WorldWideScience

Sample records for prolactin binding sites

  1. Prolactin receptors in liver, kidney, and gill of the tilapia (Oreochromis mossambicus): Characterization and effect of salinity on specific binding of iodinated ovine prolactin

    International Nuclear Information System (INIS)

    Dauder, S.; Young, G.; Hass, L.; Bern, H.A.

    1990-01-01

    Specific binding of 125 I-ovine prolactin (oPRL) to microsomal fractions from gill, kidney, and liver of adult tilapia was determined. Specific binding varied among tissues, the highest values being displayed by kidney membranes. In the liver, the binding of oPRL was not strongly displaced by tilapia prolactins (tPRL177 and tPRL188), although tPRL177 was six times more potent than tPRL188. On the other hand, in kidney and gill membranes, the two tPRLs were equipotent. Tilapia PRLs showed low potency in competing for oPRL-binding sites when pregnant rat liver membranes were utilized. Tilapia growth hormone (tGH) and human growth hormone (hGH) displaced 125 I-oPRL from liver as well as did tPRL177 but were not recognized well by renal or branchial receptors. Two 125 I-oPRL-binding sites were detected in every tissue tested. These binding sites are subject to physiological regulation since adaptation to seawater resulted in a significant decrease in specific binding

  2. Maintenance of prolactin receptors in human breast cancer

    International Nuclear Information System (INIS)

    Ben-David, M.; Dror, Y.; Biran, S.

    1981-01-01

    Breast tissue specimens of 110 women with various stages of breast cancer were tested in vitro to determine their specific binding sites for human prolactin. In contrast to the case of steroid receptors, binding sites for prolactin were found in the vast majority of breast cancer tissue. Distribution profiles giving amount of prolactin receptor and their affinity coefficients were found to be similar in the tissues of women whose ages, hormonal status, or stage of breast cancer varied. These findings show that in contrast to steroid receptors, human breast cancer tissue maintains binding sites for prolactin. The findings also indicate that there may be a higher dependency of breast cancer on prolactin than on steroids. Clinical trials must be carried out to determine the role of ''positive'' prolactin receptors in prognosis and prediction of response to future hormone therapy. (author)

  3. Development of radioimmunoassay for prolactin binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Raikar, R.S.; Sheth, A.R. (Institute for Research in Reproduction, Bombay (India))

    1982-01-01

    Using a homogenous prolactin binding protein (PBP) preparations from rat seminal vesicle secretion, a sensitive and specific radioimmunoassay (RIA) for PBP has been developed. The assay was highly specific and showed no cross-reaction with other protein hormones from various species. The antiserum had an affinity constant (Ka) of 2.66 x 10/sup 10/ M/sup -1/. The assay sensitivity was in the range of 0.5-1.0 ng of pure PBP per assay tube and the intra- and inter-assay coefficients of variations were 6-8% and 12-14.5% respectively. The overall recovery of PBP to the rat seminal vesicle secretion was 96.8%. Using this RIA, PBP levels in various biological fluids and reproductive tissues were measured. Azoospermic human semen contained significantly higher levels of PBP than normospermic semen. The seminal vesicle of rat exhibited the highest concentration of PBP. Administration of antiserum to PBP to mature male rats resulted in a significant reduction in the weight of ventral prostrate and serum prolactin levels were significantly elevated in these animals suggesting that the antibody raised against the PBP was capable of blocking prolactin receptors.

  4. Crystal structure of a prolactin receptor antagonist bound to the extracellular domain of the prolactin receptor

    DEFF Research Database (Denmark)

    Svensson, L Anders; Bondensgaard, Kent; Nørskov-Lauritsen, Leif

    2008-01-01

    The crystal structure of the complex between an N-terminally truncated G129R human prolactin (PRL) variant and the extracellular domain of the human prolactin receptor (PRLR) was determined at 2.5A resolution by x-ray crystallography. This structure represents the first experimental structure...... studies, the structural data imply that the definition of PRL binding site 1 should be extended to include residues situated in the N-terminal part of loop 1 and in the C terminus. Comparison of the structure of the receptor-bound PRL variant with the structure reported for the unbound form of a similar...... scale rearrangements and structuring occur in the flexible N-terminal part of loop 1. Hydrogen exchange mass spectrometry data imply that the dynamics of the four-helix bundle in solution generally become stabilized upon receptor interaction at binding site 1....

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

    International Nuclear Information System (INIS)

    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 125 I, a very sensitive (0.4-0.6 ng/ml) and highly specific homologous RIA for ePRL was developed. A heterologous RIA using 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 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

  6. Crystal structure of an affinity-matured prolactin complexed to its dimerized receptor reveals the topology of hormone binding site 2

    DEFF Research Database (Denmark)

    Broutin, Isabelle; Jomain, Jean-Baptiste; Tallet, Estelle

    2010-01-01

    We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely relate...... and prostate cancer.......We report the first crystal structure of a 1:2 hormone.receptor complex that involves prolactin (PRL) as the ligand, at 3.8-A resolution. Stable ternary complexes were obtained by generating affinity-matured PRL variants harboring an N-terminal tail from ovine placental lactogen, a closely related...... PRL receptor (PRLR) ligand. This structure allows one to draw up an exhaustive inventory of the residues involved at the PRL.PRLR site 2 interface, consistent with all previously reported site-directed mutagenesis data. We propose, with this description, an interaction model involving three structural...

  7. Effect of gonadotrophin inhibiting material isolated from human urine on action of prolactin at rat Leydig cell

    Energy Technology Data Exchange (ETDEWEB)

    Bagli, N P; Rajendran, K G; Shah, P N [Cancer Research Inst., Bombay (India). Div. of Endocrinology

    1980-05-01

    To study the effect of gonadotropin inhibiting material (GIM) on the binding of prolactin to Leydig cell receptors isolated Leydig cells were incubated with sup(125)I-prolactin. Presence of GIM in the incubation mixture did not inhibit the binding of sup(125)I-prolactin to Leydig cells whereas unlabelled prolactin significantly reduced the binding. In another experiment, testicular cells were incubated with FITC-tagged GIM. Binding of GIM to Leydig cells was seen by the presence of fluorescence on these cells. This binding could be inhibited by untagged GIM but not by prolactin. The results suggest the presence of separate receptors for GIM and prolactin on the Leydig cells and indicate that termination of pregnancy by GIM is not due to any interference with prolactin binding to its receptors.

  8. A pigeon crop sac radioreceptor assay for prolactin

    International Nuclear Information System (INIS)

    Forsyth, I.A.; Buntin, J.D.; Nicoll, C.S.

    1978-01-01

    Ovine prolactin, labelled with 125 I by either lactoperoxidase or a mild chloramine T method, was bound to receptors from the pigeon crop sac mucosa cells of prolactin-injected pigeons. Binding was demonstrated in a crude homogenate of mucosal cells removed from the crop by scraping and in a subcellular fraction in which 5'- nucleotidase activity was enhanced two- to three-fold. The binding was specific, dependent on time, temperature and the concentration of receptors and had a dissociation constant of 7 x 10 -10 mol/l. The binding capacity of the crop tissue was 71 fmol/mg membrane protein. Nine purified preparations of prolactin from four species were assayed by local pigeon crop sac bioassay and by radioreceptor assay. The two methods were highly correlated (r = 0.934). The regression equation was radioreceptor assay = 1.22 bioassay - 0.18 indicating a 1:1 correspondence between the two methods for prolactin purified from sheep, rat, horse and pig anterior pituitary glands. (author)

  9. Effects on prolactin secretion and binding to dopaminergic receptors in sleep-deprived lupus-prone mice

    Directory of Open Access Journals (Sweden)

    B.D. Palma

    2009-03-01

    Full Text Available Sleep disturbances have far-reaching effects on the neuroendocrine and immune systems and may be linked to disease manifestation. Sleep deprivation can accelerate the onset of lupus in NZB/NZWF1 mice, an animal model of severe systemic lupus erythematosus. High prolactin (PRL concentrations are involved in the pathogenesis of systemic lupus erythematosus in human beings, as well as in NZB/NZWF1 mice. We hypothesized that PRL could be involved in the earlier onset of the disease in sleep-deprived NZB/NZWF1 mice. We also investigated its binding to dopaminergic receptors, since PRL secretion is mainly controlled by dopamine. Female NZB/NZWF1 mice aged 9 weeks were deprived of sleep using the multiple platform method. Blood samples were taken for the determination of PRL concentrations and quantitative receptor autoradiography was used to map binding of the tritiated dopaminergic receptor ligands [³H]-SCH23390, [³H]-raclopride and [³H]-WIN35,428 to D1 and D2 dopaminergic receptors and dopamine transporter sites throughout the brain, respectively. Sleep deprivation induced a significant decrease in plasma PRL secretion (2.58 ± 0.95 ng/mL compared with the control group (25.25 ± 9.18 ng/mL. The binding to D1 and D2 binding sites was not significantly affected by sleep deprivation; however, dopamine transporter binding was significantly increased in subdivisions of the caudate-putamen - posterior (16.52 ± 0.5 vs 14.44 ± 0.6, dorsolateral (18.84 ± 0.7 vs 15.97 ± 0.7 and ventrolateral (24.99 ± 0.5 vs 22.54 ± 0.7 µCi/g, in the sleep-deprived mice when compared to the control group. These results suggest that PRL is not the main mechanism involved in the earlier onset of the disease observed in sleep-deprived NZB/NZWF1 mice and the reduction of PRL concentrations after sleep deprivation may be mediated by modifications in the dopamine transporter sites of the caudate-putamen.

  10. Effect of THIP and SL 76002, two clinically experimented GABA-mimetic compounds, on anterior pituitary GABA receptors and prolactin secretion in the rat

    International Nuclear Information System (INIS)

    Apud, J.A.; Masotto, C.; Racagni, G.

    1987-01-01

    In the present study, the ability of three direct GABA agonists, muscimol, THIP and SL 76002 to displace 3 H-GABA binding from anterior pituitary and medio-basal hypothalamus membranes was evaluated. Further, the effect of both THIP and SL 76002 on baseline prolactin levels or after stimulation of hormone release with haloperidol has been also studied. Either muscimol, THIP or SL 76002 have shown to posses 7-, 7- and 3-fold higher affinity, respectively, for the central nervous system than for the anterior pituitary 3 H-GABA binding sites. Moreover, THIP and SL 76002 have demonstrated to be respectively, 25- and 1000- fold less potent than muscimol in inhibiting 3 H- GABA binding at the level of the anterior pituitary and about 25- and 2700-fold less potent at the level of the medio-basal hypothalamus. Under basal conditions, either THIP or SL 76002 were ineffective to reduce prolactin release. However, after stimulation of prolactin secretion through blockade of the dopaminergic neurotransmission with haloperidol (0.1 mg/kg), both THIP (10 mg/kg) and SL 76002 (200 mg/kg) significantly counteracted the neuroleptic-induced prolactin rise with a potency which is in line with their ability to inhibit 3 H-GABA binding in the anterior pituitary. The present results indicate that both compounds inhibit prolactin release under specific experimental situations probably through a GABAergic mechanism. In view of the endocrine effects of these GABA-mimetic compounds, the possibility arises for an application of these type of drugs in clinical neuroendocrinology. 35 references, 3 figures, 2 tables

  11. Homologous radioimmunoassay for canine prolactin and its application in various physiological states

    International Nuclear Information System (INIS)

    Graef, K.-J.; Friedreich, E.; Matthes, S.; Hasan, S.H.

    1977-01-01

    The purification of canine prolactin and the development of an homologous radioimmunoassay including several physiological studies in the Beagle dog are described. The assay measured immunoreactive canine prolactin with a sensitivity limit of 0.6 ng/ml. Purified canine luteinizing hormone gave no significant inhibition in the assay whereas purified canine growth hormone inhibited the binding of 125 I-labelled canine prolactin to antiserum only at very high dose levels. In Beagle dogs, basal serum prolactin concentrations were in the range 1 to 2 ng/ml in normal male, normal female (metoestrus and anoestrus) and oophorectomized-hysterectomized female dogs. The prolactin concentration in one sample of amniotic fluid was in the same range, while in hypophysectomized make dogs no serum prolactin could be detected by the assay system. Serum prolactin concentrations tended to increase during late pregnancy and parturition, remaining high during the first 9 days of lactation. In consequence, a negative correlation was suggested between serum prolactin and serum progesterone concentrations. (author)

  12. Prolactins, ch. 11

    International Nuclear Information System (INIS)

    Friesen, H.; Guyda, H.; Hwang, P.

    1976-01-01

    A radioimmunoassay for primate prolactin is described. Synthesis, secretion and immunological properties of prolactin in comparison to the human growth hormone and separation of the two are studied using 3 H-L-leucine. Prolactin was labelled with 131 I by the chloramine-T method of Hunter and Greenwood. Purification was performed by a sepharose column, coupled with antibodies to ovine prolactine from which the prolactin is subsequently eluted. The assay procedure and the cross-reactivity of different materials in the assay are described. Prolactin concentrations in healthy people, patients and pregnant women (amniotic fluid) are examined

  13. Prolactin promotes breast cancer cell migration through actin cytoskeleton remodeling

    Directory of Open Access Journals (Sweden)

    Priscilla Ludovico da Silva

    2015-12-01

    Full Text Available The role of prolactin on breast cancer development and progression is debated. Breast cancer progression largely depends on cell movement and on the ability to remodel the actin cytoskeleton. In this process, actin-binding proteins are requested to achieve fibrillar actin de-polymerization and relocation at the cell membrane. Kinases such as focal adhesion kinase (FAK are later required to form actin/vinculin-enriched structures called focal adhesion complexes, which mediate firm adhesion to the extracellular matrix. These controllers are regulated by c-Src, which forms multiprotein signaling complexes with membrane receptors and is regulated by a number of hormones, including prolactin. We here show that breast cancer cells exposed to prolactin display an elevated c-Src expression and phosphorylation. In parallel, increased moesin and FAK expression and phosphorylation are found. These molecular changes are associated to relocation to the plasma membrane of cytoskeletal actin fibers and to increased horizontal cell movement. In conclusion, prolactin regulates actin remodeling and enhances breast cancer cell movement. This finding broadens the understanding of prolactin actions on breast cancer cells, highlighting new pathways that may be relevant to on breast cancer progression.

  14. Are sexual side effects of prolactin-raising antipsychotics reducible to serum prolactin?

    NARCIS (Netherlands)

    Knegtering, Henderikus; van den Bosch, Rob; Castelein, Stynke; Bruggeman, Richard; Sytema, Sjoerd; van Os, Jim

    Objective: To assess the degree to which sexual side effects (SSE) are associated with prolactin-raising antipsychotics, and to what degree such SSE are reducible to serum prolactin levels. Method: A large sample (n = 264) of patients treated for 6 weeks with protactin-raising and prolactin-sparing

  15. Differences in both glycosylation and binding properties between rat and mouse liver prolactin receptors.

    Science.gov (United States)

    Lascols, O; Cherqui, G; Munier, A; Picard, J; Capeau, J

    1994-05-01

    To investigate whether glycanic chains of prolactin receptors (PRL-R) play a role in hormone binding activity, comparison was made of rat and mouse liver solubilized receptors with respect to both their affinity for the hormone and their glycosylation properties. As compared with rat receptors, mouse receptors exhibited a 2-fold higher affinity for human growth hormone (hGH), the hormone being bound by both tissues with a lactogenic specificity. Along with this increased affinity, mouse receptors had a 2 lower M(r) relative to rat receptors (62 kDa versus 64 kDa as measured on hGH cross-linked receptors). These differences could be ascribed to different glycosylation properties of the receptors from the two species, as supported by the followings. 1) After treatment with endoglycosidase F (endo F), rat and mouse PRL-R no longer exhibited any difference in their M(r) (54 kDa for both cross-linked receptors). 2) Neuraminidase treatment increased by 37% the binding of hGH to mouse receptors, but was ineffective on the hormone-binding to rat receptors. Conversely, wheat germ agglutinin (WGA), another sialic acid specific probe, decreased hGH binding to rat receptors by 25%, but had no effect on this process for mouse ones. 3) Marked differences were observed in the recoveries of rat and mouse hormone-receptor (HR) complexes from ricin-1- (RCA1-), concanavalin A- (ConA-) and WGA-immobilized lectins. These differences were reduced (RCA1 and ConA) or abolished (WGA) after rat and mouse receptor desialylation by neuraminidase, a treatment which decreased the M(r) of both receptors by 2 kDa. Taken together, these results strongly suggest that the PRL-R from rat and mouse liver contain biantennary N-linked oligosaccharidic chains with distinct type of sialylation, which may account for their differential hormone-binding affinities.

  16. Prolactin-stimulated mitogenesis in the Nb2 rat lymphoma cell: Lack of protoporphyrin IX effects

    Energy Technology Data Exchange (ETDEWEB)

    Gerrish, K.E.; Putnam, C.W.; Laird, H.E. II (Univ. of Arizona, Tucson (USA))

    1990-01-01

    Pharmacological characterization of the Nb2 cell peripheral-type benzodiazepine receptor (PBR) was determined using selected 1,4-benzodiazepines, PK 11195, and protoporphyrin IX (PPIX) to compete for specific ({sup 3}H) Ro5-4864 binding. These data suggest that PPIX possesses an affinity for the Nb2 cell PBR. We have previously reported that the peripheral benzodiazepine ligands, Ro5-4864 and PK 11195, modulate prolactin-stimulated mitogenesis in the Nb2 cell. In contrast, PPIX, a putative endogenous ligand for the PBR had no effect on prolactin-stimulated mitogenesis in the Nb2 cell over the concentration range from 10{sup {minus}15} M to 10{sup {minus}6} M. Taken together these data show that PPIX has an affinity for the Nb2 cell PBR but does not modulate prolactin-stimulated mitogenesis at concentrations which should bind to the Nb2 cell PBR.

  17. The Beckman DxI 800 prolactin assay demonstrates superior specificity for monomeric prolactin.

    LENUS (Irish Health Repository)

    Byrne, Brendan

    2010-02-01

    Commercially available prolactin immunoassays detect macroprolactin to variable degrees. Best practice requires laboratories to assess the cross-reactivity of their prolactin assay with macroprolactin, and where appropriate, introduce a screen for the presence of macroprolactin. Our policy has been to reanalyse hyperprolactinaemic samples following polyethylene glycol (PEG) precipitation and to report the resultant value as the monomeric prolactin content of the sample. The goal of this study was to determine the need to continue PEG precipitation when prolactin measurements with the Wallac AutoDELFIA were replaced by the Beckman DxI 800.

  18. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-01-01

    High (17 nM) and low (603 nM) affinity binding sites for [ 3 ]tetrahydrotrazodone ([ 3 ] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [ 3 ]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [ 3 ] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [ 3 ]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  19. Prolactin blood test

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003718.htm Prolactin blood test To use the sharing features on this page, ... test measures the amount of prolactin in the blood. How the Test is Performed A blood sample is needed . How ...

  20. Prolactin-sensitive neurons express estrogen receptor-α and depend on sex hormones for normal responsiveness to prolactin.

    Science.gov (United States)

    Furigo, Isadora C; Kim, Ki Woo; Nagaishi, Vanessa S; Ramos-Lobo, Angela M; de Alencar, Amanda; Pedroso, João A B; Metzger, Martin; Donato, Jose

    2014-05-30

    Estrogens and prolactin share important target tissues, including the gonads, brain, liver, kidneys and some types of cancer cells. Herein, we sought anatomical and functional evidence of possible crosstalk between prolactin and estrogens in the mouse brain. First, we determined the distribution of prolactin-responsive neurons that express the estrogen receptor α (ERα). A large number of prolactin-induced pSTAT5-immunoreactive neurons expressing ERα mRNA were observed in several brain areas, including the anteroventral periventricular nucleus, medial preoptic nucleus, arcuate nucleus of the hypothalamus, ventrolateral subdivision of the ventromedial nucleus of the hypothalamus (VMH), medial nucleus of the amygdala and nucleus of the solitary tract. However, although the medial preoptic area, periventricular nucleus of the hypothalamus, paraventricular nucleus of the hypothalamus, retrochiasmatic area, dorsomedial subdivision of the VMH, lateral hypothalamic area, dorsomedial nucleus of the hypothalamus and ventral premammillary nucleus contained significant numbers of prolactin-responsive neurons, these areas showed very few pSTAT5-immunoreactive cells expressing ERα mRNA. Second, we evaluated prolactin sensitivity in ovariectomized mice and observed that sex hormones are required for a normal responsiveness to prolactin as ovariectomized mice showed a lower number of prolactin-induced pSTAT5 immunoreactive neurons in all analyzed brain nuclei compared to gonad-intact females. In addition, we performed hypothalamic gene expression analyses to determine possible post-ovariectomy changes in components of prolactin signaling. We observed no significant changes in the mRNA expression of prolactin receptor, STAT5a or STAT5b. In summary, sex hormones exert a permissive role in maintaining the brain's prolactin sensitivity, most likely through post-transcriptional mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Thioredoxin binding site of phosphoribulokinase overlaps the catalytic site

    International Nuclear Information System (INIS)

    Porter, M.A.; Hartman, F.C.

    1986-01-01

    The ATP-regulatory binding site of phosphoribulokinase was studied using bromoacetylethanolamine phosphate (BrAcNHEtOP). BrAcNHEtOP binds to the active-regulatory binding site of the protein. Following trypsin degradation of the labeled protein, fragments were separated by HPLC and sequenced. (DT)

  2. Obtention of antibodies anti prolactin from human prolactin of national production

    International Nuclear Information System (INIS)

    Caso, R.; Mosquera, M.; Perez, E.; Amanz, C.

    1996-01-01

    In this work was studied the use of the the Prolactin hormone as immuno gen, which is obtained in Cuba by the pharmaceutical institute Mario Munoz, to produce the antibody antiprolactin. Was made the validation of obtained antibody (tritatium, specificity and affinity) The produced antibody had necessary quality to be use as a component of the Kits-RIA Prolactin

  3. Serum prolactin level in patients taking olanzapine

    Directory of Open Access Journals (Sweden)

    Diganta Das

    2015-01-01

    Full Text Available Introduction: Olanzapine is a commonly used antipsychotic. Prolactin elevation is a common adverse effect of anstipsychotics, and serum prolactin elevation is seen in about 30% patients treated with olanzapine. There are confounding results about dose dependency of olanzapine and prolactin elevation, and also the duration of treatment. Method: Fifty six patients, 36 male and 20 female, who were taking olanzapine for any condition for more than a month at a constant dose were enrolled in the study. Patients’ age, weight, body mass index (BMI, serum prolactin levels, and some biochemical values were recorded. Patients were taken from the review outpatient department (OPD after due consent. Results: Five each in male and female groups showed elevation of serum prolactin (estimated to be high if >20 ng/dl for males, and >25 ng/dl for females. In females, the elevation was found at lesser dose of olanzapine (13 mg/day, in males 18 mg/day and early in the treatment (2.4 months vs. 9.7 months in males. Males tended to show raised prolactin with higher doses of olanzapine (mean 18 mg/day. Females (26.31% also showed higher prevalence of prolactin elevation compared to males (13.51%. No other parameter was found to modify the prolactin levels. Conclusion: Olanzapine causes elevation of serum prolactin, though lesser degree than some other antipsychotics. Females are more prone to have raised serum prolactin with olanzapine compared to males. However, the elevation seems to be transient. Higher doses of olanzapine tend to cause elevation of serum prolactin. Serum prolactin estimation in patients taking olanzapine may be undertaken to maintain quality life, particularly in females.

  4. LIBRA: LIgand Binding site Recognition Application.

    Science.gov (United States)

    Hung, Le Viet; Caprari, Silvia; Bizai, Massimiliano; Toti, Daniele; Polticelli, Fabio

    2015-12-15

    In recent years, structural genomics and ab initio molecular modeling activities are leading to the availability of a large number of structural models of proteins whose biochemical function is not known. The aim of this study was the development of a novel software tool that, given a protein's structural model, predicts the presence and identity of active sites and/or ligand binding sites. The algorithm implemented by ligand binding site recognition application (LIBRA) is based on a graph theory approach to find the largest subset of similar residues between an input protein and a collection of known functional sites. The algorithm makes use of two predefined databases for active sites and ligand binding sites, respectively, derived from the Catalytic Site Atlas and the Protein Data Bank. Tests indicate that LIBRA is able to identify the correct binding/active site in 90% of the cases analyzed, 90% of which feature the identified site as ranking first. As far as ligand binding site recognition is concerned, LIBRA outperforms other structure-based ligand binding sites detection tools with which it has been compared. The application, developed in Java SE 7 with a Swing GUI embedding a JMol applet, can be run on any OS equipped with a suitable Java Virtual Machine (JVM), and is available at the following URL: http://www.computationalbiology.it/software/LIBRAv1.zip. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Adaptive evolution of transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Berg Johannes

    2004-10-01

    Full Text Available Abstract Background The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. Results We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background mutation rate, the selection coefficient, and the effective population size. Conclusions The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.

  6. Prolactin receptors in Rip-cre cells, but not in AgRP neurones, are involved in energy homeostasis.

    Science.gov (United States)

    Ladyman, S R; MacLeod, M A; Khant Aung, Z; Knowles, P; Phillipps, H R; Brown, R S E; Grattan, D R

    2017-10-01

    Among its many functions, prolactin has been implicated in energy homeostasis, particularly during pregnancy and lactation. The arcuate nucleus is a key site in the regulation of energy balance. The present study aimed to examine whether arcuate nucleus neuronal populations involved in energy homeostasis are prolactin responsive and whether they can mediate the effects of prolactin on energy homeostasis. To determine whether Agrp neurones or Rip-Cre neurones are prolactin responsive, transgenic mice expressing the reporter td-tomato in Agrp neurones (td-tomato/Agrp-Cre) or Rip-Cre neurones (td-tomato/Rip-Cre) were treated with prolactin and perfused 45 minutes later. Brains were processed for double-labelled immunohistochemistry for pSTAT5, a marker of prolactin-induced intracellular signalling, and td-tomato. In addition, Agrp-Cre mice and Rip-Cre mice were crossed with mice in which the prolactin receptor gene (Prlr) was flanked with LoxP sites (Prlr lox/lox mice). The Prlr lox/lox construct was designed such that Cre-mediated recombination resulted in deletion of the Prlr and expression of green fluorescent protein (GFP) in its place. In td-tomato/Rip-Cre mice, prolactin-induced pSTAT5 was co-localised with td-tomato, indicating that there is a subpopulation of Rip-Cre neurones in the arcuate nucleus that respond to prolactin. Furthermore, mice with a specific deletion of Prlr in Rip-Cre neurones had lower body weights, increased oxygen consumption, increased running wheel activity and numerous cells in the arcuate nucleus had positive GFP staining indicating deletion of Prlr from Rip-Cre neurones. By contrast, no co-localisation of td-tomato and pSTAT5 was observed in td-tomato/Agrp-Cre mice after prolactin treatment. Moreover, Prlr lox/lox /Agrp-Cre mice had no positive GFP staining in the arcuate nucleus and did not differ in body weight compared to littermate controls. Overall, these results indicate that Rip-Cre neurones in the arcuate nucleus are

  7. Bitopic Ligands and Metastable Binding Sites

    DEFF Research Database (Denmark)

    Fronik, Philipp; Gaiser, Birgit I; Sejer Pedersen, Daniel

    2017-01-01

    of orthosteric binding sites. Bitopic ligands have been employed to address the selectivity problem by combining (linking) an orthosteric ligand with an allosteric modulator, theoretically leading to high-affinity subtype selective ligands. However, it remains a challenge to identify suitable allosteric binding...... that have been reported to date, this type of bitopic ligands would be composed of two identical pharmacophores. Herein, we outline the concept of bitopic ligands, review metastable binding sites, and discuss their potential as a new source of allosteric binding sites....

  8. Clinical indications of prolactin radioimmunoassay

    International Nuclear Information System (INIS)

    Lengyel, A.M.J.; Vieira, J.G.H.; Zanella, M.R.; Zampieri, M.; Chacra, A.R.

    1980-01-01

    Is a review is presented of the main clinical uses of prolactin measurements, including the galactorrhea-amenorrhea syndrome, an experiment employing the prolactin radioimmunoassay is related. (Author) [pt

  9. Prolactin release, oestrogens and proliferation of prolactin-secreting cells in the anterior pituitary gland of adult male rats.

    Science.gov (United States)

    Pérez, R L; Machiavelli, G A; Romano, M I; Burdman, J A

    1986-03-01

    Relationships among the release of prolactin, the effect of oestrogens and the proliferation of prolactin-secreting cells were studied under several experimental conditions. Administration of sulpiride or oestradiol released prolactin and stimulated cell proliferation in the anterior pituitary gland of adult male rats. Clomiphene completely abolished the rise in cell proliferation, but did not interfere with the sulpiride-induced release of prolactin. Treatment with oestradiol plus sulpiride significantly increased serum prolactin concentrations and the mitotic index compared with the sum of the stimulation produced by both drugs separately. Bromocriptine abolished the stimulatory effect of oestradiol on the serum prolactin concentration and on cell proliferation. In oestradiol- and/or sulpiride-treated rats, 80% of the cells in mitoses were lactotrophs. The remaining 20% did not stain with antisera against any of the pituitary hormones. The number of prolactin-secreting cells in the anterior pituitary gland significantly increased after the administration of oestradiol or sulpiride. The results demonstrate that treatment with sulpiride and/or oestradiol increases the proliferation and the number of lactotrophs in the anterior pituitary gland of the rat.

  10. Prolactin and teleost ionocytes: new insights into cellular and molecular targets of prolactin in vertebrate epithelia

    Science.gov (United States)

    Breves, Jason P.; McCormick, Stephen D.; Karlstrom, Rolf O.

    2014-01-01

    The peptide hormone prolactin is a functionally versatile hormone produced by the vertebrate pituitary. Comparative studies over the last six decades have revealed that a conserved function for prolactin across vertebrates is the regulation of ion and water transport in a variety of tissues including those responsible for whole-organism ion homeostasis. In teleost fishes, prolactin was identified as the “freshwater-adapting hormone”, promoting ion-conserving and water-secreting processes by acting on the gill, kidney, gut and urinary bladder. In mammals, prolactin is known to regulate renal, intestinal, mammary and amniotic epithelia, with dysfunction linked to hypogonadism, infertility, and metabolic disorders. Until recently, our understanding of the cellular mechanisms of prolactin action in fishes has been hampered by a paucity of molecular tools to define and study ionocytes, specialized cells that control active ion transport across branchial and epidermal epithelia. Here we review work in teleost models indicating that prolactin regulates ion balance through action on ion transporters, tight-junction proteins, and water channels in ionocytes, and discuss recent advances in our understanding of ionocyte function in the genetically and embryonically accessible zebrafish (Danio rerio). Given the high degree of evolutionary conservation in endocrine and osmoregulatory systems, these studies in teleost models are contributing novel mechanistic insight into how prolactin participates in the development, function, and dysfunction of osmoregulatory systems across the vertebrate lineage.

  11. Interactions between prolactin and kisspeptin to control reproduction.

    Science.gov (United States)

    Donato, Jose; Frazão, Renata

    2016-01-01

    Prolactin is best known for its effects of stimulating mammary gland development and lactogenesis. However, prolactin is a pleiotropic hormone that is able to affect several physiological functions, including fertility. Prolactin receptors (PRLRs) are widely expressed in several tissues, including several brain regions and reproductive tract organs. Upon activation, PRLRs may exert prolactin's functions through several signaling pathways, although the recruitment of the signal transducer and activator of transcription 5 causes most of the known effects of prolactin. Pathological hyperprolactinemia is mainly due to the presence of a prolactinoma or pharmacological effects induced by drugs that interact with the dopamine system. Notably, hyperprolactinemia is a frequent cause of reproductive dysfunction and may lead to infertility in males and females. Recently, several studies have indicated that prolactin may modulate the reproductive axis by acting on specific populations of hypothalamic neurons that express the Kiss1 gene. The Kiss1 gene encodes neuropeptides known as kisspeptins, which are powerful activators of gonadotropin-releasing hormone neurons. In the present review, we will summarize the current knowledge about prolactin's actions on reproduction. Among other aspects, we will discuss whether the interaction between prolactin and the Kiss1-expressing neurons can affect reproduction and how kisspeptins may become a novel therapeutic approach to treat prolactin-induced infertility.

  12. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    International Nuclear Information System (INIS)

    Lummis, S.C.R.; Johnston, G.A.R.; Nicoletti, G.; Holan, G.

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial [ 3 H]diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli [ 3 H]diazepam binding are those that are active in displacing [ 3 H]benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed

  13. CaMELS: In silico prediction of calmodulin binding proteins and their binding sites.

    Science.gov (United States)

    Abbasi, Wajid Arshad; Asif, Amina; Andleeb, Saiqa; Minhas, Fayyaz Ul Amir Afsar

    2017-09-01

    Due to Ca 2+ -dependent binding and the sequence diversity of Calmodulin (CaM) binding proteins, identifying CaM interactions and binding sites in the wet-lab is tedious and costly. Therefore, computational methods for this purpose are crucial to the design of such wet-lab experiments. We present an algorithm suite called CaMELS (CalModulin intEraction Learning System) for predicting proteins that interact with CaM as well as their binding sites using sequence information alone. CaMELS offers state of the art accuracy for both CaM interaction and binding site prediction and can aid biologists in studying CaM binding proteins. For CaM interaction prediction, CaMELS uses protein sequence features coupled with a large-margin classifier. CaMELS models the binding site prediction problem using multiple instance machine learning with a custom optimization algorithm which allows more effective learning over imprecisely annotated CaM-binding sites during training. CaMELS has been extensively benchmarked using a variety of data sets, mutagenic studies, proteome-wide Gene Ontology enrichment analyses and protein structures. Our experiments indicate that CaMELS outperforms simple motif-based search and other existing methods for interaction and binding site prediction. We have also found that the whole sequence of a protein, rather than just its binding site, is important for predicting its interaction with CaM. Using the machine learning model in CaMELS, we have identified important features of protein sequences for CaM interaction prediction as well as characteristic amino acid sub-sequences and their relative position for identifying CaM binding sites. Python code for training and evaluating CaMELS together with a webserver implementation is available at the URL: http://faculty.pieas.edu.pk/fayyaz/software.html#camels. © 2017 Wiley Periodicals, Inc.

  14. Mu opioid receptor binding sites in human brain

    International Nuclear Information System (INIS)

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand [ 3 H]DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of [ 3 H]DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas

  15. Prostate response to prolactin in sexually active male rats

    Directory of Open Access Journals (Sweden)

    Garcia Luis I

    2006-05-01

    Full Text Available Abstract Background The prostate is a key gland in the sexual physiology of male mammals. Its sensitivity to steroid hormones is widely known, but its response to prolactin is still poorly known. Previous studies have shown a correlation between sexual behaviour, prolactin release and prostate physiology. Thus, here we used the sexual behaviour of male rats as a model for studying this correlation. Hence, we developed experimental paradigms to determine the influence of prolactin on sexual behaviour and prostate organization of male rats. Methods In addition to sexual behaviour recordings, we developed the ELISA procedure to quantify the serum level of prolactin, and the hematoxilin-eosin technique for analysis of the histological organization of the prostate. Also, different experimental manipulations were carried out; they included pituitary grafts, and haloperidol and ovine prolactin treatments. Data were analyzed with a One way ANOVA followed by post hoc Dunnet test if required. Results Data showed that male prolactin has a basal level with two peaks at the light-dark-light transitions. Consecutive ejaculations increased serum prolactin after the first ejaculation, which reached the highest level after the second, and started to decrease after the third ejaculation. These normal levels of prolactin did not induce any change at the prostate tissue. However, treatments for constant elevations of serum prolactin decreased sexual potency and increased the weight of the gland, the alveoli area and the epithelial cell height. Treatments for transient elevation of serum prolactin did not affect the sexual behaviour of males, but triggered these significant effects mainly at the ventral prostate. Conclusion The prostate is a sexual gland that responds to prolactin. Mating-induced prolactin release is required during sexual encounters to activate the epithelial cells in the gland. Here we saw a precise mechanism controlling the release of prolactin

  16. LIGAND-BINDING SITES ON THE MYCOBACTERIUM TUBERCULOSIS UREASE

    Directory of Open Access Journals (Sweden)

    Lisnyak Yu. V.

    2017-10-01

    Full Text Available Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis that remains a serious medical and social health problem. Despite intensive efforts have been made in the past decade, there are no new efficient anti-tuberculosis drugs today, and that need is growing due to the spread of drug-resistant strains of M.tuberculosis. M. tuberculosis urease (MTU, being an important factor of the bacterium viability and virulence, is an attractive target for anti-tuberculosis drugs acting by inhibition of urease activity. However, the commercially available urease inhibitors are toxic and unstable, that prevent their clinical use. Therefore, new more potent anti-tuberculosis drugs inhibiting new targets are urgently needed. A useful tool for the search of novel inhibitors is a computational drug design. The inhibitor design is significantly easier if binding sites on the enzyme are identified in advance. This paper aimed to determine the probable ligand binding sites on the surface of M. tuberculosis urease. Methods. To identify ligand binding sites on MTU surface, сomputational solvent mapping method FTSite was applied by the use of MTU homology model we have built earlier. The method places molecular probes (small organic molecules containing various functional groups on a dense grid defined around the enzyme, and for each probe finds favorable positions. The selected poses are refined by free energy minimization, the low energy conformations are clustered, and the clusters are ranked on the basis of the average free energy. FTSite server outputs the protein residues delineating a binding sites and the probe molecules representing each cluster. To predict allosteric pockets on MTU, AlloPred and AlloSite servers were applied. AlloPred uses the normal mode analysis (NMA and models how the dynamics of a protein would be altered in the presence of a modulator at a specific pocket. Pockets on the enzyme are predicted using the Fpocket

  17. Prolactin secretion: the impact of dynamic studies

    International Nuclear Information System (INIS)

    L'Hermite, M.; Degueldre, M.; Caufriez, A.; Delvoye, P.; Robyn, C.

