoh, T; Karasaki, Y; Hirano, H; Gotoh, S; Higashi, K

    1990-04-30

    Tumor necrosis factor (TNF) caused an inhibition of 125I-labeled epidermal growth factor [( 125I]EGF) binding to its receptors of human amniotic (WISH) cells at 5 min after addition of TNF, which reached a maximal level (60-70% reduction) after 15-30 min and declined thereafter. TNF also induced a translocation of protein kinase C activity from the cytosol to the membrane, which peaked at 45-60 min after addition of TNF and almost returned to basal level at 120 min. Furthermore, prolonged incubation of WISH cells with 12-O-tetradecanoylphorbol 13 acetate (TPA) diminished the TPA effect on the inhibition of EGF binding to the cells due to the desensitization of protein kinase C; however, TNF still reduced the EGF binding to the cells pretreated with TPA for a long time. These results indicate that although TNF causes the translocation of protein kinase C to the membrane, activation of protein kinase C is not required for TNF to induce a decrease in EGF binding to the cells. PMID:2334431

  10. Binding of navy bean (Phaseolus vulgaris) lectin to the intestinal cells of the rat and its effect on the absorption of glucose

    SciTech Connect

    Donatucci, D.A.; Liener, I.E.; Gross, C.J.

    1987-12-01

    The main objectives of this investigation were to study the binding of a lectin from navy beans with the epithelial cells of the rat intestine and to assess the effect of such binding on the ability of the intestine to absorb glucose. A Scatchard plot, based on the binding of /sup 125/I-labeled lectin to isolated intestinal epithelial cells, was used to calculate an association constant (Ka) of 15 x 10(6)M-1 and the number of binding sites per cell, 12 x 10(6). Metabolic studies were conducted over a period of 5 d on groups of rats fed raw or autoclaved navy bean flour and casein with or without the purified lectin. Growth, protein digestibility, biological value and net protein utilization were significantly lower in animals that had been fed raw navy bean flour or casein plus lectin than in control groups fed diets containing autoclaved navy bean flour or casein alone. Vascular perfusion was used to measure the rate of uptake of glucose by the intestines of rats that had received the various dietary treatments. The rate of absorption of (/sup 14/C)glucose by intestines from rats fed raw navy bean flour or casein plus lectin was approximately one-half that of their counterparts fed the autoclaved flour or casein alone. These results provide evidence that the lectin, by virtue of its interference with intestinal absorption, is responsible, at least in part, for the nutritional inferiority of raw navy beans.

  11. Inhibitory effect of alpha-fetoprotein on the binding of myasthenia gravis antibody to acetylcholine receptor.

    PubMed Central

    Brenner, T; Beyth, Y; Abramsky, O

    1980-01-01

    The binding of myasthenia gravis antibody acetylcholine receptor (AcChoR) as measured in vitro by Radioimmunoassay with 125I-labeled alpha-bungarotoxin (alpha-BuTx), can be blocked by amniotic fluid, maternal serum, and umbilical cord serum. This inhibitory effect is due to alpha-fetoprotein present in high concentrations in amniotic fluid and serum, as shown by: (i) selective removal of several components from amniotic fluid and serum; (ii) selective addition of different components present in amniotic fluid and serum, including alpha-fetoprotein, to be radioimmunoassay; (iii) correlation between the inhibitory effect of both amniotic fluid and serum and between the amounts of alpha-fetoprotein they contain; (iv) blocking of the alpha-fetoprotein in vitro suggests a similar effect in vivo in pregnant women with myasthenia gravis. This effect may explain in part the variability in the development of neonatal myasthenia gravis in the babies, due to transplacental transfer of maternal anti-AcChoR antibody, only after delivery and only in the minority of the cases. It also may explain the appearnace of remissions in females with myasthenia gravis during the second and third trimesters of pregnancy. Similar phenomena observed during pregnancy in other autoimmune and immunopathogenic diseases also might be attributed to activity of alpha-fetoprotein. PMID:6158053

  12. Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

    PubMed Central

    Pierce, E J; Davison, M D; Parton, R G; Habig, W H; Critchley, D R

    1986-01-01

    Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site. Images Fig. 5. PMID:3539106

  13. A binding site for Bacillus thuringiensis Cry1Ab toxin is lost during larval development in two forest pests.

    PubMed

    Rausell, C; Martínez-Ramírez, A C; García-Robles, I; Real, M D

    2000-04-01

    The insecticidal activity and receptor binding properties of Bacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) and Lymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/microl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with (125)I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age. PMID:10742241

  14. A Binding Site for Bacillus thuringiensis Cry1Ab Toxin Is Lost during Larval Development in Two Forest Pests

    PubMed Central

    Rausell, Carolina; Martínez-Ramírez, Amparo Consuelo; García-Robles, Inmaculada; Real, María Dolores

    2000-01-01

    The insecticidal activity and receptor binding properties of Bacillus thuringiensis Cry1A toxins towards the forest pests Thaumetopoea pityocampa (processionary moth) and Lymantria monacha (nun moth) were investigated. Cry1Aa, Cry1Ab, and Cry1Ac were highly toxic (corresponding 50% lethal concentration values: 956, 895, and 379 pg/μl, respectively) to first-instar T. pityocampa larvae. During larval development, Cry1Ab and Cry1Ac toxicity decreased with increasing age, although the loss of activity was more pronounced for Cry1Ab. Binding assays with 125I-labelled Cry1Ab and brush border membrane vesicles from T. pityocampa first- and last-instar larvae detected a remarkable decrease in the overall Cry1Ab binding affinity in last-instar larvae, although saturable Cry1Ab binding to both instars was observed. Homologous competition experiments demonstrated the loss of one of the two Cry1Ab high-affinity binding sites detected in first-instar larvae. Growth inhibition assays with sublethal doses of Cry1Aa, Cry1Ab, and Cry1Ac in L. monacha showed that all three toxins were able to delay molting from second instar to third instar. Specific saturable binding of Cry1Ab was detected only in first- and second-instar larvae. Cry1Ab binding was not detected in last-instar larvae, although specific binding of Cry1Aa and Cry1Ac was observed. These results demonstrate a loss of Cry1Ab binding sites during development on the midgut epithelium of T. pityocampa and L. monacha, correlating in T. pityocampa with a decrease in Cry1Ab toxicity with increasing age. PMID:10742241

  15. Purification of a sarcoplasmic reticulum protein that binds Ca2+ and plasma lipoproteins

    SciTech Connect

    Hofmann, S.L.; Brown, M.S.; Lee, E.; Pathak, R.K.; Anderson, R.G.; Goldstein, J.L. )

    1989-05-15

    A protein in the sarcoplasmic reticulum of rabbit skeletal and cardiac muscle was identified because of its ability to bind 125I-labeled low density lipoprotein (LDL) with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, referred to as the 165-kDa protein, is restricted to striated muscle. It was not detected in 14 other tissues, including several that contain smooth muscle, but it appears in rat L6 myoblasts when they differentiate into myocytes. Immunofluorescence and immunoelectron microscopic studies revealed that the protein is present throughout the sarcoplasmic reticulum and the terminal cisternae. It binds 45Ca2+ on nitrocellulose blots and stains metachromatically with Stains-all, a cationic dye that stains Ca2+-binding proteins. It does not appear to be a glycoprotein, and it appears slightly larger than the 160-kDa glycoprotein previously described in sarcoplasmic reticulum. The 165-kDa protein binds LDL, beta-migrating very low density lipoprotein, and a cholesterol-induced high density lipoprotein particle that contains apoprotein E as its sole apoprotein with much higher affinity than it binds high density lipoprotein. The protein is stable to boiling and to treatment with sodium dodecyl sulfate, but it becomes sensitive to these treatments when its cystine residues are reduced and alkylated. The protein was purified 1300-fold to apparent homogeneity from rabbit skeletal muscle membranes. It differs from the cell surface LDL receptor in that (1) its apparent molecular weight is not changed by reduction and alkylation; (2) it is present in Watanabe-heritable hyperlipidemic rabbits, which lack functional LDL receptors; (3) binding of lipoproteins is not inhibited by EDTA; and (4) it is located within the lumen of the sarcoplasmic reticulum where it has no access to plasma lipoproteins.

  16. Alterations in insulin binding accompanying differentiation of 3T3-L1 preadipocytes.

    PubMed Central

    Reed, B C; Kaufmann, S H; Mackall, J C; Student, A K; Lane, M D

    1977-01-01

    Expression of the adipocyte phenotype by differentiating 3T3-L1 preadipocytes occurs upon exposure of the cells to insulin. Differentiation-linked changes in 125I-labeled insulin binding to 3T3-L1 cells were monitored and compared with those in nondifferentiating 3T3-C2 controls treated similarly. Without chronic insulin treatment, 3T3-L1 cells failed to express the adipocyte phenotype but maintained a level of 25,000-35,000 insulin-binding sites per cell. Treatment of 3T3-L1 cells with insulin resulted in an initial suppression of insulin binding followed by a 12-fold increase that paralleled the appearance of differentiated cells. A maximum of 170,000 insulin-binding sites per cell was attained for a population in which greater than 75% of the cells had differentiated. The increase of insulin receptor level appears to be differentiation-dependent and is not a general response of cells to the culture conditions. 3T3-C2 cells maintained in the presence of insulin for 30 days exhibited the undifferentiated phenotype and suppressed levels of insulin binding (35,000 sites per cell). The binding capacity of 3T3-L1 cells for epidermal growth factor remained unchanged between 25,000 and 40;000 sites per cell and was independent of the state of differentiation. Thus, induction by insulin in receptor-specific changes. Insulin receptors increase in number but epidermal growth factor receptors remain constant. PMID:303773

  17. The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces.

    PubMed Central

    Lai, C F; Gong, S C; Esteban, M

    1991-01-01

    The nature of interaction between vaccinia virus and the surface of host cells as the first step in virus infection is undefined. A 32-kDa virus envelope protein has been identified as a cell surface binding protein (J.-S. Maa, J. F. Rodriguez, and M. Esteban, J. Biol. Chem. 265:1569-1577, 1990). To carry out studies on the structure-function relationship of this protein, the 32-kDa protein was obtained from Escherichia coli cells harboring the expression plasmid pT7Ek32. The recombinant polypeptide was found to have structural properties similar to those of the native virus envelope protein. Binding studies of 125I-labeled 32-kDa protein to cultured cells of various origins revealed that the E. coli-produced 32-kDa protein exhibited selectivity, specificity, and saturability. Scatchard analysis indicated about 4.5 x 10(4) sites per cell with a high affinity (Kd = 1.8 x 10(-9) M), suggesting interaction of the 32-kDa protein with a specific receptor. The availability of large quantities of the 32-kDa virus protein in bacteria will permit further structural and functional studies of this virus envelope protein and facilitate identification of the specific cell surface receptor. Images PMID:1985213

  18. Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

    PubMed Central

    Sine, Steven M.; Huang, Sun; Li, Shu-Xing; daCOSTA, Corrie J. B.; Chen, Lin

    2014-01-01

    The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr184 depends on local residues, we generated mutations in an α7/5HT3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr184 and local residues contributes to high-affinity subtype-selective α-btx binding. PMID:23802200

  19. Binding of a radioiodinated 13-azapinane thromboxane antagonist to platelets: correlation with antiaggregatory activity in different species.