    1975-01-01

    Human prolactin blood levels were determined by radioimmunoassay in basal condition and in response to various inhibiting and/or stimulating agents (levodopa, water overload, insulinic hypoglycaemia, synthetic TRH, sulpiride) in cases of disturbed hypothalamo-pituitary axis (failure to lactate, prolactin-secreting pituitary adenomas, acromegaly, other pituitary tumours, clinical panhypopituitarism). A blunted prolactin response to suckling was evidenced in 2 post-partum women who were unable to breast feed. Hyperprolactinaemia, whether related to the existence of a prolactin-producing adenoma or not was associated with the disappearance of the normal circadian pattern of prolactin secretion and with a blunted relative response to TRH; the latter phenomenon occurred also in acromegaly regardless of the basal prolactinaemia, and during the last trimester of pregnancy. Water overload was unsuccessful to suppress prolactin during the last trimester of pregnancy while the acute administration of levodopa was quite effective in about half of the patients with pituitary tumour. Therefore none of the dynamic tests presently studied allowed to attribute a hyperprolactinaemia to a pituitary tumour rather than to a functional disturbance. On the contrary, stimulation tests can help to locate the level of a defect in cases of hypopituitarism [fr

  18. RBPmap: a web server for mapping binding sites of RNA-binding proteins.

    Science.gov (United States)

    Paz, Inbal; Kosti, Idit; Ares, Manuel; Cline, Melissa; Mandel-Gutfreund, Yael

    2014-07-01

    Regulation of gene expression is executed in many cases by RNA-binding proteins (RBPs) that bind to mRNAs as well as to non-coding RNAs. RBPs recognize their RNA target via specific binding sites on the RNA. Predicting the binding sites of RBPs is known to be a major challenge. We present a new webserver, RBPmap, freely accessible through the website http://rbpmap.technion.ac.il/ for accurate prediction and mapping of RBP binding sites. RBPmap has been developed specifically for mapping RBPs in human, mouse and Drosophila melanogaster genomes, though it supports other organisms too. RBPmap enables the users to select motifs from a large database of experimentally defined motifs. In addition, users can provide any motif of interest, given as either a consensus or a PSSM. The algorithm for mapping the motifs is based on a Weighted-Rank approach, which considers the clustering propensity of the binding sites and the overall tendency of regulatory regions to be conserved. In addition, RBPmap incorporates a position-specific background model, designed uniquely for different genomic regions, such as splice sites, 5' and 3' UTRs, non-coding RNA and intergenic regions. RBPmap was tested on high-throughput RNA-binding experiments and was proved to be highly accurate. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Autoradiographic localization of benzomorphan binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Crain, B.J.; Kwenjen Chang; McNamara, J.O.; Valdes, F.

    1985-07-17

    The benzomorphan subpopulation of opiate binding sites was labeled by (TH)diprenorphine in the presence of unlabeled ligands selected to quench and delta opiate binding sites. The distribution of benzomorphan binding sites was then localized autoradiographically. The distribution differs from the distributions of , delta and kappa opiate binding and is quite similar to the distribution of US -endorphin immunoreactivity. These observations support the hypothesis, based on biochemical studies in brain membranes, that benzomorphan binding sites may represent the ligand recognition sites of putative epsilon receptors. (Auth.). 34 refs.; 3 figs.

  20. Effects of light deprivation on prolactin regulation in the Golden Syrian hamster

    International Nuclear Information System (INIS)

    Massa, J.S.

    1986-01-01

    Pineal-mediated depressions in prolactin cell activity after light deprivation were studied in the male and female Golden Syrian hamster. Prolactin cell activity was determined by measuring radioimmunoassayable prolactin, newly synthesized prolactin, newly synthesized prolactin and prolactin mRNA levels in the pituitary. Serum prolactin was also measured by radioimmunoassay. Use of the recombinant DNA plasmid, pPRL-1, which contains the rat prolactin complimentary DNA sequence, was validated in this dissertation for measuring prolactin mRNA in the hamster. Male Hamsters blinded for 11, 21, or 42 days showed significant and progressively greater declines in prolactin mRNA levels which were completely prevented by pinealectomy. Female hamsters blinded for 28 days, however, showed no such decreases in prolactin cell activity if they continued to display estrous cyclicity. After 12 weeks of blinding, females were acyclic and had dramatically depressed levels of prolactin cell activity. However, pinealectomy did not completely prevent this decline due to blinding unless the females continue to display estrous cyclicity. In ovariectomized females, blinding caused a decline in prolactin cell activity. In a separate study, significant changes in prolactin cell activity during the estrous cycle were seen in untreated normally cycling female hamsters. These changes in prolactin mRNA, prolactin synthesis, and radioimmunoassayable prolactin in the pituitary were measured in the morning, when, consistent with other reports, no differences in serum prolactin were observed

  1. Evolution of Metal(Loid) Binding Sites in Transcriptional Regulators

    Energy Technology Data Exchange (ETDEWEB)

    Ordonez, E.; Thiyagarajan, S.; Cook, J.D.; Stemmler, T.L.; Gil, J.A.; Mateos, L.M.; Rosen, B.P.

    2009-05-22

    Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys{sup 15}, Cys{sup 16}, and Cys{sup 55}. This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

  2. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

    2016-01-13

    Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  3. Domain-based small molecule binding site annotation

    Directory of Open Access Journals (Sweden)

    Dumontier Michel

    2006-03-01

    Full Text Available Abstract Background Accurate small molecule binding site information for a protein can facilitate studies in drug docking, drug discovery and function prediction, but small molecule binding site protein sequence annotation is sparse. The Small Molecule Interaction Database (SMID, a database of protein domain-small molecule interactions, was created using structural data from the Protein Data Bank (PDB. More importantly it provides a means to predict small molecule binding sites on proteins with a known or unknown structure and unlike prior approaches, removes large numbers of false positive hits arising from transitive alignment errors, non-biologically significant small molecules and crystallographic conditions that overpredict ion binding sites. Description Using a set of co-crystallized protein-small molecule structures as a starting point, SMID interactions were generated by identifying protein domains that bind to small molecules, using NCBI's Reverse Position Specific BLAST (RPS-BLAST algorithm. SMID records are available for viewing at http://smid.blueprint.org. The SMID-BLAST tool provides accurate transitive annotation of small-molecule binding sites for proteins not found in the PDB. Given a protein sequence, SMID-BLAST identifies domains using RPS-BLAST and then lists potential small molecule ligands based on SMID records, as well as their aligned binding sites. A heuristic ligand score is calculated based on E-value, ligand residue identity and domain entropy to assign a level of confidence to hits found. SMID-BLAST predictions were validated against a set of 793 experimental small molecule interactions from the PDB, of which 472 (60% of predicted interactions identically matched the experimental small molecule and of these, 344 had greater than 80% of the binding site residues correctly identified. Further, we estimate that 45% of predictions which were not observed in the PDB validation set may be true positives. Conclusion By

  4. Opioid binding sites in the guinea pig and rat kidney: Radioligand homogenate binding and autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Dissanayake, V.U.; Hughes, J.; Hunter, J.C. (Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge (England))

    1991-07-01

    The specific binding of the selective {mu}-, {delta}-, and {kappa}-opioid ligands (3H)(D-Ala2,MePhe4,Gly-ol5)enkephalin ((3H) DAGOL), (3H)(D-Pen2,D-Pen5)enkephalin ((3H)DPDPE), and (3H)U69593, respectively, to crude membranes of the guinea pig and rat whole kidney, kidney cortex, and kidney medulla was investigated. In addition, the distribution of specific 3H-opioid binding sites in the guinea pig and rat kidney was visualized by autoradiography. Homogenate binding and autoradiography demonstrated the absence of {mu}- and {kappa}-opioid binding sites in the guinea pig kidney. No opioid binding sites were demonstrable in the rat kidney. In the guinea pig whole kidney, cortex, and medulla, saturation studies demonstrated that (3H)DPDPE bound with high affinity (KD = 2.6-3.5 nM) to an apparently homogeneous population of binding sites (Bmax = 8.4-30 fmol/mg of protein). Competition studies using several opioid compounds confirmed the nature of the {delta}-opioid binding site. Autoradiography experiments demonstrated that specific (3H)DPDPE binding sites were distributed radially in regions of the inner and outer medulla and at the corticomedullary junction of the guinea pig kidney. Computer-assisted image analysis of saturation data yielded KD values (4.5-5.0 nM) that were in good agreement with those obtained from the homogenate binding studies. Further investigation of the {delta}-opioid binding site in medulla homogenates, using agonist ((3H)DPDPE) and antagonist ((3H)diprenorphine) binding in the presence of Na+, Mg2+, and nucleotides, suggested that the {delta}-opioid site is linked to a second messenger system via a GTP-binding protein. Further studies are required to establish the precise localization of the {delta} binding site in the guinea pig kidney and to determine the nature of the second messenger linked to the GTP-binding protein in the medulla.

  5. Sex differences in the hypothalamic control of prolactin secretion

    International Nuclear Information System (INIS)

    Grattan, D.R.; Liu, L.; Bunn, S.J.

    2001-01-01

    Full text: Sex differences in the brain may arise from the organisational effects of exposure to sex steroids during development, or from the exposure to a differential hormonal milieu in the adult. There is a marked sex difference in the neuroendocrine mechanism that regulates prolactin secretion. Levels of prolactin in the blood are higher in females than in males. Similarly, basal activity of tuberoinfundibular dopamine (TIDA) neurons, which are involved in the tonic suppression of prolactin secretion, are two fold higher in females than in males. Prolactin is known to stimulate the activity of TIDA neurons, thereby regulating its own secretion by short-loop feedback. Hence, it is thought that elevated TIDA neuronal activity in females is induced by increased prolactin in the blood. We have recently demonstrated that prolactin stimulation of TIDA neurons requires the transcription factor, STAT5b. We have now investigated prolactin secretion in male and female STAT5b-deficient mice, to test the hypothesis that sex differences in TIDA neuronal activity are dependent on stimulation by prolactin acting through STAT5b. Prolactin levels in blood were measured by radioimmunoassay, and TIDA activity was assessed by measuring concentrations of the dopamine metabolite DOPAC in the median eminence by HPLC, and by measuring tyrosine hydroxylase mRNA in the arcuate nucleus by real-time RT-PCR. The data demonstrate marked gender differences in the activity of TIDA neurons. While TIDA activity in STAT5b-deficient mice was reduced compared to wild type, the sex difference persisted. Since STAT5b is required for the actions of prolactin on these neurons, we can conclude that the sexual dimorphism in brain function is independent of gender differences in blood levels of prolactin. It seems likely that differential exposure to gonadal steroid hormones, either during development or in adulthood, might underlie the sex difference in TIDA neuronal activity. Copyright (2001

  6. Detection of secondary binding sites in proteins using fragment screening.

    Science.gov (United States)

    Ludlow, R Frederick; Verdonk, Marcel L; Saini, Harpreet K; Tickle, Ian J; Jhoti, Harren

    2015-12-29

    Proteins need to be tightly regulated as they control biological processes in most normal cellular functions. The precise mechanisms of regulation are rarely completely understood but can involve binding of endogenous ligands and/or partner proteins at specific locations on a protein that can modulate function. Often, these additional secondary binding sites appear separate to the primary binding site, which, for example for an enzyme, may bind a substrate. In previous work, we have uncovered several examples in which secondary binding sites were discovered on proteins using fragment screening approaches. In each case, we were able to establish that the newly identified secondary binding site was biologically relevant as it was able to modulate function by the binding of a small molecule. In this study, we investigate how often secondary binding sites are located on proteins by analyzing 24 protein targets for which we have performed a fragment screen using X-ray crystallography. Our analysis shows that, surprisingly, the majority of proteins contain secondary binding sites based on their ability to bind fragments. Furthermore, sequence analysis of these previously unknown sites indicate high conservation, which suggests that they may have a biological function, perhaps via an allosteric mechanism. Comparing the physicochemical properties of the secondary sites with known primary ligand binding sites also shows broad similarities indicating that many of the secondary sites may be druggable in nature with small molecules that could provide new opportunities to modulate potential therapeutic targets.

  7. Night shift work and prolactin as a breast cancer risk factor

    Directory of Open Access Journals (Sweden)

    Agnieszka Bukowska

    2013-04-01

    Full Text Available Prolactin - a hormone secreted in a circadian rhythm acts as a regulator of growth and development of the mammary glands. It has been observed that working at night increases breast cancer risk in women. Night shift work, probably carcinogenic to humans (Group 2A IARC, can disrupt a circadian rhythm, and thus potentially alter the rhythm of prolactin secretion. The aim of our work was to review epidemiological evidence on the association between prolactin and the risk of breast cancer and the influence of work at night on prolactin secretion. Search was done in the Medline database by keywords (shift work, work at night, risk of breast cancer and prolactin. The increased proliferation of breast cells activated by prolactin can promote the development of cancer. The results of the largest epidemiological prospective studies suggest the association between prolactin levels and the risk of breast cancer in women. So far, only seven studies have investigated the association between work at night and prolactin secretion. In three studies lower concentrations of prolactin have been observed in night shift workers. No relationship between the night shift work duration and prolactin level in women have been reported. Night shift work can modify the profile of prolactin secretion in night workers, probably decreasing the secretion of this hormone at night. It is therefore unlikely that prolactin plays an important role in the development of breast cancer in women working at night. This conclusion is based on the results of a few epidemiological studies. Med Pr 2013;64(2:245–257

  8. Prolactin suppresses malonyl-CoA concentration in human adipose tissue

    DEFF Research Database (Denmark)

    Nilsson, L. A.; Roepstorff, Carsten; Kiens, Bente

    2009-01-01

    Prolactin is best known for its involvement in lactation, where it regulates mechanisms that supply nutrients for milk production. In individuals with pathological hyperprolactinemia, glucose and fat homeostasis have been reported to be negatively influenced. It is not previously known, however......, whether prolactin regulates lipogenesis in human adipose tissue. The aim of this study was to investigate the effect of prolactin on lipogenesis in human adipose tissue in vitro. Prolactin decreased the concentration of malonyl-CoA, the product of the first committed step in lipogenesis, to 77......+/-6% compared to control 100+/-5% (p=0.022) in cultured human adipose tissue. In addition, prolactin was found to decrease glucose transporter 4 ( GLUT4) mRNA expression, which may cause decreased glucose uptake. In conclusion, we propose that prolactin decreases lipogenesis in human adipose tissue...

  9. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    Energy Technology Data Exchange (ETDEWEB)

    Gangi Setty, Thanuja [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Cho, Christine [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Govindappa, Sowmya [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India); Apicella, Michael A. [Carver College of Medicine, University of Iowa, Iowa City, IA 52242-1109 (United States); Ramaswamy, S., E-mail: ramas@instem.res.in [Institute for Stem Cell Biology and Regenerative Medicine, NCBS Campus, GKVK Post, Bangalore, Karnataka 560 065 (India)

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  10. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    International Nuclear Information System (INIS)

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-01-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states

  11. An overview of the prediction of protein DNA-binding sites.

    Science.gov (United States)

    Si, Jingna; Zhao, Rui; Wu, Rongling

    2015-03-06

    Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  12. Serum prolactin revisited

    DEFF Research Database (Denmark)

    Overgaard, Martin; Pedersen, Susanne Møller

    2017-01-01

    regimes across commonly used automated immunoassay platforms. METHODS: Parametric total and monomeric gender-specific reference intervals were determined for six immunoassay methods using female (n=96) and male sera (n=127) from healthy donors. The reference intervals were validated using 27...... and macroprolactinemic; n=27) showed higher discordant classification [mean=2.8; 95% confidence interval (CI) 1.2-4.4] for the monomer reference interval method compared to the post-polyethylene glycol (PEG) recovery cutoff method (mean=1.8; 95% CI 0.8-2.8). The two monomer/macroprolactin discrimination methods did...... not differ significantly (p=0.089). Among macroprolactinemic sera evaluated by both discrimination methods, the Cobas and Architect/Kryptor prolactin assays showed the lowest and the highest number of misclassifications, respectively. CONCLUSIONS: Current automated immunoassays for prolactin testing require...

  13. Binding-site assessment by virtual fragment screening.

    Directory of Open Access Journals (Sweden)

    Niu Huang

    2010-04-01

    Full Text Available The accurate prediction of protein druggability (propensity to bind high-affinity drug-like small molecules would greatly benefit the fields of chemical genomics and drug discovery. We have developed a novel approach to quantitatively assess protein druggability by computationally screening a fragment-like compound library. In analogy to NMR-based fragment screening, we dock approximately 11,000 fragments against a given binding site and compute a computational hit rate based on the fraction of molecules that exceed an empirically chosen score cutoff. We perform a large-scale evaluation of the approach on four datasets, totaling 152 binding sites. We demonstrate that computed hit rates correlate with hit rates measured experimentally in a previously published NMR-based screening method. Secondly, we show that the in silico fragment screening method can be used to distinguish known druggable and non-druggable targets, including both enzymes and protein-protein interaction sites. Finally, we explore the sensitivity of the results to different receptor conformations, including flexible protein-protein interaction sites. Besides its original aim to assess druggability of different protein targets, this method could be used to identifying druggable conformations of flexible binding site for lead discovery, and suggesting strategies for growing or joining initial fragment hits to obtain more potent inhibitors.

  14. Prolactin and Psychopathology in Schizophrenia: A Literature Review and Reappraisal

    Directory of Open Access Journals (Sweden)

    Ravi Philip Rajkumar

    2014-01-01

    Full Text Available Secretion of the anterior pituitary hormone prolactin can be significantly increased by antipsychotic drugs, leading to a range of adverse effects in patients with schizophrenia. However, there is evidence from a variety of studies that prolactin may also be related to symptom profile and treatment response in these patients, and recent work has identified variations in prolactin secretion even in drug-free patients. In this paper, a selective review of all relevant studies pertaining to prolactin and schizophrenia, including challenge and provocation studies, is presented. The implications of this work are discussed critically. A tentative model, which synthesizes these findings and argues for a significant role for prolactin in the development of schizophrenia, is outlined.

  15. Seasonal prolactin secretion and its role in seasonal reproduction: a review.

    Science.gov (United States)

    Curlewis, J D

    1992-01-01

    The majority of seasonally breeding mammals show a seasonal pattern of prolactin secretion with peak concentrations in spring or summer and a nadir in autumn or winter. Photoperiod influences prolactin secretion via its effects on the secretion of the pineal hormone melatonin. Preliminary evidence suggests that the effects of melatonin on both prolactin and gonadotrophin secretion are via a common target area, possibly within the anterior hypothalamus, and that differences in response to photoperiod may be due to differences in the processing and/or interpretation of the melatonin signal. In contrast to seasonal gonadotrophin secretion, the seasonal changes in prolactin are not due to changes in the sensitivity of a feedback loop and so must be due to direct effects on the hypothalamic pathways that control prolactin secretion. Little else can be said with confidence about the neuroendocrine mechanisms that lead to the seasonal changes in prolactin secretion. Dopamine and noradrenaline turnover in the arcuate nucleus and median eminence decrease under short daylength. If catecholamine turnover in these structures is positively correlated with catecholamine concentrations in the long or short hypophysial portal vessels, it is unlikely that the decrease in prolactin concentration in winter is due to the effects of increased concentrations of dopamine or noradrenaline in the portal vessels. There is, however, evidence for increased pituitary sensitivity to dopamine under short daylength, so increased dopamine concentrations may not be required for suppression of prolactin secretion at this time. In addition to the diminished secretion of prolactin under short daylength, rate of prolactin synthesis and pituitary content of prolactin also decline although the mechanisms that regulate these changes are poorly understood. Although all seasonal breeders show a seasonal change in prolactin secretion, there are continuously breeding species in which prolactin secretion is

  16. An Overview of the Prediction of Protein DNA-Binding Sites

    Directory of Open Access Journals (Sweden)

    Jingna Si

    2015-03-01

    Full Text Available Interactions between proteins and DNA play an important role in many essential biological processes such as DNA replication, transcription, splicing, and repair. The identification of amino acid residues involved in DNA-binding sites is critical for understanding the mechanism of these biological activities. In the last decade, numerous computational approaches have been developed to predict protein DNA-binding sites based on protein sequence and/or structural information, which play an important role in complementing experimental strategies. At this time, approaches can be divided into three categories: sequence-based DNA-binding site prediction, structure-based DNA-binding site prediction, and homology modeling and threading. In this article, we review existing research on computational methods to predict protein DNA-binding sites, which includes data sets, various residue sequence/structural features, machine learning methods for comparison and selection, evaluation methods, performance comparison of different tools, and future directions in protein DNA-binding site prediction. In particular, we detail the meta-analysis of protein DNA-binding sites. We also propose specific implications that are likely to result in novel prediction methods, increased performance, or practical applications.

  17. Evaluation of the human prolactin of National Production for use in radioimmunoassay (RIA)

    International Nuclear Information System (INIS)

    Caso, R.; Arranz, C.

    1996-01-01

    In this work was studied the possibility of using the Prolactin hormone as raw material to produce Kits-RIA of Prolactin. Was used the prolactin, which is obtained in Cuba by the Pharmaceutical Institute Mario Munoz. Was made the labbeling of Prolactin with I-125, was used the hormone as standard and were done the probes of quality control. The Prolactin Hormone had the necesary quality to produce Kits-RIA-Prolactin

  18. Localization of gonadotropin binding sites in human ovarian neoplasms

    International Nuclear Information System (INIS)

    Nakano, R.; Kitayama, S.; Yamoto, M.; Shima, K.; Ooshima, A.

    1989-01-01

    The binding of human luteinizing hormone and human follicle-stimulating hormone to ovarian tumor biopsy specimens from 29 patients was analyzed. The binding sites for human luteinizing hormone were demonstrated in one tumor of epithelial origin (mucinous cystadenoma) and in one of sex cord-stromal origin (theca cell tumor). The binding sites for human follicle-stimulating hormone were found in three tumors of epithelial origin (serous cystadenoma and mucinous cystadenoma) and in two of sex cord-stromal origin (theca cell tumor and theca-granulosa cell tumor). The surface-binding autoradiographic study revealed that the binding sites for gonadotropins were localized in the stromal tissue. The results suggest that gonadotropic hormones may play a role in the growth and differentiation of a certain type of human ovarian neoplasms

  19. Development and characterization of a homologous radioimmunoassay for equine prolactin

    International Nuclear Information System (INIS)

    Roser, J.F.; Chang, Y.S.; Papkoff, H.; Li, C.H.

    1984-01-01

    A specific and sensitive homologous radioimmunoassay has been developed for equine prolactin, suitable for measuring prolactin concentrations in serum of horses. The sensitivity of the assay ranged from 0.4 to 0.6 ng/ml and the intra- and inter-assay coefficients of variation averaged 6.9 and 15.4%, respectively, for five doses of hormone. Cross-reactivity with other mammalian and nonmammalian prolactins and growth hormones was less than 20 and 0.3%, respectively. Cross-reactivity with equine growth hormone was less than 0.07%. Equine serum and pituitary extracts showed parallel dilution-response curves with equine prolactin. The percentage recovery of exogenous equine prolactin in serum was 89%. Preliminary analysis of several physiological samples (stallions, pregnant, and nonpregnant mares) yielded values from 0.6 to 12.0 ng/ml

  20. LHRH-pituitary plasma membrane binding: the presence of specific binding sites in other tissues.

    Science.gov (United States)

    Marshall, J C; Shakespear, R A; Odell, W D

    1976-11-01

    Two specific binding sites for LHRH are present on plasma membranes prepared from rat and bovine anterior pituitary glands. One site is of high affinity (K = 2X108 1/MOL) and the second is of lower affinity (8-5X105 1/mol) and much greater capacity. Studies on membrane fractions prepared from other tissues showed the presence of a single specific site for LHRH. The kinetics and specificity of this site were similar to those of the lower affinity pituitary receptor. These results indicate that only pituitary membranes possess the higher affinity binding site and suggest that the low affinity site is not of physiological importance in the regulation of gonadotrophin secretion. After dissociation from membranes of non-pituitary tissues 125I-LHRH rebound to pituitary membrane preparations. Thus receptor binding per se does not result in degradation of LHRH and the function of these peripheral receptors remains obscure.

  1. Transfer of milk prolactin ro the plasma of neonatal rats by intestinal absorption

    Energy Technology Data Exchange (ETDEWEB)

    Whitworth, N S; Grosvenor, C E [Tennessee Univ., Memphis (USA). Dept. of Physiology and Biophysics

    1978-11-01

    Prolactin passes from the systemic circulation of lactating rats into the milk where it can be consumed by the young rats during suckling. /sup 131/- labelled rat prolactin was detected in the plasma of 9- to 14-day-old rats after being nursed by mothers previously injected with /sup 131/I-labelled rat prolactin and after the pups had received /sup 131/I-labelled rat prolactin by gastric intubation. It was estimated that 16% of the /sup 131/I-labelled rat prolactin given by gastric intubation subsequently appeared in the plasma of the neonate. Gastric administration of 10.5 or 21.0 ..mu..g B-1 rat prolactin significantly raised the level of prolactin in the plasma of 13-day-old pups, but a similar increase was not observed when 27-day-old rats were given 46.2 ..mu..g B-1 prolactin by gastric intubation. The concentration of prolactin in the plasma of 13-to 14-day-old rats rose to 55 ng/ml 30 min after the onset of nursing by mothers whose mammary glands were full of milk, whereas the concentration in the plasma of mothers with empty mammary glands remained at basal values. It is concluded that the intestine of the newborn is permeable to prolactin and that milk may constitute an exogeneous source of prolactin for the suckled offspring.

  2. A tool for calculating binding-site residues on proteins from PDB structures

    Directory of Open Access Journals (Sweden)

    Hu Jing

    2009-08-01

    Full Text Available Abstract Background In the research on protein functional sites, researchers often need to identify binding-site residues on a protein. A commonly used strategy is to find a complex structure from the Protein Data Bank (PDB that consists of the protein of interest and its interacting partner(s and calculate binding-site residues based on the complex structure. However, since a protein may participate in multiple interactions, the binding-site residues calculated based on one complex structure usually do not reveal all binding sites on a protein. Thus, this requires researchers to find all PDB complexes that contain the protein of interest and combine the binding-site information gleaned from them. This process is very time-consuming. Especially, combing binding-site information obtained from different PDB structures requires tedious work to align protein sequences. The process becomes overwhelmingly difficult when researchers have a large set of proteins to analyze, which is usually the case in practice. Results In this study, we have developed a tool for calculating binding-site residues on proteins, TCBRP http://yanbioinformatics.cs.usu.edu:8080/ppbindingsubmit. For an input protein, TCBRP can quickly find all binding-site residues on the protein by automatically combining the information obtained from all PDB structures that consist of the protein of interest. Additionally, TCBRP presents the binding-site residues in different categories according to the interaction type. TCBRP also allows researchers to set the definition of binding-site residues. Conclusion The developed tool is very useful for the research on protein binding site analysis and prediction.

  3. Defining the plasticity of transcription factor binding sites by Deconstructing DNA consensus sequences: the PhoP-binding sites among gamma/enterobacteria.

    Directory of Open Access Journals (Sweden)

    Oscar Harari

    2010-07-01

    Full Text Available Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg(2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs using a machine learning method inspired by the "Divide & Conquer" strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target

  4. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    International Nuclear Information System (INIS)

    James, I.F.; Goldstein, A.

    1984-01-01

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [ 3 H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [ 3 H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

  5. DeepSite: protein-binding site predictor using 3D-convolutional neural networks.

    Science.gov (United States)

    Jiménez, J; Doerr, S; Martínez-Rosell, G; Rose, A S; De Fabritiis, G

    2017-10-01

    An important step in structure-based drug design consists in the prediction of druggable binding sites. Several algorithms for detecting binding cavities, those likely to bind to a small drug compound, have been developed over the years by clever exploitation of geometric, chemical and evolutionary features of the protein. Here we present a novel knowledge-based approach that uses state-of-the-art convolutional neural networks, where the algorithm is learned by examples. In total, 7622 proteins from the scPDB database of binding sites have been evaluated using both a distance and a volumetric overlap approach. Our machine-learning based method demonstrates superior performance to two other competitive algorithmic strategies. DeepSite is freely available at www.playmolecule.org. Users can submit either a PDB ID or PDB file for pocket detection to our NVIDIA GPU-equipped servers through a WebGL graphical interface. gianni.defabritiis@upf.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  6. Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.

    Directory of Open Access Journals (Sweden)

    Daniel M Dupont

    Full Text Available Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126 with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA. We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A controlling uPA activities. One of the aptamers (upanap-126 binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12 binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.

  7. Pubertal dependent effects of cadmium on episodic prolactin secretion in male rats

    Energy Technology Data Exchange (ETDEWEB)

    Lafuente, A.; Alvarez-Demanuel, E.; Marquez, N. [Fac. de Cienicas, Orense (Spain). Lab. de Toxicologia; Esquifino, A.I. [Dept. Bioquimica, Facultad de Medicina, Universidad Complutense, 28040-Madrid (Spain)

    1999-02-01

    This work was undertaken to assess if exposure to cadmium related to puberty may affect the episodic pattern of prolactin. Male rats were submitted to cadmium exposure, from day 30 to 60 or from day 60 to 90 of life respectively, at a dose of 50 ppm in the drinking water. Control age-matched rats received cadmium-free water. Prepubertal cadmium administration decreased mean serum prolactin levels and the absolute amplitude of the prolactin pulses. Subchronic exposure to cadmium of adult rats decreased mean serum prolactin levels, the absolute amplitude of the prolactin pulses and their duration, and the mean half-life of the hormone. These results suggest that subchronic cadmium exposure changes the secretory pattern of prolactin in adult male rats in a puberty-dependent way. (orig.) With 1 fig., 1 tab., 37 refs.

  8. Impact of germline and somatic missense variations on drug binding sites.

    Science.gov (United States)

    Yan, C; Pattabiraman, N; Goecks, J; Lam, P; Nayak, A; Pan, Y; Torcivia-Rodriguez, J; Voskanian, A; Wan, Q; Mazumder, R

    2017-03-01

    Advancements in next-generation sequencing (NGS) technologies are generating a vast amount of data. This exacerbates the current challenge of translating NGS data into actionable clinical interpretations. We have comprehensively combined germline and somatic nonsynonymous single-nucleotide variations (nsSNVs) that affect drug binding sites in order to investigate their prevalence. The integrated data thus generated in conjunction with exome or whole-genome sequencing can be used to identify patients who may not respond to a specific drug because of alterations in drug binding efficacy due to nsSNVs in the target protein's gene. To identify the nsSNVs that may affect drug binding, protein-drug complex structures were retrieved from Protein Data Bank (PDB) followed by identification of amino acids in the protein-drug binding sites using an occluded surface method. Then, the germline and somatic mutations were mapped to these amino acids to identify which of these alter protein-drug binding sites. Using this method we identified 12 993 amino acid-drug binding sites across 253 unique proteins bound to 235 unique drugs. The integration of amino acid-drug binding sites data with both germline and somatic nsSNVs data sets revealed 3133 nsSNVs affecting amino acid-drug binding sites. In addition, a comprehensive drug target discovery was conducted based on protein structure similarity and conservation of amino acid-drug binding sites. Using this method, 81 paralogs were identified that could serve as alternative drug targets. In addition, non-human mammalian proteins bound to drugs were used to identify 142 homologs in humans that can potentially bind to drugs. In the current protein-drug pairs that contain somatic mutations within their binding site, we identified 85 proteins with significant differential gene expression changes associated with specific cancer types. Information on protein-drug binding predicted drug target proteins and prevalence of both somatic and

  9. Primary Hypothyroidism with Exceptionally High Prolactin-A Really Big Deal.

    Science.gov (United States)

    Khorassanizadeh, Rahim; Sundaresh, Vishnu; Levine, Steven N

    2016-07-01

    Primary hypothyroidism can cause both hyperprolactinemia and pituitary hyperplasia. The degree of hyperprolactinemia is generally modest, and rarely do prolactin concentrations exceed 100 ng/mL (4.34 nmol/L). This combination of hyperprolactinemia and pituitary gland enlargement might raise suspicion for a prolactinoma or a nonfunctioning adenoma limiting the ability of hypothalamic dopamine to inhibit prolactin production, the so-called "stalk effect." We describe a 30-year-old female with headaches, galactorrhea, and hyperprolactinemia with a presumptive diagnosis of a prolactinoma. She had hypothyroidism after treatment of Graves disease at age 17 years but was noncompliant with levothyroxine replacement. Thyroid-stimulating hormone (TSH) was 263 mIU/L, FT4 was 3.01 pmol/L, and prolactin was 323 ng/mL (14.06 nmol/L). Magnetic resonance imaging (MRI) demonstrated increased volume of the pituitary gland with homogeneous enhancement with gadolinium. Levothyroxine treatment for 2 weeks decreased her TSH to130 mIU/L, but her total prolactin remained elevated at 386 ng/mL (16.78 nmol/L). Further testing identified that 76% of the total prolactin was macroprolactin. Primary hypothyroidism can cause hyperprolactinemia, and prolonged disease may lead to pituitary hyperplasia. However, a marked elevation of prolactin should raise suspicion to investigate additional etiologies for hyperprolactinemia. Our case exemplifies a dual etiology for hyperprolactinemia and pituitary hyperplasia caused by both hypothyroidism and macroprolactin. This knowledge is invaluable for clinicians to avoid unnecessary management with dopamine agonists and/or surgery. This patient's prolactin was 323 ng/mL (14.06 nmol/L). Before our case, the highest prolactin in a hypothyroid patient reported in the literature was 253 ng/mL (11.0 nmol/L). Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Multiple [3H]-nemonapride binding sites in calf brain.

    Science.gov (United States)

    Helmeste, D M; Tang, S W; Li, M; Fang, H

    1997-07-01

    [3H]-Nemonapride has been the ligand of choice to label D4 dopamine receptors. Its specificity was questioned when it was discovered that sigma (sigma) sites were also labeled by [3H]-nemonapride. To further characterize the binding of [3H]-nemonapride, three areas of calf brain (striatum, frontal cortex and cerebellum) were examined. In all three areas, [3H]-nemonapride labeled multiple sites. Dopaminergic and sigma sites were the most prominent. The sigma binding profile was sigma-1 like with a Ki binding profile as follows (in order of decreasing potency): haloperidol, PPAP, pentazocine, DTG, U-50488, R(+)-3-PPP. Experiments using sulpiride and pentazocine to block striatal dopaminergic and sigma sites, respectively, revealed additional, not previously characterized binding sites for [3H]-nemonapride. One component which was present in striatum but not in frontal cortex or cerebellum, had affinity for some neuroleptics and WB-4101, but not for typical serotonergic agents. Thus, [3H]-nemonapride has no selectivity for dopamine receptors unless stringent experimental conditions are met.

  11. Target-mediated drug disposition model for drugs with two binding sites that bind to a target with one binding site.

    Science.gov (United States)

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2017-10-01

    The paper extended the TMDD model to drugs with two identical binding sites (2-1 TMDD). The quasi-steady-state (2-1 QSS), quasi-equilibrium (2-1 QE), irreversible binding (2-1 IB), and Michaelis-Menten (2-1 MM) approximations of the model were derived. Using simulations, the 2-1 QSS approximation was compared with the full 2-1 TMDD model. As expected and similarly to the standard TMDD for monoclonal antibodies (mAb), 2-1 QSS predictions were nearly identical to 2-1 TMDD predictions, except for times of fast changes following initiation of dosing, when equilibrium has not yet been reached. To illustrate properties of new equations and approximations, several variations of population PK data for mAbs with soluble (slow elimination of the complex) or membrane-bound (fast elimination of the complex) targets were simulated from a full 2-1 TMDD model and fitted to 2-1 TMDD models, to its approximations, and to the standard (1-1) QSS model. For a mAb with a soluble target, it was demonstrated that the 2-1 QSS model provided nearly identical description of the observed (simulated) free drug and total target concentrations, although there was some minor bias in predictions of unobserved free target concentrations. The standard QSS approximation also provided a good description of the observed data, but was not able to distinguish between free drug concentrations (with no target attached and both binding site free) and partially bound drug concentrations (with one of the binding sites occupied by the target). For a mAb with a membrane-bound target, the 2-1 MM approximation adequately described the data. The 2-1 QSS approximation converged 10 times faster than the full 2-1 TMDD, and its run time was comparable with the standard QSS model.

  12. Structural Fingerprints of Transcription Factor Binding Site Regions

    Directory of Open Access Journals (Sweden)

    Peter Willett

    2009-03-01

    Full Text Available Fourier transforms are a powerful tool in the prediction of DNA sequence properties, such as the presence/absence of codons. We have previously compiled a database of the structural properties of all 32,896 unique DNA octamers. In this work we apply Fourier techniques to the analysis of the structural properties of human chromosomes 21 and 22 and also to three sets of transcription factor binding sites within these chromosomes. We find that, for a given structural property, the structural property power spectra of chromosomes 21 and 22 are strikingly similar. We find common peaks in their power spectra for both Sp1 and p53 transcription factor binding sites. We use the power spectra as a structural fingerprint and perform similarity searching in order to find transcription factor binding site regions. This approach provides a new strategy for searching the genome data for information. Although it is difficult to understand the relationship between specific functional properties and the set of structural parameters in our database, our structural fingerprints nevertheless provide a useful tool for searching for function information in sequence data. The power spectrum fingerprints provide a simple, fast method for comparing a set of functional sequences, in this case transcription factor binding site regions, with the sequences of whole chromosomes. On its own, the power spectrum fingerprint does not find all transcription factor binding sites in a chromosome, but the results presented here show that in combination with other approaches, this technique will improve the chances of identifying functional sequences hidden in genomic data.

  13. Heterogeneity of Opioid Binding Sites in Guinea Pig Spinal Cord

    Science.gov (United States)

    1984-11-30

    MEDICAL CENTER WILFORD HALL AIR FORCE MEDICAL CENTER Title of Thesis: "Heterogeneity of Opioid Binding Sites in Guinea Pig Spinal Cord" Name of...that the use of any copyrighted material in the dissertation manuscript entitled: "Heterogeneity of Opioid Binding Sites in Guinea Pig Spinal Cord...University of the Health Sciences 11 Abstract Title of Thesis: Heterogenity of Opioid Binding Sites In Guinea Pig Spinal Cord Gary Dean Zarr MAJ/ANC

  14. EFFECT OF ADRENALECTOMY ON PROLACTIN SECRETION IN PRIMIPAROUS AND MULTIPAROUS LACTATING RATS

    Directory of Open Access Journals (Sweden)

    C. D. C. Sanches

    2018-04-01

    Full Text Available The adenohypophysis produces among other hormones prolactin, which plays an important role in reproduction, especially on the mammary glands and lactation of mammals. Prolactin is tonically controlled by tufo-infundibular dopamine, but several studies indicate that prolactin secretion is altered by the action of glucocorticoids and, therefore, is related to stress. However, the exact contribution of corticosteroids in the control of prolactin secretion is poorly understood. On the other hand, it is also known that reproductive experience can modify prolactin secretion by adenohypophysis. Thus, the present study aims to study the hormonal relationships of the hypothalamic-pituitary-adrenal axis, in particular, the glucocorticoid relationship on prolactin secretion as a function of the reproductive experience in females during lactation. The results show that reproductive experience may be a factor modifying the sensitivity of the neuroendocrine response of prolactin secretion to glucocorticoids. However, more studies are needed to understand the possible mechanisms involved, as well as possible modifications in this response as a function of the reproductive status of the females.

  15. Human chorionic ganodotropin binding sites in the human endometrium

    International Nuclear Information System (INIS)

    Bhattacharya, S.; Banerjee, J.; Sen, S.; Manna, P.R.

    1993-01-01

    The existence of high-affinity and low-capacity specific binding sites for luteinizing hormone/human chorionic gonadotropin (hCG) has been reported in porcine, rabbit and rat uteri. The authors have identified the hCG binding sites in the human endometrium collected from 35-42-year-old ovulatory and anovulatory women. The binding characteristics of hCG to endometrial tissue preparations from ovulatory and anovulatory women showed saturability with high affinity and low capacity. Scatchard plot analysis showed the dissociation constant of specific binding sites in the ovulatory women to be 3.5x10 -10 mol/l and in anovulatory women to be 3.1x10 -10 mol/l. The maximum binding capacity varied considerably between ovulatory and anovulatory endometrium. Among the divalent metal ions tested Zn 2+ effected a remarkable increase in [ 125 I]hCG binding to the endometrium, whereas Mn 2+ showed a marginal increase and other metal ions did not have any effect. Data obtained with human endometrium indicate an influence of the functional state of the ovary on [ 125 I]hCG binding to endometrium. 14 refs., 3 figs

  16. Transcription factor binding sites prediction based on modified nucleosomes.

    Directory of Open Access Journals (Sweden)

    Mohammad Talebzadeh

    Full Text Available In computational methods, position weight matrices (PWMs are commonly applied for transcription factor binding site (TFBS prediction. Although these matrices are more accurate than simple consensus sequences to predict actual binding sites, they usually produce a large number of false positive (FP predictions and so are impoverished sources of information. Several studies have employed additional sources of information such as sequence conservation or the vicinity to transcription start sites to distinguish true binding regions from random ones. Recently, the spatial distribution of modified nucleosomes has been shown to be associated with different promoter architectures. These aligned patterns can facilitate DNA accessibility for transcription factors. We hypothesize that using data from these aligned and periodic patterns can improve the performance of binding region prediction. In this study, we propose two effective features, "modified nucleosomes neighboring" and "modified nucleosomes occupancy", to decrease FP in binding site discovery. Based on these features, we designed a logistic regression classifier which estimates the probability of a region as a TFBS. Our model learned each feature based on Sp1 binding sites on Chromosome 1 and was tested on the other chromosomes in human CD4+T cells. In this work, we investigated 21 histone modifications and found that only 8 out of 21 marks are strongly correlated with transcription factor binding regions. To prove that these features are not specific to Sp1, we combined the logistic regression classifier with the PWM, and created a new model to search TFBSs on the genome. We tested the model using transcription factors MAZ, PU.1 and ELF1 and compared the results to those using only the PWM. The results show that our model can predict Transcription factor binding regions more successfully. The relative simplicity of the model and capability of integrating other features make it a superior method

  17. Variation in prolactin is related to variation in sexual behavior and contact affiliation.

    Directory of Open Access Journals (Sweden)

    Charles T Snowdon

    Full Text Available Prolactin is associated with both maternal and paternal care and appears important in developing a bond between parent and infant. In contrast with oxytocin, another hormone important in infant care, there is scant information on the role of prolactin in maintaining adult heterosexual relationships. We present here the first results demonstrating a relationship between prolactin levels and sexual and contact affiliation behavior in a pair-bonded species. We studied cotton-top tamarins, a socially-monogamous, cooperatively-breeding primate. We measured chronic urinary prolactin levels over a four week period to include the entire female ovulatory cycle and correlated prolactin levels in males and females with simultaneous measures of contact affiliation and sexual behavior. Current mothers who were no longer nursing displayed lower amounts of sexual behavior and proximity than non-breeding females and also had marginally lower levels of prolactin. The prolactin levels of males and females were similar within pairs, and variation in prolactin levels for both sexes was explained both by the amount of sexual behavior and contact affiliation. The results parallel a previous study that compared oxytocin levels with sociosexual behavior in the same species, and supports the hypothesis that both prolactin and oxytocin are involved in pair-bonding as well as in infant care.

  18. Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively.

    Science.gov (United States)

    Clifford, Jacob; Adami, Christoph

    2015-09-02

    Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

    Specific oxytocin (OT)-binding sites were localized in the rat kidney with use of a selective 125 I-labeled OT antagonist ( 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 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 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Stoeckel, M.E.; Freund-Mercier, M.J. (Centre National de la Recherche Scientifique, Strasbourg (France))

    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. Activation of protein kinase C inhibits synthesis and release of decidual prolactin

    International Nuclear Information System (INIS)

    Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

    1986-01-01

    Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

  2. Receptor-ligand binding sites and virtual screening.

    Science.gov (United States)

    Hattotuwagama, Channa K; Davies, Matthew N; Flower, Darren R

    2006-01-01

    Within the pharmaceutical industry, the ultimate source of continuing profitability is the unremitting process of drug discovery. To be profitable, drugs must be marketable: legally novel, safe and relatively free of side effects, efficacious, and ideally inexpensive to produce. While drug discovery was once typified by a haphazard and empirical process, it is now increasingly driven by both knowledge of the receptor-mediated basis of disease and how drug molecules interact with receptors and the wider physiome. Medicinal chemistry postulates that to understand a congeneric ligand series, or set thereof, is to understand the nature and requirements of a ligand binding site. Likewise, structural molecular biology posits that to understand a binding site is to understand the nature of ligands bound therein. Reality sits somewhere between these extremes, yet subsumes them both. Complementary to rules of ligand design, arising through decades of medicinal chemistry, structural biology and computational chemistry are able to elucidate the nature of binding site-ligand interactions, facilitating, at both pragmatic and conceptual levels, the drug discovery process.

  3. Probing binding hot spots at protein-RNA recognition sites.

    Science.gov (United States)

    Barik, Amita; Nithin, Chandran; Karampudi, Naga Bhushana Rao; Mukherjee, Sunandan; Bahadur, Ranjit Prasad

    2016-01-29

    We use evolutionary conservation derived from structure alignment of polypeptide sequences along with structural and physicochemical attributes of protein-RNA interfaces to probe the binding hot spots at protein-RNA recognition sites. We find that the degree of conservation varies across the RNA binding proteins; some evolve rapidly compared to others. Additionally, irrespective of the structural class of the complexes, residues at the RNA binding sites are evolutionary better conserved than those at the solvent exposed surfaces. For recognitions involving duplex RNA, residues interacting with the major groove are better conserved than those interacting with the minor groove. We identify multi-interface residues participating simultaneously in protein-protein and protein-RNA interfaces in complexes where more than one polypeptide is involved in RNA recognition, and show that they are better conserved compared to any other RNA binding residues. We find that the residues at water preservation site are better conserved than those at hydrated or at dehydrated sites. Finally, we develop a Random Forests model using structural and physicochemical attributes for predicting binding hot spots. The model accurately predicts 80% of the instances of experimental ΔΔG values in a particular class, and provides a stepping-stone towards the engineering of protein-RNA recognition sites with desired affinity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Using TESS to predict transcription factor binding sites in DNA sequence.

    Science.gov (United States)

    Schug, Jonathan

    2008-03-01

    This unit describes how to use the Transcription Element Search System (TESS). This Web site predicts transcription factor binding sites (TFBS) in DNA sequence using two different kinds of models of sites, strings and positional weight matrices. The binding of transcription factors to DNA is a major part of the control of gene expression. Transcription factors exhibit sequence-specific binding; they form stronger bonds to some DNA sequences than to others. Identification of a good binding site in the promoter for a gene suggests the possibility that the corresponding factor may play a role in the regulation of that gene. However, the sequences transcription factors recognize are typically short and allow for some amount of mismatch. Because of this, binding sites for a factor can typically be found at random every few hundred to a thousand base pairs. TESS has features to help sort through and evaluate the significance of predicted sites.

  5. Pactamycin binding site on archaebacterial and eukaryotic ribosomes

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  6. Bifunctional avidin with covalently modifiable ligand binding site.

    Directory of Open Access Journals (Sweden)

    Jenni Leppiniemi

    Full Text Available The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (streptavidin to improve the existing applications. Even so, (streptavidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.

  7. Distinct roles of beta1 metal ion-dependent adhesion site (MIDAS), adjacent to MIDAS (ADMIDAS), and ligand-associated metal-binding site (LIMBS) cation-binding sites in ligand recognition by integrin alpha2beta1.

    Science.gov (United States)

    Valdramidou, Dimitra; Humphries, Martin J; Mould, A Paul

    2008-11-21

    Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as alpha2beta1, ligand recognition takes place exclusively at the alpha subunit I domain. However, activation of the alphaI domain depends on its interaction with a structurally similar domain in the beta subunit known as the I-like or betaI domain. The top face of the betaI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the alphaI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to alpha2beta1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between alphaI and betaI, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of betaI. An activating mutation in the alpha2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca(2+), Mg(2+), and Mn(2+) on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn(2+) stimulates ligand binding, whereas the LIMBS is a stimulatory Ca(2+)-binding site, occupancy of which increases the affinity of Mg(2+) for the MIDAS.

  8. Nucleotide Interdependency in Transcription Factor Binding Sites in the Drosophila Genome.

    Science.gov (United States)

    Dresch, Jacqueline M; Zellers, Rowan G; Bork, Daniel K; Drewell, Robert A

    2016-01-01

    A long-standing objective in modern biology is to characterize the molecular components that drive the development of an organism. At the heart of eukaryotic development lies gene regulation. On the molecular level, much of the research in this field has focused on the binding of transcription factors (TFs) to regulatory regions in the genome known as cis-regulatory modules (CRMs). However, relatively little is known about the sequence-specific binding preferences of many TFs, especially with respect to the possible interdependencies between the nucleotides that make up binding sites. A particular limitation of many existing algorithms that aim to predict binding site sequences is that they do not allow for dependencies between nonadjacent nucleotides. In this study, we use a recently developed computational algorithm, MARZ, to compare binding site sequences using 32 distinct models in a systematic and unbiased approach to explore nucleotide dependencies within binding sites for 15 distinct TFs known to be critical to Drosophila development. Our results indicate that many of these proteins have varying levels of nucleotide interdependencies within their DNA recognition sequences, and that, in some cases, models that account for these dependencies greatly outperform traditional models that are used to predict binding sites. We also directly compare the ability of different models to identify the known KRUPPEL TF binding sites in CRMs and demonstrate that a more complex model that accounts for nucleotide interdependencies performs better when compared with simple models. This ability to identify TFs with critical nucleotide interdependencies in their binding sites will lead to a deeper understanding of how these molecular characteristics contribute to the architecture of CRMs and the precise regulation of transcription during organismal development.

  9. Long chain fatty acids alter the interactive binding of ligands to the two principal drug binding sites of human serum albumin.

    Directory of Open Access Journals (Sweden)

    Keishi Yamasaki

    Full Text Available A wide variety of drugs bind to human serum albumin (HSA at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand and ibuprofen (site II ligand at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a 'competitive-like' allosteric interaction in the case of the defatted HSA conformer to a 'nearly independent' binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a 'competitive-like' allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to 'nearly independent' binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

  10. Oligomycin frames a common drug-binding site in the ATP synthase

    Energy Technology Data Exchange (ETDEWEB)

    Symersky, Jindrich; Osowski, Daniel; Walters, D. Eric; Mueller, David M. (Rosalind)

    2015-12-01

    We report the high-resolution (1.9 {angstrom}) crystal structure of oligomycin bound to the subunit c10 ring of the yeast mitochondrial ATP synthase. Oligomycin binds to the surface of the c10 ring making contact with two neighboring molecules at a position that explains the inhibitory effect on ATP synthesis. The carboxyl side chain of Glu59, which is essential for proton translocation, forms an H-bond with oligomycin via a bridging water molecule but is otherwise shielded from the aqueous environment. The remaining contacts between oligomycin and subunit c are primarily hydrophobic. The amino acid residues that form the oligomycin-binding site are 100% conserved between human and yeast but are widely different from those in bacterial homologs, thus explaining the differential sensitivity to oligomycin. Prior genetics studies suggest that the oligomycin-binding site overlaps with the binding site of other antibiotics, including those effective against Mycobacterium tuberculosis, and thereby frames a common 'drug-binding site.' We anticipate that this drug-binding site will serve as an effective target for new antibiotics developed by rational design.

  11. Demonstration of specific binding sites for 3H-RRR-alpha-tocopherol on human erythrocytes

    International Nuclear Information System (INIS)

    Kitabchi, A.E.; Wimalasena, J.

    1982-01-01

    Previous work from our laboratory demonstrated specific binding sites for 3 H-RRR-alpha-tocopherol ( 3 H-d alpha T) in membranes of rat adrenal cells. As tocopherol deficiency is associated with increased susceptibility of red blood cells to hemolysis, we investigated tocopherol binding sites in human RBCs. Erythrocytes were found to have specific binding sites for 3 H-d alpha T that exhibited saturability and time and cell-concentration dependence as well as reversibility of binding. Kinetic studies of binding demonstrated two binding sites--one with high affinity (Ka of 2.6 x 10(7) M-1), low capacity (7,600 sites per cell) and the other with low affinity (1.2 x 10(6) M-1), high capacity (150,000 sites per cell). In order to localize the binding sites further, RBCs were fractionated and greater than 90% of the tocopherol binding was located in the membranes. Similar to the findings in intact RBCs, the membranes exhibited two binding sites with a respective Ka of 3.3 x 10(7) M-1 and 1.5 x 10(6) M-1. Specificity data for binding demonstrated 10% binding for RRR-gamma-tocopherol, but not other tocopherol analog exhibited competition for 3 H-d alpha T binding sites. Instability data suggested a protein nature for these binding sites. Preliminary studies on Triton X-100 solubilized fractions resolved the binding sites to a major component with an Mr of 65,000 and a minor component with an Mr of 125,000. We conclude that human erythrocyte membranes contain specific binding sites for RRR-alpha-tocopherol. These sites may be of physiologic significance in the function of tocopherol on the red blood cell membrane

  12. Position specific variation in the rate of evolution intranscription factor binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Moses, Alan M.; Chiang, Derek Y.; Kellis, Manolis; Lander, EricS.; Eisen, Michael B.

    2003-08-28

    The binding sites of sequence specific transcription factors are an important and relatively well-understood class of functional non-coding DNAs. Although a wide variety of experimental and computational methods have been developed to characterize transcription factor binding sites, they remain difficult to identify. Comparison of non-coding DNA from related species has shown considerable promise in identifying these functional non-coding sequences, even though relatively little is known about their evolution. Here we analyze the genome sequences of the budding yeasts Saccharomyces cerevisiae, S. bayanus, S. paradoxus and S. mikataeto study the evolution of transcription factor binding sites. As expected, we find that both experimentally characterized and computationally predicted binding sites evolve slower than surrounding sequence, consistent with the hypothesis that they are under purifying selection. We also observe position-specific variation in the rate of evolution within binding sites. We find that the position-specific rate of evolution is positively correlated with degeneracy among binding sites within S. cerevisiae. We test theoretical predictions for the rate of evolution at positions where the base frequencies deviate from background due to purifying selection and find reasonable agreement with the observed rates of evolution. Finally, we show how the evolutionary characteristics of real binding motifs can be used to distinguish them from artifacts of computational motif finding algorithms. As has been observed for protein sequences, the rate of evolution in transcription factor binding sites varies with position, suggesting that some regions are under stronger functional constraint than others. This variation likely reflects the varying importance of different positions in the formation of the protein-DNA complex. The characterization of the pattern of evolution in known binding sites will likely contribute to the effective use of comparative

  13. Characterization of a second ligand binding site of the insulin receptor

    International Nuclear Information System (INIS)

    Hao Caili; Whittaker, Linda; Whittaker, Jonathan

    2006-01-01

    Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the α subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K d of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site

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

    International Nuclear Information System (INIS)

    Nye, J.S.

    1988-01-01

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

  15. Ontogenic studies of the neural control of adenohypophyseal hormones in the rat. II. Prolactin.

    Science.gov (United States)

    Becú-Villalobos, D; Lacau-Mengido, I M; Díaz-Torga, G S; Libertun, C

    1992-02-01

    1. Serum prolactin levels are low during the first 20 days of life and gradually increase toward puberty, in both male and female rats. 2. There is an age-related increase in the cell population engaged in prolactin secretion, as well as an increase in the synthesis of prolactin and of the amount of prolactin secreted from individual lactotropes. 3. The gradual increase in prolactin levels in the third week of life is not related to a decrease in dopaminergic inhibition but to an increase in the efficiency of prolactin releasing factors such as estrogen, serotonin, opiates, and posterior pituitary extracts. 4. Prolactin release induced by physiological factors, such as stress, cervical stimulation, or the expression of spontaneous diurnal and nocturnal surges, requires maturational events within the hypothalamic-pituitary axis which are evident at the end of the third week of life. 5. In the female rat the steadily increasing levels of prolactin are involved in the timing of puberty eclosion acting at the ovary and at the brain. 6. In the prepubertal male rat increasing titers of prolactin may be involved in testicular and accessory organ development and may facilitate the actions of luteinizing hormone, follicle stimulating hormone, and testosterone on male sexual organs.

  16. Correlation between FSH, LH and prolactin serum levels. [Radioimmunoassay of hormones

    Energy Technology Data Exchange (ETDEWEB)

    Krause, W [Giessen Univ. (Germany, F.R.)

    1978-01-01

    In 188 males FSH, LH, and prolactin serum levels determined by radioimmunoassay from a single blood sample were found to be closely correlated. No correlation appeared to testosterone levels. The same correlation is observed, if serum levels of FSH, LH, and prolactin are measured after stimulation with LH-RH and TRH. In order to explain the close correlation, in five young men hormone levels were measured at 2-min-intervals over a period of 2 hours. Peaks of prolactin often correspond to those of FSH and LH, and a statistical correlation was found in two cases between FSH and prolactin. Results suggest a common releasing mechanism, which is superposed to the main mediating mechanism.

  17. [Plasma prolactin concentration and the effect of metergoline in pseudopregnant Afghan hounds].

    Science.gov (United States)

    Okkens, A C; Dieleman, S J; Kooistra, H S; Bevers, M M

    2000-02-01

    The effects of metergoline, a 5-hydroxytryptamine (serotinin) antagonist, on the plasma concentrations of prolactin in overtly pseudopregnant Afghan hounds and on the clinical symptoms of overt pseudopregnancy were studied. Plasma concentrations of prolactin and progesterone were determined in six Afghan hounds with signs of overt pseudopregnancy for 2-3 weeks and in three Afghan hounds that were not pseudopregnant at the time of blood sampling. In the overtly pseudopregnant bitches the plasma concentrations of prolactin before treatment (35.5 +/- 8.5 micrograms l-1) were significantly higher than the plasma concentrations of prolactin of the three bitches that were not pseudopregnant (6.3 +/- 0.5 micrograms l-1); the latter values were similar to those of non-psueodopregnant beagle bitches during the total luteal phase. The six pseudopregnant Afghan hounds were treated for 10 days with the antiserotoninergic drug metergoline. At 2 h after the onset of treatment with metergoline, the mean plasma concentration of prolactin had decreased to 10.8 +/- 2.9 micrograms l-1. The plasma concentrations of prolactin continued to decline to 5.4 +/- 1.0 micrograms l-1 at 4 h and to 1.0 +/- 0.1 microgram l-1 during treatment days 3-10. Signs of pseudopregnancy, such as swelling of the mammary glands and digging, decreased during the treatment period. The treatment was associated with mild behavioural side effects such as whimpering and aggressiveness. These side effects are probably not related to suppression of prolactin but are due to a direct effect on serotoninergic pathways in the brain. It is concluded that high plasma concentrations of prolactin are associated with the development and maintenance of pseudopregnancy. The serotonin antagonist metergoline strongly suppresses plasma concentration of prolactine in pseudopregnant dogs and decreases the clinical signs of pseudopregnancy.

  18. CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins.

    Science.gov (United States)

    Khorshid, Mohsen; Rodak, Christoph; Zavolan, Mihaela

    2011-01-01

    The stability, localization and translation rate of mRNAs are regulated by a multitude of RNA-binding proteins (RBPs) that find their targets directly or with the help of guide RNAs. Among the experimental methods for mapping RBP binding sites, cross-linking and immunoprecipitation (CLIP) coupled with deep sequencing provides transcriptome-wide coverage as well as high resolution. However, partly due to their vast volume, the data that were so far generated in CLIP experiments have not been put in a form that enables fast and interactive exploration of binding sites. To address this need, we have developed the CLIPZ database and analysis environment. Binding site data for RBPs such as Argonaute 1-4, Insulin-like growth factor II mRNA-binding protein 1-3, TNRC6 proteins A-C, Pumilio 2, Quaking and Polypyrimidine tract binding protein can be visualized at the level of the genome and of individual transcripts. Individual users can upload their own sequence data sets while being able to limit the access to these data to specific users, and analyses of the public and private data sets can be performed interactively. CLIPZ, available at http://www.clipz.unibas.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements.

  19. Methylmercury inhibits prolactin release in a cell line of pituitary origin

    Directory of Open Access Journals (Sweden)

    L.A.L. Maués

    2015-08-01

    Full Text Available Heavy metals, such as methylmercury, are key environmental pollutants that easily reach human beings by bioaccumulation through the food chain. Several reports have demonstrated that endocrine organs, and especially the pituitary gland, are potential targets for mercury accumulation; however, the effects on the regulation of hormonal release are unclear. It has been suggested that serum prolactin could represent a biomarker of heavy metal exposure. The aim of this study was to evaluate the effect of methylmercury on prolactin release and the role of the nitrergic system using prolactin secretory cells (the mammosomatotroph cell line, GH3B6. Exposure to methylmercury (0-100 μM was cytotoxic in a time- and concentration-dependent manner, with an LC50 higher than described for cells of neuronal origin, suggesting GH3B6 cells have a relative resistance. Methylmercury (at exposures as low as 1 μM for 2 h also decreased prolactin release. Interestingly, inhibition of nitric oxide synthase by N-nitro-L-arginine completely prevented the decrease in prolactin release without acute neurotoxic effects of methylmercury. These data indicate that the decrease in prolactin production occurs via activation of the nitrergic system and is an early effect of methylmercury in cells of pituitary origin.

  20. In vitro site selection of a consensus binding site for the Drosophila melanogaster Tbx20 homolog midline.

    Directory of Open Access Journals (Sweden)

    Nima Najand

    Full Text Available We employed in vitro site selection to identify a consensus binding sequence for the Drosophila melanogaster Tbx20 T-box transcription factor homolog Midline. We purified a bacterially expressed T-box DNA binding domain of Midline, and used it in four rounds of precipitation and polymerase-chain-reaction based amplification. We cloned and sequenced 54 random oligonucleotides selected by Midline. Electromobility shift-assays confirmed that 27 of these could bind the Midline T-box. Sequence alignment of these 27 clones suggests that Midline binds as a monomer to a consensus sequence that contains an AGGTGT core. Thus, the Midline consensus binding site we define in this study is similar to that defined for vertebrate Tbx20, but differs from a previously reported Midline binding sequence derived through site selection.

  1. Opioid binding site in EL-4 thymoma cell line

    International Nuclear Information System (INIS)

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of [ 3 H] bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10 6 cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of [ 3 H] bremazocine with an IC 50 value = 0.57μM. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, [D-Pen 2 , D-Pen 5 ] enkephalin and β-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC 50 = 60μM, that was similar to naloxone. 32 references, 3 figures, 2 tables

  2. Cloud computing for protein-ligand binding site comparison.

    Science.gov (United States)

    Hung, Che-Lun; Hua, Guan-Jie

    2013-01-01

    The proteome-wide analysis of protein-ligand binding sites and their interactions with ligands is important in structure-based drug design and in understanding ligand cross reactivity and toxicity. The well-known and commonly used software, SMAP, has been designed for 3D ligand binding site comparison and similarity searching of a structural proteome. SMAP can also predict drug side effects and reassign existing drugs to new indications. However, the computing scale of SMAP is limited. We have developed a high availability, high performance system that expands the comparison scale of SMAP. This cloud computing service, called Cloud-PLBS, combines the SMAP and Hadoop frameworks and is deployed on a virtual cloud computing platform. To handle the vast amount of experimental data on protein-ligand binding site pairs, Cloud-PLBS exploits the MapReduce paradigm as a management and parallelizing tool. Cloud-PLBS provides a web portal and scalability through which biologists can address a wide range of computer-intensive questions in biology and drug discovery.

  3. Enhanced prolactin levels in opium smokers.

    Science.gov (United States)

    Moshtaghi-Kashanian, Ghollam-Reza; Esmaeeli, Farzaneh; Dabiri, Shahriar

    2005-12-01

    In Iran, opium is smoked for pleasure or as a medication by some people. It is a complex mixture of 40 different alkaloids, including morphine and codeine along with many impurities. Although it is well established that opioids or tobacco affect many physiological functions in humans, to our knowledge there has been no specific study looking at these effects in opium smokers. To assess that, we investigated the circulating levels of prolactin, TSH, LH, FSH and testosterone in male opium smokers who also smoke cigarettes (n=23, aged 28.4+/- 4.1 years), and comparing this with the corresponding values for nicotine abusers (n=12, 15-25 cigarettes/day) or a healthy control group (n=20) of the same age. Our results showed that 86.96% of the opium-dependent and 41.67 % of the nicotine-dependent group displayed high prolactin values (popium and the plasma prolactin level of opium dependents (p=0.748, popium smokers and 50% of the cigarette smokers (popium smokers was also lower than that of the other two groups (popium and cigarette smoking may synergistically influence pituitary hormone production through the effects on neuropeptides produced either locally or systemic.

  4. Prolactin and Dehydroepiandrosterone Levels in Women with Systemic Lupus Erythematosus: The Role of the Extrapituitary Prolactin Promoter Polymorphism at −1149G/T

    Directory of Open Access Journals (Sweden)

    Edward L. Treadwell

    2015-01-01

    Full Text Available Systemic lupus erythematosus (SLE has shown an association with high levels of prolactin, low levels of dehydroepiandrosterone (DHEA, and induction of inflammatory cytokines in the serum of patients with the disease. This preliminary study examined the relevance of a −1149G/T functional single-nucleotide polymorphism (SNP (rs1341239 in the promoter of the extrapituitary prolactin gene in a cohort of African American and European American women with lupus. Examination of this SNP revealed that the −1149TT genotype was correlated with higher levels of prolactin in serum and prolactin gene expression (p=0.0001 in peripheral blood mononuclear cells (PBMCs. Lower levels of DHEA in serum were demonstrated in lupus patients (p=0.001; those with the −1149TT genotype had the lowest levels of DHEA. Furthermore, a small subset of women who were on DHEA therapy and had a TT genotype showed a significant decrease in prolactin gene expression and lower disease activity scores (SLEDAI. Lupus patients, particularly African Americans, had significantly higher levels of IL-6 (p=0.0001 and TNF-α (p=0.042. This study suggests that the −1149TT genotype may be a risk factor for lupus and may predict who could possibly benefit from DHEA therapy; therefore, these results should be validated in a larger cohort with all ethnic groups.

  5. The RNA-Binding Site of Poliovirus 3C Protein Doubles as a Phosphoinositide-Binding Domain.

    Science.gov (United States)

    Shengjuler, Djoshkun; Chan, Yan Mei; Sun, Simou; Moustafa, Ibrahim M; Li, Zhen-Lu; Gohara, David W; Buck, Matthias; Cremer, Paul S; Boehr, David D; Cameron, Craig E

    2017-12-05

    Some viruses use phosphatidylinositol phosphate (PIP) to mark membranes used for genome replication or virion assembly. PIP-binding motifs of cellular proteins do not exist in viral proteins. Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using nuclear magnetic resonance spectroscopy. The PIP-binding site was located on a highly dynamic α helix, which also functions in RNA binding. Broad PIP-binding activity was observed in solution using a fluorescence polarization assay or in the context of a lipid bilayer using an on-chip, fluorescence assay. All-atom molecular dynamics simulations of the 3C protein-membrane interface revealed PIP clustering and perhaps PIP-dependent conformations. PIP clustering was mediated by interaction with residues that interact with the RNA phosphodiester backbone. We conclude that 3C binding to membranes will be determined by PIP abundance. We suggest that the duality of function observed for 3C may extend to RNA-binding proteins of other viruses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Lack of effect of naloxone on prolactin and seizures in electroconvulsive therapy.

    Science.gov (United States)

    Sperling, M R; Melmed, S; McAllister, T; Price, T R

    1989-01-01

    Both opiate agonist and antagonist injection have been reported to modulate prolactin secretion, alter brain excitability and produce seizures, and modify the postictal state. We studied the effects of administration of high-dose naloxone, an opiate antagonist, on postictal prolactin levels, seizure duration, and postictal behavior, using patients undergoing electroconvulsive therapy (ECT) as a seizure model. Seven patients had 8 mg naloxone injected prior to one ECT treatment and saline injected prior to another treatment, with the order of injection randomized. Before ECT and 15 min after ECT, prolactin levels were drawn, and no blunting of the expected postictal prolactin elevation by naloxone injection was observed. We found no evidence that endogenous opiates trigger prolactin secretion during seizures. Seizure duration was also similar in saline and naloxone groups, and naloxone did not reverse postictal depression, as has been reported in an animal model.

  7. Differential Modulation of Annexin I Binding Sites on Monocytes and Neutrophils

    Directory of Open Access Journals (Sweden)

    H. S. Euzger

    1999-01-01

    Full Text Available Specific binding sites for the anti-inflammatory protein annexin I have been detected on the surface of human monocytes and polymorphonuclear leukocytes (PMN. These binding sites are proteinaceous in nature and are sensitive to cleavage by the proteolytic enzymes trypsin, collagenase, elastase and cathepsin G. When monocytes and PMN were isolated independently from peripheral blood, only the monocytes exhibited constitutive annexin I binding. However PMN acquired the capacity to bind annexin I following co-culture with monocytes. PMN incubation with sodium azide, but not protease inhibitors, partially blocked this process. A similar increase in annexin I binding capacity was also detected in PMN following adhesion to endothelial monolayers. We propose that a juxtacrine activation rather than a cleavage-mediated transfer is involved in this process. Removal of annexin I binding sites from monocytes with elastase rendered monocytes functionally insensitive to full length annexin I or to the annexin I-derived pharmacophore, peptide Ac2-26, assessed as suppression of the respiratory burst. These data indicate that the annexin I binding site on phagocytic cells may have an important function in the feedback control of the inflammatory response and their loss through cleavage could potentiate such responses.

  8. Prolactin and cortisol levels in women with endometriosis

    Directory of Open Access Journals (Sweden)

    A.P. Lima

    2006-08-01

    Full Text Available Endometriosis is a progressive estrogen-dependent disease affecting women during their reproductive years. The objective of the present study was to investigate whether endometriosis is associated with stress parameters. We determined cortisol and prolactin levels in serum, peritoneal and follicular fluid from infertile women with endometriosis and fertile women without the disease. The extent of the disease was staged according to the revised American Fertility Society classification (1997. Serum and peritoneal fluid were collected from 49 women aged 19 to 39 years undergoing laparoscopy. Eighteen women had stage I-II endometriosis and 10 had stage III-IV. Controls were 21 women undergoing laparoscopy for tubal sterilization. Follicular fluid was obtained from 39 women aged 25-39 years undergoing in vitro fertilization (21 infertile women with endometriosis and 18 infertile women without endometriosis. Serum prolactin levels were significantly higher in infertile women with stage III-IV endometriosis (28.9 ± 2.1 ng/mL than in healthy controls (13.2 ± 2.1 ng/mL. Serum cortisol levels were significantly higher in infertile women with stage III-IV endometriosis (20.1 ± 1.3 ng/mL than in controls (10.5 ± 1.4 ng/mL. Cortisol and prolactin levels in follicular fluid and peritoneal fluid did not differ significantly between groups. The high levels of cortisol and prolactin in the serum from women with endometriosis might contribute to the subfertility frequently associated with the disease. Moreover, since higher levels of cortisol and prolactin are often associated with stress, it is probable that stress might contribute to the development of endometriosis and its progression to advanced stages of the disease.

  9. PocketMatch: A new algorithm to compare binding sites in protein structures

    Directory of Open Access Journals (Sweden)

    Chandra Nagasuma

    2008-12-01

    Full Text Available Abstract Background Recognizing similarities and deriving relationships among protein molecules is a fundamental requirement in present-day biology. Similarities can be present at various levels which can be detected through comparison of protein sequences or their structural folds. In some cases similarities obscure at these levels could be present merely in the substructures at their binding sites. Inferring functional similarities between protein molecules by comparing their binding sites is still largely exploratory and not as yet a routine protocol. One of the main reasons for this is the limitation in the choice of appropriate analytical tools that can compare binding sites with high sensitivity. To benefit from the enormous amount of structural data that is being rapidly accumulated, it is essential to have high throughput tools that enable large scale binding site comparison. Results Here we present a new algorithm PocketMatch for comparison of binding sites in a frame invariant manner. Each binding site is represented by 90 lists of sorted distances capturing shape and chemical nature of the site. The sorted arrays are then aligned using an incremental alignment method and scored to obtain PMScores for pairs of sites. A comprehensive sensitivity analysis and an extensive validation of the algorithm have been carried out. A comparison with other site matching algorithms is also presented. Perturbation studies where the geometry of a given site was retained but the residue types were changed randomly, indicated that chance similarities were virtually non-existent. Our analysis also demonstrates that shape information alone is insufficient to discriminate between diverse binding sites, unless combined with chemical nature of amino acids. Conclusion A new algorithm has been developed to compare binding sites in accurate, efficient and high-throughput manner. Though the representation used is conceptually simplistic, we demonstrate that

  10. Brain prolactin is involved in stress-induced REM sleep rebound.

    Science.gov (United States)

    Machado, Ricardo Borges; Rocha, Murilo Ramos; Suchecki, Deborah

    2017-03-01

    REM sleep rebound is a common behavioural response to some stressors and represents an adaptive coping strategy. Animals submitted to multiple, intermittent, footshock stress (FS) sessions during 96h of REM sleep deprivation (REMSD) display increased REM sleep rebound (when compared to the only REMSD ones, without FS), which is correlated to high plasma prolactin levels. To investigate whether brain prolactin plays a role in stress-induced REM sleep rebound two experiments were carried out. In experiment 1, rats were either not sleep-deprived (NSD) or submitted to 96h of REMSD associated or not to FS and brains were evaluated for PRL immunoreactivity (PRL-ir) and determination of PRL concentrations in the lateral hypothalamus and dorsal raphe nucleus. In experiment 2, rats were implanted with cannulas in the dorsal raphe nucleus for prolactin infusion and were sleep-recorded. REMSD associated with FS increased PRL-ir and content in the lateral hypothalamus and all manipulations increased prolactin content in the dorsal raphe nucleus compared to the NSD group. Prolactin infusion in the dorsal raphe nucleus increased the time and length of REM sleep episodes 3h after the infusion until the end of the light phase of the day cycle. Based on these results we concluded that brain prolactin is a major mediator of stress-induced REMS. The effect of PRL infusion in the dorsal raphe nucleus is discussed in light of the existence of a bidirectional relationship between this hormone and serotonin as regulators of stress-induced REM sleep rebound. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Opioid binding site in EL-4 thymoma cell line

    Energy Technology Data Exchange (ETDEWEB)

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  12. Effect of Changes in Prolactin RIA Reactants on the Validity of the Results

    International Nuclear Information System (INIS)

    Ahmed, A.M.; Megahed, Y.M.; El Mosallamy, M.A.F.; El-Khoshnia, R.A.M.