    PubMed Central

    Narumiya, S.; Okuma, M.; Ushikubi, F.

    1986-01-01

    Binding of a 125I-labelled derivative of the 13-azapinane thromboxane antagonist (ONO-11120), [125I]-9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyp hen yl)-13, 14-dihydro-13-aza-15-beta-omega-tetranor-thromboxane A2 ([125I]-PTA-OH), to washed platelets of human, dog and rabbit was studied. Results were compared with the in vitro inhibitory potency of ONO-11120 on platelet aggregation induced by arachidonate and a thromboxane agonist, 9,11-epithio-11,12-methano-thromboxane A2 (STA2). [125I]-PTA-OH bound to washed human platelets in a reversible, saturable and temperature-dependent manner, and specific binding displaced by 20 microM ONO-11120 constituted about 40% of the total binding. Scatchard analyses revealed a single class of specific binding and the equilibrium dissociation constant (KD) and maximal concentration of binding sites (Bmax) were 22 nM and 390 fmol per 10(8) platelets (about 2,300 sites per platelet), respectively. In addition to ONO-11120, STA2 and another thromboxane receptor agonist, (15S)-hydroxy-11,9-epoxymethano-prosta-5Z,13E-dienoic acid (U-46619), effectively displaced the binding with IC50 values of 44 and 125 nM respectively. Prostaglandin D2 (PGD2) partially displaced the binding only at a concentration above 1 microM. PGE1 and thromboxane B2 (TXB2) were without effect up to 100 microM. Similar binding of [125I]-PTA-OH was observed on dog platelets. The KD and Bmax were 12 nM and 110 fmol per 10(8) platelets (about 680 sites per platelet), respectively, and these values did not change significantly after adrenaline treatment which potentiated arachidonate-induced aggregation of platelets in this species.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3730697

  20. In vitro binding properties and autoradiographic imaging of 3-iodobenzamide ((/sup 125/I)-IBZM): a potential imaging ligand for D-2 dopamine receptors in SPECT

    SciTech Connect

    Bruecke, T.; Tsai, Y.F.; McLellan, C.; Singhanyom, W.; Kung, H.F.; Cohen, R.M.; Chiueh, C.C.

    1988-01-01

    The in vitro binding properties of the (/sup 125/I) labeled benzamide, (S(-)-N-((1-ethyl-2-pyrrolidinyl)-methyl)-2-hydroxy-3-iodo-6-methoxy-benzamide, IBZM) were determined in bovine and mouse caudate membrane homogenates and by autoradiography of mouse brain slices. (/sup 125/I)-IBZM binding is saturable and reversible with B/sub max/ of 373 +/- 51 fmol/mg protein and a K/sub d/ of 3.1 +/- 0.62 nM and 0.56 nM as calculated by association and dissociation time constants. In competition experiments, K/sub i/ values for the D-2 antagonists YM-09151-2 and spiperone are 4 orders of magnitude lower than the K/sub i/ value for the D-1 antagonist SCH-23390 and S(-)-IBZM is ten-fold more potent than R(+)-IBZM. (/sup 125/I)-IBZM has a low affinity for serotonin S-2 and for alpha receptors. Therefore, it is a highly selective ligand for dopamine D-2 receptors. Autoradiographic images of brain sections incubated with (/sup 125/I)-IBZM show the dopamine D-2 receptors of the striatum, nucleus accumbens and olfactory tubercle with a high ratio of specific to nonspecific binding. Thus, S(-)-IBZM, when labeled with (/sup 125/I), may be useful for in vivo imaging of dopamine D-2 receptors by single photon emission computerized tomography (SPECT).

  1. Reactions of immunoglobulin G-binding ligands with platelets and platelet-associated immunoglobulin G.

    PubMed Central

    Rosse, W F; Devine, D V; Ware, R

    1984-01-01

    Immunoglobulin G (IgG) bound to platelets is usually detected by one of two general methods: binding of labeled anti-IgG or consumption of anti-IgG. The latter method gives, in general, values 5-10-fold greater than the former under the same conditions. To investigate these discrepancies, we have compared the detection of platelet-bound IgG by a labeled anti-IgG binding assay and by a quantitative antiglobulin consumption test using the same antibodies. The interaction of 125I-labeled monoclonal anti-IgG or polyclonal anti-IgG with washed and IgG-coated platelets was studied. The binding of these ligands to washed normal platelets was largely (50-80%) nonspecific; the binding was not saturable and was only partially inhibitable by excess unlabeled anti-IgG. The binding of anti-IgG to platelets coated with anti-PIA1, a platelet-specific IgG antibody, appeared to be saturable and inhibitable; the dissociation constant (KD) of this IgG-anti-IgG reaction was 4.9 X 10(-9) for monoclonal and 1.4 X 10(-7) for polyclonal anti-IgG. The ratio of sites present on the membrane (determined by 131I-labeled anti-PIA1) to the number of binding sites for anti-IgG determined by Scatchard analysis was 0.53 for monoclonal anti-IgG and 1.3 for polyclonal anti-IgG. The binding of monoclonal anti-IgG to platelet-bound immune complexes or IgG aggregates appeared to be complex. 131I-Labeled IgG was affixed to platelets and was detected by three tests: direct binding of radiolabeled monoclonal anti-IgG and quantitative antiglobulin consumption (QAC) tests, which were quantitated either by measuring directly the amount of radiolabeled anti-IgG consumed from fluid phase (direct QAC), or indirectly by reference to a calibration curve relating the consumption of anti-IgG by known amounts of fluid-phase, non-immune IgG (indirect QAC). The amount of platelet-bound IgG detected by the direct binding of 125I-labeled monoclonal anti-IgG and by the direct QAC approximated that known to be bound to

  2. Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

    SciTech Connect

    Rivkees, S.A.; Carlson, L.L.; Reppert, S.M. )

    1989-05-01

    Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using {sup 125}I-labeled melatonin ({sup 125}I-Mel), a potent melatonin agonist. {sup 125}I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K{sub d} of 2.3 {plus minus} 1.0 {times} 10{sup {minus}11} M and 2.06 {plus minus} 0.43 {times} 10{sup {minus}10} M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)), significantly reduced the number of high-affinity receptors and increased the dissociation rate of {sup 125}I-Mel from its receptor. Furthermore, GTP({gamma}S) treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of {sup 125}I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M{sub r} > 400,000 and M{sub r} ca. 110,000. This elution profile was markedly altered by pretreatment with GTP({gamma}S) before solubilization; only the M{sub r} 110,000 peak was present in GTP({gamma}S)-pretreated membranes. The results strongly suggest that {sup 125}I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.

  3. Group A Streptococci Bind to Mucin and Human Pharyngeal Cells through Sialic Acid-Containing Receptors

    PubMed Central

    Ryan, Patricia A.; Pancholi, Vijaykumar; Fischetti, Vincent A.

    2001-01-01

    The first step in the colonization of group A streptococci (Streptococcus pyogenes) is adherence to pharyngeal epithelial cells. Prior to adherence to their target tissue, the first barrier that the streptococci encounter is the mucous layer of the respiratory tract. The present study was undertaken to characterize the interaction between mucin, the major glycoprotein component of mucus, and streptococci. We report here that S. pyogenes is able to bind to bovine submaxillary mucin in solid-phase microtiter plate assays. Western blots probed with 125I-labeled mucin and a panel of monoclonal antibodies revealed that the streptococcal M protein is one of two cell wall-associated proteins responsible for this binding. The binding was further localized to the N-terminal portion of the M molecule. Further analysis revealed that the M protein binds to the sialic acid moieties on mucin, and this interaction seems to be based on M-protein conformation rather than specific amino acid sequences. We found that sialic acid also plays a critical role in the adherence of an M6 streptococcal strain to the Detroit 562 human pharyngeal cell line and have identified α2-6-linked sialic acid as an important sialylated linkage for M-protein recognition. Western blot analysis of extracted pharyngeal cell membrane proteins identified three potential sialic acid-containing receptors for the M protein. The results are the first to show that sialic acid not only is involved in the binding of the streptococci to mucin but also plays an important role in adherence of group A streptococci to the pharyngeal cell surface. PMID:11705914

  4. Binding and degradation of 3,5,3'-triiodothyronine and thyroxine by rat intestinal bacteria.

    PubMed

    DiStefano, J J; de Luze, A; Nguyen, T T

    1993-06-01

    Intestinal bacteria hydrolyze conjugates of thyroxine (T4) and 3,5,3'-triiodothyronine (T3) secreted in bile, but it is not clear whether they have any other role in metabolism, storage, transport, or action of thyroid hormone in the intestines. We have examined aspects of T3 and T4 binding and degradation processes in fresh feces and cecum contents, obtained from normal control rats and from rats partially decontaminated by treatment with oral antibiotics for 2-3 wk. Samples were homogenized in phosphate buffer, fractionated, and subjected to various test conditions and incubated at 37 degrees C with 125I-labeled T3 (T3*) or T4 (T4*) for 2 or 24 h. Supernatants of high-speed centrifuged incubates were chromatographed to test for degradation products, and percentage binding was measured in the pellets. Substantial binding of T3* and T4* was found in all control rat feces and cecum content samples by 2 h, but binding was absent or significantly reduced in partially decontaminated rat samples. Bacterial binding of T3* and T4* were further shown to be competitive with graded doses of bovine serum albumin. Considerable degradation of T3* and T4* to labeled iodide (I*) only was also observed in feces and cecum content samples and was much greater in control rat than in corresponding partially decontaminated rat samples. Light had no effects in our system and heat reduced I* production. Propylthiouracil and sodium ipodate had little effect or equivocal effects, but dithiothreitol substantially inhibited I* production.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8333521

  5. Simple, rapid /sup 125/I-labeled cyclosporine double antibody/polyethylene glycol radioimmunoassay used in a pediatric cardiac transplant program

    SciTech Connect

    Berk, L.S.; Webb, G.; Imperio, N.C.; Nehlsen-Cannarella, S.L.; Eby, W.C.