    1998-01-01

    Human prolactin plays an essential role in the secretion of milk and has the ability to suppress gonadal function. This study is considered as atrial to discuss some technical problems which made by operator in the RIA technique to select an optimized reliable and valid parameters for the measurement of prolactin concentration in human sera. Prolactin concentration was measured in normal control group and chronic renal failure group using the optimized technique. Finally the present optimized technique is very suitable selected one for measurement of prolactin

  13. Two distinct affinity binding sites for IL-1 on human cell lines

    International Nuclear Information System (INIS)

    Bensimon, C.; Wakasugi, N.; Tagaya, Y.; Takakura, K.; Yodoi, J.; Tursz, T.; Wakasugi, H.

    1989-01-01

    We used two human cell lines, NK-like YT-C3 and an EBV-containing B cell line, 3B6, as models to study the receptor(s) for IL-1. Two distinct types of saturable binding sites were found on both cell lines at 37 degrees C. Between 1 pM and 100 pM of 125I-IL-1-alpha concentration, saturable binding sites were detected on the YT-C3 cells with a K of 4 x 10(-11) M. The K found for the IL-1-alpha binding sites on 3B6 cells was 7.5 x 10(-11) M. An additional binding curve was detected above 100 pM on YT-C3 cells with a K of 7 x 10(-9) M and on 3B6 cells with a K of 5 x 10(-9) M. Scatchard plot analysis revealed 600 sites/cell with high affinity binding and 7000 sites/cell with low affinity for YT-C3 cells and 300 sites/cell with high affinity binding and 6000 sites/cell with low affinity for 3B6 cells. At 37 degrees C, the internalization of 125I-labeled IL-1 occurred via both high and low affinity IL-1R on both YT-C3 and 3B6 cells, whereas the rates of internalization for high affinity binding sites on YT-C3 cells were predominant in comparison to that of low affinity binding sites. In chemical cross-linking studies of 125 I-IL-1-alpha to 3B6 and YT-C3 cells, two protein bands were immunoprecipitated with Mr around 85 to 90 kDa leading to an estimation of the Mr of the IL-1R around 68 to 72 kDa. In similar experiments, the Mr found for the IL-1R expressed on the murine T cell line EL4 was slightly higher (around 80 kDa). Whether these distinct affinity binding sites are shared by a single molecule or by various chains remains to be elucidated

  14. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    Science.gov (United States)

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

  15. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...

  16. Polymorphism of the prolactin gene and its association with egg ...

    African Journals Online (AJOL)

    p2492989

    In this study, polymorphism of the prolactin gene was screened in six Chinese native ... Prolactin (PRL) is a single-chain polypeptide hormone that belongs to the growth hormone gene ..... Enhance the efficiency of single-strand conformation polymorphism analysis by short polyacrylamide gel and modified silver staining.

  17. Mammalian Prolactin – An Ancient But Still A Mysterious Hormone

    Indian Academy of Sciences (India)

    Table of contents. Mammalian Prolactin – An Ancient But Still A Mysterious Hormone · Prolactin inhibits LHRH action during lactational ammenorrhoea · Slide 3 · Slide 4 · REDUCTIONIST VIEW OF HORMONES · CONCERN · PURIFICATION PROTOCOLS · CHARACTERIZATION OF HORMONES · Slide 9 · Slide 10.

  18. (-)PPAP: a new and selective ligand for sigma binding sites.

    Science.gov (United States)

    Glennon, R A; Battaglia, G; Smith, J D

    1990-11-01

    Most agents employed for the investigation of sigma (sigma) binding sites display relatively low affinity for these sites, bind both at sigma sites and at either phencyclidine (PCP) sites or dopamine receptors with similar affinity, and/or produce some dopaminergic activity in vivo. We describe a new agent, (-)PPAP or R(-)-N-(3-phenyl-n-propyl)-1-phenyl-2-aminopropane hydrochloride, that binds with high affinity and selectivity at sigma (IC50 = 24 nM) versus either PCP sites (IC50 greater than 75,000 nM) or D1 and D2 dopamine receptors (IC50 greater than 5,000 nM). The sigma affinity of this agent is comparable to that of the standard ligands (+)-3-PPP and DTG. Furthermore, although (-)PPAP is structurally related to amphetamine, it neither produces nor antagonizes amphetamine-like stimulus effect in rats trained to discriminate 1 mg/kg of S(+)amphetamine from saline.

  19. Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

    Energy Technology Data Exchange (ETDEWEB)

    Makyio, Hisayoshi [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan); Shimabukuro, Junpei; Suzuki, Tatsuya [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Imamura, Akihiro; Ishida, Hideharu [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Kiso, Makoto [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Ando, Hiromune, E-mail: hando@gifu-u.ac.jp [Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193 (Japan); Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501 (Japan); Kato, Ryuichi, E-mail: ryuichi.kato@kek.jp [Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), 1-1 Oho, Tsukuba, Ibaraki, 305-0801 (Japan)

    2016-08-26

    The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

  20. Six independent fucose-binding sites in the crystal structure of Aspergillus oryzae lectin

    International Nuclear Information System (INIS)

    Makyio, Hisayoshi; Shimabukuro, Junpei; Suzuki, Tatsuya; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Ando, Hiromune; Kato, Ryuichi

    2016-01-01

    The crystal structure of AOL (a fucose-specific lectin of Aspergillus oryzae) has been solved by SAD (single-wavelength anomalous diffraction) and MAD (multi-wavelength anomalous diffraction) phasing of seleno-fucosides. The overall structure is a six-bladed β-propeller similar to that of other fucose-specific lectins. The fucose moieties of the seleno-fucosides are located in six fucose-binding sites. Although the Arg and Glu/Gln residues bound to the fucose moiety are common to all fucose-binding sites, the amino-acid residues involved in fucose binding at each site are not identical. The varying peak heights of the seleniums in the electron density map suggest that each fucose-binding site has a different carbohydrate binding affinity. - Highlights: • The six-bladed β-propeller structure of AOL was solved by seleno-sugar phasing. • The mode of fucose binding is essentially conserved at all six binding sites. • The seleno-fucosides exhibit slightly different interactions and electron densities. • These findings suggest that the affinity for fucose is not identical at each site.

  1. 2[125I]Iodomelatonin binding sites in spleens of guinea pigs

    International Nuclear Information System (INIS)

    Poon, A.M.S.; Pang, S.F.

    1992-01-01

    2-[ 125 I]Iodomelatonin was found to bind specifically to the membrane preparations of the spleens of guinea pigs with high affinity. The binding was rapid, stable, saturable and reversible. Scatchard analysis of the binding assays revealed an equilibrium dissociation constant (Kd) of 49.8±4.12 pmol/l and binding site density (Bmax) of 0.69±0.082 fmol/mg protein at mid-light. There was no significant change in the Kd or the Bmax at mid-dark. Kinetic analysis showed a Kd of 23.13±4.81 pmol/l, in agreement to that derived from the saturation studies. The 2-[ 125 I]iodomelatonin binding sites have the following order of potency: 2-iodomelatonin > melatonin > 6-chloromelatonin much-gt N-acetylserotonin, 6-hydroxymelatonin > 5-methoxytryptamine, 5-methoxytryptophol > serotonin, 5-methoxyindole-3-acetic acid > 5-hydroxytryptophol, 3-acetylindole, 1-acetylindole-3-carboxyaldehyde, L-tryptophan > tryptamine, 5-hydroxyindole-3-acetic acid. Differential centrifugation studies showed that the binding sites are localized mainly in the nuclear fraction, the rest are distributed in the microsomal fraction, mitochondrial fraction and cytosolic fraction. The demonstration of 2-[ 125 I]iodomelatonin binding sites in the spleen suggests the presence of melatonin receptors and a direct mechanism of action of melatonin on the immune system

  2. Effects of chronic alternating cadmium exposure on the episodic secretion of prolactin in male rats

    Energy Technology Data Exchange (ETDEWEB)

    Esquifino, A.I. [Madrid Univ. (Spain). Facultad de Medicina Complutense; Marquez, N.; Alvarez-Demanuel, E.; Lafuente, A. [Vigo Univ., Orense (Spain). Lab. de Toxicologia

    1998-07-01

    Cadmium increases or decreases prolactin secretion depending on the dose and duration of the exposure to the metal. However, whether there are cadmium effects on the episodic prolactin secretion is less well known. This study was undertaken to address whether chronic alternating exposure to two different doses of cadmium affects the episodic pattern of prolactin and to what extent the effects of cadmium are age-dependent. Male rats were treated s.c. with cadmium chloride (0.5 or 1.0 mg/kg) from day 30 to 60, or from day 60 to 90 of age, with alteration of the doses every 4 days, starting with the smaller dose. Controls received vehicle every 4 days. The last dose of cadmium was given 48 h prior to the pulsatility study. Prolactin secretion in the 4 experimental groups studied was episodic and changed significantly after cadmium exposure. Cadmium administration from day 30 to 60 of life significantly decreased the mean half-life of prolactin. On the other hand, when administered from day 60 to 90 cadmium significantly decreased the mean as well as serum prolactin levels and the absolute amplitude of the prolactin pulses, their duration, the relative amplitude or the mean half-life of the hormone. The frequency of prolactin peaks was not changed by cadmium administration. The results indicate that low intermittent doses of cadmium chronically administered change the episodic secretion pattern of prolactin in rats. The effects of cadmium on prolactin secretion were age dependent. (orig.)

  3. Synthesis of human prolactin in Chinese hamster ovary (CHO) cells

    International Nuclear Information System (INIS)

    Soares, Carlos Roberto Jorge

    2000-01-01

    Three different eukaryotic expression vectors, based on the same selectable gene marker (dhfr), have been used for dhf- CHO cells transfection to rapidly isolate stable cell lines capable of secreting high levels of recombinant human prolactin (rec-hPRL). Two vectors, one codifying a human prolactin (p658-hPRL) and the other a tag-prolactin (p658-tagPRL), contain the complete hepatitis B virus-X (HBV-X) gene coding for a viral transactivator and a sequence derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF) that mediates selective dhfr mRNA degradation. These vectors have the advantage of rapidly obtaining stable cell lines without methotrexate amplification. The highest secretion obtained by these vectors was of approximately 10 μg hPRU10 6 cells/day. The other vector (pEDdc-hPRL) is based on a dicistronic expression system, containing an internal ribosome entry site isolated from the encephalomyocarditis (EMC) virus. This vector before amplification provided secretion levels at least 10 fold lower than that obtained with the other two vectors. However, after three steps of methotrexate amplification, it provided some clones able to secrete up to 30 μg hPRU10 6 cells/day. This is the first report describing the production and purification of rec-hPRL from CHO cells, obtaining secretion levels with both vectors higher than those reported so far for this hormone in other eukaryotic systems. CHO-derived rec-hPRL contained approximately 10 % of the glycosylated form, a value that is consistent with results reported for hPRL purified from the pituitary or from transformed murine C-127 cells. CHO-derived rec-hPRL was purified with good yield, obtaining also a good resolution between non-glycosylated and glycosylated prolactin. The latter, when its potency was determined via an in vitro bioassay, presented a 47 % lower bioactivity. A qualitative and quantitative analysis of these forms was also possible thanks to the setting up of a reversed

  4. Lack of hormone binding in COS-7 cells expressing a mutated growth hormone receptor found in Laron dwarfism.

    Science.gov (United States)

    Edery, M; Rozakis-Adcock, M; Goujon, L; Finidori, J; Lévi-Meyrueis, C; Paly, J; Djiane, J; Postel-Vinay, M C; Kelly, P A

    1993-01-01

    A single point mutation in the growth hormone (GH) receptor gene generating a Phe-->Ser substitution in the extracellular binding domain of the receptor has been identified in one family with Laron type dwarfism. The mutation was introduced by site-directed mutagenesis into cDNAs encoding the full-length rabbit GH receptor and the extracellular domain or binding protein (BP) of the human and rabbit GH receptor, and also in cDNAs encoding the full length and the extracellular domain of the related rabbit prolactin (PRL) receptor. All constructs were transiently expressed in COS-7 cells. Both wild type and mutant full-length rabbit GH and PRL receptors, as well as GH and prolactin BPs (wild type and mutant), were detected by Western blot in cell membranes and concentrated culture media, respectively. Immunofluorescence studies showed that wild type and mutant full-length GH receptors had the same cell surface and intracellular distribution and were expressed with comparable intensities. In contrast, all mutant forms (full-length receptors or BPs), completely lost their modify the synthesis ligand. These results clearly demonstrate that this point mutation (patients with Laron syndrome) does not modify the synthesis or the intracellular pathway of receptor proteins, but rather abolishes ability of the receptor or BP to bind GH and is thus responsible for the extreme GH resistance in these patients. Images PMID:8450064

  5. Conversion of MyoD to a Neurogenic Factor: Binding Site Specificity Determines Lineage

    Directory of Open Access Journals (Sweden)

    Abraham P. Fong

    2015-03-01

    Full Text Available MyoD and NeuroD2, master regulators of myogenesis and neurogenesis, bind to a “shared” E-box sequence (CAGCTG and a “private” sequence (CAGGTG or CAGATG, respectively. To determine whether private-site recognition is sufficient to confer lineage specification, we generated a MyoD mutant with the DNA-binding specificity of NeuroD2. This chimeric mutant gained binding to NeuroD2 private sites but maintained binding to a subset of MyoD-specific sites, activating part of both the muscle and neuronal programs. Sequence analysis revealed an enrichment for PBX/MEIS motifs at the subset of MyoD-specific sites bound by the chimera, and point mutations that prevent MyoD interaction with PBX/MEIS converted the chimera to a pure neurogenic factor. Therefore, redirecting MyoD binding from MyoD private sites to NeuroD2 private sites, despite preserved binding to the MyoD/NeuroD2 shared sites, is sufficient to change MyoD from a master regulator of myogenesis to a master regulator of neurogenesis.

  6. Estrogen effects on angiotensin receptors are modulated by pituitary in female rats

    International Nuclear Information System (INIS)

    Douglas, J.G.

    1987-01-01

    The present studies were designed to test the hypothesis that changes in angiotensin II (ANG II) receptors might modulate the layered target tissue responsiveness accompanying estradiol administration. Estradiol was infused continuously in oophorectomized female rats. Aldosterone was also infused in control and experimental animals to avoid estrogen-induced changes in renin and ANG II. ANG II binding constants were determined in radioreceptor assays. Estradiol increased binding site concentration in adrenal glomerulosa by 76% and decreased binding sites of uterine myometrium and glomeruli by 45 and 24%, respectively. There was an accompanying increase in the affinity of ANG II binding to adrenal glomerulosa and uterine myometrium. Because estrogen is a potent stimulus of prolactin release from the pituitary of rodents, studies were also designed to test the hypothesis that prolactin may mediate some or all of the estrogen-induced effects observed. Hypophysectomy abolished estradiol stimulation of prolactin release and most ANG II receptor changes. Prolactin administration to pituitary intact rats was associated with a 50% increase in receptor density of adrenal glomerulosa simulating estradiol administration. However, the changes in glomeruli and uterine myometrium were opposite in that both tissues also increased receptor density, suggesting that prolactin was not the sole mediator of the estrogen-induced receptor changes. In conclusion, regulation of ANG II receptors in a number of diverse target tissues by estradiol is complex with contributions from estrogens and pituitary factors, which include but do not exclusively involve prolactin

  7. Agnus castus extracts inhibit prolactin secretion of rat pituitary cells.

    Science.gov (United States)

    Sliutz, G; Speiser, P; Schultz, A M; Spona, J; Zeillinger, R

    1993-05-01

    In our studies on prolactin inhibition by plant extracts we focused on the effects of extracts of Vitex agnus castus and its preparations on rat pituitary cells under basal and stimulated conditions in primary cell culture. Both extracts from Vitex agnus castus as well as synthetic dopamine agonists (Lisuride) significantly inhibit basal as well as TRH-stimulated prolactin secretion of rat pituitary cells in vitro and as a consequence inhibition of prolactin secretion could be blocked by adding a dopamine receptor blocker. Therefore because of its dopaminergic effect Agnus castus could be considered as an efficient alternative phytotherapeutic drug in the treatment of slight hyperprolactinaemia.

  8. Suppression of prolactin gene expression in GH cells correlates with site-specific DNA methylation.

    Science.gov (United States)

    Zhang, Z X; Kumar, V; Rivera, R T; Pasion, S G; Chisholm, J; Biswas, D K

    1989-10-01

    Prolactin- (PRL) producing and nonproducing subclones of the GH line of (rat) pituitary tumor cells have been compared to elucidate the regulatory mechanisms of PRL gene expression. Particular emphasis was placed on delineating the molecular basis of the suppressed state of the PRL gene in the prolactin-nonproducing (PRL-) GH subclone (GH(1)2C1). We examined six methylatable cytosine residues (5, -CCGG- and 1, -GCGC-) within the 30-kb region of the PRL gene in these subclones. This analysis revealed that -CCGG-sequences of the transcribed region, and specifically, one in the fourth exon of the PRL gene, were heavily methylated in the PRL-, GH(1)2C1 cells. Furthermore, the inhibition of PRL gene expression in GH(1)2C1 was reversed by short-term treatment of the cells with a sublethal concentration of azacytidine (AzaC), an inhibitor of DNA methylation. The reversion of PRL gene expression by AzaC was correlated with the concurrent demethylation of the same -CCGG- sequences in the transcribed region of PRL gene. An inverse correlation between PRL gene expression and the level of methylation of the internal -C- residues in the specific -CCGG-sequence of the transcribed region of the PRL gene was demonstrated. The DNase I sensitivity of these regions of the PRL gene in PRL+, PRL-, and AzaC-treated cells was also consistent with an inverse relationship between methylation state, a higher order of structural modification, and gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Down-regulation of endothelin binding sites in rat vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Roubert, P.; Gillard, V.; Plas, P.; Chabrier, P.E.; Braquet, P.

    1990-01-01

    In cultured rat aortic smooth muscle cells, [ 125 I]endothelin (ET-1) bound to an apparent single class of high affinity recognition sites with a dissociation constant of 1.84 +/- 0.29 nmol/L and a maximum binding of 62 +/- 10.5 fmol/10(6) cells. The binding was not affected by calcium antagonists or vasoactive substances, including angiotensin II, arginine vasopressin, atrial natriuretic factor and bradykinin. Exposure of the cells to ET-1 (0.01 nmol/L to 10 nmol/L) resulted in an apparent dose-dependent reduction of the number of endothelin binding sites with no significant modification of its binding affinity. The time course of the down-regulation of ET-1 binding sites showed that this effect was present after 30 min incubation and persisted after 18 h. This indicates that down-regulation of ET-1 binding sites can modulate the activity of ET-1 and suggests a rapid internalization of ET-1 in vascular cells

  10. Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

    Science.gov (United States)

    Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

    2011-05-31

    Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

  11. RELATIONSHIP BETWEEN PROLACTIN HORMONE LEVEL, MOLTING AND DUCK EGG PRODUCTION

    Directory of Open Access Journals (Sweden)

    T. Susanti

    2012-09-01

    Full Text Available The aims of this study were to obtain information on the mechanism of molting and the prolactin hormone levels affecting egg production. The study utilized AP (crossbred of Alabio ♂ with Peking ♀ and PA (crossbred of Peking ♂ and Alabio ♀ ducks with a total of 180 birds. The observed variables were the duration of cessation of egg production before and after molting, the prolactin hormone level in the period of molting, the egg production period before and after molting. The data was analyzed using ANOVA, regression and correlation. The results showed that AP crossbred had fewer molting (23.33% compared to PA (50.00%. The mechanism of molting is always preceded by cessation of egg production, molting and relaying. The prolactin hormone concentrations of AP and PA in the period before and after molting were significantly higher than in the period of molting. At the egg production period before molting, the prolactin hormone concentration of AP ducks was higher than the PA ducks. So that the egg production of AP before molting (0-16 weeks was higher than the PA. The egg production of AP was higher than PA, 256.66±6.00 vs 232.22±6.64 eggs for 48 weeks. So it can be concluded that the prolactin hormone affects the molting and egg production.

  12. Obtention of antibodies anti prolactin from human prolactin of national production; Obtencion de anticuerpos anti-prolactina a partir de prolactina humana de produccion nacional

    Energy Technology Data Exchange (ETDEWEB)

    Caso, R; Mosquera, M [Centro de Isotopos, La Habana (Cuba); Perez, E [Centro de Investigaciones de Mejoramiento Animal, La Habana (Cuba); Amanz, C [Instituto Nacional de Endocrinologia, La Habana (Cuba)

    1996-07-01

    In this work was studied the use of the the Prolactin hormone as immuno gen, which is obtained in Cuba by the pharmaceutical institute Mario Munoz, to produce the antibody antiprolactin. Was made the validation of obtained antibody (tritatium, specificity and affinity) The produced antibody had necessary quality to be use as a component of the Kits-RIA Prolactin.

  13. Exploring the composition of protein-ligand binding sites on a large scale.

    Directory of Open Access Journals (Sweden)

    Nickolay A Khazanov

    Full Text Available The residue composition of a ligand binding site determines the interactions available for diffusion-mediated ligand binding, and understanding general composition of these sites is of great importance if we are to gain insight into the functional diversity of the proteome. Many structure-based drug design methods utilize such heuristic information for improving prediction or characterization of ligand-binding sites in proteins of unknown function. The Binding MOAD database if one of the largest curated sets of protein-ligand complexes, and provides a source of diverse, high-quality data for establishing general trends of residue composition from currently available protein structures. We present an analysis of 3,295 non-redundant proteins with 9,114 non-redundant binding sites to identify residues over-represented in binding regions versus the rest of the protein surface. The Binding MOAD database delineates biologically-relevant "valid" ligands from "invalid" small-molecule ligands bound to the protein. Invalids are present in the crystallization medium and serve no known biological function. Contacts are found to differ between these classes of ligands, indicating that residue composition of biologically relevant binding sites is distinct not only from the rest of the protein surface, but also from surface regions capable of opportunistic binding of non-functional small molecules. To confirm these trends, we perform a rigorous analysis of the variation of residue propensity with respect to the size of the dataset and the content bias inherent in structure sets obtained from a large protein structure database. The optimal size of the dataset for establishing general trends of residue propensities, as well as strategies for assessing the significance of such trends, are suggested for future studies of binding-site composition.

  14. ACTIONS OF PROLACTIN IN THE BRAIN: FROM PHYSIOLOGICAL ADAPTATIONS TO STRESS AND NEUROGENESIS TO PSYCHOPATHOLOGY

    Directory of Open Access Journals (Sweden)

    Luz eTorner

    2016-03-01

    Full Text Available Prolactin is one of the most versatile hormones known. It is considered an adaptive hormone due to the key roles it plays in the modulation of the stress response and during pregnancy and lactation. Within the brain, prolactin acts as a neuropeptide to promote physiological responses related to reproduction, stress adaptation, neurogenesis, and neuroprotection. The action of prolactin on the nervous system contributes to the wide array of changes that occur in the female brain during pregnancy and result in the attenuation of the hypothalamic pituitary adrenal axis. Together, all these changes promote behavioral and physiological adaptations of the new mother to enable reproductive success. Brain adaptations driven by prolactin are also important for the regulation of maternal emotionality and wellbeing Prolactin also affects the male brain during the stress response but its effects have been less studied. Prolactin regulates neurogenesis both in the subventricular zone and in the hippocampus. Therefore, alterations in the prolactin system due to stress, or exposure to substances that reduce neurogenesis or other conditions, could contribute to maladaptive responses and pathological behavioral outcomes. Here we review the prolactin system and the role it plays in the modulation of stress response and emotion regulation. We discuss the effects of prolactin on neurogenesis and neuroprotection, the putative neuronal mechanisms underlying these effects, and their contribution to the onset of psychopathological states like depression.

  15. Location and nature of calcium-binding sites in salivary acidic proline-rich phosphoproteins

    International Nuclear Information System (INIS)

    Bennick, A.; McLaughlin, A.C.; Grey, A.A.; Madapallimattam, G.

    1981-01-01

    The location of the calcium-binding sites in the human acidic proline-rich proteins, salivary proteins A and C, was determined by equilibrium dialysis of the tryptic peptides with buffers containing 45 Ca. All the calcium-binding sites are located in the NH 2 -terminal tryptic peptide (TX peptide). The nature of the calcium binding sites in the TX peptide and native salivary proteins A and C, as well as dephosphorylated proteins was compared. Two types of sites can be distinguished in peptide TX. Type I sites have an apparent dissociation constant (K) of 38 μM and are responsible for the binding of 2.6 mol of Ca/mol of peptide. The corresponding figures for Type II sites are 780 μM and 5.3 mol of Ca/mol of peptide. In the native proteins, the amount of calcium bound at the type II sites decreases to 3.9 mol of Ca/mol of proteins A and C and K increases to 1100 μM. The amount of calcium bound at type I sites decreases to 1.5 mol/mol of protein A and 0.6 mol/mol of protein C, but there is no change in K. Dephosphorylation affects the calcium binding at both types of sites. The experiments indicate that the COOH-terminal parts of the native proteins affect the number and the nature of the protein calcium-binding sites. Proton and phosphorous NMR data demonstrate that β-COOH in aspartic acid, as well as phosphoserine, are part of the calcium-binding sites. The difference in calcium binding to salivary proteins A and C may be due at least partially to differences in the environment of one or more aspartic acids

  16. Follow-up of prolactin levels in patients with breast cancer metastases treated by cryohypophysectomy

    International Nuclear Information System (INIS)

    Rozsival, V.; Petr, R.; Kubicek, J.; Hajek, P.; Fingerova, H.; Talas, M.; Janouskova, M.

    1981-01-01

    In the years 1977 to 1979, prolactin levels were examined in the blood of 39 patients with breast cancer metastases in the skeleton. In 27 patients undergoing surgery, prolactin values were obtained prior to the operation and on the 7th day after stereotactic cryohypophysectomy; in 19 patients the values were obtained also at later intervals. Prolactin was examined using RIA. Prior to surgery, the prolactin levels ranged between 4.3 and above 100 μg/l, with an average of 24.69. Seven days after cryohypophysectomy, the average was 14.01 μg/l, i.e., a remarkable decrease was observed showing considerable significance in the pair test. Prolactin examination in patients with breast cancer metastases showed increased levels above the menopausal standard in almost 80% of the group of patients prior to hypophysectomy. After surgery, a prolactin level decrease was observed in 60% of patients, which confirmed that the intervention in the hypophysis was effective. (author)

  17. SERUM PROLACTIN LEVEL IN ACNE PATIENTS VERSUS CONTROL GROUP: STUDY ON 14 TO 35 YEARS OLD WOMEN

    Directory of Open Access Journals (Sweden)

    G FAGHIHI

    2002-03-01

    Full Text Available Introduction. Androgens have a main role in acne pathogenesis. The interaction between prolactin and androgens generate the hypothesis of prolactin role in acne pathogenesis. Methods. In a case - control study, 71 women with modearte to severe acne were compaired with 71 healthy women about their serum prolactin levels. Results. Mean of serum prolactin level was 533 632 miu/lit in cases. Mean of serum prolactin level was 365 363 in control group (P > 0.05. There was a significant correlation between serum prolactin level and severity of acne. Discussion. Despite the non significant difference between serum prolactin level in acne patients and healthy women, thare was a significant relationship between serum prolactin level and severity of acne. It may be due to our small sample size. However, the more powerful studies is needed.

  18. Radiotracers for per studies of neurotransmitter binding sites: Design considerations

    International Nuclear Information System (INIS)

    Kilbourn, M.R.

    1991-01-01

    Neurotransmitter binding sites, such as receptors, neuronal uptake systems, and vesicular uptake systems, are important targets for new radiopharmaceutical design. Selection of potential radioligands can be guided by in vitro laboratory data including such characteristics as selectivity and affinity for specific binding sites. However, development of PET radiotracers for use in vivo must include considerations of in vivo pharmacokinetics and metabolism. Introduction of potential radioligands is further narrowed by the demands of the radiochemical synthesis, which must produce radioligands of high chemical and radiochemical purity and of high specific activity. This paper will review examples of previous and current attempts by radiopharmaceutical chemists to meet these demands for new positron emitter-labeled radioligands for PET studies of a wide array of neurotransmitter binding sites

  19. Increased prolactin levels are associated with impaired processing speed in subjects with early psychosis.

    Directory of Open Access Journals (Sweden)

    Itziar Montalvo

    Full Text Available Hyperprolactinaemia, a common side effect of some antipsychotic drugs, is also present in drug-naïve psychotic patients and subjects at risk for psychosis. Recent studies in non-psychiatric populations suggest that increased prolactin may have negative effects on cognition. The aim of our study was to explore whether high plasma prolactin levels are associated with poorer cognitive functioning in subjects with early psychoses. We studied 107 participants: 29 healthy subjects and 78 subjects with an early psychosis (55 psychotic disorders with <3 years of illness, 23 high-risk subjects. Cognitive assessment was performed with the MATRICS Cognitive Consensus Cognitive Battery, and prolactin levels were determined as well as total cortisol levels in plasma. Psychopathological status was assessed and the use of psychopharmacological treatments (antipsychotics, antidepressants, benzodiazepines recorded. Prolactin levels were negatively associated with cognitive performance in processing speed, in patients with a psychotic disorder and high-risk subjects. In the latter group, increased prolactin levels were also associated with impaired reasoning and problem solving and poorer general cognition. In a multiple linear regression analysis conducted in both high-risk and psychotic patients, controlling for potential confounders, prolactin and benzodiazepines were independently related to poorer cognitive performance in the speed of processing domain. A mediation analysis showed that both prolactin and benzodiazepine treatment act as mediators of the relationship between risperidone/paliperidone treatment and speed of processing. These results suggest that increased prolactin levels are associated with impaired processing speed in early psychosis. If these results are confirmed in future studies, strategies targeting reduction of prolactin levels may improve cognition in this population.

  20. Development Of Radioimmunoassay For Prolactin HORMONE Using Solid Phase Magnetic Particles

    International Nuclear Information System (INIS)

    SHAFIK, H.M.; MEHANY, N.L.

    2009-01-01

    The preparation and development of primary reagents of prolactin (PRL) radioimmunoassay (RIA) technique using solid phase magnetic particles with low cost is considered to be the main objective of the present study. The production of polyclonal antibodies was undertaken by immunizing four female New-Zealand rabbits through primary injection and four booster doses subcutaneously. The preparation of 125 I-prolactin radiotracer was carried out using chloramine-T. The preparation of standard prolactin was undertaken by preparing stock standard solution of prolactin and diluted with assay buffer. Activation and coupling of low magnetizable particles with the purified anti-PRL was carried out. Optimization and validation of the assay were carried out. The results obtained provide a highly sensitive, specific and accurate RIA system of PRL. In conclusion, this assay could be used in diagnosis of galactorrhea, prolactinoma, visual impairment and diagnosis of infertility in males and females.

  1. Flow-cytometric determination of high-density-lipoprotein binding sites on human leukocytes

    International Nuclear Information System (INIS)

    Schmitz, G.; Wulf, G.; Bruening, T.A.; Assmann, G.

    1987-01-01

    In this method, leukocytes were isolated from 6 mL of EDTA-blood by density-gradient centrifugation and subsequently incubated with rhodamine isothiocyanate (RITC)-conjugated high-density lipoproteins (HDL). The receptor-bound conjugate particles were determined by fluorescent flow cytometry and compared with 125 I-labeled HDL binding data for the same cells. Human granulocytes express the highest number of HDL binding sites (9.4 x 10(4)/cell), followed by monocytes (7.3 x 10(4)/cell) and lymphocytes (4.0 x 10(4)/cell). Compared with conventional analysis of binding of 125 I-labeled HDL in tissue-culture dishes, the present determination revealed significantly lower values for nonspecific binding. In competition studies, the conjugate competes for the same binding sites as 125 I-labeled HDL. With the use of tetranitromethane-treated HDL3, which fails to compete for the HDL receptor sites while nonspecific binding is not affected, we could clearly distinguish between 37 degrees C surface binding and specific 37 degrees C uptake of RITC-HDL3, confirming that the HDL receptor leads bound HDL particles into an intracellular pathway rather than acting as a docking type of receptor. Patients with familial dysbetalipoproteinemia showed a significantly higher number of HDL binding sites in the granulocyte population but normal in lymphocytes and monocytes, indicating increased uptake of cholesterol-containing lipoproteins. In patients with familial hypercholesterolemia, HDL binding was increased in all three cell types, indicating increased cholesterol uptake and increased cholesterol synthesis. The present method allows rapid determination of HDL binding sites in leukocytes from patients with various forms of hyper- and dyslipoproteinemias

  2. Increased prolactin levels are associated with impaired processing speed in subjects with early psychosis.

    Science.gov (United States)

    Montalvo, Itziar; Gutiérrez-Zotes, Alfonso; Creus, Marta; Monseny, Rosa; Ortega, Laura; Franch, Joan; Lawrie, Stephen M; Reynolds, Rebecca M; Vilella, Elisabet; Labad, Javier

    2014-01-01

    Hyperprolactinaemia, a common side effect of some antipsychotic drugs, is also present in drug-naïve psychotic patients and subjects at risk for psychosis. Recent studies in non-psychiatric populations suggest that increased prolactin may have negative effects on cognition. The aim of our study was to explore whether high plasma prolactin levels are associated with poorer cognitive functioning in subjects with early psychoses. We studied 107 participants: 29 healthy subjects and 78 subjects with an early psychosis (55 psychotic disorders with levels were determined as well as total cortisol levels in plasma. Psychopathological status was assessed and the use of psychopharmacological treatments (antipsychotics, antidepressants, benzodiazepines) recorded. Prolactin levels were negatively associated with cognitive performance in processing speed, in patients with a psychotic disorder and high-risk subjects. In the latter group, increased prolactin levels were also associated with impaired reasoning and problem solving and poorer general cognition. In a multiple linear regression analysis conducted in both high-risk and psychotic patients, controlling for potential confounders, prolactin and benzodiazepines were independently related to poorer cognitive performance in the speed of processing domain. A mediation analysis showed that both prolactin and benzodiazepine treatment act as mediators of the relationship between risperidone/paliperidone treatment and speed of processing. These results suggest that increased prolactin levels are associated with impaired processing speed in early psychosis. If these results are confirmed in future studies, strategies targeting reduction of prolactin levels may improve cognition in this population.

  3. Distribution of [3H]diadenosine tetraphosphate binding sites in rat brain

    International Nuclear Information System (INIS)

    Miras-Portugal, M.T.; Palacios, J.M.; Torres, M.; Cortes, R.; Rodriguez-Pascual, F.

    1997-01-01

    The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [ 3 H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation.The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  4. Association of Thyroid Profile and Prolactin Level in Patient with Secondary Amenorrhea

    Science.gov (United States)

    Shrestha, Sujata; Neupane, Sunita; Gautam, Narayan; Dubey, Raju Kumar; Jha, Amit Chandra; Doshi, Nilesh Raj; Jayan, Archana

    2016-01-01

    Background Amenorrhea is the absence of menstrual periods. It has multiple social consequences as it may leads to infertility. This case control study was conducted for determining the association of thyroid hormones with hyperprolactinemia in patient with amenorrhea. Methods We investigated 50 women with diagnosed cases of secondary amenorrhoea, who attended UCMS hospital, for hormonal evaluations. Fifty two healthy women were taken as the controls. The thyroid dysfunction and serum prolactin level were reviewed in cases and in the controls. Results Mean serum prolactin level was found to be significantly higher in the cases as compared to the controls. Mean serum fT3 and fT4 level in the hyperprolactinemic cases (mean = 2.67, SD = 1.04 pg/ml) and (mean = 1.38, SD = 0.51 ng/dl respectively) were slightly lower as compared to normoprolactinemic cases (mean = 3.21, SD = 1.86 pg/ml) and (mean = 1.73, SD = 1.37 ng/dl) respectively. Mean TSH of normoprolactinemic and hyperprolactinemic cases were comparable (P = 0.049). There was positive correlation between prolactin, BMI and TSH whereas negative correlation of prolactin was seen with fT3, fT4 and age. In hyperprolactainemic cases, prolactin was found to be negatively correlated with TSH (r = −0.155, P = 0.491) whereas prolactin was positively correlated with TSH (r = 0.296, P = 0.126) in normoprolactainemic cases. Conclusions Thus, hyperprolactinemia with thyroid dysfunction may be contributory hormonal factor in patient with amenorrhoea and as such, estimation of prolactin, fT3, fT4 and TSH should be included for diagnostic evaluation of amenorrhea. PMID:27904425

  5. Fundamental studies, reference values and relationship to menstrual cycle on Prolactin RIA BEAD II

    International Nuclear Information System (INIS)

    Kobayashi, Midori; Sakai, Rinko; Satoh, Shigekiyo; Maruyama, Kiyoji; Kanai, Masamitsu

    1989-01-01

    We have tried fundamental studies, reference values and relationship to menstrual cycle on Prolactin RIA BEAD II kit which has a method of IRMA using monocronal antibody. On clinical studies, we investigated change of serum prolactin level during the menstrual cycle and relationship to other hormones (LH, FSH, estradiol, progesterone). It was the result that prolactin level of follicular phase was lower than that of preavulatory phase and luteal phase. We conclude that change of prolactin level during the menstrual cycle is related with change of estradiol level. (author)

  6. Fundamental studies, reference values and relationship to menstrual cycle on Prolactin RIA BEAD II

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Midori; Sakai, Rinko; Satoh, Shigekiyo; Maruyama, Kiyoji; Kanai, Masamitsu (Shinshu Univ., Matsumoto, Nagano (Japan). Faculty of Medicine)

    1989-06-01

    We have tried fundamental studies, reference values and relationship to menstrual cycle on Prolactin RIA BEAD II kit which has a method of IRMA using monocronal antibody. On clinical studies, we investigated change of serum prolactin level during the menstrual cycle and relationship to other hormones (LH, FSH, estradiol, progesterone). It was the result that prolactin level of follicular phase was lower than that of preavulatory phase and luteal phase. We conclude that change of prolactin level during the menstrual cycle is related with change of estradiol level. (author).