    1986-01-01

    We modified the Sandoz cyclosporine radioimmunoassay because of our need for frequent clinical monitoring of cyclosporine drug levels in allo- and xenograft pediatric cardiac transplant patients. With application of a commercially available (/sup 125/I)cyclosporine label in place of (/sup 3/H)cyclosporine and a second antibody/polyethylene glycol (PEG) method of separation in place of charcoal separation, we simplified and enhanced the speed and precision of assay performance. Studies of 140 whole blood samples comparing this new method to the (/sup 3/H)cyclosporine radioimmunoassay (RIA) method of Berk and colleagues yielded a coefficient of correlation of 0.96 (p less than 0.00001) with means of 626 and 667 ng/ml for (/sup 3/H)RIA and (/sup 125/I)RIA, respectively, and a regression equation of y = 28 + 1.02x. The major advantages are that total assay time is reduced to approximately 1 h; (/sup 125/I)cyclosporine label is used, avoiding the problems associated with liquid scintillation counting; and precision is enhanced by separating bound and free fractions with second antibody/PEG. These modifications should provide for greater ease of assay performance and improved clinical utility of cyclosporine monitoring not only in the pediatric but also in the adult transplant patient.

  6. Localization of /sup 111/In- and /sup 125/I-labeled monoclonal antibody in guinea pigs bearing line 10 hepatocarcinoma tumors

    SciTech Connect

    Bernhard, M.I.; Hwang, K.M.; Foon, K.A.; Keenan, A.M.; Kessler, R.M.; Frincke, J.M.; Tallam, D.J.; Hanna, M.G. Jr.; Peters, L.; Oldham, R.K.

    1983-09-01

    A murine monoclonal antibody (D3) with demonstrated specificity for the guinea pig line 10 hepatocarcinoma (L10) was radiolabeled with either /sup 125/I or /sup 111/In and used to image dermal tumors in vivo. In one set of experiments, L10 tumors were established middorsally in one group of animals, and the similarly derived, antigenically distinct line 1 tumor was established in another group of animals. In spite of background imaging of liver, kidney, and spleen, L10 tumors were visualized clearly. Incorporation of radiolabel was demonstrated to predominate in the L10 tumor. In a separate set of experiments, L10 and line 1 tumors were established in contralateral thighs in the same animals. L10 tumors were visualized clearly, and tissue uptake of radiolabel was demonstrated to reside predominantly in the L10 tumor.

  7. Accuracy of blood volume estimations in critically ill children using 125I-labelled albumin and 51Cr-labelled red cells.

    PubMed

    Linderkamp, O; Holthausen, H; Seifert, J; Butenandt, I; Riegel, K P

    1977-06-01

    Blood volume was estimated using 51chromium labelled red cells and 125iodinated human serum albumin in 5 children with sepsis, in 6 burned children and 7 children with acute lymphoblastic leukaemia. Studies of the equilibration pattern demonstrated that the mixing time of labelled red cells was prolonged to 40 minutes or more in 5 children, indicating the existence of slowly circulating red cells. Mixing of labelled albumin was complete within 10 minutes in 15 patients and within 20 minutes in all the children studied. In a burned patient with severe sepsis, exchange transfusion improved the clinical state and normalized the equilibration pattern of labelled red cells. The mean body/venous haematocrit ratio was 0.893+/-0.018 (SD) in the children with sepsis, 0.859+/-0.052 in the burned patients, and 0.916+/-0.078 in the children with acute lymphoblastic leukaemia, increasing with spleen size in the latter group. PMID:267010

  8. Effects of Peripheral Neurotensin on Appetite Regulation and Its Role in Gastric Bypass Surgery.

    PubMed

    Ratner, Cecilia; Skov, Louise J; Raida, Zindy; Bächler, Thomas; Bellmann-Sickert, Kathrin; Le Foll, Christelle; Sivertsen, Bjørn; Dalbøge, Louise S; Hartmann, Bolette; Beck-Sickinger, Annette G; Madsen, Andreas N; Jelsing, Jacob; Holst, Jens J; Lutz, Thomas A; Andrews, Zane B; Holst, Birgitte

    2016-09-01

    Neurotensin (NT) is a peptide expressed in the brain and in the gastrointestinal tract. Brain NT inhibits food intake, but the effects of peripheral NT are less investigated. In this study, peripheral NT decreased food intake in both mice and rats, which was abolished by a NT antagonist. Using c-Fos immunohistochemistry, we found that peripheral NT activated brainstem and hypothalamic regions. The anorexigenic effect of NT was preserved in vagotomized mice but lasted shorter than in sham-operated mice. This in combination with a strong increase in c-Fos activation in area postrema after ip administration indicates that NT acts both through the blood circulation and the vagus. To improve the pharmacokinetics of NT, we developed a pegylated NT peptide, which presumably prolonged the half-life, and thus, the effect on feeding was extended compared with native NT. On a molecular level, the pegylated NT peptide increased proopiomelanocortin mRNA in the arcuate nucleus. We also investigated the importance of NT for the decreased food intake after gastric bypass surgery in a rat model of Roux-en-Y gastric bypass (RYGB). NT was increased in plasma and in the gastrointestinal tract in RYGB rats, and pharmacological antagonism of NT increased food intake transiently in RYGB rats. Taken together, our data suggest that NT is a metabolically active hormone, which contributes to the regulation of food intake. PMID:27580810

  9. Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

    PubMed Central

    Demeule, Michel; Beaudet, Nicolas; Régina, Anthony; Besserer-Offroy, Élie; Murza, Alexandre; Tétreault, Pascal; Belleville, Karine; Ché, Christian; Larocque, Alain; Thiot, Carine; Béliveau, Richard; Longpré, Jean-Michel; Marsault, Éric; Leduc, Richard; Lachowicz, Jean E.; Gonias, Steven L.; Castaigne, Jean-Paul; Sarret, Philippe

    2014-01-01

    Neurotensin (NT) has emerged as an important modulator of nociceptive transmission and exerts its biological effects through interactions with 2 distinct GPCRs, NTS1 and NTS2. NT provides strong analgesia when administered directly into the brain; however, the blood-brain barrier (BBB) is a major obstacle for effective delivery of potential analgesics to the brain. To overcome this challenge, we synthesized chemical conjugates that are transported across the BBB via receptor-mediated transcytosis using the brain-penetrant peptide Angiopep-2 (An2), which targets LDL receptor–related protein-1 (LRP1). Using in situ brain perfusion in mice, we found that the compound ANG2002, a conjugate of An2 and NT, was transported at least 10 times more efficiently across the BBB than native NT. In vitro, ANG2002 bound NTS1 and NTS2 receptors and maintained NT-associated biological activity. In rats, i.v. ANG2002 induced a dose-dependent analgesia in the formalin model of persistent pain. At a dose of 0.05 mg/kg, ANG2002 effectively reversed pain behaviors induced by the development of neuropathic and bone cancer pain in animal models. The analgesic properties of ANG2002 demonstrated in this study suggest that this compound is effective for clinical management of persistent and chronic pain and establish the benefits of this technology for the development of neurotherapeutics. PMID:24531547

  10. Differential expression and tumorigenic function of neurotensin receptor 1 in neuroendocrine tumor cells

    PubMed Central

    Kim, Ji Tae; Li, Jing; Song, Jun; Lee, Eun Y.; Weiss, Heidi L.; Townsend, Courtney M.; Evers, B. Mark

    2015-01-01

    Neurotensin (NTS), localized predominantly to the small bowel, stimulates the growth of a variety of cancers, including neuroendocrine tumors (NETs), mainly through its interaction with the high-affinity NTS receptor 1 (NTSR1). Here, we observed increased expression of NTSR1 in almost all tested clinical NET samples, but not in normal tissues. Through RT-PCR analysis, we found that the expression of NTSR1 and NTSR2 was either variable (NTSR1) or absent (NTSR2) in human NET cell lines. In contrast, NTSR3 and NTS were expressed in all NET cells. Treatment with 5-aza-2′-deoxycytidine, a demethylating agent, increased levels of NTSR1 and NTSR2 suggesting that DNA methylation contributes to NTSR1/2 expression patterns, which was confirmed by methylation analyses. In addition, we found that knockdown of NTSR1 decreased proliferation, expression levels of growth-related proteins, and anchorage-independent growth of BON human carcinoid cells. Moreover, stable silencing of NTSR1 suppressed BON cell growth, adhesion, migration and invasion. Our results show that high expression of NTSR1 is found in clinical NETs and that promoter methylation is an important mechanism controlling the differential expression of NTSR1 and silencing of NTSR2 in NET cells. Furthermore, knockdown of NTSR1 in BON cells suppressed oncogenic functions suggesting that NTSR1 contributes to NET tumorigenesis. PMID:26298774

  11. Structure-Based Evolution of Subtype-Selective Neurotensin Receptor Ligands

    PubMed Central

    Schaab, Carolin; Kling, Ralf Christian; Einsiedel, Jürgen; Hübner, Harald; Clark, Tim; Seebach, Dieter; Gmeiner, Peter

    2014-01-01

    Subtype-selective agonists of the neurotensin receptor NTS2 represent a promising option for the treatment of neuropathic pain, as NTS2 is involved in the mediation of μ-opioid-independent anti-nociceptive effects. Based on the crystal structure of the subtype NTS1 and previous structure–activity relationships (SARs) indicating a potential role for the sub-pocket around Tyr11 of NT(8–13) in subtype-specific ligand recognition, we have developed new NTS2-selective ligands. Starting from NT(8–13), we replaced the tyrosine unit by β2-amino acids (type 1), by heterocyclic tyrosine bioisosteres (type 2) and peptoid analogues (type 3). We were able to evolve an asymmetric synthesis of a 5-substituted azaindolylalanine and its application as a bioisostere of tyrosine capable of enhancing NTS2 selectivity. The S-configured test compound 2 a, [(S)-3-(pyrazolo[1,5-a]pyridine-5-yl)-propionyl11]NT(8–13), exhibits substantial NTS2 affinity (4.8 nm) and has a nearly 30-fold NTS2 selectivity over NTS1. The (R)-epimer 2 b showed lower NTS2 affinity but more than 600-fold selectivity over NTS1. PMID:25478316

  12. A neurotensin analog blocks cocaine-conditioned place preference and reinstatement.

    PubMed

    Boules, Mona; Netz, Rebecca; Fredrickson, Paul A; Richelson, Elliott

    2016-04-01

    Neurotensin (NT) is a neuropeptide that acts as a neurotransmitter and neuromodulator in the central nervous system. Several studies suggest a therapeutic role for NT analogs in nicotine and other psychostimulant addictions. We studied the effects of the nonselective NT receptor agonist NT69L, which has equal affinity for the two major NT receptors, NTS1 and NTS2, on the expression of cocaine-conditioned place preference (cocaine-CPP) and reinstatement after extinction. Robust cocaine-CPP was obtained after 5 days of conditioning. Extinction was induced using eight repeated daily injections of saline. Reinstatement was prompted by priming with one injection of cocaine (12 mg/kg intraperitoneally). On the test day, NT69L (1 mg/kg intraperitoneally) was administered 30 min before assessing cocaine-CPP. Extinction led to the loss of cocaine-CPP. One injection of cocaine (12 mg/kg intraperitoneally) for cocaine priming reinstated cocaine-CPP. NT69L blocked cocaine-CPP reinstatement in cocaine-primed animals. In addition, NT69L blocked cocaine-CPP reinstatement when administered before priming with cocaine. Thus, the NT agonist NT69L blocked both cocaine-CPP and reinstatement to cocaine preference. NT69L may exert this action by modulating the mesocorticolimbic dopamine and glutamatergic pathways involved in addiction and relapse processes. Therefore, NT agonists may represent a novel therapy for the treatment of addiction to cocaine and possibly to other psychostimulants. PMID:26901162

  13. MiRNA mimic screen for improved functional expression of neurotensin receptor

    PubMed Central

    Xiao, Su; Chen, Yu-Chi; Betenbaugh, Michael J.; Martin, Scott E.; Shiloach, Joseph

    2015-01-01

    Obtaining adequate quantities of functional mammalian membrane proteins has been a bottleneck in their structural and functional studies because the expression of these proteins from mammalian cells is relatively low. To explore the possibility of enhancing expression of these proteins using miRNA, a stable T-REx-293 cell line expressing the neurotensin receptor type 1 (NTSR1), a hard-to-express G protein-coupled receptor (GPCR), was constructed. The cell line was then subjected to human miRNA mimic library screening. In parallel, an HEK293 cell line expressing luciferase was also screened with the same human miRNA mimic library. Five microRNA mimics: hsa-miR-22-5p, hsa-miR-18a-5p, hsa-miR-22-3p, hsa-miR-429 and hsa-miR-2110 were identified from both screens. They led to 48% increase in the expression of functional NTSR1 and to 239% increase of luciferase expression. These miRNAs were also effective in enhancing the expression of secreted glypican-3 hFc-fusion protein from HEK293 cells. The results indicate that these molecules may have a wide role in enhancing the production of proteins with biomedical interest. PMID:25676429

  14. Antipsychotic-like effects of a neurotensin receptor type 1 agonist.

    PubMed

    Vadnie, Chelsea A; Ayers-Ringler, Jennifer; Oliveros, Alfredo; Abulseoud, Osama A; Choi, Sun; Hitschfeld, Mario J; Choi, Doo-Sup

    2016-05-15

    Although neurotensin (NT) analogs are known to produce antipsychotic-like effects, the therapeutic possibility of a brain penetrant NTS1 agonist in treating psychiatric disorders has not been well studied. Here, we examined whether PD149163, a brain-penetrant NTS1-specific agonist, displays antipsychotic-like effects in C57BL/6J mice by investigating the effect of PD149163 on amphetamine-mediated hyperactivity and amphetamine-induced disruption of prepulse inhibition. In addition, we assessed the effect of PD149163 on glycogen synthase kinase-3 (GSK-3) activity, a downstream molecular target of antipsychotics and mood stabilizers, using phospho-specific antibodies. PD149163 (0.1 and 0.5mg/kg) inhibited amphetamine-induced hyperactivity in mice, indicating that NTS1 activation inhibits psychomotor agitation. PD149163 (0.5mg/kg) also increased prepulse inhibition, suggesting that NTS1 activation reduces prepulse inhibition deficits which often co-occur with psychosis in humans. Interestingly, PD149163 increased the inhibitory serine phosphorylation on both GSK-3α and GSK-3β in a dose- and time-dependent manner in the nucleus accumbens and medial prefrontal cortex of the mice. Moreover, PD149163 inhibited GSK-3 activity in the nucleus accumbens and medial prefrontal cortex in the presence of amphetamine. Thus, like most current antipsychotics and mood stabilizers, PD149163 inhibited GSK-3 activity in cortico-striatal circuitry. Together, our findings indicate that PD149163 may be a novel antipsychotic. PMID:26909848

  15. Role of endogenous somatostatin in postprandial hypersecretion of neurotensin in patients after gastrectomy.

    PubMed Central

    Sakamoto, T; Miyata, M; Izukura, M; Tanaka, Y; Iwase, K; Imabun, S; Matsuda, H

    1997-01-01

    OBJECTIVE: The purpose of this report is to elucidate the mechanism of the hypersecretion of neurotensin (NT) after gastrectomy. SUMMARY BACKGROUND DATA: NT secretion induced by fat ingestion is increased after pancreatoduodenectomy or distal gastrectomy. The hypersecretion of NT in the patients undergoing resection of the upper gastrointestinal tract is suppressed by an exogenous somatostatin (SST) analog. METHODS: We observed simultaneously the secretion of NT and SST in the same patients before and after gastrectomy (n = 7). We also observed the secretion of these hormones induced by intraduodenal (ID) fat infusion in the normal volunteers (n = 6). RESULTS: The response of plasma NT to fat ingestion was significantly increased after gastrectomy compared with that before gastrectomy. The response of plasma SST after gastrectomy was significantly suppressed. The response of plasma NT and SST after ID fat infusion in the normal volunteers was similar to the gastrectomized state. CONCLUSION: Diminution of SST secretion, probably caused by the lack of SST cells in the distal part of the stomach, may play a role in augmenting NT secretion after gastrectomy. PMID:9114796

  16. Influence of Molecular Structure on O2-Binding Properties and Blood Circulation of Hemoglobin‒Albumin Clusters.

    PubMed

    Yamada, Kana; Yokomaku, Kyoko; Haruki, Risa; Taguchi, Kazuaki; Nagao, Saori; Maruyama, Toru; Otagiri, Masaki; Komatsu, Teruyuki

    2016-01-01

    A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, is an artificial O2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater than that of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity compared to the others because of the chemical linkage between the Cys-93(β) residues in Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4. Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior. PMID:26895315

  17. Influence of Molecular Structure on O2-Binding Properties and Blood Circulation of Hemoglobin‒Albumin Clusters

    PubMed Central

    Yamada, Kana; Yokomaku, Kyoko; Haruki, Risa; Taguchi, Kazuaki; Nagao, Saori; Maruyama, Toru; Otagiri, Masaki; Komatsu, Teruyuki

    2016-01-01

    A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, is an artificial O2-carrier with the potential to function as a red blood cell substitute. This paper describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i) bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant). Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater than that of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity compared to the others because of the chemical linkage between the Cys-93(β) residues in Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated by assay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4. Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior. PMID:26895315

  18. Central action of dendrotoxin: selective reduction of a transient K conductance in hippocampus and binding to localized acceptors

    SciTech Connect

    Halliwell, J.V.; Othman, I.B.; Pelchen-Matthews, A.; Dolly, J.O.

    1986-01-01

    Dendrotoxin, a small single-chain protein from the venom of Dendroaspis angusticeps, is highly toxic following intracerebroventricular injection into rats. Voltage-clamp analysis of CA/sub 1/ neurons in hippocampal slices, treated with tetrodotoxin, revealed that nanomolar concentrations of dendrotoxin reduce selectively a transient, voltage-dependent K conductance. Epileptiform activity known to be induced by dendrotoxin can be attributed to such an action. Membrane currents not affected directly by the toxin include (i) Ca-activated K conductance; (ii) noninactivating voltage-dependent K conductance; (iii) inactivating the noninactivating Ca conductances; (iv) persistent inward (anomalous) rectifier current. Persistence of the effects of the toxin when Cd was included to suppress spontaneous transmitter release indicates a direct action on the neuronal membrane. Using biologically active, /sup 125/I-labeled dendrotoxin, protein acceptor sites of high affinity were detected on cerebrocortical synapotosomal membranes and sections of rat brain. In hippocampus, toxin binding was shown autoradiographically to reside in synapse-rich and white matter regions, with lower levels in cell body layers. This acceptor is implicated in the action of toxin because its affinities for dendrotoxin congeners are proportional to their central neurotoxicities and potencies in reducing the transient, voltage-dependent K conductance.

  19. Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct.

    PubMed

    McLick, J; Hakam, A; Bauer, P I; Kun, E; Zacharias, D E; Glusker, J P

    1987-06-01

    The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide

  20. Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts.

    PubMed Central

    Allen-Hoffmann, B L; Crankshaw, C L; Mosher, D F

    1988-01-01

    Transforming growth factor beta (TGF-beta) enhances the cell surface binding of 125I-fibronectin by cultured human fibroblasts. The effect of TGF-beta on cell surface binding was maximal after 2 h of exposure to TFG-beta and did not require epidermal growth factor or protein synthesis. The enhancement was dose dependent and was found with the 125I-labeled 70-kilodalton amino-terminal fragment of fibronectin as well as with 125I-fibronectin. Treatment of cultures with TGF-beta for 6 h resulted in a threefold increase in the estimated number of fibronectin binding sites. The increase in number of binding sites was accompanied by an increased accumulation of labeled fibronectin in detergent-insoluble extracellular matrix. The effect of TGF-beta was biphasic; after 6 h of exposure, less labeled fibronectin bound to treated cultures than to control cultures. Exposure of cells to TGF-beta for greater than 6 h caused a two- to threefold increase in the accumulation of cellular fibronectin in culture medium as detected by a quantitative enzyme-linked immunosorbent assay. The second phase of the biphasic effect and the increase in soluble cellular fibronectin were blocked by cycloheximide. Immunofluorescence staining of fibroblast cultures with antifibronectin revealed that TGF-beta caused a striking increase in fibronectin fibrils. The 70-kilodalton amino-terminal fragment of fibronectin, which blocks incorporation of fibronectin into extracellular matrix, blocked anchorage-independent growth of NRK-49F cells in the presence of epidermal growth factor. Our results show that an increase in the binding and rate of assembly of exogenous fibronectin is an early event preceding the increase in expression of extracellular matrix proteins. Such an early increase in cell surface binding of exogenous fibronectin may be a mechanism whereby TGF-beta can modify extracellular matrix characteristics rapidly after tissue injury or during embryonic morphogenesis. Images PMID:3054513

  1. The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the binding of PDGF to its receptors

    SciTech Connect

    Raines, E.W.; Lane, T.F.; Iruela-Arispe, M.L.; Ross, R.; Sage, E.H. )

    1992-02-15

    Interactions among growth factors, cells, and extracellular matrix are critical to the regulation of directed cell migration and proliferation associated with development wound healing, and pathologic processes. Here the authors report the association of PDGF-AB and -BB, but not PDGF-AA, with the extracellular glycoprotein SPARC. Complexes of SPARC and {sup 125}I-labeled PDGF-BB or -AB were specifically immunoprecipitated by anti-SPARC immunoglobulins. {sup 125}I-PDGF-BB and -AB also bound specifically to SPARC that was immobilized on microtiter wells or bound to nitrocellulose after transfer from SDS/polyacrylamide gels. The binding of PDGF-BB to SPARC was pH-dependent; significant binding was detectable only above pH 6.6. Enhanced expression of both PDGF-B chain and SPARC was seen in advanced lesions of atherosclerosis. They suggest that the coordinate expression of SPARC and PDGF-B-containing dimers following vascular injury may regulate the activity of specific dimeric forms of PDGF in vivo.