  7. A disposable electrochemical immunosensor for prolactin involving affinity reaction on streptavidin-functionalized magnetic particles

    International Nuclear Information System (INIS)

    Moreno-Guzman, Maria; Gonzalez-Cortes, Araceli; Yanez-Sedeno, Paloma; Pingarron, Jose M.

    2011-01-01

    A novel electrochemical immunosensor was developed for the determination of the hormone prolactin. The design involved the use of screen-printed carbon electrodes and streptavidin-functionalized magnetic particles. Biotinylated anti-prolactin antibodies were immobilized onto the functionalized magnetic particles and a sandwich-type immunoassay involving prolactin and anti-prolactin antibody labelled with alkaline phosphatase was employed. The resulting bio-conjugate was trapped on the surface of the screen-printed electrode with a small magnet and prolactin quantification was accomplished by differential pulse voltammetry of 1-naphtol formed in the enzyme reaction using 1-naphtyl phosphate as alkaline phosphatase substrate. All variables involved in the preparation of the immunosensor and in the electrochemical detection step were optimized. The calibration plot for prolactin exhibited a linear range between 10 and 2000 ng mL -1 with a slope value of 7.0 nA mL ng -1 . The limit of detection was 3.74 ng mL -1 . Furthermore, the modified magnetic beads-antiprolactin conjugates showed an excellent stability. The immunosensor exhibited also a high selectivity with respect to other hormones. The analytical usefulness of the immnunosensor was demonstrated by analyzing human sera spiked with prolactin at three different concentration levels.

  8. A disposable electrochemical immunosensor for prolactin involving affinity reaction on streptavidin-functionalized magnetic particles

    Energy Technology Data Exchange (ETDEWEB)

    Moreno-Guzman, Maria; Gonzalez-Cortes, Araceli [Department of Analytical Chemistry, Faculty of Chemistry, University Computense of Madrid, 28040 Madrid (Spain); Yanez-Sedeno, Paloma, E-mail: yseo@quim.ucm.es [Department of Analytical Chemistry, Faculty of Chemistry, University Computense of Madrid, 28040 Madrid (Spain); Pingarron, Jose M. [Department of Analytical Chemistry, Faculty of Chemistry, University Computense of Madrid, 28040 Madrid (Spain)

    2011-04-29

    A novel electrochemical immunosensor was developed for the determination of the hormone prolactin. The design involved the use of screen-printed carbon electrodes and streptavidin-functionalized magnetic particles. Biotinylated anti-prolactin antibodies were immobilized onto the functionalized magnetic particles and a sandwich-type immunoassay involving prolactin and anti-prolactin antibody labelled with alkaline phosphatase was employed. The resulting bio-conjugate was trapped on the surface of the screen-printed electrode with a small magnet and prolactin quantification was accomplished by differential pulse voltammetry of 1-naphtol formed in the enzyme reaction using 1-naphtyl phosphate as alkaline phosphatase substrate. All variables involved in the preparation of the immunosensor and in the electrochemical detection step were optimized. The calibration plot for prolactin exhibited a linear range between 10 and 2000 ng mL{sup -1} with a slope value of 7.0 nA mL ng{sup -1}. The limit of detection was 3.74 ng mL{sup -1}. Furthermore, the modified magnetic beads-antiprolactin conjugates showed an excellent stability. The immunosensor exhibited also a high selectivity with respect to other hormones. The analytical usefulness of the immnunosensor was demonstrated by analyzing human sera spiked with prolactin at three different concentration levels.

  9. Predicting Flavin and Nicotinamide Adenine Dinucleotide-Binding Sites in Proteins Using the Fragment Transformation Method

    Directory of Open Access Journals (Sweden)

    Chih-Hao Lu

    2015-01-01

    Full Text Available We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified.

  10. Quantitative autoradiography of [3H]ouabain binding sites in rat brain

    International Nuclear Information System (INIS)

    Spyropoulos, A.C.; Rainbow, T.C.

    1984-01-01

    In vitro quantitative autoradiography was used to localize in rat brain binding sites for [ 3 H]ouabain, an inhibitor of the Na + ,K + -ATPase. High levels of [ 3 H]ouabain sites were found in the superior and inferior colliculi, the mammillary nucleus, the interpeduncular nucleus, and in various divisions of the olfactory, auditory and somatomotor systems. The heterogeneous distribution of [ 3 H]ouabain binding closely parallels the regional brain glucose consumption as determined by the [ 14 C]deoxyglucose method. Lesion studies of the rat hippocampus using the excitotoxin, ibotenic acid, showed both a marked decrease of neuronal cell types on the injected side and a corresponding decrease in [ 3 H]ouabain binding, indicating that some of the [ 3 H]ouabain binding sites are localized to neurons. The close correlation between [ 3 H]ouabain binding and regional glucose utilization provides further evidence for a linkage between glucose utilization and the neuronal Na + ,K + -ATPase. (Auth.)

  11. [Adenylate cyclase from rabbit heart: substrate binding site].

    Science.gov (United States)

    Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

    1981-08-01

    The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

  12. Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

    International Nuclear Information System (INIS)

    Mueller, H.; Fehr, J.

    1986-01-01

    The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

  13. Serum prolactin profiles of normal human conception cycles

    Energy Technology Data Exchange (ETDEWEB)

    Adejuwon, C A [Ibadan Univ. (Nigeria). Coll. of Medicine; Faundes, Anibal [Universidade Estadual de Campinas (Brazil). Faculdade de Medicina; Segal, S J [Rockefeller Foundation, New York (USA); Alvarez-Sanchez, Francisco [Hospital Moscoso Puello, Santo Domingo (Dominican Republic). Dept. of Obstet. and Gynaecol.

    1984-06-01

    Commencing on day 10 of the menstrual cycle through onset of subsequent menses, or confirmation of pregnancy, daily sera collected from 15 women planning pregnancy were analyzed by radioimmunoassays (RIA) for prolactin (hPRL), estradiol-17..beta.. and luteinizing hormone (hLH). Two of the observed subjects became pregnant in the single cycles studied. The profiles of these hormones during the early gestation following spontaneous ovulation were established. No distinct midcycle peaks of hPEL were observed in either subject. Enormous spikes were observed in daily prolactin values, with wide variations between subjects.

  14. Plasma concentrations of prolactin in overtly pseudopregnant Afghan hounds and the effect of metergoline.

    Science.gov (United States)

    Okkens, A C; Dieleman, S J; Kooistra, H S; Bevers, M M

    1997-01-01

    The effect of metergoline, a 5-hydroxytryptamine (serotonin) antagonist, on the plasma concentrations of prolactin in overtly pseudopregnant Afghan hounds and on the clinical symptoms of overt pseudopregnancy were studied. Plasma concentrations of prolactin and progesterone were determined in six Afghan hounds with signs of overt pseudopregnancy for 2-3 weeks and in three Afghan hounds that were not pseudopregnant at the time of blood sampling. In the overtly pseudopregnant bitches the plasma concentrations of prolactin before treatment (35.5 +/- 8.5 micrograms l-1) were significantly higher than the plasma concentrations of prolactin of the three bitches that were not pseudopregnant (6.3 +/- 0.5 micrograms l-1); the latter values were similar to those of non-pseudopregnant beagle bitches during the total luteal phase. The six pseudopregnant Afghan hounds were treated for 10 days with the antiserotoninergic drug metergoline. At 2 h after the onset of treatment with metergoline, the mean plasma concentration of prolactin had decreased to 10.8 +/- 2.9 micrograms l-1. The plasma concentrations of prolactin continued to decline to 5.4 +/- 1.0 micrograms l-1 at 4 h and to 1.0 +/- 0.1 microgram l-1 during treatment days 3-10. Signs of pseudopregnancy, such as swelling of the mammary glands and digging, decreased during the treatment period. The treatment was associated with mild behavioural side effects such as whimpering and aggressiveness. These side effects are probably not related to suppression of prolactin but are due to a direct effect on serotoninergic pathways in the brain. It is concluded that high plasma concentrations of prolactin are associated with the development and maintenance of pseudopregnancy. The serotonin antagonist metergoline strongly suppresses plasma concentrations of prolactin in pseudopregnant dogs and decreases the clinical signs of pseudopregnancy.

  15. Regulatory role of melatonin and BMP-4 in prolactin production by rat pituitary lactotrope GH3 cells.

    Science.gov (United States)

    Ogura-Ochi, Kanako; Fujisawa, Satoshi; Iwata, Nahoko; Komatsubara, Motoshi; Nishiyama, Yuki; Tsukamoto-Yamauchi, Naoko; Inagaki, Kenichi; Wada, Jun; Otsuka, Fumio

    2017-08-01

    The effects of melatonin on prolactin production and its regulatory mechanism remain uncertain. We investigated the regulatory role of melatonin in prolactin production using rat pituitary lactotrope GH3 cells by focusing on the bone morphogenetic protein (BMP) system. Melatonin receptor activation, induced by melatonin and its receptor agonist ramelteon, significantly suppressed basal and forskolin-induced prolactin secretion and prolactin mRNA expression in GH3 cells. The melatonin MT2 receptor was predominantly expressed in GH3 cells, and the inhibitory effects of melatonin on prolactin production were reversed by treatment with the receptor antagonist luzindole, suggesting functional involvement of MT2 action in the suppression of prolactin release. Melatonin receptor activation also suppressed BMP-4-induced prolactin expression by inhibiting phosphorylation of Smad and transcription of the BMP-target gene Id-1, while BMP-4 treatment upregulated MT2 expression. Melatonin receptor activation suppressed basal, BMP-4-induced and forskolin-induced cAMP synthesis; however, BtcAMP-induced prolactin mRNA expression was not affected by melatonin or ramelteon, suggesting that MT2 activation leads to inhibition of prolactin production through the suppression of Smad signaling and cAMP synthesis. Experiments using intracellular signal inhibitors revealed that the ERK pathway is, at least in part, involved in prolactin induction by GH3 cells. Thus, a new regulatory role of melatonin involving BMP-4 in prolactin secretion was uncovered in lactotrope GH3 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Computational design of trimeric influenza-neutralizing proteins targeting the hemagglutinin receptor binding site

    Energy Technology Data Exchange (ETDEWEB)

    Strauch, Eva-Maria; Bernard, Steffen M.; La, David; Bohn, Alan J.; Lee, Peter S.; Anderson, Caitlin E.; Nieusma, Travis; Holstein, Carly A.; Garcia, Natalie K.; Hooper, Kathryn A.; Ravichandran, Rashmi; Nelson, Jorgen W.; Sheffler, William; Bloom, Jesse D.; Lee, Kelly K.; Ward, Andrew B.; Yager, Paul; Fuller, Deborah H.; Wilson, Ian A.; Baker , David (UWASH); (Scripps); (FHCRC)

    2017-06-12

    Many viral surface glycoproteins and cell surface receptors are homo-oligomers1, 2, 3, 4, and thus can potentially be targeted by geometrically matched homo-oligomers that engage all subunits simultaneously to attain high avidity and/or lock subunits together. The adaptive immune system cannot generally employ this strategy since the individual antibody binding sites are not arranged with appropriate geometry to simultaneously engage multiple sites in a single target homo-oligomer. We describe a general strategy for the computational design of homo-oligomeric protein assemblies with binding functionality precisely matched to homo-oligomeric target sites5, 6, 7, 8. In the first step, a small protein is designed that binds a single site on the target. In the second step, the designed protein is assembled into a homo-oligomer such that the designed binding sites are aligned with the target sites. We use this approach to design high-avidity trimeric proteins that bind influenza A hemagglutinin (HA) at its conserved receptor binding site. The designed trimers can both capture and detect HA in a paper-based diagnostic format, neutralizes influenza in cell culture, and completely protects mice when given as a single dose 24 h before or after challenge with influenza.

  17. Characterization of [3H] oxymorphone binding sites in mouse brain

    DEFF Research Database (Denmark)

    Yoo, Ji Hoon; Borsodi, Anna; Tóth, Géza

    2017-01-01

    Oxymorphone, one of oxycodone's metabolic products, is a potent opioid receptor agonist which is thought to contribute to the analgesic effect of its parent compound and may have high potential abuse liability. Nonetheless, the in vivo pharmacological binding profile of this drug is still unclear....... This study uses mice lacking mu (MOP), kappa (KOP) or delta (DOP) opioid receptors as well as mice lacking all three opioid receptors to provide full characterisation of oxymorphone binding sites in the brain. Saturation binding studies using [3H]oxymorphone revealed high affinity binding sites in mouse......]Oxymorphone binding was completely abolished across the majority of the brain regions in mice lacking MOP as well as in mice lacking all three opioid receptors. DOP and KOP knockout mice retained [3H]oxymorphone binding sites suggesting oxymorphone may not target DOP or KOP. These results confirm that the MOP...

  18. Nonequivalence of alpha-bungarotoxin binding sites in the native nicotinic receptor molecule

    International Nuclear Information System (INIS)

    Conti-Tronconi, B.M.; Tang, F.; Walgrave, S.; Gallagher, W.

    1990-01-01

    In the native, membrane-bound form of the nicotinic acetylcholine receptor (M-AcChR) the two sites for the cholinergic antagonist alpha-bungarotoxin (alpha-BGT) have different binding properties. One site has high affinity, and the M-AcChR/alpha-BGT complexes thus formed dissociate very slowly, similar to the complexes formed with detergent-solubilized AcChR (S-AcChR). The second site has much lower affinity (KD approximately 59 +/- 35 nM) and forms quickly reversible complexes. The nondenaturing detergent Triton X-100 is known to solubilize the AcChR in a form unable, upon binding of cholinergic ligands, to open the ion channel and to become desensitized. Solubilization of the AcChR in Triton X-100 affects the binding properties of this second site and converts it to a high-affinity, slowly reversible site. Prolonged incubation of M-AcChR at 4 degrees C converts the low-affinity site to a high-affinity site similar to those observed in the presence of Triton X-100. Although the two sites have similar properties when the AcChR is solubilized in Triton X-100, their nonequivalence can be demonstrated by the effect on alpha-BGT binding of concanavalin A, which strongly reduces the association rate of one site only. The Bmax of alpha-BGT to either Triton-solubilized AcChR or M-AcChR is not affected by the presence of concanavalin A. Occupancy of the high-affinity, slowly reversible site in M-AcChR inhibits the Triton X-100 induced conversion to irreversibility of the second site. At difference with alpha-BGT, the long alpha-neurotoxin from Naja naja siamensis venom (alpha-NTX) binds with high affinity and in a very slowly reversible fashion to two sites in the M-AcChR. We confirm here that Triton-solubilized AcChR or M-AcChR binds in a very slowly reversible fashion the same amount of alpha-NTX

  19. Effects of bovine prolactin gene polymorphism within exon 4 on milk ...

    African Journals Online (AJOL)

    In this study, polymorphism of prolactin gene was analyzed as a candidate gene responsible for variation and genetic trends in milk yield and composition traits. Genomic DNAs were extracted from 268 semen samples belonged to Iranian Holstein bulls. Genotyping for the prolactin gene using PCRRFLP technique and RsaI ...

  20. Effects of LHRH and ANG II on prolactin stimulation are mediated by hypophysial AT1 receptor subtype.

    Science.gov (United States)

    Becú-Villalobos, D; Lacau-Mengido, I M; Thyssen, S M; Díaz-Torga, G S; Libertun, C

    1994-02-01

    We have used the nonpeptide angiotensin II (ANG II) receptor antagonists losartan (receptor subtype AT1) and PD-123319 (AT2) to determine the participation of ANG II receptor subtypes in luteinizing hormone-releasing hormone (LHRH)-induced prolactin release in a perifusion study using intact pituitaries in vitro. LHRH (1.85 x 10(-7) M) released prolactin consistently, whereas losartan (10(-5) M) abolished prolactin response without modifying basal prolactin or luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release. PD-123319 (10(-5) M) had no effect on basal or LHRH-induced prolactin, LH, or FSH release. We also determined that the effect of ANG II on prolactin release was mediated by the same receptor subtype. In adenohypophysial cells dispersed in vitro ANG II (10(-8) M) released prolactin. Losartan (10(-7) and 10(-6) M), but not PD-123319, inhibited this effect. We conclude that in intact hypophyses of 15-day-old female rats the effect of LHRH on prolactin release is readily demonstrated. LHRH-induced prolactin release appears to be mediated by ANG II acting in a paracrine manner on AT1 receptors located on lactotrophs.

  1. Excitatory and inhibitory effects of prolactin release activated by nerve stimulation in rat anterior pituitary

    Directory of Open Access Journals (Sweden)

    Gao Li-Zhi

    2009-12-01

    Full Text Available Abstract Background A series of studies showed the presence of substantial amount of nerve fibers and their close relationship with the anterior pituitary gland cells. Our previous studies have suggested that aside from the classical theory of humoral regulation, the rat anterior pituitary has direct neural regulation on adrenocorticotropic hormone release. In rat anterior pituitary, typical synapses are found on every type of the hormone-secreting cells, many on lactotrophs. The present study was aimed at investigating the physiological significance of this synaptic relationship on prolactin release. Methods The anterior pituitary of rat was sliced and stimulated with electrical field in a self-designed perfusion chamber. The perfusate was continuously collected in aliquots and measured by radioimmunoassay for prolactin levels. After statistic analysis, differences of prolactin concentrations within and between groups were outlined. Results The results showed that stimulation at frequency of 2 Hz caused a quick enhancement of prolactin release, when stimulated at 10 Hz, prolactin release was found to be inhibited which came slower and lasted longer. The effect of nerve stimulation on prolactin release is diphasic and frequency dependent. Conclusions The present in vitro study offers the first physiological evidence that stimulation of nerve fibers can affect prolactin release in rat anterior pituitary. Low frequency stimulation enhances prolactin release and high frequency mainly inhibits it.

  2. Pineal-mediated inhibition of prolactin cell activity: Investigation of dopaminergic involvement

    International Nuclear Information System (INIS)

    Burns, D.M.

    1989-01-01

    The present studies in the male Syrian hamster addressed two issues. First, it was of interest to determine if anterior pituitaries of long photoperiod-exposed male hamsters possess dopamine receptors, which are presumably necessary for responsiveness to dopamine. This was accomplished by analysis of 3 H-spiperone binding to anterior pituitary membranes. Second, possible changes in pituitary sensitivity to dopamine were assessed by comparison of dose response curves for the inhibition by dopamine of prolactin release from hemipituitaries incubated in vitro from both long and short photoperiod-exposed animals over a series of time points from three to fifteen weeks. In the second series of experiments, adult female F344 rats received daily injection of melatonin or saline vehicle. After two weeks, half of the animals were sacrificed for analysis of 3 H-spiperone binding to anterior pituitary membranes, measurement of hypothalamic dopamine turnover and analysis of in vitro pituitary sensitivity to dopamine. The remaining animals received subcutaneous implants containing DES and injections were continued on the same schedule until sacrifice four weeks later for measurement of the same parameters

  3. AutoSite: an automated approach for pseudo-ligands prediction—from ligand-binding sites identification to predicting key ligand atoms

    Science.gov (United States)

    Ravindranath, Pradeep Anand; Sanner, Michel F.

    2016-01-01

    Motivation: The identification of ligand-binding sites from a protein structure facilitates computational drug design and optimization, and protein function assignment. We introduce AutoSite: an efficient software tool for identifying ligand-binding sites and predicting pseudo ligand corresponding to each binding site identified. Binding sites are reported as clusters of 3D points called fills in which every point is labelled as hydrophobic or as hydrogen bond donor or acceptor. From these fills AutoSite derives feature points: a set of putative positions of hydrophobic-, and hydrogen-bond forming ligand atoms. Results: We show that AutoSite identifies ligand-binding sites with higher accuracy than other leading methods, and produces fills that better matches the ligand shape and properties, than the fills obtained with a software program with similar capabilities, AutoLigand. In addition, we demonstrate that for the Astex Diverse Set, the feature points identify 79% of hydrophobic ligand atoms, and 81% and 62% of the hydrogen acceptor and donor hydrogen ligand atoms interacting with the receptor, and predict 81.2% of water molecules mediating interactions between ligand and receptor. Finally, we illustrate potential uses of the predicted feature points in the context of lead optimization in drug discovery projects. Availability and Implementation: http://adfr.scripps.edu/AutoDockFR/autosite.html Contact: sanner@scripps.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27354702

  4. Pharmacophore screening of the protein data bank for specific binding site chemistry.

    Science.gov (United States)

    Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

    2010-03-22

    A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

  5. Prolactin effect on the insulin content of albino rats in different physiological states

    International Nuclear Information System (INIS)

    Megahed, Y.M.; Abdel-Wahab, M.F.; El-Mougi, S.M.; El-Sayed, F.B.; Kuwait Univ.)

    1980-01-01

    The metabolic action of prolactin on insulin levels in plasma and pancreas has been studied. Prolactin was injected in a single dose or single daily doses on 4 successive days into albino rats in six different physiological states. Insulin was determined by radioimmunoassay using 125 I insulin. From the results it is concluded that prolactin injected i.p. influences the output of insulin and stimulates the pancreas to secrete insulin into the plasma. (author)

  6. Effect of exogenous prolactin on ultrastructure of pinealocyte in female pigs during puberty

    International Nuclear Information System (INIS)

    Przybylska, B.; Dusza, L.; Lewczuk, B.; Ciesielska-Myszka, L.

    1994-01-01

    Influence of the administration of prolactin to female swine during puberty on the ultrastructure of pinealocytes has been examined by means of morphometric analysis. Prolactin administration for 15 consecutive days resulted in a decrease in the cytoplasmic dense bodies type MBB-2, lysosomes and multivesicular bodies. Some differences in structure of pinealocytes were also observed. Prolactin appeared to stimulate the process of transformation of cytoplasmic dense bodies. (author). 28 refs, 5 figs

  7. Effect of exogenous prolactin on ultrastructure of pinealocyte in female pigs during puberty

    Energy Technology Data Exchange (ETDEWEB)

    Przybylska, B.; Dusza, L.; Lewczuk, B.; Ciesielska-Myszka, L. [Akademia Rolniczo-Technicza, Olsztyn (Poland)

    1994-12-31

    Influence of the administration of prolactin to female swine during puberty on the ultrastructure of pinealocytes has been examined by means of morphometric analysis. Prolactin administration for 15 consecutive days resulted in a decrease in the cytoplasmic dense bodies type MBB-2, lysosomes and multivesicular bodies. Some differences in structure of pinealocytes were also observed. Prolactin appeared to stimulate the process of transformation of cytoplasmic dense bodies. (author). 28 refs, 5 figs.

  8. Prolactin and Male Fertility: The Long and Short Feedback Regulation

    Directory of Open Access Journals (Sweden)

    M. K. Gill-Sharma

    2009-01-01

    Full Text Available In the last 20 years, a pituitary-hypothalamus tissue culture system with intact neural and portal connections has been developed in our lab and used to understand the feedback mechanisms that regulate the secretions of adenohypophyseal hormones and fertility of male rats. In the last decade, several in vivo rat models have also been developed in our lab with a view to substantiate the in vitro findings, in order to delineate the role of pituitary hormones in the regulation of fertility of male rats. These studies have relied on both surgical and pharmacological interventions to modulate the secretions of gonadotropins and testosterone. The interrelationship between the circadian release of reproductive hormones has also been ascertained in normal men. Our studies suggest that testosterone regulates the secretion of prolactin through a long feedback mechanism, which appears to have been conserved from rats to humans. These studies have filled in a major lacuna pertaining to the role of prolactin in male reproductive physiology by demonstrating the interdependence between testosterone and prolactin. Systemic levels of prolactin play a deterministic role in the mechanism of chromatin condensation during spermiogenesis.

  9. Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

    DEFF Research Database (Denmark)

    Cuyvers, Sven; Dornez, Emmie; Abou Hachem, Maher

    2012-01-01

    Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first...

  10. Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

    Science.gov (United States)

    Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

    2017-10-01

    While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. A high pressure liquid chromatography method for separation of prolactin forms.

    Science.gov (United States)

    Bell, Damon A; Hoad, Kirsten; Leong, Lillian; Bakar, Juwaini Abu; Sheehan, Paul; Vasikaran, Samuel D

    2012-05-01

    Prolactin has multiple forms and macroprolactin, which is thought not to be bioavailable, can cause a raised serum prolactin concentration. Gel filtration chromatography (GFC) is currently the gold standard method for separating macroprolactin, but is labour-intensive. Polyethylene glycol (PEG) precipitation is suitable for routine use but may not always be accurate. We developed a high pressure liquid chromatography (HPLC) assay for macroprolactin measurement. Chromatography was carried out using an Agilent Zorbax GF-250 (9.4 × 250 mm, 4 μm) size exclusion column and 50 mmol/L Tris buffer with 0.15 mmol/L NaCl at pH 7.2 as mobile phase, with a flow rate of 1 mL/min. Serum or plasma was diluted 1:1 with mobile phase and filtered and 100 μL injected. Fractions of 155 μL were collected for prolactin measurement and elution profile plotted. The area under the curve of each prolactin peak was calculated to quantify each prolactin form, and compared with GFC. Clear separation of monomeric-, big- and macroprolactin forms was achieved. Quantification was comparable to GFC and precision was acceptable. Total time from injection to collection of the final fraction was 16 min. We have developed an HPLC method for quantification of macroprolactin, which is rapid and easy to perform and therefore can be used for routine measurement.

  12. Self-Assembly of Coordinative Supramolecular Polygons with Open Binding Sites.

    Science.gov (United States)

    Zheng, Yao-Rong; Wang, Ming; Kobayashi, Shiho; Stang, Peter J

    2011-04-27

    The design and synthesis of coordinative supramolecular polygons with open binding sites is described. Coordination-driven self-assembly of 2,6-bis(pyridin-4-ylethynyl)pyridine with 60° and 120° organoplatinum acceptors results in quantitative formation of a supramolecular rhomboid and hexagon, respectively, both bearing open pyridyl binding sites. The structures were determined by multinuclear ((31)P and (1)H) NMR spectroscopy and electrospray ionization (ESI) mass spectrometry, along with a computational study.

  13. Substance P and substance K receptor binding sites in the human gastrointestinal tract: localization by autoradiography

    International Nuclear Information System (INIS)

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

    1988-01-01

    Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for 125 I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: (1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; (2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; (3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and (4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells

  14. Identification of an allosteric binding site for RORγt inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Scheepstra, Marcel; Leysen, Seppe; vanAlmen, Geert C.; Miller, J. Richard; Piesvaux, Jennifer; Kutilek, Victoria; van Eenennaam, Hans; Zhang, Hongjun; Barr, Kenneth; Nagpal, Sunil; Soisson, Stephen M.; Kornienko, Maria; Wiley, Kristen; Elsen, Nathaniel; Sharma, Sujata; Correll, Craig C.; Trotter, B. Wesley; van der Stelt, Mario; Oubrie, Arthur; Ottmann, Christian; Parthasarathy, Gopal; Brunsveld, Luc (Merck); (Eindhoven)

    2015-12-07

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of small-molecule antagonists demonstrates occupancy of a previously unreported allosteric binding pocket. Binding at this non-canonical site induces an unprecedented conformational reorientation of helix 12 in the RORγt LBD, which blocks cofactor binding. The functional consequence of this allosteric ligand-mediated conformation is inhibition of function as evidenced by both biochemical and cellular studies. RORγt function is thus antagonized in a manner molecularly distinct from that of previously described orthosteric RORγt ligands. This brings forward an approach to target RORγt for the treatment of Th17-mediated autoimmune diseases. The elucidation of an unprecedented modality of pharmacological antagonism establishes a mechanism for modulation of nuclear receptors.

  15. Resonance energy transfer study on the proximity relationship between the GTP binding site and the rifampicin binding site of Escherichia coli RNA polymerase

    International Nuclear Information System (INIS)

    Kumar, K.P.; Chatterji, D.

    1990-01-01

    Terbium(III) upon complexation with guanosine 5'-triphosphate showed remarkable enhancement of fluorescence emission at 488 and 545 nm when excited at 295 nm. Analysis of the binding data yielded a value for the mean K d between Tb(III) and GTP of 0.2 μM, with three binding sites for TB(III) on GTP. 31 P and 1 H NMR measurements revealed that Tb(III) mainly binds the phosphate moiety of GTP. Fluorescence titration of the emission signals of the TbGTP complex with varying concentrations of Escherichia coli RNA polymerase resulted in a K d values of 4 μM between the TbGTP and the enzyme. It was observed that TbGTP can be incorporated in the place of GTP during E. coli RNA polymerase catalyzed abortive synthesis of dinucleotide tetraphosphate at T7A2 promoter. Both the substrate TbGTP and the inhibitor of the initiation of transcription rifampicin bind to the β-subunit of E. coli RNA polymerase. This allows the measurement of the fluorescence excited-state energy transfer from the donor TbGTP-RNA polymerase to the acceptor rifampicin. Both emission bands of Tb(III) overlap with the rifampicin absorption, and the distances at 50% efficiency of energy transfer were calculated to be 28 and 24 angstrom for the 488- and 545-nm emission bands, respectively. The distance between the substrate binding site and the rifampicin binding site on the β-subunit of E. coli RNA polymerase was measured to be around 30 angstrom. This suggest that the nature of inhibition of transcription by rifampicin is essentially noncompetitive with the substrate

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

    Science.gov (United States)

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

    1988-08-01

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

  17. Prolactin modulates luteal activity in the short-nosed fruit bat, Cynopterus sphinx during delayed embryonic development.

    Science.gov (United States)

    Anuradha; Krishna, Amitabh

    2017-07-01

    The aim of this study was to evaluate the role of prolactin as a modulator of luteal steroidogenesis during the period of delayed embryonic development in Cynopterus sphinx. A marked decline in circulating prolactin levels was noted during the months of November through December coinciding with the period of decreased serum progesterone and delayed embryonic development. The seasonal changes in serum prolactin levels correlated positively with circulating progesterone (P) level, but inversely with circulating melatonin level during first pregnancy showing delayed development in Cynopterus sphinx. The results also showed decreased expression of prolactin receptor-short form (PRL-RS) both in the corpus luteum and in the utero-embryonic unit during the period of delayed embryonic development. Bats treated in vivo with prolactin during the period of delayed development showed significant increase in serum progesterone and estradiol levels together with significant increase in the expression of PRL-RS, luteinizing hormone receptor (LH-R), steroidogenic acute receptor protein (STAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) in the ovary. Prolactin stimulated ovarian angiogenesis (vascular endothelial growth factor) and cell survival (B-cell lymphoma 2) in vivo. Significant increases in ovarian progesterone production and the expression of prolactin-receptor, LH-R, STAR and 3β-HSD proteins were noted following the exposure of LH or prolactin in vitro during the delayed period. In conclusion, short-day associated increased melatonin level may be responsible for decreased prolactin release during November-December. The decline in prolactin level might play a role in suppressing P and estradiol-17β (E2) estradiol levels thereby causing delayed embryonic development in C. sphinx. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. eMatchSite: sequence order-independent structure alignments of ligand binding pockets in protein models.

    Directory of Open Access Journals (Sweden)

    Michal Brylinski

    2014-09-01

    Full Text Available Detecting similarities between ligand binding sites in the absence of global homology between target proteins has been recognized as one of the critical components of modern drug discovery. Local binding site alignments can be constructed using sequence order-independent techniques, however, to achieve a high accuracy, many current algorithms for binding site comparison require high-quality experimental protein structures, preferably in the bound conformational state. This, in turn, complicates proteome scale applications, where only various quality structure models are available for the majority of gene products. To improve the state-of-the-art, we developed eMatchSite, a new method for constructing sequence order-independent alignments of ligand binding sites in protein models. Large-scale benchmarking calculations using adenine-binding pockets in crystal structures demonstrate that eMatchSite generates accurate alignments for almost three times more protein pairs than SOIPPA. More importantly, eMatchSite offers a high tolerance to structural distortions in ligand binding regions in protein models. For example, the percentage of correctly aligned pairs of adenine-binding sites in weakly homologous protein models is only 4-9% lower than those aligned using crystal structures. This represents a significant improvement over other algorithms, e.g. the performance of eMatchSite in recognizing similar binding sites is 6% and 13% higher than that of SiteEngine using high- and moderate-quality protein models, respectively. Constructing biologically correct alignments using predicted ligand binding sites in protein models opens up the possibility to investigate drug-protein interaction networks for complete proteomes with prospective systems-level applications in polypharmacology and rational drug repositioning. eMatchSite is freely available to the academic community as a web-server and a stand-alone software distribution at http://www.brylinski.org/ematchsite.

  19. Effects of bovine prolactin gene polymorphism within exon 4 on milk ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-05

    Oct 5, 2009 ... In this study, polymorphism of prolactin gene was analyzed as a candidate gene responsible for ... studied. Based on important role of prolactin gene in milk related traits and their genetic trends in dairy cattle, the aims of this study were, to screen ..... isolation and selection towards high fat and protein per-.

  20. N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary.

    Science.gov (United States)

    Ferraris, Jimena; Radl, Daniela Betiana; Zárate, Sandra; Jaita, Gabriela; Eijo, Guadalupe; Zaldivar, Verónica; Clapp, Carmen; Seilicovich, Adriana; Pisera, Daniel

    2011-01-01

    The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this

  1. N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary.

    Directory of Open Access Journals (Sweden)

    Jimena Ferraris

    Full Text Available The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry. In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic

  2. A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel

    DEFF Research Database (Denmark)

    Long, Katherine S; Porse, Bo T

    2003-01-01

    , of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site...... on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome....

  3. Phyloscan: locating transcription-regulating binding sites in mixed aligned and unaligned sequence data.

    Science.gov (United States)

    Palumbo, Michael J; Newberg, Lee A

    2010-07-01

    The transcription of a gene from its DNA template into an mRNA molecule is the first, and most heavily regulated, step in gene expression. Especially in bacteria, regulation is typically achieved via the binding of a transcription factor (protein) or small RNA molecule to the chromosomal region upstream of a regulated gene. The protein or RNA molecule recognizes a short, approximately conserved sequence within a gene's promoter region and, by binding to it, either enhances or represses expression of the nearby gene. Since the sought-for motif (pattern) is short and accommodating to variation, computational approaches that scan for binding sites have trouble distinguishing functional sites from look-alikes. Many computational approaches are unable to find the majority of experimentally verified binding sites without also finding many false positives. Phyloscan overcomes this difficulty by exploiting two key features of functional binding sites: (i) these sites are typically more conserved evolutionarily than are non-functional DNA sequences; and (ii) these sites often occur two or more times in the promoter region of a regulated gene. The website is free and open to all users, and there is no login requirement. Address: (http://bayesweb.wadsworth.org/phyloscan/).

  4. Dopamine inhibits maitotoxin-stimulated pituitary 45Ca2+ efflux and prolactin release

    International Nuclear Information System (INIS)

    Login, I.S.; Judd, A.M.; MacLeod, R.M.

    1986-01-01

    The authors examined the hypothesis that dopaminergic inhibition of prolactin release is coupled to modulation of cellular calcium flux. Dispersed female rat pituitary cells were prelabeled in 45 Ca 2+ and perifused to determine simultaneously fractional calcium efflux and prolactin release, as stimulated by maitotoxin, a calcium channel activator. The integrated response of each parameter to 5 ng/ml maitotoxin was obtained in individual perifusion columns in the absence or presence of various concentrations of dopamine. Maitotoxin-stimulated calcium efflux was suppressed by dopamine concentrations of 0.01 μM and greater and achieved a maximal effect at ∼0.1 μM, at which calcium efflux was reduced by 50%. Maitotoxin-stimulated prolactin release was inhibited by 0.03 μM dopamine and greater concentrations, and at a concentration of ∼10.0 μM dopamine the effect became maximal at ∼85% suppression. Haloperidol (0.1 μM) blocked the effects of 0.1 μM dopamine on both parameters. Simultaneous suppression of maitotoxin-stimulated calcium efflux and prolactin release by concentrations of dopamine within the nonomolar range suggests that dopamine receptor activation is negatively coupled to modulation of calcium flux in the physiological regulation of prolactin secretion

  5. Gephyrin-binding peptides visualize postsynaptic sites and modulate neurotransmission

    DEFF Research Database (Denmark)

    Maric, Hans Michael; Hausrat, Torben Johann; Neubert, Franziska

    2017-01-01

    is associated with perturbation of the basic physiological action. Here we pursue a fundamentally different approach, by instead targeting the intracellular receptor-gephyrin interaction. First, we defined the gephyrin peptide-binding consensus sequence, which facilitated the development of gephyrin super......-binding peptides and later effective affinity probes for the isolation of native gephyrin. Next, we demonstrated that fluorescent super-binding peptides could be used to directly visualize inhibitory postsynaptic sites for the first time in conventional and super-resolution microscopy. Finally, we demonstrate...

  6. Nucleos: a web server for the identification of nucleotide-binding sites in protein structures.

    Science.gov (United States)

    Parca, Luca; Ferré, Fabrizio; Ausiello, Gabriele; Helmer-Citterich, Manuela

    2013-07-01

    Nucleos is a web server for the identification of nucleotide-binding sites in protein structures. Nucleos compares the structure of a query protein against a set of known template 3D binding sites representing nucleotide modules, namely the nucleobase, carbohydrate and phosphate. Structural features, clustering and conservation are used to filter and score the predictions. The predicted nucleotide modules are then joined to build whole nucleotide-binding sites, which are ranked by their score. The server takes as input either the PDB code of the query protein structure or a user-submitted structure in PDB format. The output of Nucleos is composed of ranked lists of predicted nucleotide-binding sites divided by nucleotide type (e.g. ATP-like). For each ranked prediction, Nucleos provides detailed information about the score, the template structure and the structural match for each nucleotide module composing the nucleotide-binding site. The predictions on the query structure and the template-binding sites can be viewed directly on the web through a graphical applet. In 98% of the cases, the modules composing correct predictions belong to proteins with no homology relationship between each other, meaning that the identification of brand-new nucleotide-binding sites is possible using information from non-homologous proteins. Nucleos is available at http://nucleos.bio.uniroma2.it/nucleos/.

  7. Using sequence-specific chemical and structural properties of DNA to predict transcription factor binding sites.

    Directory of Open Access Journals (Sweden)

    Amy L Bauer

    2010-11-01

    Full Text Available An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF. Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate.

  8. Distribution of [{sup 3}H]diadenosine tetraphosphate binding sites in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Miras-Portugal, M.T. [Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid (Spain); Palacios, J.M. [Laboratorios Almirall, Research Center, Cardener 68, 08024 Barcelona (Spain); Torres, M. [Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid (Spain); Cortes, R. [Departamento de Neuroquimica, Centro de Investigacion y Desarrollo, CSIC Jordi Girona 18-26, 08034 Barcelona (Spain); Rodriguez-Pascual, F. [Departamento de Bioquimica, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid (Spain)

    1997-01-06

    The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [{sup 3}H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation.The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  9. Rotating night shift work, sleep quality, selected lifestyle factors and prolactin concentration in nurses and midwives.

    Science.gov (United States)

    Bukowska, Agnieszka; Sobala, Wojciech; Peplonska, Beata

    2015-04-01

    The pattern of secretion of many hormones, including prolactin, is dependent on the circadian rhythm. Night shift work involves exposure to artificial light at night and sleep deficiency, which in turn can affect prolactin synthesis. The aim of this study was to evaluate a possible association between night shift work characteristics, sleep quality, lifestyle factors and prolactin concentration, using data from a cross-sectional study of nurses and midwives. A cross-sectional study was conducted among 327 nurses and midwives currently working on rotating night shifts, and 330 nurses and midwives working during the day (aged 40-60 years) (388 premenopausal and 269 postmenopausal). Information about night shift work characteristics, lifestyle, reproductive factors, sleep pattern and other covariates was collected through a face-to-face interview, and from a one-week work and sleep diary completed by the subjects. Weight and height were measured. Prolactin concentration was measured in the morning blood sample using the electrochemiluminesence immunoassay method. Associations were analyzed using linear regression models adjusted for important confounders. Analyses were carried out separately in pre- and postmenopausal women. None of the night shift work or sleep characteristics was significantly associated with prolactin concentration. Prolactin concentration was significantly (p night shift work is not associated with prolactin concentration. Smoking, parity, time of blood collection and age among postmenopausal women were significant determinants of prolactin.

  10. Quantitative autoradiographic distribution of L-[3H]glutamate-binding sites in rat central nervous system

    International Nuclear Information System (INIS)

    Greenamyre, J.T.; Young, A.B.; Penney, J.B.

    1984-01-01

    Quantitative autoradiography was used to determine the distribution of L-[3H]glutamate-binding sites in the rat central nervous system. Autoradiography was carried out in the presence of Cl- and Ca2+ ions. Scatchard plots and Hill coefficients of glutamate binding suggested that glutamate was interacting with a single population of sites having a K-D of about 300 nM and a capacity of 14.5 pmol/mg of protein. In displacement studies, ibotenate also appeared to bind to a single class of non-interacting sites with a KI of 28 microM. However, quisqualate displacement of [3H]glutamate binding revealed two well-resolved sites with KIS of 12 nM and 114 microM in striatum. These sites were unevenly distributed, representing different proportions of specific glutamate binding in different brain regions. The distribution of glutamate-binding sites correlated very well with the projection areas of putative glutamatergic pathways. This technique provides an extremely sensitive assay which can be used to gather detailed pharmacological and anatomical information about L-[3H]glutamate binding in the central nervous system

  11. Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat

    International Nuclear Information System (INIS)

    Lopez-Gonzalez, M.A.; Calvo, J.R.; Rubio, A.; Goberna, R.; Guerrero, J.M.

    1991-01-01

    The characterization of specific melatonin binding sites in the Harderian gland (HG) and median eminence (ME) of the rat was studied using [ 125 I]melatonin. Binding of melatonin to membrane crude preparations of both tissues was dependent on time and temperature. Thus, maximal binding was obtained at 37 degree C after 30-60 min incubation. Binding was also dependent on protein concentration. The specific binding of [ 125 I]melatonin was saturable, exhibiting only the class of binding sites in both tissues. The dissociation constants (Kd) were 170 and 190 pM for ME and HG, respectively. The concentration of the binding sites in ME was 8 fmol/mg protein, and in the HG 4 fmol/mg protein. In competition studies, binding of [ 125 I]melatonin to ME or HG was inhibited by increasing concentration of native melatonin; 50% inhibition was observed at about 702 and 422 nM for ME and HG, respectively. Additionally, the [ 125 I]melatonin binding to the crude membranes was not affected by the addition of different drugs such as norepinephrine, isoproterenol, phenylephrine, propranolol, or prazosin. The results confirm the presence of melatonin binding sites in median eminence and show, for the first time, the existence of melatonin binding sites in the Harderian gland

  12. Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography

    International Nuclear Information System (INIS)

    Gillberg, P.-G.; Aquilonius, S.-M.

    1985-01-01

    Binding sites for the receptor ligands 3 H-quinuclidinylbenzilate, 3 H-alpha-bungarotoxin ( 3 H-alpha-Btx), 3 H-etorphine and 3 H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3 H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distribute within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. (author)

  13. Non-opiate [beta]-endorphin fragments and dopamine--V [gamma]-type endorphins and prolactin secretion in rats

    NARCIS (Netherlands)

    Lamberts, S.W.J.; De Quijada, M.; Ree, J.M. van; Wied, D. de

    1982-01-01

    The effects on prolactin secretion of three peptide-derivatives of β-endorphin which show neuroleptic-like activities in rats were studied. Intravenous administration of γ-endorphin (β-endorphin (βE) 1–17) enhanced plasma prolactin levels. γ-Endorphin did not affect the prolactin secretion by

  14. Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

    International Nuclear Information System (INIS)

    Gil, D.W.; Wolfe, B.B.

    1986-01-01

    Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [ 3 H]quinuclidinyl benzilate or [ 3 H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [ 3 H]quinuclidinyl benzilate in a biphasic manner

  15. A web server for analysis, comparison and prediction of protein ligand binding sites.

    Science.gov (United States)

    Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

    2016-03-25

    One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

  17. A heterologous radioimmunoassay for avian prolactin: Application to the measurement of prolactin in the turkey

    International Nuclear Information System (INIS)

    McNeilly, A.S.; Etches, R.J.; Friesen, H.G.

    1978-01-01

    A specific heterologous double-antibody radioimmunoassay has been developed to measure turkey prolactin (PRL) using a guinea pig anti-hPRL antiserum and 125 I-labelled ovine PRL [ 125 I]oPRL. Turkey pituitary prolactin and serum give parallel dose-response curves and no cross-rection is seen with turkey growth hormone, LH or FSH, or mammalian LH, FSH, TSH, GH or placental lactogens. The RIA is accurate and precise and is sufficiently sensitive to measure PRL in all physiological situations investigated in the turkey. The RIA will measure PRL in several avian species including the chicken, duck, goose, pheasant, pheasant x chicken F 1 hybrid, pigeon, quail, and rook. Plasma PRL concentrations in laying and broody turkey hens were not significantly different (46.5 +- 2.5 vs. 39.7 +- 3.8 ng/ml) but both were significantly higher (P < 0.001) than in non-laying turkey hens (4.6 +- 0.7 ng/ml). Oestradiol injection into laying hens did not alter PRL levels while the same injection in non-laying hens caused a significant three-fold increse in plasma PRL levels. (author)

  18. Regulatory role of prolactin in paternal behavior in male parents: A narrative review

    Directory of Open Access Journals (Sweden)

    F Hashemian

    2016-01-01

    Full Text Available In all mammalian species, a combination of neuroendocrine and experiential factors contributes to the emergence of remarkable behavioral changes observed in parental behavior. Yet, our understanding of neuroendocrine bases of paternal behavior in humans is still preliminary and more research is needed in this area. In the present review, the authors summarized hormonal bases of paternal behavior in both human and nonhuman mammalian species and focused on studies on the regulatory role of prolactin in occurrence of paternal behavior. All peer-reviewed journal articles published before 2015 for each area discussed (parental brain, hormonal bases of maternal behavior, hormonal bases of paternal behavior and the role of prolactin in regulation of paternal behavior in nonhuman mammalian species, hormonal bases of paternal behavior and the role of prolactin in regulation of paternal behavior in humans were searched by PubMed, Medline, and Scopus for original research and review articles. Publications between 1973 and 2015 were included. Similar to female parents, elevated prolactin levels in new fathers most probably contribute to child-caring behavior and facilitate behavioral and emotional states attributed to child care. Moreover, elevated parental prolactin levels after childbirth decrease the parents′ libidos so that they invest more in parental care than in fertility behavior. According to the available clinical studies, elevation in the amounts of prolactin levels after childbirth in male parents are probably associated with paternal behavior observed in humans.

  19. Autoradiographic localization of peptide YY and neuropeptide Y binding sites in the medulla oblongata

    International Nuclear Information System (INIS)

    Leslie, R.A.; McDonald, T.J.; Robertson, H.A.

    1988-01-01

    Peptide YY is a highly potent emetic when given intravenously in dogs. We hypothesized that the area postrema, a small brain stem nucleus that acts as a chemoreceptive trigger zone for vomiting and lies outside the blood-brain barrier, might have receptors that PYY would bind to, in order to mediate the emetic response. We prepared [ 125 I]PYY and used autoradiography to show that high affinity binding sites for this ligand were highly localized in the area postrema and related nuclei of the dog medulla oblongata. Furthermore, the distribution of [ 125 I]PYY binding sites in the rat medulla oblongata was very similar to that in the dog; the distribution of [ 125 I]PYY binding sites throughout the rat brain was seen to be similar to the distribution of [ 125 I]NPY binding sites

  20. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

  1. Analysis of functional importance of binding sites in the Drosophila gap gene network model.

    Science.gov (United States)

    Kozlov, Konstantin; Gursky, Vitaly V; Kulakovskiy, Ivan V; Dymova, Arina; Samsonova, Maria

    2015-01-01

    The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. We elaborate a two-layer model for the Drosophila gap gene network and include in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. We show that the new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. We quantify the functional importance of binding sites by calculating their impact on gene expression in the model and calculate how these impacts correlate across all sites under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains.

  2. Pituitary Macroprolactinoma with Mildly Elevated Serum Prolactin: Hook Effect

    Directory of Open Access Journals (Sweden)

    Mahnaz Pejman-Sani

    2018-04-01

    Full Text Available A 45-year-old man was admitted in our department with complaints of severe headache for over 6 months period. He also suffered from several problems such as visual field defect, decreased energy and libido, body hair loss, cold intolerance, decreased appetite and dry skin. On physical examination, he was afebrile: BP (blood pressure: 110/70 mm/Hg, PR (pulse rate :65 beat/min, BMI (body mass index: 24. He had no terminal hair on face or chest and subcutaneous adipose tissue mass had been decreased substantially. Laboratory tests revealed; Hb: 12 g/dL (N: 14–17 g/dL, Total testosterone: 1.2 ng/mL (N:–-10 ng/mL, Luteinizing hormone (LH:3.3MIU/mL (N:1–8 MIU/mL, Follicle Stimulating hormone (FSH:1.3 MIU/mL (N:1–7 MIU/mL, T4:3.4 micg/dL (N:4–12 micg/dL, TSH:0.6 MIU/mL (N:0.5–5 MIU/mL, Prolactin:100 ng/mL (2–24 ng/mL, serum cortisol:6 MIU/mL (N:4–21 MIU/mL, IGF1:162 ng/mL (50–245. Pituitary MRI showed macroadenoma (29*16*14 mm in left side of sella turcica which bulged to suprasellar cistern with pressure effect on left optic nerve (Figure 1, 2. Visual field examination revealed mild temporal hemianopia. These findings are consistent with macroadenoma and mild prolactin elevation. We also observed a discrepancy between pituitary tumor size and prolactin level. The correct estimate of serum prolactin was obtained after serial dilutional measurement. Serum prolactin after dilution was 6470 ng/mL. With these findings pituitary macroadenoma was diagnosed and treatment with cabergoline (dopamine agonist 0.5 mg/week was started. After one month follow-up he had no symptoms, visual field defect was improved and pituitary MRI showed significant shrinkage of tumor.

  3. Characterization of 6-mercaptopurine binding to bovine serum albumin and its displacement from the binding sites by quercetin and rutin

    Energy Technology Data Exchange (ETDEWEB)

    Ehteshami, Mehdi [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rasoulzadeh, Farzaneh [Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Mahboob, Soltanali [Nutrition Research Center, School of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of); Rashidi, Mohammad-Reza, E-mail: rashidi@tbzmed.ac.ir [Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz 51644-14766 (Iran, Islamic Republic of)

    2013-03-15

    Binding of a drug to the serum albumins as major serum transport proteins can be influenced by other ligands leading to alteration of its pharmacological properties. In the present study, binding characteristics of 6-mercaptopurine (6-MP) with bovine serum albumin (BSA) together with its displacement from its binding site by quercetin and rutin have been investigated by the spectroscopic method. According to the binding parameters, a static quenching component in overall dynamic quenching process is operative in the interaction between 6-MP and BSA. The binding of 6-MP to BSA occurred spontaneously due to entropy-driven hydrophobic interactions. The synchronous fluorescence spectroscopy study revealed that the secondary structure of BSA is changed in the presence of 6-MP and both Tyr and Trp residues participate in the interaction between 6-MP and BSA with the later one being more dominant. The binding constant value of 6-MP-BSA in the presence of quercetin and rutin increased. 6-MP was displaced by ibuprofen indicating that the binding site of 6-MP on albumin is site II. Therefore, the change of the pharmacokinetic and pharmacodynamic properties of 6-MP by quercetin and rutin through alteration of binding capacity of 6-MP to the serum albumin cannot be ruled out. In addition, the displacement study showed that 6-MP is located in site II of BSA. - Highlights: Black-Right-Pointing-Pointer Participation of both Tyr and particularly Trp residues in the interaction between 6-MP and BSA. Black-Right-Pointing-Pointer Involvement of a static quenching component in an overall dynamic quenching process. Black-Right-Pointing-Pointer Ability of quercetin and rutin to change the binding constants of 6-MP-BSA complex. Black-Right-Pointing-Pointer Binding of 6-MP to BSA through entropy-driven hydrophobic interactions.

  4. Targeting Stromal Androgen Receptor Suppresses Prolactin-Driven Benign Prostatic Hyperplasia (BPH)

    Science.gov (United States)

    Lai, Kuo-Pao; Huang, Chiung-Kuei; Fang, Lei-Ya; Izumi, Kouji; Lo, Chi-Wen; Wood, Ronald; Kindblom, Jon; Yeh, Shuyuan

    2013-01-01

    Stromal-epithelial interaction plays a pivotal role to mediate the normal prostate growth, the pathogenesis of benign prostatic hyperplasia (BPH), and prostate cancer development. Until now, the stromal androgen receptor (AR) functions in the BPH development, and the underlying mechanisms remain largely unknown. Here we used a genetic knockout approach to ablate stromal fibromuscular (fibroblasts and smooth muscle cells) AR in a probasin promoter-driven prolactin transgenic mouse model (Pb-PRL tg mice) that could spontaneously develop prostate hyperplasia to partially mimic human BPH development. We found Pb-PRL tg mice lacking stromal fibromuscular AR developed smaller prostates, with more marked changes in the dorsolateral prostate lobes with less proliferation index. Mechanistically, prolactin mediated hyperplastic prostate growth involved epithelial-stromal interaction through epithelial prolactin/prolactin receptor signals to regulate granulocyte macrophage-colony stimulating factor expression to facilitate stromal cell growth via sustaining signal transducer and activator of transcription-3 activity. Importantly, the stromal fibromuscular AR could modulate such epithelial-stromal interacting signals. Targeting stromal fibromuscular AR with the AR degradation enhancer, ASC-J9®, led to the reduction of prostate size, which could be used in future therapy. PMID:23893956

  5. Effects of melatonin and prolactin in reproduction: review of literature

    OpenAIRE

    Tenorio, Fernanda das Chagas Angelo Mendes; Simões, Manuel de Jesus; Teixeira, Valéria Wanderley; Teixeira, Álvaro Aguiar Coelho

    2015-01-01

    Summary The pineal gland is responsible for producing a hormone called melatonin (MEL), and is accepted as the gland that regulates reproduction in mammals. Prolactin (PRL) also exhibits reproductive activity in animals in response to photoperiod. It is known that the concentrations of PRL are high in the summer and reduced during winter, the opposite of what is seen with melatonin in these seasons. In placental mammals, both prolactin and melatonin affect implantation, which is considered a ...

  6. Ecological and physiological factors affecting brood patch area and prolactin levels in arctic-nesting geese

    Science.gov (United States)

    Jonsson, J.E.; Afton, A.D.; Alisauskas, R.T.; Bluhm, C.K.; El Halawani, M.E.

    2006-01-01

    We investigated effects of ecological and physiological factors on brood patch area and prolactin levels in free-ranging Lesser Snow Geese (Chen caerulescens caerulescens; hereafter “Snow Geese”) and Ross's Geese (C. rossii). On the basis of the body-size hypothesis, we predicted that the relationships between prolactin levels, brood patch area, and body condition would be stronger in Ross's Geese than in the larger Snow Geese. We found that brood patch area was positively related to clutch volume and inversely related to prolactin levels in Ross's Geese, but not in Snow Geese. Nest size, nest habitat, and first egg date did not affect brood patch area in either species. Prolactin levels increased as incubation progressed in female Snow Geese, but this relationship was not significant in Ross's Geese. Prolactin levels and body condition (as indexed by size-adjusted body mass) were inversely related in Ross's Geese, but not in Snow Geese. Our findings are consistent with the prediction that relationships between prolactin levels, brood patch area, and body condition are relatively stronger in Ross's Geese, because they mobilize endogenous reserves at faster rates than Snow Geese.

  7. Circulating prolactin level in systemic lupus erythematosus and its correlation with disease activity: a meta-analysis.

    Science.gov (United States)

    Song, G G; Lee, Y H

    2017-10-01

    Objective This study aimed to evaluate the relationship between circulating prolactin level and systemic lupus erythematosus (SLE), and to establish a correlation between plasma/serum prolactin levels and SLE activity. Methods We performed a meta-analysis comparing the plasma/serum prolactin levels in patients with SLE to controls, and examined correlation coefficients between circulating prolactin level and SLE disease activity. Results Twenty-five studies with a total of 1056 SLE patients and 426 controls were included. Prolactin levels were significantly higher overall in the SLE group than in the control group (standardized mean difference (SMD) = 0.987, 95% CI = 0.512-1.463, p = 4.7 × 10 -5 ). Stratification by ethnicity showed significantly elevated prolactin levels in the SLE group in Asian, Latin American, and mixed populations (SMD = 0.813, 95% CI = 0.137-1.490, p = 0.018; SMD = 0.981, 95% CI = 0.307-1.655, p = 0.004; SMD = 1.469, 95% CI = 0.443-2.495, p = 0.005, respectively), but not in the European population. Subgroup analysis by sample size showed significantly higher prolactin levels in the SLE group by small ( n  30). Meta-analysis of correlation coefficients showed a significantly positive correlation between circulating prolactin level and SLE activity (correlation coefficient = 0.379, 95% CI = 0.026-0.487, p = 4.0 × 10 -9 ). Circulating prolactin levels were positively associated with SLE activity in European, Asian, and mixed populations (SMD = 0.532, 95% CI = 0.443-0.609  p < 1.0 × 10 -8 ; SMD = 0.427, 95% CI = 0.240-0.583, p = 2.4 × 10 -5 ; SMD = 0.433, 95% CI = 0.212-0.591, p = 2.7 × 10 -5 , respectively). Conclusions Our meta-analysis demonstrated that circulating prolactin levels are higher in patients with SLE, and that a significantly positive correlation exists between prolactin levels and SLE activity.

  8. Serotoninergic receptors in brain tissue: properties and identification of various 3H-ligand binding sites in vitro

    International Nuclear Information System (INIS)

    Leysen, J.E.

    1981-01-01

    In vitro binding studies to serotoninergic receptors were performed using 3 H-LSD, 3 H-5-HT and 3 H-spiperone. An overwiew is given on findings using these three ligands with respect to the following: localization of specific binding sites, in various animal species, the regional distribution in the brain and periphery, the subcellular and cellular distribution. Properties of the binding sites, influence of the composition of the assay medium, binding kinetic properties, receptor regulation in vivo. Identity of the binding sites, differences between site for various 3 H-ligands, pharmacological specificity of the membranous binding sites, chemical composition of the macromolecular complex constituting the binding site. Function of the receptor. Binding affinities of 44 compounds were measured in binding assays using 3 H-spiperone and 3 H-LSD with rat frontal cortex membrane preparations and using 3 H-5-HT and 3 H-LSD with rat hippocampal membrane preparations

  9. Involvement of arachidonate metabolism in neurotensin-induced prolactin release in vitro

    International Nuclear Information System (INIS)

    Canonico, P.L.; Speciale, C.; Sortino, M.A.; Scapagnini, U.

    1985-01-01

    Neurotensin increased in a concentration-dependent manner the level of hypophyseal [ 3 H]arachidonic acid in vitro as well as prolactin release from hemipituitary glands. The effect of 1 microM neurotensin on arachidonate release was already present at 2.5 min, maximal at 5, and disappeared after a 10-min incubation. Neurotensin analogues produced an enhancement of hypophyseal arachidonate similar to their relative potencies in other cellular systems, whereas other peptides (somatostatin and vasoactive intestinal peptide) were devoid of any effect on the concentration of the fatty acid in the pituitary. Seventy micromoles RHC 80267, a rather selective inhibitor of diacylglycerol lipase, completely prevented the neurotensin-stimulated prolactin release and decreased arachidonate release both in basal or in neurotensin-induced conditions. Similar results were obtained with 50 microM quinacrine, a phospholipase A2 inhibitor. To clarify whether arachidonate released by neurotensin requires a further metabolism through specific pathways to stimulate prolactin release, the authors used indomethacin and BW 755c, two blockers of cyclooxygenase and lipoxygenase pathways. Thirty micromoles indomethacin, a dose active to inhibit cyclooxygenase, did not affect unesterified arachidonate levels either in basal or in neurotensin-induced conditions; moreover, the drug did not modify basal prolactin release but slightly potentiated the stimulatory effect of neurotensin on the release of the hormone. On the other hand, 250 microM BW 755c, an inhibitor of both cyclooxygenase and lipoxygenase pathways, significantly inhibited both basal and neurotensin-stimulated prolactin release and further potentiated the increase of the fatty acid concentrations produced by 1 microM neurotensin

  10. Mathematical description of drug-target interactions: application to biologics that bind to targets with two binding sites.

    Science.gov (United States)

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2018-02-01

    The emerging discipline of mathematical pharmacology occupies the space between advanced pharmacometrics and systems biology. A characteristic feature of the approach is application of advance mathematical methods to study the behavior of biological systems as described by mathematical (most often differential) equations. One of the early application of mathematical pharmacology (that was not called this name at the time) was formulation and investigation of the target-mediated drug disposition (TMDD) model and its approximations. The model was shown to be remarkably successful, not only in describing the observed data for drug-target interactions, but also in advancing the qualitative and quantitative understanding of those interactions and their role in pharmacokinetic and pharmacodynamic properties of biologics. The TMDD model in its original formulation describes the interaction of the drug that has one binding site with the target that also has only one binding site. Following the framework developed earlier for drugs with one-to-one binding, this work aims to describe a rigorous approach for working with similar systems and to apply it to drugs that bind to targets with two binding sites. The quasi-steady-state, quasi-equilibrium, irreversible binding, and Michaelis-Menten approximations of the model are also derived. These equations can be used, in particular, to predict concentrations of the partially bound target (RC). This could be clinically important if RC remains active and has slow internalization rate. In this case, introduction of the drug aimed to suppress target activity may lead to the opposite effect due to RC accumulation.

  11. Eel calcitonin binding site distribution and antinociceptive activity in rats

    International Nuclear Information System (INIS)

    Guidobono, F.; Netti, C.; Sibilia, V.; Villa, I.; Zamboni, A.; Pecile, A.

    1986-01-01

    The distribution of binding site for [ 125 I]-eel-calcitonin (ECT) to rat central nervous system, studied by an autoradiographic technique, showed concentrations of binding in the diencephalon, the brain stem and the spinal cord. Large accumulations of grains were seen in the hypothalamus, the amygdala, in the fasciculus medialis prosencephali, in the fasciculus longitudinalis medialis, in the ventrolateral part of the periventricular gray matter, in the lemniscus medialis and in the raphe nuclei. The density of grains in the reticular formation and in the nucleus tractus spinalis nervi trigemini was more moderate. In the spinal cord, grains were scattered throughout the dorsal horns. Binding of the ligand was displaced equally by cold ECT and by salmon CT(sCT), indicating that both peptides bind to the same receptors. Human CT was much weaker than sCT in displacing [ 125 I]-ECT binding. The administration of ECT into the brain ventricles of rats dose-dependently induced a significant and long-lasting enhancement of hot-plate latencies comparable with that obtained with sCT. The antinociceptive activity induced by ECT is compatible with the topographical distribution of binding sites for the peptide and is a further indication that fish CTs are active in the mammalian brain

  12. GABAA [gamma-aminobutyric acid] type binding sites on membranes of spermatozoa

    International Nuclear Information System (INIS)

    Erdoe, S.L.; Wekerle, L.

    1990-01-01

    The binding of [ 3 H] gamma-aminobutyric acid (GABA) to seminal membranes of swines and rams was examined. Specific, GABA binding was demonstrated in both species, which showed the features of GABA A type receptors. The affinity of binding was similar in both species, whereas the density of seminal GABA binding sites was 5 times higher in swine. Our findings suggest that GABA may have a direct effect on spermatozoa

  13. Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

    Science.gov (United States)

    Marsh, Lorraine

    2015-01-01

    Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

  14. Prolactin-inducible proteins in human breast cancer cells

    International Nuclear Information System (INIS)

    Shiu, R.P.; Iwasiow, B.M.

    1985-01-01

    The mechanism of action of prolactin in target cells and the role of prolactin in human breast cancer are poorly understood phenomena. The present study examines the effect of human prolactin (hPRL) on the synthesis of unique proteins by a human breast cancer cell line, T-47D, in serum-free medium containing bovine serum albumin. [ 35 S]Methionine-labeled proteins were analysed by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and fluorography. Treatment of cells with hPRL (1-1000 ng/ml) and hydrocortisone (1 microgram/ml) for 36 h or longer resulted in the synthesis and secretion of three proteins having molecular weights of 11,000, 14,000, and 16,000. Neither hPRL nor hydrocortisone alone induced these proteins. Of several other peptide hormones tested, only human growth hormone, a hormone structurally and functionally similar to hPRL, could replace hPRL in causing protein induction. These three proteins were, therefore, referred to as prolactin-inducible proteins (PIP). Each of the three PIPs was purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and specific antibodies were generated to them in rabbits. By immunoprecipitation and immunoblotting (Western blot) of proteins secreted by T-47D cells, it was demonstrated that the three PIPs were immunologically identical to one another. In addition, the 16-kDa and 14-kDa proteins (PIP-16 and PIP-14), and not the 11-kDa protein (PIP-11), incorporated [ 3 H]glycosamine. Furthermore, 2-deoxyglucose (2 mM) and tunicamycin (0.5 micrograms/ml), two compounds known to inhibit glycosylation, blocked the production of PIP-16 and PIP-14, with a concomitant increase in the accumulation of PIP-11

  15. [Effect of estradiol on the prolactin content in the adenohypophysis of sexually mature and immature rats].

    Science.gov (United States)

    Arse, Kh A

    1979-01-01

    Gel electrophoresis was used in a comparative study of prolactin content in the hypophysis of rats of different age and sex, and at various stages of the estral cycle. The hormone level in the pubertal rats was twice or thrice greater than in the immature ones; it was by 16% less at the diestrus than at the estrus stage. There was no change in the hypophysis prolactin content in male rats at puberty. Ovariectomy was accompained by a sharp reduction of prolactin in the hypophysis. Replacing estradiol therapy increased the amount of prolactin in the hypophysis, without bringing it, however, to the level characteristic of intact rats. Estrogens are responsible for the maintenance of prolactin level, but apparently other factors influencing its content in the hypophysis also exist.

  16. Prolactin as a Marker of Successful Catheterization during IPSS in Patients with ACTH-Dependent Cushing's Syndrome

    Science.gov (United States)

    Sharma, S. T.; Raff, H.

    2011-01-01

    Context: Anomalous venous drainage can lead to false-negative inferior petrosal sinus sampling (IPSS) results. Baseline inferior petrosal sinus to peripheral (IPS/P) prolactin ratio higher than 1.8 ipsilateral to the highest ACTH ratio has been proposed to verify successful catheterization. Prolactin-normalized ACTH IPS/P ratios may differentiate Cushing's disease (CD) from ectopic ACTH syndrome (EAS). Objective: Our objective was to examine the utility of prolactin measurement during IPSS. Design, Setting, and Participants: We conducted a retrospective analysis of prolactin levels in basal and CRH-stimulated IPSS samples in ACTH-dependent Cushing's syndrome (2007–2010). Results: Twenty-five of 29 patients had a pathologically proven diagnosis (17 CD and eight EAS). IPSS results were partitioned into true positive for CD (n = 16), true negative (n = 7), false negative (n = 1), and false positive (n = 1). Prolactin IPS/P ratio suggested successful IPSS in eight of 11 with abnormal venograms. Baseline prolactin IPS/P ratio was helpful in two patients with abnormal venograms and false-negative (catheterization unsuccessful) or true-negative (catheterization successful) IPSS results; the normalized ratio correctly diagnosed their disease. Normalized ACTH IPS/P ratio was at least 1.3 in all with CD, but prolactin IPS/P ratios were misleadingly low in two. One patient with cyclic EAS had a false-positive IPSS when eucortisolemic (baseline prolactin IPS/P = 1.7; normalized ratio = 5.6). All other EAS patients had normalized ratios no higher than 0.7. Conclusion: Prolactin measurement and evaluation of the venogram can improve diagnostic accuracy when IPSS results suggest EAS but is not necessary with positive IPSS results. Confirmation of hypercortisolemia remains a prerequisite for IPSS. A normalized ratio of 0.7–1.3 was not diagnostic. PMID:22031511

  17. Recognition of AT-Rich DNA Binding Sites by the MogR Repressor

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Aimee; Higgins, Darren E.; Panne, Daniel; (Harvard-Med); (EMBL)

    2009-07-22

    The MogR transcriptional repressor of the intracellular pathogen Listeria monocytogenes recognizes AT-rich binding sites in promoters of flagellar genes to downregulate flagellar gene expression during infection. We describe here the 1.8 A resolution crystal structure of MogR bound to the recognition sequence 5' ATTTTTTAAAAAAAT 3' present within the flaA promoter region. Our structure shows that MogR binds as a dimer. Each half-site is recognized in the major groove by a helix-turn-helix motif and in the minor groove by a loop from the symmetry-related molecule, resulting in a 'crossover' binding mode. This oversampling through minor groove interactions is important for specificity. The MogR binding site has structural features of A-tract DNA and is bent by approximately 52 degrees away from the dimer. The structure explains how MogR achieves binding specificity in the AT-rich genome of L. monocytogenes and explains the evolutionary conservation of A-tract sequence elements within promoter regions of MogR-regulated flagellar genes.

  18. Ropizine concurrently enhances and inhibits [3H] dextromethorpan binding to different structures of the guinea pig brain: Autoradiographic evidence for multiple binding sites

    International Nuclear Information System (INIS)

    Canoll, P.D.; Smith, P.R.; and Musacchio, J.M.

    1990-01-01

    Ropizine produces a simultaneous enhancement and inhibition of [ 3 H] dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity [ 3 H]DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhances [ 3 H]DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+)- pentazocine, has not been fully characterized. This study demonstrates that the biphasic effects to ropizine are due, at least in part, to the effects of ropizine on two different types of [ 3 H]DM binding sites. However, this study does not rule out that the common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine

  19. Evidence for a non-opioid sigma binding site din the guinea-pig myenteric plexus

    International Nuclear Information System (INIS)

    Roman, F.; Pascaud, X.; Vauche, D.; Junien, J.

    1988-01-01

    The presence of a binding site to (+)-( 3 H)SKF 10,047 was demonstrated in a guinea-pig myenteric plexus (MYP) membrane preparation. Specific binding to this receptor was saturable, reversible, linear with protein concentration and consisted of two components, a high affinity site and a low affinity site. Morphine and naloxone 10 -4 M were unable to displace (+)-( 3 H)SKF 10,047 binding. Haloperidol, imipramine, ethylketocyclazocine and propranolol were among the most potent compounds to inhibit this specific binding. These results suggest the presence of a non-opioid haloperidol sensitive sigma receptor in the MYP of the guinea-pig

  20. Identification of an allosteric binding site for RORγt inhibition

    NARCIS (Netherlands)

    Scheepstra, M.; Leysen, S.; van Almen, G.; Miller, J.R.; Piesvaux, J.; Kutilek, V.; van Eenennaam, H.; Zhang, H.; Barr, K.; Nagpal, S.; Soisson, S.M.; Kornienko, M.; Wiley, K.; Elsen, N.; Sharma, S.; Correll, C.C.; Trotter, B.W.; Stelt, van der M.; Oubrie, A.; Ottmann, C.; Parthasarathy, G.; Brunsveld, L.

    2015-01-01

    RORγt is critical for the differentiation and proliferation of Th17 cells associated with several chronic autoimmune diseases. We report the discovery of a novel allosteric binding site on the nuclear receptor RORγt. Co-crystallization of the ligand binding domain (LBD) of RORγt with a series of

  1. Copper(II) Binding Sites in N-Terminally Acetylated α-Synuclein: A Theoretical Rationalization.

    Science.gov (United States)

    Ramis, Rafael; Ortega-Castro, Joaquín; Vilanova, Bartolomé; Adrover, Miquel; Frau, Juan

    2017-08-03

    The interactions between N-terminally acetylated α-synuclein and Cu(II) at several binding sites have been studied with DFT calculations, specifically with the M06 hybrid functional and the ωB97X-D DFT-D functional. In previous experimental studies, Cu(II) was shown to bind several α-synuclein residues, including Met1-Asp2 and His50, forming square planar coordination complexes. Also, it was determined that a low-affinity binding site exists in the C-terminal domain, centered on Asp121. However, in the N-terminally acetylated protein, present in vivo, the Met1 site is blocked. In this work, we simplify the representation of the protein by modeling each experimentally found binding site as a complex between an N-terminally acetylated α-synuclein dipeptide (or several independent residues) and a Cu(II) cation, and compare the results with a number of additional, structurally analogous sites not experimentally found. This way of representing the binding sites, although extremely simple, allows us to reproduce experimental results and to provide a theoretical rationale to explain the preference of Cu(II) for certain sites, as well as explicit geometrical structures for the complexes formed. These results are important to understand the interactions between α-synuclein and Cu(II), one of the factors inducing structural changes in the protein and leading to aggregated forms of it which may play a role in neurodegeneration.

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  3. Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  4. Importance of prolactin for lactation in the ewe

    Energy Technology Data Exchange (ETDEWEB)

    Hooley, R D; Campbell, J J; Findlay, J K [Reproduction Research Section, Melbourne Univ., C/- Animal Research Inst., Werribee (Australia)

    1978-12-01

    The effect of 2-bromo-..cap alpha..-ergocryptine (bromocriptine) on the induction and maintenance of milk secretion was studied in post-parturient ewes and in ovariectomized ewes artificially induced to lactate by treatment with oestrogen plus progesterone and then dexamethasone. Treatment with bromocriptine (about 0.4 mk/kg every 3 days) lowered and maintained the plasma concentration of prolactin at < 12 ng/ml. Ewes receiving bromocriptine concurrently with oestrogen plus progesterone during the priming phase had a significantly lower (P < 0.05) mean cumulative milk yield than control ewes, although the milk of the treated ewes contained normal amounts of fat, protein and lactose. Administration of bromocriptine during dexamethasone-induced lactogenesis had no significant effect on the mean cumulative milk yield but significantly (P < 0.05) increased the milk fat and protein content. In establishing lactation, bromocriptine markedly reduced the milk yield in both intact and ovariectomised ewes. The concentration of protein was not significantly affected although the milk fat content was higher in the bromocriptine-treated than in the control ewes. The effects of bromocriptine on milk yield and composition during galactopoiesis could be reversed by concurrent infusion of prolactin and the results suggest that prolactin is an important hormone during mammogenesis and galactopoiesis in sheep.