  2. Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells

    SciTech Connect

    Barnwell, J.W.; Ockenhouse, C.F.; Knowles, D.M. II

    1985-11-01

    Plasmodium falciparum-infected erythrocytes bind in vitro to human endothelial cells, monocytes, and a certain melanoma cell line. Evidence suggests that this interaction is mediated by similar mechanisms which lead to the sequestration of parasitized erythrocytes in vivo through their attachment to endothelial cells of small blood vessels. They show here the monoclonal antibody OKM5, previously shown to react with the membranes of endothelial cells, monocyte,s and platelets, also reacts with the C32 melanoma cell line which also binds P. falciparum-infected erythrocytes. At relatively low concentrations, OKM5 inhibits and reverses the in vitro adherence of infected erythrocytes to target cells. As with monocytes, OKM5 antibody recognizes an /sup 125/I-labeled protein of approximately 88 Kd on the surface of C32 melanoma cells. It seems likely, therefore, that the 88 Kd polypeptide plays a role in cytoadherence, possibly as the receptor or part of a receptor for a ligand on the surface of infected erythrocytes.

  3. Stimulation of oval cell and hepatocyte proliferation by exogenous bombesin and neurotensin in partially hepatectomized rats

    PubMed Central

    Assimakopoulos, Stelios F; Tsamandas, Athanassios C; Alexandris, Ilias H; Georgiou, Christos; Vagianos, Constantine E; Scopa, Chrisoula D

    2011-01-01

    AIM: To investigate the effect of the neuropeptides bombesin (BBS) and neurotensin (NT) on oval cell proliferation in partially hepatectomized rats not pretreated with a known hepatocyte inhibitor. METHODS: Seventy male Wistar rats were randomly divided into five groups: I = controls, II = sham operated, III = partial hepatectomy 70% (PHx), IV = PHx + BBS (30 μg/kg per day), V = PHx + NT (300 μg/kg per day). Forty eight hours after liver resection, portal endotoxin levels and hepatic glutathione redox state were determined. α-fetoprotein (AFP) mRNA (in situ hybridisation), cytokeratin-19 and Ki67 antigen expression (immunohistochemistry) and apoptosis (TUNEL) were evaluated on liver tissue samples. Cells with morphological features of oval cells that were cytokeratin-19 (+) and AFP mRNA (+) were scored in morphometric analysis and their proliferation was recorded. In addition, the proliferation and apoptotic rates of hepatocytes were determined. RESULTS: In the control and sham operated groups, oval cells were significantly less compared to groups III, IV and V (P < 0.001). The neuropeptides BBS and NT significantly increased the proliferation of oval cells compared to group III (P < 0.001). In addition, BBS and NT induced a significant increase of hepatocyte proliferation (P < 0.001), whereas it decreased their apoptotic activity (P < 0.001) compared to group III. BBS and NT significantly decreased portal endotoxemia (P < 0.001) and increased the hepatic GSH: GSSG ratio (P < 0.05 and P < 0.001, respectively) compared to group III. CONCLUSION: BBS and NT stimulated oval cell proliferation in a model of liver regeneration, without use of concomitant suppression of hepatocyte proliferation as oval cell activation stimuli, and improved the hepatocyte regenerative response. This peptides-induced combined stimulation of oval cell and hepatocyte proliferation might serve as a possible treatment modality for several liver diseases. PMID:22180848

  4. Neurotensin-loaded collagen dressings reduce inflammation and improve wound healing in diabetic mice.

    PubMed

    Moura, Liane I F; Dias, Ana M A; Suesca, Edward; Casadiegos, Sergio; Leal, Ermelindo C; Fontanilla, Marta R; Carvalho, Lina; de Sousa, Hermínio C; Carvalho, Eugénia

    2014-01-01

    Impaired wound healing is an important clinical problem in diabetes mellitus and results in failure to completely heal diabetic foot ulcers (DFUs), which may lead to lower extremity amputations. In the present study, collagen based dressings were prepared to be applied as support for the delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. The performance of NT alone and NT-loaded collagen matrices to treat wounds in streptozotocin (STZ) diabetic induced mice was evaluated. Results showed that the prepared dressings were not-cytotoxic up to 72h after contact with macrophages (Raw 264.7) and human keratinocyte (HaCaT) cell lines. Moreover, those cells were shown to adhere to the collagen matrices without noticeable change in their morphology. NT-loaded collagen dressings induced faster healing (17% wound area reduction) in the early phases of wound healing in diabetic wounded mice. In addition, they also significantly reduced inflammatory cytokine expression namely, TNF-α (p<0.01) and IL-1β (p<0.01) and decreased the inflammatory infiltrate at day 3 post-wounding (inflammatory phase). After complete healing, metalloproteinase 9 (MMP-9) is reduced in diabetic skin (p<0.05) which significantly increased fibroblast migration and collagen (collagen type I, alpha 2 (COL1A2) and collagen type III, alpha 1 (COL3A1)) expression and deposition. These results suggest that collagen-based dressings can be an effective support for NT release into diabetic wound enhancing the healing process. Nevertheless, a more prominent scar is observed in diabetic wounds treated with collagen when compared to the treatment with NT alone. PMID:24161538

  5. Neurotensin releases norepinephrine differentially from perfused hypothalamus of sated and fasted rat

    SciTech Connect

    Lee, T.F.; Rezvani, A.H.; Hepler, J.R.; Myers, R.D.

    1987-01-01

    The central injection of neurotensin (NT) has been reported to attenuate the intake of food in the fasted animal. To determine whether endogenous norepinephrine (NE) is involved in the satiating effect of NT, the in vivo activity of NE in circumscribed sites in the hypothalamus of the unanesthetized rat was examined. Bilateral guide tubes for push-pull perfusion were implanted stereotaxically to rest permanently above one of several intended sites of perfusion, which included the paraventricular nucleus (PVN), ventromedial nucleus (VMN), and the lateral hypothalamic (LH) area. After endogenous stores of NE at a specific hypothalamic locus were radiolabeled by microinjection of 0.02-0.5 ..mu..Ci of (/sup 3/H)NE, an artificial cerebrospinal fluid was perfused at the site at a rate of 20 ..mu..l/min over successive intervals of 5.0 min. When 0.05 or 0.1 ..mu..g/..mu..l NT was added to the perfusate, the peptide served either to enhance or educe the local release of NE at 50% of the sites of perfusion. In these experiments, the circumscribed effect of NT on the characteristics of catecholamine efflux depended entirely on the state of hunger or satiety of the rat. That is, when NT was perfused in the fully satiated rat, NE release was augmented within the PVn or VMN; conversely, NE release was inhibited in the LH. in the animal fasted for 18-22 h, NT exerted an opposite effect on the activity of NE within the same anatomical loci in that the efflux of NE was enhanced in the LH but attenuated or unaffected in the PVN or VMN. Taken together, these observations provide experimental support for the view-point that NT could act as a neuromodulator of the activity of hypothalamic noradrenergic neurons that are thought to play a functional role in the regulation of food intake.

  6. Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination.

    PubMed

    Shibata, Yoko; Gvozdenovic-Jeremic, Jelena; Love, James; Kloss, Brian; White, Jim F; Grisshammer, Reinhard; Tate, Christopher G

    2013-04-01

    Conformational thermostabilisation of G protein-coupled receptors is a successful approach for their structure determination. We have recently determined the structure of a thermostabilised neurotensin receptor NTS1 in complex with its peptide agonist and here we describe the strategy for the identification and combination of the 6 thermostabilising mutations essential for crystallisation. First, thermostability assays were performed on a panel of 340 detergent-solubilised Ala/Leu NTS1 mutants and the best 16 thermostabilising mutations were identified. These mutations were combined pair-wise in nearly all combinations (119 out of a possible 120 combinations) and each mutant was expressed and its thermostability was experimentally determined. A theoretical stability score was calculated from the sum of the stabilities measured for each double mutant and applied to develop 24 triple mutants, which in turn led to the construction of 14 quadruple mutants. Use of the thermostability data for the double mutants to predict further mutant combinations resulted in a greater percentage of the triple and quadruple mutants showing improved thermostability than if only the thermostability data for the single mutations was considered. The best quadruple mutant (NTS1-Nag36k) was further improved by including an additional 2 mutations (resulting in NTS1-GW5) that were identified from a complete Ala/Leu scan of Nag36k by testing the thermostability of the mutants in situ in whole bacteria. NTS1-GW5 had excellent stability in short chain detergents and could be readily purified as a homogenous sample that ultimately allowed crystallisation and structure determination. PMID:23337476

  7. Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

    SciTech Connect

    Clark, G.F.; Krivan, H.C.; Wilkins, T.D.; Smith, D.F.

    1987-08-15

    The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using /sup 125/I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. /sup 125/I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.

  8. Receptor-binding region in human choriogonadotropin/lutropin. beta. subunit

    SciTech Connect

    Keutmann, H.T.; Charlesworth, M.C.; Mason, K.A.; Ostrea, T.; Johnson, L.; Ryan, R.J.

    1987-04-01

    Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. The authors prepared a series of peptides representing the intercysteine loop sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) ..beta.. subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCG..beta..-(38-57) and hLH..beta..-(38-57) peptides inhibited binding of /sup 125/I-labeled hCG half-maximally at 1.51 x 10/sup -4/ and 2.03 x 10/sup -5/ M, respectively, while other peptide hormones and fragments from elsewhere in the ..beta.. subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 x 10/sup -5/ M (hCG) and 2.18 x 10/sup -5/ M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCG..beta..-(38-57) peptide, native hCG and ..beta.. subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. These results indicate that the 38-57 region of ..beta.. subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones.

  9. Putative melatonin receptors in a human biological clock

    SciTech Connect

    Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.; Stopa, E.G.

    1988-10-07

    In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

  10. Primary and secondary structural determinants in the receptor binding sequence. beta. -(38-57) from human luteinizing hormone

    SciTech Connect

    Keutmann, H.T.; Charlesworth, M.C.; Kitzmann, K.; Mason, K.A.; Johnson, L.; Ryan, R.J.

    1988-12-13

    The intercysteine loop sequence 38-57 in the ..beta.. subunit has been shown to be a determinant for expression of biological activity in human lutropin (hLH) and choriogonadotropin (hCG). Together with other sequences, the 38-57 region may contribute to a multicomponent receptor binding domain in hLH/hCG. Because the structural features influencing activity in this important region are not easy to evaluate in the full-length subunit, the authors have used analogues of hLH..beta..-(38-57) prepared by solid-phase synthesis. The peptides were tested for inhibition of /sup 125/I-labeled hCG binding to rat ovarian membrane receptors. Secondary structure was analyzed by circular dichroism (CD) and by reactivity with antibodies to the native 38-57 peptide. An analogue lacking the 38-57 disulfide linkage retained 20% receptor binding and full immunoreactivity. Far-ultraviolet CD profiles were essentially identical with those of the disulfide-intact peptide; a transition from 10% to 30% ..cap alpha..-helix in 90% trifluoroethanol was characteristic of both. The peptide thus appears not to require the disulfide bridge to retain a looped conformation with amphipathic secondary structure. An essential positive charge at position 43 was shown by complete loss of activity upon substitution of Asp or Ala for the Arg found in all known species of LH. These results indicate that the 38-57 sequence is a relatively rigid and structurally autonomous region, not merely a series of residues constrained passively into a loop by a disulfide linkage. It includes segments of ordered structure, probably including both amphipathic helical and turn sequences. Evidence from studies of other hormones suggests that this region may be important to binding and specificity in the glycoprotein hormones as a group.

  11. Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

    PubMed Central

    Machold, J; Utkin, Y; Kirsch, D; Kaufmann, R; Tsetlin, V; Hucho, F

    1995-01-01

    A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis. Images Fig. 2 PMID:7543679

  12. An obligatory role for neurotensin in high-fat-diet-induced obesity.

    PubMed

    Li, Jing; Song, Jun; Zaytseva, Yekaterina Y; Liu, Yajuan; Rychahou, Piotr; Jiang, Kai; Starr, Marlene E; Kim, Ji Tae; Harris, Jennifer W; Yiannikouris, Frederique B; Katz, Wendy S; Nilsson, Peter M; Orho-Melander, Marju; Chen, Jing; Zhu, Haining; Fahrenholz, Timothy; Higashi, Richard M; Gao, Tianyan; Morris, Andrew J; Cassis, Lisa A; Fan, Teresa W-M; Weiss, Heidi L; Dobner, Paul R; Melander, Olle; Jia, Jianhang; Evers, B Mark

    2016-05-19

    Obesity and its associated comorbidities (for example, diabetes mellitus and hepatic steatosis) contribute to approximately 2.5 million deaths annually and are among the most prevalent and challenging conditions confronting the medical profession. Neurotensin (NT; also known as NTS), a 13-amino-acid peptide predominantly localized in specialized enteroendocrine cells of the small intestine and released by fat ingestion, facilitates fatty acid translocation in rat intestine, and stimulates the growth of various cancers. The effects of NT are mediated through three known NT receptors (NTR1, 2 and 3; also known as NTSR1, 2, and NTSR3, respectively). Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with increased risk of diabetes, cardiovascular disease and mortality; however, a role for NT as a causative factor in these diseases is unknown. Here we show that NT-deficient mice demonstrate significantly reduced intestinal fat absorption and are protected from obesity, hepatic steatosis and insulin resistance associated with high fat consumption. We further demonstrate that NT attenuates the activation of AMP-activated protein kinase (AMPK) and stimulates fatty acid absorption in mice and in cultured intestinal cells, and that this occurs through a mechanism involving NTR1 and NTR3 (also known as sortilin). Consistent with the findings in mice, expression of NT in Drosophila midgut enteroendocrine cells results in increased lipid accumulation in the midgut, fat body, and oenocytes (specialized hepatocyte-like cells) and decreased AMPK activation. Remarkably, in humans, we show that both obese and insulin-resistant subjects have elevated plasma concentrations of pro-NT, and in longitudinal studies among non-obese subjects, high levels of pro-NT denote a doubling of the risk of developing obesity later in life. Our findings directly link NT with increased fat absorption and obesity and

  13. Quantitative radiommunoassay for DNA-binding antibodies. [Iodine 131, Iodine 125

    SciTech Connect

    Smith, L.H.; Guyer, R.L.; Minami, R.M.; Teplitz, R.L.

    1981-09-01

    A radioimmunoassay (RIA) is described for the measurement of serum immunoglobulins capable of binding to double-standard or single-standard DNA. DNA attached to Sephadex G-50 by ultraviolet radiation was used as a solid- phase immunoabsorbent for DNA-binding proteins from serum. Goat anti-human (GAH) IgG (/sup 125/I-labeled) were used to detect the human immunoglobulins bound onto the washed DNA-Sephadex. The quantities of immunoglobulins bound were determined by comparison with a standard curve constructed by dilution of a plasma from an systemic lupus erythematosus (SLE) patient containing known amounts of bound, DNA-specific IgM and IgG. Another RIA was employed for measuring levels of IgG and IgM. In combination with measurements of the total serum IgM and IgG, the RIA allowed for the determination of the fraction of the total serum IgM or IgG that was specific for double- or single-standard DNA. For a pool of normal human sera the quantities were as follows: 0.04% of the total IgM and 0.001% of the total IgG bound double-standard DNA; 0.22% of the total IgM and 0.05% of the total IgG bound single-stranded DNA. This capability is important because information regarding the quantitative measurement of antibodies to DNA and their class determination may be of significance in monitoring the status of subjects with SLE.

  14. D-Arg1,D-Phe5,D-Trp7,9,Leu11 substance P, a neuropeptide antagonist, blocks binding, Ca2(+)-mobilizing, and mitogenic effects of endothelin and vasoactive intestinal contractor in mouse 3T3 cells

    SciTech Connect

    Fabregat, I.; Rozengurt, E. )

    1990-10-01

    Endothelin (ET1) and vasoactive intestinal contractor (VIC) stimulate quiescent Swiss 3T3 cells to resume DNA synthesis acting synergistically with epidermal growth factors (EGF) and other mitogens. The peptide (D-Arg1,D-Phe5,D-Trp7,9,Leu11) substance P has been identified as a broad spectrum neuropeptide antagonist which blocks the binding and biological effects of the Ca2(+)-mobilizing neuropeptides bombesin, vasopressin, and bradykinin. In the present study we show that (D-Arg1,D-Phe5,D-Trp7,9,Leu11) substance P also acts as an ET1/VIC antagonist as judged by the following criteria: (a) inhibition of specific 125I-labelled ET1 binding to a ET1/VIC receptor in a competitive and dose-dependent manner; (b) blocking of the rapid increase in the cytosolic Ca2+ concentration promoted by ET1 or VIC; and (c) inhibition of DNA synthesis stimulated by VIC in the presence of EGF. The inhibitory effects of (D-Arg1,D-Phe5,D-Trp7,9,Leu 11) substance P on Ca2+ mobilization and DNA synthesis were reversed by increasing the concentration of VIC. This is the first time that a peptide structurally unrelated to ET1 or VIC is shown to block the binding and mitogenic effects of peptides of the endothelin family.

  15. Murine interleukin 1 receptor. Direct identification by ligand blotting and purification to homogeneity of an interleukin 1-binding glycoprotein

    SciTech Connect

    Bird, T.A.; Gearing, A.J.; Saklatvala, J.

    1988-08-25

    Functional receptors (IL1-R) for the proinflammatory cytokine interleukin 1 (IL1) were solubilized from plasma membranes of the NOB-1 subclone of murine EL4 6.1 thymoma cells using the zwitterionic detergent 3((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS). Membrane extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and ligand blotted with /sup 125/I-labeled recombinant human IL1 alpha in order to reveal proteins capable of specifically binding IL1. A single polydisperse polypeptide of Mr approximately equal to 80,000 was identified in this way, which bound IL1 alpha and IL1 beta with the same affinity as the IL1-R on intact NOB-1 cells (approximately equal to 10(-10) M). The IL1-binding polypeptide was only seen in membranes from IL1-R-bearing cells and did not react with interleukin 2, tumor necrosis factor alpha, or interferon. IL1-R was purified to apparent homogeneity from solubilized NOB-1 membranes by affinity chromatography on wheat germ agglutinin-Sepharose and IL1 alpha-Sepharose. Gel electrophoresis and silver staining of purified preparations revealed a single protein of Mr approximately equal to 80,000 which reacted positively in the ligand-blotting procedure and which we identify as the ligand-binding moiety of the murine IL1-R. Purified IL1-R exhibited the same affinity and specificity as the receptor on intact cells. The relationship of this protein to proteins identified by covalent cross-linking studies is discussed.

  16. IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein

    SciTech Connect

    Victoria, E.J.; Pierce, S.W.; Branks, M.J.; Masouredis, S.P. )

    1990-01-01

    Red blood cell (RBC) autoantibodies from patients with IgG warm-type autoimmune hemolytic anemia were labeled with iodine 125 and their RBC binding behavior characterized. Epitope-bearing RBC membrane polypeptides were identified after autoantibody immunoprecipitation of labeled membranes and immunoblotting. Immunoaffinity isolation of labeled membrane proteins with 12 different IgG hemolytic autoantibodies with protein A-agarose revealed a major polypeptide at Mr 95 to 110 kd, which coelectrophoresed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with a membrane component isolated with sheep IgG anti-band 3. Immunoprecipitation studies with chymotrypsinized RBCs resulted in the recovery of two labeled membrane polypeptides with molecular weights characteristically resulting from the chymotryptic fragmentation of band 3. Immunoblotting with sheep IgG anti-band 3 of the immunoprecipitated polypeptides confirmed that hemolytic autoantibody binding led to recovery of band 3 or its fragments. Two 125I-labeled IgG hemolytic autoantibodies showed binding behavior consistent with epitope localization on band 3. The labeled RBC autoantibodies bound immunospecifically to all types of human RBC tested, including those of rare Rh type (Rh-null, D--) at a site density of approximately 10(6) per RBC. The 125I-IgG in two labeled autoantibodies was 84% and 92% adsorbable by human and higher nonhuman primate RBCs. Antigen-negative animal RBC bound less than 10%, consistent with immunospecific RBC binding. IgG-1 was the major subclass in five autoantibodies tested; one of six fixed complement; and autoantibody IgG appeared polyclonal by isoelectric focusing. We conclude that IgG eluted from RBCs of patients with autoimmune hemolytic anemia consists predominantly of a single totally RBC-adsorbable antibody population that binds to antigenic determinants on band 3.

  17. Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

    SciTech Connect

    Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

    1988-05-05

    A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of /sup 125/I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10/sup -8/ to 1 x 10/sup -6/ M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca/sup 2 +/, Mg/sup 2 +/, or Mn/sup 2 +/ partially inhibited binding of /sup 125/I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved ..gamma..-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins.

  18. Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters.