  5. GenProBiS: web server for mapping of sequence variants to protein binding sites.

    Science.gov (United States)

    Konc, Janez; Skrlj, Blaz; Erzen, Nika; Kunej, Tanja; Janezic, Dusanka

    2017-07-03

    Discovery of potentially deleterious sequence variants is important and has wide implications for research and generation of new hypotheses in human and veterinary medicine, and drug discovery. The GenProBiS web server maps sequence variants to protein structures from the Protein Data Bank (PDB), and further to protein-protein, protein-nucleic acid, protein-compound, and protein-metal ion binding sites. The concept of a protein-compound binding site is understood in the broadest sense, which includes glycosylation and other post-translational modification sites. Binding sites were defined by local structural comparisons of whole protein structures using the Protein Binding Sites (ProBiS) algorithm and transposition of ligands from the similar binding sites found to the query protein using the ProBiS-ligands approach with new improvements introduced in GenProBiS. Binding site surfaces were generated as three-dimensional grids encompassing the space occupied by predicted ligands. The server allows intuitive visual exploration of comprehensively mapped variants, such as human somatic mis-sense mutations related to cancer and non-synonymous single nucleotide polymorphisms from 21 species, within the predicted binding sites regions for about 80 000 PDB protein structures using fast WebGL graphics. The GenProBiS web server is open and free to all users at http://genprobis.insilab.org. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

    Science.gov (United States)

    Tavagnacco, Letizia; Mason, Philip E; Schnupf, Udo; Pitici, Felicia; Zhong, Linghao; Himmel, Michael E; Crowley, Michael; Cesàro, Attilio; Brady, John W

    2011-05-01

    Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of β-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Comprehensive human transcription factor binding site map for combinatory binding motifs discovery.

    Directory of Open Access Journals (Sweden)

    Arnoldo J Müller-Molina

    Full Text Available To know the map between transcription factors (TFs and their binding sites is essential to reverse engineer the regulation process. Only about 10%-20% of the transcription factor binding motifs (TFBMs have been reported. This lack of data hinders understanding gene regulation. To address this drawback, we propose a computational method that exploits never used TF properties to discover the missing TFBMs and their sites in all human gene promoters. The method starts by predicting a dictionary of regulatory "DNA words." From this dictionary, it distills 4098 novel predictions. To disclose the crosstalk between motifs, an additional algorithm extracts TF combinatorial binding patterns creating a collection of TF regulatory syntactic rules. Using these rules, we narrowed down a list of 504 novel motifs that appear frequently in syntax patterns. We tested the predictions against 509 known motifs confirming that our system can reliably predict ab initio motifs with an accuracy of 81%-far higher than previous approaches. We found that on average, 90% of the discovered combinatorial binding patterns target at least 10 genes, suggesting that to control in an independent manner smaller gene sets, supplementary regulatory mechanisms are required. Additionally, we discovered that the new TFBMs and their combinatorial patterns convey biological meaning, targeting TFs and genes related to developmental functions. Thus, among all the possible available targets in the genome, the TFs tend to regulate other TFs and genes involved in developmental functions. We provide a comprehensive resource for regulation analysis that includes a dictionary of "DNA words," newly predicted motifs and their corresponding combinatorial patterns. Combinatorial patterns are a useful filter to discover TFBMs that play a major role in orchestrating other factors and thus, are likely to lock/unlock cellular functional clusters.

  8. Discovery and validation of information theory-based transcription factor and cofactor binding site motifs.

    Science.gov (United States)

    Lu, Ruipeng; Mucaki, Eliseos J; Rogan, Peter K

    2017-03-17

    Data from ChIP-seq experiments can derive the genome-wide binding specificities of transcription factors (TFs) and other regulatory proteins. We analyzed 765 ENCODE ChIP-seq peak datasets of 207 human TFs with a novel motif discovery pipeline based on recursive, thresholded entropy minimization. This approach, while obviating the need to compensate for skewed nucleotide composition, distinguishes true binding motifs from noise, quantifies the strengths of individual binding sites based on computed affinity and detects adjacent cofactor binding sites that coordinate with the targets of primary, immunoprecipitated TFs. We obtained contiguous and bipartite information theory-based position weight matrices (iPWMs) for 93 sequence-specific TFs, discovered 23 cofactor motifs for 127 TFs and revealed six high-confidence novel motifs. The reliability and accuracy of these iPWMs were determined via four independent validation methods, including the detection of experimentally proven binding sites, explanation of effects of characterized SNPs, comparison with previously published motifs and statistical analyses. We also predict previously unreported TF coregulatory interactions (e.g. TF complexes). These iPWMs constitute a powerful tool for predicting the effects of sequence variants in known binding sites, performing mutation analysis on regulatory SNPs and predicting previously unrecognized binding sites and target genes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Serum Prolactin in Diagnosis of Epileptic Seizures

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2005-09-01

    Full Text Available The results of studies in databases and references concerning serum prolactin levels (PRL in patients with suspected seizures were rated for quality and analyzed by members of the Therapeutics Subcommittee of the American Academy of Neurology.

  10. Radioimmunoassay of human homologous prolactin in serum with commercially available reagents

    International Nuclear Information System (INIS)

    Kao, P.C.; Jiang, N.S.; Abboud, C.F.

    1977-01-01

    A clinically useful and reproducible radioimmunoassay for human homologous prolactin, established with commercially available reagents, was studied and validated. We present detailed conditions for iodination and purification of labeled prolactin and the optimal conditions for the assay. By the method, we found values (μg/liter) as follows for serum prolactin: normal men, 8.9 +- 5.2 (mean +- SD); normal women, 11.8 +- 5.5; normal women taking contraceptive pills, 9.2 +- 5.0; pregnant women in the third trimester, 188 +- 69.5; patients with various diseases other than of the hypothalamic-pituitary axis, 9.3 +- 6.3; in some patients with amenorrhea and galactorrhea of diverse origin, 78.2 +- 87.4; and in some patients with surgically proven pituitary tumor, 1414 +- 1980. Results under provocative testing are also presented for a patient with normal hypothalamic-pituitary function

  11. Biomimetic conformation-specific assembly of proteins at artificial binding sites nano-patterned on silicon

    Science.gov (United States)

    de la Rica, Roberto; Matsui, Hiroshi

    2009-01-01

    Biomolecules such as enzymes and antibodies possess binding sites where the molecular architecture and the physicochemical properties are optimum for their interaction with a particular target, in some cases even differentiating between stereoisomers. Here, we mimic this exquisite specificity via the creation of a suitable chemical environment by fabricating artificial binding sites for the protein calmodulin (CaM). By downscaling well-known surface chemical modification methodologies to the nanometer scale via silicon nanopatterning, the Ca2+-CaM conformer was found to selectively bind the biomimetic binding sites. The methodology could be adapted to mimic other protein-receptor interactions for sensing and catalysis. PMID:19757782

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    Autoradiography using 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

  13. The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes.

    Science.gov (United States)

    Atambayeva, Shara; Niyazova, Raigul; Ivashchenko, Anatoliy; Pyrkova, Anna; Pinsky, Ilya; Akimniyazova, Aigul; Labeit, Siegfried

    2017-06-01

    Normally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications. The search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3'UTR (214 sites), CDS (3 sites), and 5'UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3'UTRs. The miR-619-5p binding site in the 5'UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3'UTRs of all human target genes are also present in the 3'UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes. The majority of miR-619-5p binding sites are located in the 3'UTRs but some genes have miRNA binding sites in the 5'UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p

  14. Cortisol decreases 2[125I] iodomelatonin binding sites in the duck thymus

    International Nuclear Information System (INIS)

    Poon, A.M.S.; Liu, Z.M.; Tang, F.; Pang, S.F.

    1994-01-01

    The immunosuppressive effect of chronic glucocorticoid treatment on 2[ 125 I] iodomelatonin binding in the duck thymus was studied. Two-week-old ducks were injected intraperitoneally with either 1 mg of cortisol per day (experimental group) or an equivalent volume of vehicle (control group) in the middle of the light period for seven days. 2[ 125 I] iodomelatonin binding assays were performed on thymic membranes. Cortisol injection reduced the body weight gain, size of the bursa of Fabricius and absolute weights of the primary lymphoid organs but had no effect on the spleen weights. The relative weights of the spleen were increased while those of the primary lymphoid organs were unchanged. The density of the thymus 2[ 125 I] iodomelatonin binding sites was decreased while the affinity was not affected. The modulation of the thymic 2[ 125 I] iodomelatonin binding sites by changes in the immune status of the duck suggests that these binding sites represent physiologically relevant melatonin receptors and that melatonin exerts its action on the lymphoid tissues directly. The authors findings support the hypothesis that the thymus is the target site for the immunomodulatory interactions between the pineal melatonin and the adrenal steroids. A possible inhibitory influence of adrenal steroids on the immuno-enhancing effect of melatonin is also suggested. 34 refs., 3 tabs

  15. Cholesterol-Binding Sites in GIRK Channels: The Devil is in the Details.

    Science.gov (United States)

    Rosenhouse-Dantsker, Avia

    2018-01-01

    In recent years, it has become evident that cholesterol plays a direct role in the modulation of a variety of ion channels. In most cases, cholesterol downregulates channel activity. In contrast, our earlier studies have demonstrated that atrial G protein inwardly rectifying potassium (GIRK) channels are upregulated by cholesterol. Recently, we have shown that hippocampal GIRK currents are also upregulated by cholesterol. A combined computational-experimental approach pointed to putative cholesterol-binding sites in the transmembrane domain of the GIRK2 channel, the primary subunit in hippocampal GIRK channels. In particular, the principal cholesterol-binding site was located in the center of the transmembrane domain in between the inner and outer α-helices of 2 adjacent subunits. Further studies pointed to a similar cholesterol-binding site in GIRK4, a major subunit in atrial GIRK channels. However, a close look at a sequence alignment of the transmembrane helices of the 2 channels reveals surprising differences among the residues that interact with the cholesterol molecule in these 2 channels. Here, we compare the residues that form putative cholesterol-binding sites in GIRK2 and GIRK4 and discuss the similarities and differences among them.

  16. The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide-Localization of a putative binding site.

    Science.gov (United States)

    Dayan, Avraham; Babin, Gilad; Ganoth, Assaf; Kayouf, Nivin Samir; Nitoker Eliaz, Neta; Mukkala, Srijana; Tsfadia, Yossi; Fleminger, Gideon

    2017-08-01

    Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high-affinity titanium dioxide (TiO 2 )-binding protein (TiBP), purified from Rhodococcus ruber, has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO 2 -binding capabilities with TiBP. Intrigued by the unique TiO 2 -binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti-based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein-TiO 2 surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO 2 , indicating that neither the enzyme undergoes major conformational changes nor the TiO 2 binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO 2 through similar regions located far from the active site and the dimerization sites. The putative TiO 2 -binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal-binding sites in proteins. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Crystal structure of equine serum albumin in complex with cetirizine reveals a novel drug binding site.

    Science.gov (United States)

    Handing, Katarzyna B; Shabalin, Ivan G; Szlachta, Karol; Majorek, Karolina A; Minor, Wladek

    2016-03-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in human serum albumin (HSA), and suggest that binding of cetirizine to HSA will be similar. In support of that hypothesis, we show that the dissociation constants for cetirizine binding to CBS2 in ESA and HSA are identical using tryptophan fluorescence quenching. Presence of lysine and arginine residues that have been previously reported to undergo nonenzymatic glycosylation in CBS1 and CBS2 suggests that cetirizine transport in patients with diabetes could be altered. A review of all available SA structures from the PDB shows that in addition to the novel drug binding site we present here (CBS1), there are two pockets on SA capable of binding drugs that do not overlap with fatty acid binding sites and have not been discussed in published reviews. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Ligand-bound Structures and Site-directed Mutagenesis Identify the Acceptor and Secondary Binding Sites of Streptomyces coelicolor Maltosyltransferase GlgE*

    Science.gov (United States)

    Syson, Karl; Stevenson, Clare E. M.; Miah, Farzana; Barclay, J. Elaine; Tang, Minhong; Gorelik, Andrii; Rashid, Abdul M.; Lawson, David M.; Bornemann, Stephen

    2016-01-01

    GlgE is a maltosyltransferase involved in α-glucan biosynthesis in bacteria that has been genetically validated as a target for tuberculosis therapies. Crystals of the Mycobacterium tuberculosis enzyme diffract at low resolution so most structural studies have been with the very similar Streptomyces coelicolor GlgE isoform 1. Although the donor binding site for α-maltose 1-phosphate had been previously structurally defined, the acceptor site had not. Using mutagenesis, kinetics, and protein crystallography of the S. coelicolor enzyme, we have now identified the +1 to +6 subsites of the acceptor/product, which overlap with the known cyclodextrin binding site. The sugar residues in the acceptor subsites +1 to +5 are oriented such that they disfavor the binding of malto-oligosaccharides that bear branches at their 6-positions, consistent with the known acceptor chain specificity of GlgE. A secondary binding site remote from the catalytic center was identified that is distinct from one reported for the M. tuberculosis enzyme. This new site is capable of binding a branched α-glucan and is most likely involved in guiding acceptors toward the donor site because its disruption kinetically compromises the ability of GlgE to extend polymeric substrates. However, disruption of this site, which is conserved in the Streptomyces venezuelae GlgE enzyme, did not affect the growth of S. venezuelae or the structure of the polymeric product. The acceptor subsites +1 to +4 in the S. coelicolor enzyme are well conserved in the M. tuberculosis enzyme so their identification could help inform the design of inhibitors with therapeutic potential. PMID:27531751

  19. Penicillin-binding site on the Escherichia coli cell envelope

    International Nuclear Information System (INIS)

    Amaral, L.; Lee, Y.; Schwarz, U.; Lorian, V.

    1986-01-01

    The binding of 35 S-labeled penicillin to distinct penicillin-binding proteins (PBPs) of the cell envelope obtained from the sonication of Escherichia coli was studied at different pHs ranging from 4 to 11. Experiments distinguishing the effect of pH on penicillin binding by PBP 5/6 from its effect on beta-lactamase activity indicated that although substantial binding occurred at the lowest pH, the amount of binding increased with pH, reaching a maximum at pH 10. Based on earlier studies, it is proposed that the binding at high pH involves the formation of a covalent bond between the C-7 of penicillin and free epsilon amino groups of the PBPs. At pHs ranging from 4 to 8, position 1 of penicillin, occupied by sulfur, is considered to be the site that establishes a covalent bond with the sulfhydryl groups of PBP 5. The use of specific blockers of free epsilon amino groups or sulfhydryl groups indicated that wherever the presence of each had little or no effect on the binding of penicillin by PBP 5, the presence of both completely prevented binding. The specific blocker of the hydroxyl group of serine did not affect the binding of penicillin

  20. Effects of short- and long-term risperidone treatment on prolactin levels in children with autism.

    Science.gov (United States)

    Anderson, George M; Scahill, Lawrence; McCracken, James T; McDougle, Christopher J; Aman, Michael G; Tierney, Elaine; Arnold, L Eugene; Martin, Andrés; Katsovich, Liliya; Posey, David J; Shah, Bhavik; Vitiello, Benedetto

    2007-02-15

    The effects of short- and long-term risperidone treatment on serum prolactin were assessed in children and adolescents with autism. Patients with autism (N = 101, 5-17 years of age) were randomized to an 8-week trial of risperidone or placebo and 63 then took part in a 4-month open-label follow-up phase. Serum samples were obtained at Baseline and Week-8 (N = 78), and at 6-month (N = 43) and 22-month (N = 30) follow-up. Serum prolactin was determined by immunoradiometric assay; dopamine type-2 receptor (DRD2) polymorphisms were genotyped. Baseline prolactin levels were similar in the risperidone (N = 42) and placebo (N = 36) groups (9.3 +/- 7.5 and 9.3 +/- 7.6 ng/ml, respectively). After 8 weeks of risperidone, prolactin increased to 39.0 +/- 19.2 ng/ml, compared with 10.1 +/- 8.8 ng/ml for placebo (p autism. Although risperidone-induced increases tended to diminish with time, further research on the consequences of long-term prolactin elevations in children and adolescents is needed.

  1. The binding sites for cocaine and dopamine in the dopamine transporter overlap

    DEFF Research Database (Denmark)

    Beuming, Thijs; Kniazeff, Julie; Bergmann, Marianne L

    2008-01-01

    Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog Leu......T. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed...... inhibition of dopamine transport by cocaine....

  2. Asap: a framework for over-representation statistics for transcription factor binding sites

    DEFF Research Database (Denmark)

    Marstrand, Troels T; Frellsen, Jes; Moltke, Ida

    2008-01-01

    -founded choice. METHODOLOGY: We introduce a software package, Asap, for fast searching with position weight matrices that include several standard methods for assessing over-representation. We have compared the ability of these methods to detect over-represented transcription factor binding sites in artificial......BACKGROUND: In studies of gene regulation the efficient computational detection of over-represented transcription factor binding sites is an increasingly important aspect. Several published methods can be used for testing whether a set of hypothesised co-regulated genes share a common regulatory...... regime based on the occurrence of the modelled transcription factor binding sites. However there is little or no information available for guiding the end users choice of method. Furthermore it would be necessary to obtain several different software programs from various sources to make a well...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. 21 CFR 862.1625 - Prolactin (lactogen) test system.

    Science.gov (United States)

    2010-04-01

    ... anterior pituitary gland or of the hypothalamus portion of the brain. (b) Classification. Class I (general... system is a device intended to measure the anterior pituitary polypeptide hormone prolactin in serum and...

  5. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

    Science.gov (United States)

    Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas

    2017-01-01

    The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. PMID:28949297

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

    International Nuclear Information System (INIS)

    John, D.; Fox, I.V.

    1986-01-01

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

  7. 8-anilino-1-naphthaline sulfonate binds at the hemoglobin allosteric regulatory sites: inhibitory analyses

    International Nuclear Information System (INIS)

    Bokut', S.B.; Parul', D.A.; Yachnik, N.N.; Milyutin, A.A.

    2001-01-01

    The present study focused on the localization at least one of the ANS binding sites in the major form of human hemoglobin HbA. High-resolution docking predict ANS binding to the hemoglobin central cavity. Steady-state fluorescence titration data obtained in the absence/presence of natural effector inositol hexaphosphate (IHP) allowed to conclude that IHP competitively inhibited ANS binding to HbA. Thus, we must conclude that one of the ANS binding sites is central cavity, which makes it possible to monitor changes at this region upon ligation/deligation, effector binding and changes in hemoglobin structure

  8. Sex differences in the relationship between prolactin levels and impaired processing speed in early psychosis.

    Science.gov (United States)

    Montalvo, Itziar; Nadal, Roser; Armario, Antonio; Gutiérrez-Zotes, Alfonso; Creus, Marta; Cabezas, Ángel; Solé, Montse; Algora, Maria José; Sánchez-Gistau, Vanessa; Vilella, Elisabet; Labad, Javier

    2018-06-01

    Hyperprolactinaemia is commonly observed in people with psychotic disorders due to D2 receptor blockade by antipsychotic drugs, although it may also exist in drug-naïve patients with first-episode psychosis. Recent studies suggest that hyperprolactinaemia may have a negative impact on cognitive function in people with early psychosis. We aimed to explore whether there are sex differences in the association between prolactin levels and cognitive performance in early psychosis patients. We studied 60 young patients with early psychosis (aged 18-35 years, 35% females) and a sex- and age-matched control group of 50 healthy subjects. Cognitive assessment was performed with the MATRICS Consensus Cognitive Battery. Prolactin, total cortisol, follicular-stimulating hormone, luteal hormone and sex steroids (testosterone in men, oestradiol and progesterone in women) were measured in plasma. Salivary cortisol was measured at different sampling times (awakening response, 10:00 and 23:00). Psychopathological status was assessed, and antipsychotic treatment was registered. Multiple linear regression analyses were used to explore the relationship between prolactin and cognitive tasks while adjusting for covariates. Prolactin levels were associated with impaired processing speed in men, and this association was independent of cortisol and testosterone. In women, prolactin levels were not associated with processing speed tasks, although we observed a negative effect of prolactin on verbal learning and spatial working memory in female healthy subjects. The male-dependent effect maintained its significance after adjusting for education status, antipsychotic treatment and negative symptoms. Our study demonstrates that the previously reported association between high prolactin levels and impaired cognitive processes in early psychosis is restricted to men.

  9. Studies on the regulation of anuran metamorphosis by thyroid hormones and prolactin

    International Nuclear Information System (INIS)

    Ray, L.B.

    1985-01-01

    Resorption of the tail of the anuran larva during metamorphosis is induced by the thyroid hormones. In contrast, the pituitary hormone prolactin favors growth of the tail fin and inhibits resorption. The present investigations were designed to explore the mechanisms by which the thyroid hormones and prolactin bring about their cellular effects. Incubation of explants of tail fin with derivatives of cAMP was shown to inhibit T 4 -induced resorption of explants in a manner similar to that of prolactin. Likewise, inhibition of phosphodiesterases also inhibited resorption. Prolactin, however, failed to alter the levels of cAMP in cultured explants of tail fin. Although cAMP antagonizes the resorptive effects of T 4 , prolactin apparently does not act by elevating cellular levels of that cyclic nucleotide. Newly synthesized proteins from explants of tail fin were examined by isotopical labeling followed by two-dimensional gel electrophoresis and fluorography. Incorporation of 35 S-methionine into four proteins was increased within 8 to 48 hours after exposure of explants to T 4 . Three of the same proteins appeared to be synthesized more rapidly in explants of fin from tadpoles at metamorphic climax than in fin from tadpoles of premetamorphic stages. These results indicate that treatment of explants with T 4 or elevation of endogenous levels of thyroid hormones during spontaneous metamorphosis increased the relative rates of synthesis of several proteins. Those proteins are potentially involved in initiating the effects of T 4 which lead to cell death and resorption of the tail

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

    Energy Technology Data Exchange (ETDEWEB)

    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. High-Affinity Quasi-Specific Sites in the Genome: How the DNA-Binding Proteins Cope with Them

    Science.gov (United States)

    Chakrabarti, J.; Chandra, Navin; Raha, Paromita; Roy, Siddhartha

    2011-01-01

    Many prokaryotic transcription factors home in on one or a few target sites in the presence of a huge number of nonspecific sites. Our analysis of λ-repressor in the Escherichia coli genome based on single basepair substitution experiments shows the presence of hundreds of sites having binding energy within 3 Kcal/mole of the OR1 binding energy, and thousands of sites with binding energy above the nonspecific binding energy. The effect of such sites on DNA-based processes has not been fully explored. The presence of such sites dramatically lowers the occupation probability of the specific site far more than if the genome were composed of nonspecific sites only. Our Brownian dynamics studies show that the presence of quasi-specific sites results in very significant kinetic effects as well. In contrast to λ-repressor, the E. coli genome has orders of magnitude lower quasi-specific sites for GalR, an integral transcription factor, thus causing little competition for the specific site. We propose that GalR and perhaps repressors of the same family have evolved binding modes that lead to much smaller numbers of quasi-specific sites to remove the untoward effects of genomic DNA. PMID:21889449

  12. New human erythrocyte protein with binding sites for both spectrin and calmodulin

    International Nuclear Information System (INIS)

    Gardner, K.; Bennett, V.

    1986-01-01

    A new cytoskeletal protein that binds calmodulin has been purified to greater than 95% homogeneity from human erythrocyte cytoskeletons. The protein is a heterodimer with subunits of 103,000 and 97,000 and M/sub r/ = 197,000 calculated from its Stokes radius of 6.9 nm and sedimentation coefficient of 6.8. A binding affinity of this protein for calmodulin has been estimated to be 230 nM by displacement of two different concentrations of 125 I-azidocalmodulin with increasing concentrations of unmodified calmodulin followed by Dixon plot analysis. This protein is present in red cells at approximately 30,000 copies per cell and contains a very tight binding site(s) on cytoskeletons. The protein can be only partially solubilized from isolated cytoskeletons in buffers containing high salt, but can be totally solubilized from red cell ghost membranes by extraction in low ionic strength buffers. Affinity purified IgG against this calmodulin-binding protein identifies crossreacting polypeptide(s) in brain, kidney, testes and retina. Visualization of the calmodulin-binding protein by negative staining, rotary shadowing and unidirectional shadowing indicate that it is a flattened circular molecule with molecular height of 5.4 nm and a diameter of 12.4 nm. Preliminary cosedimentation studies with purified spectrin and F-actin indicate that the site of interaction of this calmodulin-binding protein with the cytoskeleton resides on spectrin

  13. Na-K pump site density and ouabain binding affinity in cultured chick heart cells

    International Nuclear Information System (INIS)

    Lobaugh, L.A.; Lieberman, M.

    1987-01-01

    The possible existence of multiple [ 3 H]ouabain binding sites and the relationship between ouabain binding and Na-K pump inhibition in cardiac muscle were studied using cultured embryonic chick heart cells. [ 3 H]ouabain bound to a single class of sites in 0.5 mM K (0.5 Ko) with an association rate constant (k+1) of 3.4 X 10(4) M-1.s-1 and a dissociation rate constant (k-1) of 0.0095 s. Maximal specific [ 3 H]ouabain binding RT to myocyte-enriched cultures is 11.7 pmol/mg protein and Kd is 0.43 microM in 0.5 Ko, whereas Kd,apparent is 6.6 microM in 5.4 Ko. The number of binding sites per myocyte was calculated by correcting for the contribution of fibroblasts in myocyte-enriched cultures using data from homogeneous fibroblast cultures (RT = 3.3 pmol/mg protein; Kd = 0.19 microM in 0.5 Ko). Equivalence of [ 3 H]ouabain binding sites and Na-K pumps was implied by agreement between maximal specific binding of [ 3 H]ouabain and 125 I-labeled monoclonal antibody directed against Na+-K+-ATPase (approximately 2 X 10(6) sites/cell). However, [ 3 H]ouabain binding occurred at lower concentrations than inhibition of ouabain-sensitive 42 K uptake in 0.5 Ko. Further studies in both 0.5 K and 5.4 Ko showed that ouabain caused cell Na content Nai to increase over the same range of concentrations that binding occurred, implying that increased Nai may stimulate unbound Na-K pumps and prevent a proportional decrease in 42 K uptake rate. The results show that Na-K pump inhibition occurs as a functional consequence of specific ouabain binding and indicate that the Na-K pump is the cardiac glycoside receptor in cultured heart cells

  14. Identification of the quinolinedione inhibitor binding site in Cdc25 phosphatase B through docking and molecular dynamics simulations

    Science.gov (United States)

    Ge, Yushu; van der Kamp, Marc; Malaisree, Maturos; Liu, Dan; Liu, Yi; Mulholland, Adrian J.

    2017-11-01

    Cdc25 phosphatase B, a potential target for cancer therapy, is inhibited by a series of quinones. The binding site and mode of quinone inhibitors to Cdc25B remains unclear, whereas this information is important for structure-based drug design. We investigated the potential binding site of NSC663284 [DA3003-1 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5, 8-dione] through docking and molecular dynamics simulations. Of the two main binding sites suggested by docking, the molecular dynamics simulations only support one site for stable binding of the inhibitor. Binding sites in and near the Cdc25B catalytic site that have been suggested previously do not lead to stable binding in 50 ns molecular dynamics (MD) simulations. In contrast, a shallow pocket between the C-terminal helix and the catalytic site provides a favourable binding site that shows high stability. Two similar binding modes featuring protein-inhibitor interactions involving Tyr428, Arg482, Thr547 and Ser549 are identified by clustering analysis of all stable MD trajectories. The relatively flexible C-terminal region of Cdc25B contributes to inhibitor binding. The binding mode of NSC663284, identified through MD simulation, likely prevents the binding of protein substrates to Cdc25B. The present results provide useful information for the design of quinone inhibitors and their mechanism of inhibition.

  15. Identification of the quinolinedione inhibitor binding site in Cdc25 phosphatase B through docking and molecular dynamics simulations.

    Science.gov (United States)

    Ge, Yushu; van der Kamp, Marc; Malaisree, Maturos; Liu, Dan; Liu, Yi; Mulholland, Adrian J

    2017-11-01

    Cdc25 phosphatase B, a potential target for cancer therapy, is inhibited by a series of quinones. The binding site and mode of quinone inhibitors to Cdc25B remains unclear, whereas this information is important for structure-based drug design. We investigated the potential binding site of NSC663284 [DA3003-1 or 6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5, 8-dione] through docking and molecular dynamics simulations. Of the two main binding sites suggested by docking, the molecular dynamics simulations only support one site for stable binding of the inhibitor. Binding sites in and near the Cdc25B catalytic site that have been suggested previously do not lead to stable binding in 50 ns molecular dynamics (MD) simulations. In contrast, a shallow pocket between the C-terminal helix and the catalytic site provides a favourable binding site that shows high stability. Two similar binding modes featuring protein-inhibitor interactions involving Tyr428, Arg482, Thr547 and Ser549 are identified by clustering analysis of all stable MD trajectories. The relatively flexible C-terminal region of Cdc25B contributes to inhibitor binding. The binding mode of NSC663284, identified through MD simulation, likely prevents the binding of protein substrates to Cdc25B. The present results provide useful information for the design of quinone inhibitors and their mechanism of inhibition.

  16. Computational prediction of cAMP receptor protein (CRP binding sites in cyanobacterial genomes

    Directory of Open Access Journals (Sweden)

    Su Zhengchang

    2009-01-01

    Full Text Available Abstract Background Cyclic AMP receptor protein (CRP, also known as catabolite gene activator protein (CAP, is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed. Results We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution. Conclusion The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The

  17. A homologous radioimmunoassay for canine prolactin: plasma levels huring the reproductive cycle

    International Nuclear Information System (INIS)

    Coster, R. De; Beerens, D.; Mey, J. De; Beckers, J.-F.

    1983-01-01

    A method is described for the purification of canine prolactin, involving preparative isoelectrofocusing. Canine prolactin has a molecular weight of 23 000 daltons, an isoelectric point of 5.7 and exhibits a high degree of homogeneity in polyacrylamide gels stained by means of a silver method. A specific, homologous radioimmunoassay is described using the Bolton-Hunter method for preparation of the labelled ligand, with a sensitivity of 0.1 ng/tube. Basal plasma prolactin levels of 2-4 ng/ml obtained through the oestrous cycle remained fairly constant but a rise of 9 ng/ml was found at the end of dioestrus in non-pregnant bitches. Level also rose 30 days after mating to reach a peak of about 50 ng/ml near parturition and during early lactation. (author)

  18. De-novo discovery of differentially abundant transcription factor binding sites including their positional preference.

    Science.gov (United States)

    Keilwagen, Jens; Grau, Jan; Paponov, Ivan A; Posch, Stefan; Strickert, Marc; Grosse, Ivo

    2011-02-10

    Transcription factors are a main component of gene regulation as they activate or repress gene expression by binding to specific binding sites in promoters. The de-novo discovery of transcription factor binding sites in target regions obtained by wet-lab experiments is a challenging problem in computational biology, which has not been fully solved yet. Here, we present a de-novo motif discovery tool called Dispom for finding differentially abundant transcription factor binding sites that models existing positional preferences of binding sites and adjusts the length of the motif in the learning process. Evaluating Dispom, we find that its prediction performance is superior to existing tools for de-novo motif discovery for 18 benchmark data sets with planted binding sites, and for a metazoan compendium based on experimental data from micro-array, ChIP-chip, ChIP-DSL, and DamID as well as Gene Ontology data. Finally, we apply Dispom to find binding sites differentially abundant in promoters of auxin-responsive genes extracted from Arabidopsis thaliana microarray data, and we find a motif that can be interpreted as a refined auxin responsive element predominately positioned in the 250-bp region upstream of the transcription start site. Using an independent data set of auxin-responsive genes, we find in genome-wide predictions that the refined motif is more specific for auxin-responsive genes than the canonical auxin-responsive element. In general, Dispom can be used to find differentially abundant motifs in sequences of any origin. However, the positional distribution learned by Dispom is especially beneficial if all sequences are aligned to some anchor point like the transcription start site in case of promoter sequences. We demonstrate that the combination of searching for differentially abundant motifs and inferring a position distribution from the data is beneficial for de-novo motif discovery. Hence, we make the tool freely available as a component of the open

  19. Vasoactive intestinal peptide (VIP) binds to guinea pig peritoneal eosinophils: A single class of binding sites with low affinity and high capacity

    International Nuclear Information System (INIS)

    Sakakibara, H.; Shima, K.; Takamatsu, J.; Said, S.I.

    1990-01-01

    VIP binds to specific receptors on lymphocytes and mononuclear cells and exhibits antiinflammatory properties. Eosinophils (Eos) contribute to inflammatory reactions but the regulation of Eos function is incompletely understood. The authors examined the binding of monoradioiodinated VIP, [Tyr( 125 I) 10 ] VIP ( 125 I-VIP), to Eos in guinea pigs. The interaction of 125 i-VIP with Eos was rapid, reversible, saturable and linearly dependent on the number of cells. At equilibrium the binding was competitively inhibited by native peptide or by the related peptide helodermin. Scatchard analysis suggested the presence of a single class of VIP binding sites with a low affinity and a high capacity. In the presence of isobutyl-methylxanthine, VIP, PHI or helodermin did not stimulate cyclic AMP accumulation in intact Eos, while PGE 2 or 1-isoproterenol did. VIP also did not inhibit superoxide anion generation from Eos stimulated by phorbol myristate acetate. The authors conclude that: (1) VIP binds to low-affinity, specific sites on guinea pig peritoneal eosinophils; (2) this binding is not coupled to stimulation of adenylate cyclase; and (3) the possible function of these binding sites is at present unknown

  20. Incorporating evolution of transcription factor binding sites into ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Identifying transcription factor binding sites (TFBSs) is essential to elucidate ... alignments with parts annotated as gap lessly aligned TFBSs (pair-profile hits) are generated. Moreover, the pair- profile related parameters are derived in a sound statistical framework. ... Much research has gone into the study of the evolution of.

  1. Cation binding at the node of Ranvier: I. Localization of binding sites during development.

    Science.gov (United States)

    Zagoren, J C; Raine, C S; Suzuki, K

    1982-06-17

    Cations are known to bind to the node of Ranvier and the paranodal regions of myelinated fibers. The integrity of these specialized structures is essential for normal conduction. Sites of cation binding can be microscopically identified by the electrondense histochemical reaction product formed by the precipitate of copper sulfate/potassium ferrocyanide. This technique was used to study the distribution of cation binding during normal development of myelinating fibers. Sciatic nerves of C57B1 mice, at 1, 3, 5, 6, 7, 8, 9, 13, 16, 18, 24 and 30 days of age, were prepared for electron microscopy following fixation in phosphate-buffered 2.5% glutaraldehyde and 1% osmic acid, microdissection and incubation in phosphate-buffered 0.1 M cupric sulfate followed by 0.1 M potassium ferrocyanide. Localization of reaction product was studied by light and electron microscopy. By light microscopy, no reaction product was observed prior to 9 days of age. At 13 days, a few nodes and paranodes exhibited reaction product. This increased in frequency and intensity up to 30 days when almost all nodes or paranodes exhibited reaction product. Ultrastructurally, diffuse reaction product was first observed at 3 days of age in the axoplasm of the node, in the paranodal extracellular space of the terminal loops, in the Schwann cell proper and in the terminal loops of Schwann cell cytoplasm. When myelinated axons fulfilled the criteria for mature nodes, reaction product was no longer observed in the Schwann cell cytoplasm, while the intensity of reaction product in the nodal axoplasm and paranodal extracellular space of the terminal loops increased. Reaction product in the latter site appeared to be interrupted by the transverse bands. These results suggest that cation binding accompanies nodal maturity and that the Schwann cell may play a role in production or storage of the cation binding substance during myelinogenesis and development.

  2. Quantitative analysis of EGR proteins binding to DNA: assessing additivity in both the binding site and the protein

    Directory of Open Access Journals (Sweden)

    Stormo Gary D

    2005-07-01

    Full Text Available Abstract Background Recognition codes for protein-DNA interactions typically assume that the interacting positions contribute additively to the binding energy. While this is known to not be precisely true, an additive model over the DNA positions can be a good approximation, at least for some proteins. Much less information is available about whether the protein positions contribute additively to the interaction. Results Using EGR zinc finger proteins, we measure the binding affinity of six different variants of the protein to each of six different variants of the consensus binding site. Both the protein and binding site variants include single and double mutations that allow us to assess how well additive models can account for the data. For each protein and DNA alone we find that additive models are good approximations, but over the combined set of data there are context effects that limit their accuracy. However, a small modification to the purely additive model, with only three additional parameters, improves the fit significantly. Conclusion The additive model holds very well for every DNA site and every protein included in this study, but clear context dependence in the interactions was detected. A simple modification to the independent model provides a better fit to the complete data.

  3. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    DEFF Research Database (Denmark)

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    is not universal and is in fact rare among some families of enzymes. In some cases an alternative to possessing a CBM is for the enzyme to bind to the substrate at a site on the catalytic domain, but away from the active site. Such a site is termed a surface (or secondary) binding site (SBS). SBSs have been...