    PubMed Central

    Wilson, P T; Gershoni, J M; Hawrot, E; Lentz, T L

    1984-01-01

    Proteolytic fragments of the alpha subunit of the acetylcholine receptor retain the ability to bind alpha-bungarotoxin following resolution by polyacrylamide gel electrophoresis and immobilization on protein transfers. The alpha subunit of the acetylcholine receptor of Torpedo electric organ was digested with four proteases: Staphylococcus aureus V-8 protease, papain, bromelain, and proteinase K. The proteolytic fragments resolved on 15% polyacrylamide gels were electrophoretically transferred onto positively charged nylon membrane filters. When incubated with 0.3 nM 125I-labeled alpha-bungarotoxin and autoradiographed, the transfers yielded patterns of labeled bands characteristic for each protease. The molecular masses of the fragments binding toxin ranged from 7 to 34 kDa, with major groupings in the 8-, 18-, and 28-kDa ranges. The apparent affinity of the fragments for alpha-bungarotoxin as determined from the IC50 value was 6.7 X 10(-8) M. The labeling of fragments with alpha-bungarotoxin could be inhibited by prior affinity alkylation of receptor-containing membranes with 4-(N-maleimido)-alpha-benzyltrimethylammonium iodide. These findings demonstrate that immobilized proteolytic fragments as small as 1/5 the size of the alpha subunit retain the structural characteristics necessary for binding alpha-bungarotoxin, although the toxin is bound to the fragments with lower affinity than to the native receptor. The effect of affinity ligand alkylation demonstrates that the alpha-bungarotoxin binding site detected on the proteolytic fragments is the same as the affinity-labeled acetylcholine binding site on the intact acetylcholine receptor. Images PMID:6371817

  19. Binding pattern of 125iodine thyroxine and tri-iodothyronine in skin and liver tissues of spotted munia, Lonchura punctulata: co-relation to seasonal cycles of breeding and molting.

    PubMed

    Thapliyal, Ashish; Chandola-Saklani, Asha; Bhatt, Dinesh; Anthwal, Prashant

    2014-05-01

    Prevalent notion about thyroid hormones is that thyroxine (T4) is a mere precursor and physiological effects of thyroid hormones are elicited by tri-iodothyronine (T3) after mono-deiodination of T4. Earlier studies on feather regeneration and molt done on spotted munia L. punctulata suggest that T4 (mono-deiodination suppressed by iopanoic acid and thyroidectomized birds) is more effective than T3 in inducing feather regeneration. The binding pattern of 125I labeled T4 and T3 has been investigated in the nuclei prepared from skin and liver tissues (samples obtained during different months) of spotted munia using scatchard plot analysis. The results show that binding capacity (B(max)--pmole/80 microgm DNA) of 125I-T3 to nuclei of skin was significantly higher in November as compared to April and June, whereas the binding affinity (Kd-10(-9)M(-1)) was significantly lower in November as compared to April and June. During November, B(max) for binding of T3 and T4 did not vary in liver and skin nuclei but Kd varied significantly. Binding capacity of 125I- T3 to skin and liver did not vary but binding affinity of 125I- T4 to skin was approximately 7 times higher than that of liver. The results suggest that T4 does show a variation in binding pattern that co-relates to the molting pattern of spotted munia. These variations might play important role in different physiological phenomenon in this tropical bird. The experiments do point towards the possibility of independent role of T4 as a hormone, however, further experiments need to be done to ascertain the role of T4 in this model and work out the exact molecular mechanism of action. PMID:24851410

  20. Effects of cholestyramine on low density lipoprotein binding sites on liver membranes from rabbits with endogenous hypercholesterolemia induced by a wheat starch-casein diet.

    PubMed

    Chao, Y; Yamin, T T; Alberts, A W

    1982-04-10

    Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia with a lipoprotein distribution similar to that of humans. Approximately 76% of the total cholesterol is carried in the low density lipoprotein (LDL) fraction (1.006 less than d less than 1.063 g/ml). Inclusion of 1% cholestyramine in the diet prevents the increase in plasma cholesterol. The cholestyramine effect is mediated through an increased fractional catabolic rate of 125I-LDL. In order to determine the potential role of hepatic LDL receptors in the removal of LDL from the plasma, binding of 125I-LDL and 125I-beta-VLDL (beta-migrating very low density lipoproteins) to hepatic membranes prepared from livers of rabbits fed the wheat starch-casein diet with or without cholestyramine supplementation was investigated. Membranes from livers of the cholestyramine-supplemented animals exhibit high levels of specific EDTA-sensitive binding of either of the 125I-labeled lipoproteins. Very little EDTA-sensitive binding occurs on liver membranes from wheat starch-casein-fed rabbits that have not been treated with cholestyramine. These results indicate that the hypercholesterolemia in rabbits associated with the wheat starch-casein diet is wholly or partially the result of a decreased number of specific hepatic LDL receptors and thus a decreased catabolism of plasma cholesterol. The response of the liver to the inclusion in the diet of the bile acid sequestrant, cholestyramine, is to maintain or increase the number of specific LDL binding sites, thus promoting catabolism of plasma cholesterol. PMID:6277940

  1. Evidence for monomeric and oligomeric hormone-binding domains in affinity-purified gonadotropin receptor from rat ovary

    SciTech Connect

    Zhang, Q.Y.; Menon, K.M.J. )

    1989-11-01

    Rat ovarian lutropin/choriogonadotropin receptor was purified from a Triton X-100-solubilized membrane preparation by affinity chromatography with Affi-Gel 10 coupled to purified human choriogonadotropin. The affinity-purified receptor preparations contained a single class of high-affinity binding sites for {sup 125}I-labeled human choriogonadotropin, with an equilibrium dissociation constant (K{sub d}) of 2.5 {times} 10{sup {minus}9} M, which is comparable to the K{sub d} values for membrane-bound and solubilized receptors. The purified receptor appeared as two dominant bands with molecular weights of 135,000 and 92,000 after sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) under nonreducing conditions. When the individual affinity-purified receptor bands were electroeluted from the gel and analyzed again by SDS/PAGE under nonreducing conditions, both the M{sub r} 92,000 and the 135,000 proteins retained their original molecular form even when 8 M urea was included in the gel. However, when the electrophoretically purified M{sub r} 92,000 and 135,000 bands were subjected to SDS/PAGE under reducing conditions, the M{sub r} 135,000 species was almost completely converted to a M{sub r} 92,000 band, but the M{sub r} 92,000 species did not undergo any alteration in molecular weight. The results suggest that the lutropin/choriogonadotropin receptor from rat ovary exists in two molecular forms, and the higher molecular weight form appears to be composed of disulfide-linked M{sup r} 92,000 subunit, which comprises the hormone-binding domain.

  2. OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope.

    PubMed Central

    Lemaire, M; Ohayon, H; Gounon, P; Fujino, T; Béguin, P

    1995-01-01

    Several proteins of Clostridium thermocellum possess a C-terminal triplicated sequence related to bacterial cell surface proteins. This sequence was named the SLH domain (for S-layer homology), and it was proposed that it might serve to anchor proteins to the cell surface (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). This hypothesis was investigated by using the SLH-containing protein ORF1p from C. thermocellum as a model. Subcellular fractionation, immunoblotting, and electron microscopy of immunocytochemically labeled cells indicated that ORF1p was located on the surface of C. thermocellum. To detect C. thermocellum components interacting with the SLH domains of ORF1p, a probe was constructed by grafting these domains on the C terminus of the MalE protein of Escherichia coli. The SLH domains conferred on the chimeric protein (MalE-ORF1p-C) the ability to bind noncovalently to the peptidoglycan of C. thermocellum. In addition, 125I-labeled MalE-ORF1p-C was shown to bind to SLH-bearing proteins transferred onto nitrocellulose, and to a 26- to 28-kDa component of the cell envelope. These results agree with the hypothesis that SLH domains contribute to the binding of exocellular proteins to the cell surface of bacteria. The gene carrying ORF1 and its product, ORF1p, are renamed olpB and OlpB (for outer layer protein B), respectively. PMID:7730277

  3. Effect of amphetamine on extracellular concentrations of amino acids in striatum in neurotensin subtype 1 and 2 receptor null mice: a possible interaction between neurotensin receptors and amino acid systems for study of schizophrenia.

    PubMed

    Li, Zhimin; Liang, Yanqi; Boules, Mona; Gordillo, Andres; Richelson, Elliott

    2010-06-01

    Neurotensin (NT) is a tridecapeptide that acts as a neuromodulator in the central nervous system mainly through two NT receptors: NTS1 and NTS2. The present study was done to determine the roles of NTS1 and NTS2 on amino acid release in striatum with the use of NTS1 or NTS2 knockout ((-/-)) mice given d-amphetamine. Both NTS1(-/-) and NTS2(-/-) mice had lower extracellular concentrations of D-serine in striatum than did wild type (WT) mice. NTS2(-/-) but not NTS1(-/-) mice also had significantly lower basal concentrations of glutamate in striatum as compared to that for WT mice. Systemic administration of d-amphetamine (4 mg/kg, ip) increased glutamate release by 500% in WT mice, as compared to 300% in NTS2(-/-) mice, and 250% in NTS1(-/-) mice. Additionally, d-amphetamine injection caused a 4-fold increase in GABA release in both WT and NTS2(-/-) mice, but only a 2-fold increase in NTS1(-/-) mice. Therefore, NTS1 and NTS2 modulate basal release of D-serine and glutamate, and also d-amphetamine-induced GABA and glutamate release in striatum. These results provide further support for the involvement of NT receptors in the pathogenesis of schizophrenia and provide a better understanding of the imbalance of amino acid systems through investigation of a DA-based animal model. PMID:20193696

  4. Opiate-mediated inhibition of the release of cholecystokinin and substance P, but not neurotensin from cat hypothalamic slices.

    PubMed

    Micevych, P E; Yaksh, T L; Go, V L

    1982-11-01

    The neuroactive peptides neurotensin (NT), substance P (SP) and cholecystokinin (CCK) have been shown to be distributed in the hypothalamus. These peptides may be part of hypothalamic mechanisms which regulate the release of pituitary hormones and feeding behavior. Numerous experiments have demonstrated opiate modulation of anterior pituitary hormone release. These effects have been reported to be mediated via a hypothalamic mechanism, which modulates the secretion of releasing, release inhibiting factors or other neuroactive peptides such as SP, CCK and NT. We have examined the effects of morphine on the potassium-stimulated, calcium-dependent release of SP, CCK and NT from cat hypothalamic slices. The potassium-stimulated release of SP and CCK was profoundly depressed by the addition of morphine (10(-5) M) in a naloxone-reversible manner. This morphine inhibition was shown to be stereospecific, levorphanol (10(-7) M) depressed the release, while dextrophan (10(-7) M) was inactive. Gel filtration chromatography of the potassium-stimulated release was determined to be isographic with authentic NT, SP and CCK-8, respectively. There was no indication of any gastrin-like activity. These data may suggest a regulatory mechanism through which opiates exert some of their neuroendocrine or feeding regulatory effects. PMID:6184121

  5. Ventral Tegmental Area Neurotensin Signaling Links the Lateral Hypothalamus to Locomotor Activity and Striatal Dopamine Efflux in Male Mice

    PubMed Central

    Patterson, Christa M.; Wong, Jenny-Marie T.; Leinninger, Gina M.; Allison, Margaret B.; Mabrouk, Omar S.; Kasper, Chelsea L.; Gonzalez, Ian E.; Mackenzie, Alexander; Jones, Justin C.

    2015-01-01

    Projections from the lateral hypothalamic area (LHA) innervate components of the mesolimbic dopamine (MLDA) system, including the ventral tegmental area (VTA) and nucleus accumbens (NAc), to modulate motivation appropriately for physiologic state. Neurotensin (NT)-containing LHA neurons respond to multiple homeostatic challenges and project to the VTA, suggesting that these neurons could link such signals to MLDA function. Indeed, we found that pharmacogenetic activation of LHA NT neurons promoted prolonged DA-dependent locomotor activity and NAc DA efflux, suggesting the importance of VTA neurotransmitter release by LHA NT neurons for the control of MLDA function. Using a microdialysis-mass spectrometry technique that we developed to detect endogenous NT in extracellular fluid in the mouse brain, we found that activation of LHA NT cells acutely increased the extracellular concentration of NT (a known activator of VTA DA cells) in the VTA. In contrast to the prolonged elevation of extracellular NAc DA, however, VTA NT concentrations rapidly returned to baseline. Intra-VTA infusion of NT receptor antagonist abrogated the ability of LHA NT cells to increase extracellular DA in the NAc, demonstrating that VTA NT promotes NAc DA release. Thus, transient LHA-derived NT release in the VTA couples LHA signaling to prolonged changes in DA efflux and MLDA function. PMID:25734363

  6. Chitosan-based dressings loaded with neurotensin--an efficient strategy to improve early diabetic wound healing.

    PubMed

    Moura, Liane I F; Dias, Ana M A; Leal, Ermelindo C; Carvalho, Lina; de Sousa, Hermínio C; Carvalho, Eugénia

    2014-02-01

    One important complication of diabetes mellitus is chronic, non-healing diabetic foot ulcers (DFUs). This study aims to develop and use dressings based on chitosan derivatives for the sustained delivery of neurotensin (NT), a neuropeptide that acts as an inflammatory modulator in wound healing. Three different derivatives, namely N-carboxymethyl chitosan, 5-methyl pyrrolidinone chitosan (MPC) and N-succinyl chitosan, are presented as potential biomaterials for wound healing applications. Our results show that MPC has the best fluid handling capacity and delivery profile, also being non-toxic to Raw 264.7 and HaCaT cells. NT-loaded and non-loaded MPC dressings were applied to control/diabetic wounds to evaluate their in vitro/in vivo performance. The results show that the former induced more rapid healing (50% wound area reduction) in the early phases of wound healing in diabetic mice. A NT-loaded MPC foam also reduced expression of the inflammatory cytokine TNF-α (P<0.001) and decreased the amount of inflammatory infiltrate on day 3. On day 10 MMP-9 was reduced in diabetic skin (P<0.001), significantly increasing fibroblast migration and collagen (COL1A1, COL1A2 and COL3A1) expression and deposition. These results suggest that MPC-based dressings may work as an effective support for sustained NT release to reduce DFUs. PMID:24121197

  7. Properties of follicle-stimulating-hormone receptor in cell membranes of bovine testis.

    PubMed Central

    Cheng, K W

    1975-01-01

    A simple method for preparing plasma membranes from bovine testes is described. Bovine testicular receptor has a high affinity and specificity for 125I-labelled human FSH (follicle-stimulating hormone). The specific binding of 125I-labelled human FSH to the plasma membranes is a saturable process with respect to the amounts of receptor protein and FSH added. The association and dissociation of 125I-labelled human FSH are time- and temperature-dependent, and the binding of labelled human FSH to bovine testicular receptor is strong and not readily reversible. Scatchard [Ann. N.Y. Acad. Sci. (1949) 51, 660-672] analysis indicates a dissociation constant, Kd, of 9.8 X10(-11)M, and 5.9 X 10(-14)mol of binding sites/mg of membrane protein. The testicular membrane receptor is heat-labile. Preheating at 40 degrees C for 15 min destroyed 30% of the binding activity. Specific binding is pH-dependent, with an optimum between pH 7.0 and 7.5. Brief exposure to extremes of pH caused irreversible damage to the receptors. The ionic strength of the incubation medium markedly affects the association of 125I-labelled human FSH with its testicular receptor. Various cations at concentrations of 0.1M inhibit almost completely the binding of 125I-labelled human FSH. Nuclectides and steroid hormones at concentrations of 1mM and 5mu/ml respectively have no effect on the binding of FSH to its receptor. Incubation of membranes with and chymotrypsin resulted in an almost complete loss of binding activity, suggesting that protein moieties are essential for the binding of 125I-labelled human FSH. Binding of 125I-labelled human FSH to bovine testicular receptor does not result in destruction or degradation of the hormone. PMID:242318

  8. Repeated effects of the neurotensin receptor agonist PD149163 in three animal tests of antipsychotic activity: assessing for tolerance and cross-tolerance to clozapine

    PubMed Central

    Chou, Shinnyi; Davis, Collin; Jones, Sean; Li, Ming

    2014-01-01

    Neurotensin is an endogenous neuropeptide closely associated with the mesolimbic dopaminergic system and shown to possess antipsychotic-like effects. In particular, acute neurotensin receptor activation can inhibit conditioned avoidance response (CAR), attenuate phencyclidine (PCP)-induced prepulse inhibition (PPI) disruptions, and reverse PCP-induced hyperlocomotion. However, few studies have examined the long term effects of repeated neurotensin receptor activation and results are inconsistent. Since clinical administration of antipsychotic therapy often requires a prolonged treatment schedule, here we assessed the effects of repeated activation of neurotensin receptors using an NTS1 receptor selective agonist, PD149163, in 3 behavioral tests of antipsychotic activity. We also investigated whether reactivity to the atypical antipsychotic clozapine was altered following prior PD149163 treatment. Using both normal and prenatally immune activated rats generated through maternal immune activation with polyinosinic:polycytidilic acid, we tested PD149163 in CAR, PCP (1.5 mg/kg)-induced PPI disruption, and PCP (3.2 mg/kg)-induced hyperlocomotion. For each paradigm, rats were first repeatedly tested with vehicle or PD149163 (1.0, 4.0, 8.0 mg/kg, sc) along with vehicle or PCP for PPI and hyperlocomotion tests, then challenged with PD149163 after 2 drug-free days. All rats were then challenged with clozapine (5.0 mg/kg, sc). During the repeated test period, PD149163 exhibited antipsychotic-like effects in all three models. On the PD149163 challenge day, prior drug treatment only caused a tolerance effect in CAR. This tolerance in CAR was transferrable to clozapine, as it enhanced clozapine tolerance in the same group of animals. Although no tolerance effect was seen in the PD149163 challenge for the PCP-induced hyperlocomotion test, the clozapine challenge showed increased sensitivity in groups previously exposed to repeated PD149163 treatment. Our findings suggest

  9. Suppression of Neurotensin Receptor Type 1 Expression and Function by Histone Deacetylase Inhibitors in Human Colorectal Cancers*

    PubMed Central

    Wang, Xiaofu; Jackson, Lindsey N.; Johnson, Sara M.; Wang, Qingding; Evers, B. Mark

    2010-01-01

    Neurotensin (NT), a gut peptide, stimulates growth of colorectal cancers (CRCs) which possess the high affinity NT receptor (NTR1). Sodium butyrate (NaBT) is a potent histone deacetylase inhibitor (HDACi) which induces growth arrest, differentiation and apoptosis of CRCs. Previously, we showed that NaBT increases nuclear GSK-3β expression and kinase activity; GSK-3β functions as a negative regulator of ERK signaling. The purpose of our current study was to determine: (a) whether HDACi alters NTR1 expression and function, and (b) the role of GSK-3β/ERK in NTR1 regulation. Human CRCs with NTR1 were treated with various HDACi and NTR1 expression and function were assessed. Treatment with HDACi dramatically decreased endogenous NTR1 mRNA, protein and promoter activity. Overexpression of GSK-3β decreased NTR1 promoter activity (> 30%); inhibition of GSK-3β increased NTR1 expression in CRC cells, indicating that GSK-3β is a negative regulator of ERK and NTR1. Consistent with our previous findings, HDACi significantly decreased phosphorylated ERK while increasing GSK-3β. Selective MEK/ERK inhibitors suppressed NTR1 mRNA expression in a time- and dose-dependent fashion, and reduced NTR1 promoter activity by ~70%. Finally, pretreatment with NaBT prevented NT-mediated COX-2 and c-myc expression and attenuated NT-induced IL-8 expression. HDACi suppresses endogenous NTR1 expression and function in CRC cell lines; this effect is mediated through, at least in part, the GSK-3β/ERK pathway. The down-regulation of NTR1 in CRCs may represent an important mechanism for the anti-cancer effects of HDACi. PMID:20663927

  10. Neurotensin immunolabeling relates to sexually-motivated song and other social behaviors in male European starlings (Sturnus vulgaris)

    PubMed Central

    Merullo, Devin P.; Cordes, Melissa A.; Stevenson, Sharon A.; Riters, Lauren V.

    2015-01-01

    The brain regions involved in vocal communication are well described for some species, including songbirds, but less is known about the neural mechanisms underlying motivational aspects of communication. Mesolimbic dopaminergic projections from the ventral tegmental area (VTA) are central to mediating motivated behaviors. In songbirds, VTA provides dopaminergic innervation to brain regions associated with motivation and social behavior that are also involved in sexually-motivated song production. Neurotensin (NT) is a neuropeptide that strongly modulates dopamine activity, co-localizes with dopamine in VTA, and is found in regions where dopaminergic cells project from VTA. Yet, little is known about how NT contributes to vocal communication or other motivated behaviors. We examined the relationships between sexually-motivated song produced by male European starlings (Sturnus vulgaris) and NT immunolabeling in brain regions involved in social behavior and motivation. Additionally, we observed relationships between NT labeling, non-vocal courtship behaviors (another measure of sexual motivation), and agonistic behavior to begin to understand NT’s role in socially-motivated behaviors. NT labeling in VTA, lateral septum, and bed nucleus of the stria terminalis correlated with sexually-motivated singing and non-vocal courtship behaviors. NT labeling in VTA, lateral septum, medial preoptic nucleus, and periaqueductal gray was associated with agonistic behavior. This study is the first to suggest NT’s involvement in song, and one of the few to implicate NT in social behaviors more generally. Additionally, our results are consistent with the idea that distinct patterns of neuropeptide activity in brain areas involved in social behavior and motivation underlie differentially motivated behaviors. PMID:25595421