  4. Identification of metal ion binding sites based on amino acid sequences.

    Science.gov (United States)

    Cao, Xiaoyong; Hu, Xiuzhen; Zhang, Xiaojin; Gao, Sujuan; Ding, Changjiang; Feng, Yonge; Bao, Weihua

    2017-01-01

    The identification of metal ion binding sites is important for protein function annotation and the design of new drug molecules. This study presents an effective method of analyzing and identifying the binding residues of metal ions based solely on sequence information. Ten metal ions were extracted from the BioLip database: Zn2+, Cu2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Na+, K+ and Co2+. The analysis showed that Zn2+, Cu2+, Fe2+, Fe3+, and Co2+ were sensitive to the conservation of amino acids at binding sites, and promising results can be achieved using the Position Weight Scoring Matrix algorithm, with an accuracy of over 79.9% and a Matthews correlation coefficient of over 0.6. The binding sites of other metals can also be accurately identified using the Support Vector Machine algorithm with multifeature parameters as input. In addition, we found that Ca2+ was insensitive to hydrophobicity and hydrophilicity information and Mn2+ was insensitive to polarization charge information. An online server was constructed based on the framework of the proposed method and is freely available at http://60.31.198.140:8081/metal/HomePage/HomePage.html.

  5. Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

    Directory of Open Access Journals (Sweden)

    Jose L S Lopes

    Full Text Available Diacylglycerol acyltransferase 1 (DGAT1 is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

  6. Binding sites for luminescent amyloid biomarkers from non-biased molecular dynamics simulations.

    Science.gov (United States)

    König, Carolin; Skånberg, Robin; Hotz, Ingrid; Ynnerman, Anders; Norman, Patrick; Linares, Mathieu

    2018-03-25

    A very stable binding site for the interaction between a pentameric oligothiophene and an amyloid-β(1-42) fibril has been identified by means of non-biased molecular dynamics simulations. In this site, the probe is locked in an all-trans conformation with a Coulombic binding energy of 1200 kJ mol -1 due to the interactions between the anionic carboxyl groups of the probe and the cationic ε-amino groups in the lysine side chain. Upon binding, the conformationally restricted probes show a pronounced increase in molecular planarity. This is in line with the observed changes in luminescence properties that serve as the foundation for their use as biomarkers.

  7. (+)- and (-)-N-allylnormetazocine binding sites in mouse brain: in vitro and in vivo characterization and regional distribution

    International Nuclear Information System (INIS)

    Compton, D.R.; Bagley, R.B.; Katzen, J.S.; Martin, B.R.

    1987-01-01

    In vivo and in vitro binding studies, both in whole brain and in selected areas, indicate that non-identical (+)- and (-)-NANM sites exist in the mouse brain, and each exhibits a different regional distribution. The in vivo binding of (+)- 3 H-NANM was found to be saturable at pharmacologically relevant doses, and represents a relatively small (10 - 22%) portion of total brain (+)- 3 H-NANM concentrations. The in vivo binding of (+)- 3 H-NANM was selectively displaced by (+)-NANM and PCP, and more sensitive to haloperidol and (+)-ketocyclazocine than the (-)- 3 H-NANM site. The in vivo binding of (-)- 3 H-NANM was selectively displaced by (-)-NANM, and more sensitive to naloxone and (-) ketocyclazocine than the (+)- 3 H-NANM site, and insensitive to PCP. This study indicates that the investigation of NANM binding sites is possible using in vivo binding techniques, and that each isomer apparently binds, in the mouse brain, to a single class of distinct sites. 32 references, 4 figures, 2 tables

  8. A systems biology approach to transcription factor binding site prediction.

    Directory of Open Access Journals (Sweden)

    Xiang Zhou

    2010-03-01

    Full Text Available The elucidation of mammalian transcriptional regulatory networks holds great promise for both basic and translational research and remains one the greatest challenges to systems biology. Recent reverse engineering methods deduce regulatory interactions from large-scale mRNA expression profiles and cross-species conserved regulatory regions in DNA. Technical challenges faced by these methods include distinguishing between direct and indirect interactions, associating transcription regulators with predicted transcription factor binding sites (TFBSs, identifying non-linearly conserved binding sites across species, and providing realistic accuracy estimates.We address these challenges by closely integrating proven methods for regulatory network reverse engineering from mRNA expression data, linearly and non-linearly conserved regulatory region discovery, and TFBS evaluation and discovery. Using an extensive test set of high-likelihood interactions, which we collected in order to provide realistic prediction-accuracy estimates, we show that a careful integration of these methods leads to significant improvements in prediction accuracy. To verify our methods, we biochemically validated TFBS predictions made for both transcription factors (TFs and co-factors; we validated binding site predictions made using a known E2F1 DNA-binding motif on E2F1 predicted promoter targets, known E2F1 and JUND motifs on JUND predicted promoter targets, and a de novo discovered motif for BCL6 on BCL6 predicted promoter targets. Finally, to demonstrate accuracy of prediction using an external dataset, we showed that sites matching predicted motifs for ZNF263 are significantly enriched in recent ZNF263 ChIP-seq data.Using an integrative framework, we were able to address technical challenges faced by state of the art network reverse engineering methods, leading to significant improvement in direct-interaction detection and TFBS-discovery accuracy. We estimated the accuracy

  9. Radiolabelling of phoneutria nigriventer spider toxin (Tx1): a tool to study its binding site

    International Nuclear Information System (INIS)

    Santos, Raquel Gouvea dos; Diniz, Carlos Roberto; Nascimento, Marta Cordeiro; Lima, Maria Elena de

    1996-01-01

    The neurotoxin Tx1, isolated from the venom of the South American spider Phoneutria nigriventer produces tail elevation and spastic paralysis of posterior limbs after intracerebral ventricular injection in mice. Tx1 also produces ileum contraction in bioassay. We have investigated the binding of radioiodinated-Tx1 ( 125 I-Tx1) on the preparation of myenteric plexus-longitudinal muscle membrane from guinea pig ileum (MPLM) as a tool to characterize the interaction of this neurotoxin with its site. The neurotoxin Tx1 was radioiodinated with Na 125 I by the lactoperoxidase method. 125 I-Tx1 specifically binds to a single class of noninteracting binding sites of high affinity (Kd= 3.5 x 10 -10 M) and low capacity (1.2 pmol/mg protein). The specific binding increased in parallel with the protein concentration. In competition experiments the ligands of ionic channels used (sodium, potassium and calcium) did not affect the binding of 125 I-Tx1 to MPLM neither did the cholinergic ligands (hemicholinium-3, hexamethonium, d-tubocurarine and atropine). Another neurotoxin (Tx2-6, one of the isoforms of Tx2 pool) decreased toxin with MPLM and showed that toxin has a specific and saturable binding site in guinea pig ileum and this binding site appears to be related to the Tx2 site. (author)

  10. 2-[125I]iodomelatonin binding sites in hamster brain membranes: pharmacological characteristics and regional distribution

    International Nuclear Information System (INIS)

    Duncan, M.J.; Takahashi, J.S.; Dubocovich, M.L.

    1988-01-01

    Studies in a variety of seasonally breeding mammals have shown that melatonin mediates photoperiodic effects on reproduction. Relatively little is known, however, about the site(s) or mechanisms of action of this hormone for inducing reproductive effects. Although binding sites for [3H]melatonin have been reported previously in bovine, rat, and hamster brain, the pharmacological selectivity of these sites was never demonstrated. In the present study, we have characterized binding sites for a new radioligand, 2-[125I]iodomelatonin, in brains from a photoperiodic species, the Syrian hamster. 2-[125I]Iodomelatonin labels a high affinity binding site in hamster brain membranes. Specific binding of 2-[125I]iodomelatonin is rapid, stable, saturable, and reversible. Saturation studies demonstrated that 2-[125I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 3.3 +/- 0.5 nM and a total binding capacity (Bmax) of 110.2 +/- 13.4 fmol/mg protein (n = 4). The Kd value determined from kinetic analysis (3.1 +/- 0.9 nM; n = 5) was very similar to that obtained from saturation experiments. Competition experiments showed that the relative order of potency of a variety of indoles for inhibition of 2-[125I]iodomelatonin binding site to hamster brain membranes was as follows: 6-chloromelatonin greater than or equal to 2-iodomelatonin greater than N-acetylserotonin greater than or equal to 6-methoxymelatonin greater than or equal to melatonin greater than 6-hydroxymelatonin greater than or equal to 6,7-dichloro-2-methylmelatonin greater than 5-methoxytryptophol greater than 5-methoxytryptamine greater than or equal to 5-methoxy-N,N-dimethyltryptamine greater than N-acetyltryptamine greater than serotonin greater than 5-methoxyindole (inactive)

  11. Endogenously generated plasmin at the vascular wall injury site amplifies lysine binding site-dependent plasminogen accumulation in microthrombi.

    Directory of Open Access Journals (Sweden)

    Tomasz Brzoska

    Full Text Available The fibrinolytic system plays a pivotal role in the regulation of hemostasis; however, it remains unclear how and when the system is triggered to induce thrombolysis. Using intra-vital confocal fluorescence microscopy, we investigated the process of plasminogen binding to laser-induced platelet-rich microthrombi generated in the mesenteric vein of transgenic mice expressing green fluorescent protein (GFP. The accumulation of GFP-expressing platelets as well as exogenously infused Alexa Fluor 568-labeled Glu-plasminogen (Glu-plg on the injured vessel wall was assessed by measuring the increase in the corresponding fluorescence intensities. Glu-plg accumulated in a time-dependent manner in the center of the microthrombus, where phosphatidylserine is exposed on platelet surfaces and fibrin formation takes place. The rates of binding of Glu-plg in the presence of ε-aminocaproic acid and carboxypeptidase B, as well as the rates of binding of mini-plasminogen lacking kringle domains 1-4 and lysine binding sites, were significantly lower than that of Glu-plg alone, suggesting that the binding was dependent on lysine binding sites. Furthermore, aprotinin significantly suppressed the accumulation of Glu-plg, suggesting that endogenously generated plasmin activity is a prerequisite for the accumulation. In spite of the endogenous generation of plasmin and accumulation of Glu-plg in the center of microthrombi, the microthrombi did not change in size during the 2-hour observation period. When human tissue plasminogen activator was administered intravenously, Glu-plg further accumulated and the microthrombi were lysed. Glu-plg appeared to accumulate in the center of microthrombi in the early phase of microthrombus formation, and plasmin activity and lysine binding sites were required for this accumulation.

  12. The importance of prolactin for lactation in the ewe

    International Nuclear Information System (INIS)

    Hooley, R.D.; Campbell, J.J.; Findlay, J.K.

    1978-01-01

    The effect of 2-bromo-α-ergocryptine (bromocriptine) on the induction and maintenance of milk secretion was studied in post-parturient ewes and in ovariectomized ewes artificially induced to lactate by treatment with oestrogen plus progesterone and then dexamethasone. Treatment with bromocriptine (about 0.4 mk/kg every 3 days) lowered and maintained the plasma concentration of prolactin at < 12 ng/ml. Ewes receiving bromocriptine concurrently with oestrogen plus progesterone during the priming phase had a significantly lower (P < 0.05) mean cumulative milk yield than control ewes, although the milk of the treated ewes contained normal amounts of fat, protein and lactose. Administration of bromocriptine during dexamethasone-induced lactogenesis had no significant effect on the mean cumulative milk yield but significantly (P < 0.05) increased the milk fat and protein content. In establishing lactation, bromocriptine markedly reduced the milk yield in both intact and ovariectomised ewes. The concentration of protein was not significantly affected although the milk fat content was higher in the bromocriptine-treated than in the control ewes. The effects of bromocriptine on milk yield and composition during galactopoiesis could be reversed by concurrent infusion of prolactin and the results suggest that prolactin is an important hormone during mammogenesis and galactopoiesis in sheep. (author)

  13. Solid Phase Radioimmunoassay for Measuring Serum Prolactin Using Antibody Coupled Magnetizable Particles

    International Nuclear Information System (INIS)

    El-Bayoumy, A.S.A.

    2012-01-01

    The objective of the present work was to prepare solid phase radioimmunoassay (RIA) reagents. Development as well as optimization and validation of RIA system using solid phase magnetic particles for the measurement of prolactin (PRL) in human serum are described. The production of polyclonal antibodies was carried out by immunizing three Balb/C mice intraperitoneal through primary injection and two booster doses. Low density magnetizable cellulose iron oxide particles have been used to couple covalently to the IgG fraction of polyclonal anti-prolactin using carbonyl diimidazole activation method and applied as a solid phase separating agent for RIA of serum prolactin. Preparation of 125 I-PRL tracer was prepared using lactoperoxidase method and it was purified by gel filtration using sephadex G-100. The PRL standards were prepared using a highly purified PRL antigen with assay buffer as standard matrix. Optimization and validation of the assay were carried out. The results obtained provide a low cost, simple, sensitive, specific and accurate RIA system of prolactin based on magnetizable solid phase separation. These magnetic particles retain their characteristics during storage for 6 months at 4 degree C. In conclusion, this assay could be used as a useful diagnostic tool for pituitary dysfunction and possible reproductive disability.

  14. Autoradiographic localization of GABA-regulated chloride ionophore binding site using t-[3H]butylbicycloorthobenzoate (TBOB)

    International Nuclear Information System (INIS)

    O'Connor, L.H.; McEwen, B.S.

    1986-01-01

    t-Butylbicycloorthobenzoate (TBOB) has been shown to bind with high affinity to sites on or near the chloride ionophore in rat brain membrane preparations. The present study used in vitro quantitative autoradiography to localize the regional distribution of [ 3 H]TBOB binding sites in rat forebrain. Receptors were labelled with 10 nM [ 3 H]TBOB. Nonspecific binding was determined by adding 10 μM picrotoxin to the incubation. Autoradiograms were generated using LKB Ultrofilm and then quantitated using computer-assisted spot-densitometry. The highest specific binding was found in frontal cortex layer 4, islands of Calleja, and ventral palladium. High binding was also found in many regions including anterior hypothalamic n., ventromedial hypothalamic n., dentate gyrus, stratum oriens and stratum lacunosum moleculare of hippocampus, and substantia nigra. Nonspecific binding represented 5 to 15% of total binding and was uniformly low throughout all brain regions. Thus, this selective probe for GABA-regulated chloride ionophore binding sites should provide a useful tool for characterizing this system and its relationship to convulsant and depressant drug action

  15. Growth hormone, prolactin and cortisol response to exercise in patients with depression

    DEFF Research Database (Denmark)

    Krogh, Jesper; Nordentoft, Merete; Mohammad-Nezhad, Mahdi

    2010-01-01

    BACKGROUND: A blunted growth hormone and prolactin response to pharmacological stress test have previously been found in depressed patients, as well as an increased cortisol response to psychosocial stress. This study investigated these hormones in response to acute exercise using an incremental...... bicycle test. METHOD: A cross-sectional comparison of cortisol, growth hormone, and prolactin in depressed (n=137) and healthy (n=44) subjects during rest and in response to an incremental bicycle test. Secondly, we tested the depressed patients again after a 4-month randomized naturalistic exercise...... intervention. RESULTS: Resting plasma levels of growth hormone (GH), cortisol, or prolactin (PRL) did not differ between depressed and healthy subjects (all p-values>.12). In response to an incremental bicycle test the GH (p=.02) and cortisol (p=.05) response in depressed was different compared to healthy...

  16. Hoogsteen base pairs proximal and distal to echinomycin binding sites on DNA

    International Nuclear Information System (INIS)

    Mendel, D.; Dervan, P.B.

    1987-01-01

    Forms of the DNA double helix containing non-Watson-Crick base-pairing have been discovered recently based on x-ray diffraction analysis of quionoxaline antibiotic-oligonucleotide complexes. In an effort to find evidence for Hoogsteen base-pairing at quinoxaline-binding sites in solution, chemical footprinting (differential cleavage reactivity) of echinomycin bound to DNA restriction fragments was examined. The authors report that purines (A>G) in the first and/or fourth base-pair positions of occupied echinomycin-binding sites are hyperreactive to diethyl pyrocarbonate. The correspondence of the solid-state data and the sites of diethyl pyrocarbonate hyperreactivity suggests that diethyl pyrocarbonate may be a sensitive reagent for the detection of Hoogsteen base-pairing in solution. Moreover, a 12-base-pair segment of alternating A-T DNA, which is 6 base pairs away from the nearest strong echinomycin-binding site, is also hyperreactive to diethyl pyrocarbonate in the presence of echinomycin. This hyperreactive segment may be an altered form of right-handed DNA that is entirely Hoogsteen base-paired

  17. Characterization of [125I]endothelin-1 binding sites in rat cardiac membrane fragments

    International Nuclear Information System (INIS)

    Gu, X.H.; Casley, D.J.; Nayler, W.G.

    1989-01-01

    Standard binding and displacement techniques were used to identify high-affinity binding sites for [ 125 I]-labeled endothelin-1 (ET-1) in membranes harvested from the hearts of adult female Sprague-Dawley rats. A single population of binding sites was identified, with a KD of 0.20 +/- 0.03 nM at 37 degrees C, and a Bmax of 93.5 +/- 6.4 fmol/mg protein. Bound [ 125 I]ET-1 was displaced by ET-1 (10(-13)-10(-8) M), with a Ki of 0.08 nM. Neither (-)Bay K 8644 (10(-11)-10(-5) M), prenylamine (10(-11)-10(-5) M), (+)-cis-diltiazem (10(-10)-10(-5) M), (-)D888 (10(-10)-10(-5) M), nicardipine (10(-10)-10(-5) M), lidoflazine (10(-11)-10(-5) M), flunarizine (10(-11)-10(-5) M), omega-conotoxin (10(-13)-10(-7) M), nor prazosin (10(-10)-10(-5) M) displaced the bound ligand. Binding occurred in the absence of Ca2+ and was absent in heat-denatured membranes. These results are interpreted to mean that [ 125 I]ET-1 binds to a single class of high-affinity binding sites that differ from those occupied by known regulators of voltage activated L- and N-type Ca2+ channels

  18. Prolactin levels during short- and long-term cross-sex hormone treatment: an observational study in transgender persons.

    Science.gov (United States)

    Nota, N M; Dekker, M J H J; Klaver, M; Wiepjes, C M; van Trotsenburg, M A; Heijboer, A C; den Heijer, M

    2017-08-01

    The cause of prolactin alterations in transgender persons is often assigned to oestrogens, but the precise cause and time course during different phases of cross-sex hormone treatment (CHT) remain unclear. In this study, we prospectively examined prolactin levels in 55 female-to-males (FtMs) and 61 male-to-females (MtFs) during the first year of CHT. Because long-term prolactin data were not available in this population, we studied these levels in a retrospective population of 25 FtMs and 38 MtFs who underwent gonadectomy. FtMs were treated with testosterone and MtFs with estradiol, with or without the anti-androgen cyproterone acetate (CPA) (after gonadectomy CPA is cessated). During the first year of CHT, prolactin decreased with 25% (95CI: -33%, -12%) in FtMs and increased with 193% (95CI: 156%, 219%) in MtFs. Eighteen MtFs developed hyperprolactinemia (≥0.6 IU L -1 ). In the retrospective population, post-gonadectomy levels in FtMs were lower than baseline levels (-39%; 95CI: -51%, -20%) while in MtFs post-gonadectomy levels and baseline levels were comparable (-6%; 95CI: -24%, 15%). No hyperprolactinemia was found after gonadectomy. In conclusion, in FtMs, prolactin decreased consistently during CHT and in MtFs, prolactin increased during pre-surgical CHT but normalised after gonadectomy. It is likely that CPA induces increasing prolactin levels in MtFs. © 2016 Blackwell Verlag GmbH.

  19. SP-A binding sites on bovine alveolar macrophages.

    Science.gov (United States)

    Plaga, S; Plattner, H; Schlepper-Schaefer, J

    1998-11-25

    Surfactant protein A (SP-A) binding to bovine alveolar macrophages was examined in order to characterize SP-A binding proteins on the cell surface and to isolate putative receptors from these cells that could be obtained in large amounts. Human SP-A, unlabeled or labeled with gold particles, was bound to freshly isolated macrophages and analyzed with ELISA or the transmission electron microscope. Binding of SP-A was inhibited by Ca2+ chelation, by an excess of unlabeled SP-A, or by the presence of 20 mg/ml mannan. We conclude that bovine alveolar macrophages expose binding sites for SP-A that are specific and that depend on Ca2+ and on mannose residues. For isolation of SP-A receptors with homologous SP-A as ligand we isolated SP-A from bovine lung lavage. SDS-PAGE analysis of the purified SP-A showed a protein of 32-36 kDa. Functional integrity of the protein was demonstrated. Bovine SP-A bound to Dynabeads was used to isolate SP-A binding proteins. From the fractionated and blotted proteins of the receptor preparation two proteins bound SP-A in a Ca2+-dependent manner, a 40-kDa protein showing mannose dependency and a 210-kDa protein, showing no mannose sensitivity. Copyright 1998 Academic Press.

  20. Number of active transcription factor binding sites is essential for the Hes7 oscillator

    Directory of Open Access Journals (Sweden)

    de Angelis Martin

    2006-02-01

    Full Text Available Abstract Background It is commonly accepted that embryonic segmentation of vertebrates is regulated by a segmentation clock, which is induced by the cycling genes Hes1 and Hes7. Their products form dimers that bind to the regulatory regions and thereby repress the transcription of their own encoding genes. An increase of the half-life of Hes7 protein causes irregular somite formation. This was shown in recent experiments by Hirata et al. In the same work, numerical simulations from a delay differential equations model, originally invented by Lewis, gave additional support. For a longer half-life of the Hes7 protein, these simulations exhibited strongly damped oscillations with, after few periods, severely attenuated the amplitudes. In these simulations, the Hill coefficient, a crucial model parameter, was set to 2 indicating that Hes7 has only one binding site in its promoter. On the other hand, Bessho et al. established three regulatory elements in the promoter region. Results We show that – with the same half life – the delay system is highly sensitive to changes in the Hill coefficient. A small increase changes the qualitative behaviour of the solutions drastically. There is sustained oscillation and hence the model can no longer explain the disruption of the segmentation clock. On the other hand, the Hill coefficient is correlated with the number of active binding sites, and with the way in which dimers bind to them. In this paper, we adopt response functions in order to estimate Hill coefficients for a variable number of active binding sites. It turns out that three active transcription factor binding sites increase the Hill coefficient by at least 20% as compared to one single active site. Conclusion Our findings lead to the following crucial dichotomy: either Hirata's model is correct for the Hes7 oscillator, in which case at most two binding sites are active in its promoter region; or at least three binding sites are active, in which

  1. Prolactin releasing effect of sulpiride isomers in rats and man

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, E E; Stefanini, E; Spano, P F [Cagliari Univ. (Italy). Inst. of Pharmacology and Pharmacognosy; Camanni, F; Massara, F [Turin Univ. (Italy). Chair of Endocrinology; Locatelli, V; Cocchi, D

    1979-01-01

    Sulpiride, an antipsychotropic drug of the benzamide class, reportedly displaces stereospecifically (/sup 3/H)-butyrophenones from putative dopamine (DA) binding sites in rat striatum. To evaluate if sulpiride displays the same stereospecifity in the inhibition of pituitary DA receptors, the effect of the two(-)-and (+)-sulpiride isomers was tested with regard to their ability to stimulate prolactin (PRL) secretion in rats and man and to displace (/sup 3/H)-spiroperidol bound to rat anterior pituitary receptors. In male rats, (-)-sulpiride at doses of 0.1 and 0.1 mg/kg i.p., induced a maximum PRL-releasing effect, not different from that evoked by a dose of 10 mg/kg of the compound. (+)-Sulpiride was active only at the dose of 10mg/kg i.p., and its PRL-releasing effect was superimposable to that evoked by the same dose of (-)-sulpiride. Similarily, in 8 normal subjects (4 men and 4 women) only (-)-sulpiride was active as PRL releaser when the low dose of 0.25 mg i.v. was used; when the higher dose of sulpiride was used (4.0 mg i.v.), it induced a rise in plasma PRL of the same entity for both isomers at early post-injection times (15-30 min) but greater with the (-)-isomer at the following time intervals (45-120 min). (-)-Sulpiride displaced (/sup 3/H)-spiroperidol bound to rat anterior pituitary homogenates with a potency about 100 times greater as that showed by (+)-sulpiride. In all, these data indicate that sulpiride isomers display at the level of pituitary DA receptors for PRL control the same stereospecifity exhibited on a population of striatal DA receptors.

  2. Mapping the heparin-binding site of the BMP antagonist gremlin by site-directed mutagenesis based on predictive modelling.

    Science.gov (United States)

    Tatsinkam, Arnold Junior; Mulloy, Barbara; Rider, Christopher C

    2015-08-15

    Gremlin is a member of the CAN (cerberus and DAN) family of secreted BMP (bone morphogenetic protein) antagonists and also an agonist of VEGF (vascular endothelial growth factor) receptor-2. It is critical in limb skeleton and kidney development and is re-expressed during tissue fibrosis. Gremlin binds strongly to heparin and heparan sulfate and, in the present study, we sought to investigate its heparin-binding site. In order to explore a putative non-contiguous binding site predicted by computational molecular modelling, we substituted a total of 11 key arginines and lysines located in three basic residue sequence clusters with homologous sequences from cerberus and DAN (differential screening selected gene abberative in neuroblastoma), CAN proteins which lack basic residues in these positions. A panel of six Myc-tagged gremlin mutants, MGR-1-MGR-6 (MGR, mutant gremlin), each containing different combinations of targeted substitutions, all showed markedly reduced affinity for heparin as demonstrated by their NaCl elution on heparin affinity chromatography, thus verifying our predictions. Both MGR-5 and MGR-6 retained BMP-4-binding activity comparable to that of wild-type gremlin. Low-molecular-mass heparin neither promoted nor inhibited BMP-4 binding. Finally, glutaraldehyde cross-linking demonstrated that gremlin forms non-covalent dimers, similar behaviour to that of DAN and also PRDC (protein related to cerberus and DAN), another CAN protein. The resulting dimer would possess two heparin-binding sites, each running along an exposed surface on the second β-strand finger loop of one of the monomers. © 2015 Authors; published by Portland Press Limited.

  3. The conserved WW-domain binding sites in Dystroglycan C-terminus are essential but partially redundant for Dystroglycan function

    Directory of Open Access Journals (Sweden)

    Deng W-M

    2009-02-01

    Full Text Available Abstract Background Dystroglycan (Dg is a transmembrane protein that is a part of the Dystrophin Glycoprotein Complex (DGC which connects the extracellular matrix to the actin cytoskeleton. The C-terminal end of Dg contains a number of putative SH3, SH2 and WW domain binding sites. The most C-terminal PPXY motif has been established as a binding site for Dystrophin (Dys WW-domain. However, our previous studies indicate that both Dystroglycan PPXY motives, WWbsI and WWbsII can bind Dystrophin protein in vitro. Results We now find that both WW binding sites are important for maintaining full Dg function in the establishment of oocyte polarity in Drosophila. If either WW binding site is mutated, the Dg protein can still be active. However, simultaneous mutations in both WW binding sites abolish the Dg activities in both overexpression and loss-of-function oocyte polarity assays in vivo. Additionally, sequence comparisons of WW binding sites in 12 species of Drosophila, as well as in humans, reveal a high level of conservation. This preservation throughout evolution supports the idea that both WW binding sites are functionally required. Conclusion Based on the obtained results we propose that the presence of the two WW binding sites in Dystroglycan secures the essential interaction between Dg and Dys and might further provide additional regulation for the cytoskeletal interactions of this complex.

  4. Cyclical Changes in Prolactin Levels among Infertile Women ...

    African Journals Online (AJOL)

    Michael Horsfall

    investigation of infertility while the elevation of prolactin in the 3 study groups might be responsible for the infertility observed. ... dopamine and enhanced by some other hormones. In ... also appears to have a role in the immune response,.

  5. Identification of nucleic acid binding sites on translin-associated factor X (TRAX protein.

    Directory of Open Access Journals (Sweden)

    Gagan Deep Gupta

    Full Text Available Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity.

  6. Identification of Nucleic Acid Binding Sites on Translin-Associated Factor X (TRAX) Protein

    Science.gov (United States)

    Gupta, Gagan Deep; Kumar, Vinay

    2012-01-01

    Translin and TRAX proteins play roles in very important cellular processes such as DNA recombination, spatial and temporal expression of mRNA, and in siRNA processing. Translin forms a homomeric nucleic acid binding complex and binds to ssDNA and RNA. However, a mutant translin construct that forms homomeric complex lacking nucleic acid binding activity is able to form fully active heteromeric translin-TRAX complex when co-expressed with TRAX. A substantial progress has been made in identifying translin sites that mediate its binding activity, while TRAX was thought not to bind DNA or RNA on its own. We here for the first time demonstrate nucleic acid binding to TRAX by crosslinking radiolabeled ssDNA to heteromeric translin-TRAX complex using UV-laser. The TRAX and translin, photochemically crosslinked with ssDNA, were individually detected on SDS-PAGE. We mutated two motifs in TRAX and translin, designated B2 and B3, to help define the nucleic acid binding sites in the TRAX sequence. The most pronounced effect was observed in the mutants of B3 motif that impaired nucleic acid binding activity of the heteromeric complexes. We suggest that both translin and TRAX are binding competent and contribute to the nucleic acid binding activity. PMID:22427937

  7. Deciphering common recognition principles of nucleoside mono/di and tri-phosphates binding in diverse proteins via structural matching of their binding sites.

    Science.gov (United States)

    Bhagavat, Raghu; Srinivasan, Narayanaswamy; Chandra, Nagasuma

    2017-09-01

    Nucleoside triphosphate (NTP) ligands are of high biological importance and are essential for all life forms. A pre-requisite for them to participate in diverse biochemical processes is their recognition by diverse proteins. It is thus of great interest to understand the basis for such recognition in different proteins. Towards this, we have used a structural bioinformatics approach and analyze structures of 4677 NTP complexes available in Protein Data Bank (PDB). Binding sites were extracted and compared exhaustively using PocketMatch, a sensitive in-house site comparison algorithm, which resulted in grouping the entire dataset into 27 site-types. Each of these site-types represent a structural motif comprised of two or more residue conservations, derived using another in-house tool for superposing binding sites, PocketAlign. The 27 site-types could be grouped further into 9 super-types by considering partial similarities in the sites, which indicated that the individual site-types comprise different combinations of one or more site features. A scan across PDB using the 27 structural motifs determined the motifs to be specific to NTP binding sites, and a computational alanine mutagenesis indicated that residues identified to be highly conserved in the motifs are also most contributing to binding. Alternate orientations of the ligand in several site-types were observed and rationalized, indicating the possibility of some residues serving as anchors for NTP recognition. The presence of multiple site-types and the grouping of multiple folds into each site-type is strongly suggestive of convergent evolution. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. Proteins 2017; 85:1699-1712. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Hypergravity and estrogen effects on avian anterior pituitary growth hormone and prolactin levels

    Science.gov (United States)

    Fiorindo, R. P.; Negulesco, J. A.

    1980-01-01

    Developing female chicks with fractured right radii were maintained for 14 d at either earth gravity (1 g) or a hypergravity state (2 g). The birds at 1 g were divided into groups which received daily injections of (1) saline, (2) 200 micrograms estrone, and (3) 400 micrograms estrone for 14 d. The 2-g birds were divided into three similarly treated groups. All 2-g birds showed significantly lower body weights than did 1-g birds. Anterior pituitary (AP) glands were excised and analyzed for growth hormone and prolactin content by analytical electrophoresis. The 1-g chicks receiving either dose of daily estrogen showed increased AP growth hormone levels, whereas hypergravity alone did not affect growth hormone content. Chicks exposed to daily estrogen and hypergravity displayed reduced growth hormone levels. AP prolactin levels were slightly increased by the lower daily estrogen dose in 1-g birds, but markedly reduced in birds exposed only to hypergravity. Doubly-treated chicks displayed normal prolactin levels. Reduced growth in 2-g birds might be due, in part, to reduced AP levels of prolactin and/or growth hormone.

  9. The prolactin response to an acute stressor in relation to parental care and corticosterone in a short-lived bird, the Eurasian hoopoe.

    Science.gov (United States)

    Schmid, Baptiste; Chastel, Olivier; Jenni, Lukas

    2011-10-01

    Prolactin plays an important role in mediating parental care in birds, but little is known about changes in prolactin levels when animals disrupt their reproductive behaviour during emergency life-history stages. We investigated the variation of prolactin levels with breeding stage, sex, body condition and as a response to a standardized acute stressor in a small short-lived bird, the Eurasian hoopoe Upupa epops under natural field conditions. We found higher baseline levels of prolactin in females during the brooding phase than in their mates which feed them and their chicks at this stage. Moreover, this is the first report of a differential prolactin stress-response between sexes with contrasting parental care within a breeding phase. Capture, handling and restraint induced a clear decrease of prolactin levels which was less pronounced in females at the very early stage of brooding compared to females in later stages. In contrast, the prolactin stress response in males remained nearly constant over the breeding stages and was stronger than in females. Baseline levels of prolactin, but not handling-induced levels, were positively correlated with body condition. We found a weak relationship between the decrease in prolactin due to acute handling stress and handling-induced levels of corticosterone. Taken together, both baseline and stress response levels of prolactin were related to the amount of parental care, although we found no relationship with reproductive success. It appears that the response to an acute stressor in prolactin levels is finely tuned to parental duties and investment. Hence, prolactin appears to be involved in mediating the trade-off between current reproduction versus self-maintenance and future reproduction. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Binding Sites for Amyloid-β Oligomers and Synaptic Toxicity

    Science.gov (United States)

    Smith, Levi M.; Strittmatter, Stephen M.

    2017-01-01

    In Alzheimer’s disease (AD), insoluble and fibrillary amyloid-β (Aβ) peptide accumulates in plaques. However, soluble Aβ oligomers are most potent in creating synaptic dysfunction and loss. Therefore, receptors for Aβ oligomers are hypothesized to be the first step in a neuronal cascade leading to dementia. A number of cell-surface proteins have been described as Aβ binding proteins, and one or more are likely to mediate Aβ oligomer toxicity in AD. Cellular prion protein (PrPC) is a high-affinity Aβ oligomer binding site, and a range of data delineates a signaling pathway leading from Aβ complexation with PrPC to neuronal impairment. Further study of Aβ binding proteins will define the molecular basis of this crucial step in AD pathogenesis. PMID:27940601

  11. Binding sites for 3H-LTC4 in membranes from guinea pig ileal longitudinal muscle

    International Nuclear Information System (INIS)

    Nicosia, S.; Crowley, H.J.; Oliva, D.; Welton, A.F.

    1984-01-01

    Leutriene (LTC4) is one of the components of Slow Reacting Substance of Anaphylaxis (SRS-A) and is a potent constrictor of guinea pig ilea. The contraction is likely to be a receptor-mediated process. Here the authors report the existence of specific binding sites for 3 H-LTC4 in a crude membrane preparation from guinea pig ileal longitudinal muscle. At 4 degrees C in the presence of 20 mM Serine-borate, binding increases linearly with protein concentration, reaches equilibrium in 10 minutes, and is reversible upon addition of 3 x 10(-5) M unlabelled LTC4. The dissociation curve is consistent with the existence of more than one class of binding site. Ca++ and Mg++ greatly enhance the binding of 3 H-LTC4 at equilibrium. In the presence of 5 mM CaCl 2 and MgCl 2 not only LTC4 (IC50 10(-7)M), but also LTD4 and the SRS-A antagonist FPL 55712 can compete with 3 H-LTC4 for its binding sites. FPL 55712 only displaces 60-70% of the total amount bound, while LTC4 displaces 90-95%. These studies indicate that multiple classes of binding sites exist for 3 H-LTC4 in guinea pig ileal longitudinal muscle, and that at least part of these binding sites might be related to the ability of LTC4 to contract guinea pig ilea

  12. The post-orgasmic prolactin increase following intercourse is greater than following masturbation and suggests greater satiety.

    Science.gov (United States)

    Brody, Stuart; Krüger, Tillmann H C

    2006-03-01

    Research indicates that prolactin increases following orgasm are involved in a feedback loop that serves to decrease arousal through inhibitory central dopaminergic and probably peripheral processes. The magnitude of post-orgasmic prolactin increase is thus a neurohormonal index of sexual satiety. Using data from three studies of men and women engaging in masturbation or penile-vaginal intercourse to orgasm in the laboratory, we report that for both sexes (adjusted for prolactin changes in a non-sexual control condition), the magnitude of prolactin increase following intercourse is 400% greater than that following masturbation. The results are interpreted as an indication of intercourse being more physiologically satisfying than masturbation, and discussed in light of prior research reporting greater physiological and psychological benefits associated with coitus than with any other sexual activities.

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

    OpenAIRE

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

    1988-01-01

    1. The interaction of substituted benzamides with brain benzodiazepine (BDZ) binding sites was examined by their ability to displace [3H]-flunitrazepam ([3H]-FNM) from specific binding sites in bovine cortical membranes in vitro. 2. Clebopride, Delagrange 2674, Delagrange 2335 and BRL 20627 displayed concentration-dependent displacement of [3H]-FNM with IC50 values of 73 nM, 132 nM, 7.7 microM and 5.9 microM, respectively. Other substituted benzamides including metoclopramide, sulpiride, tiap...

  14. PolyaPeak: Detecting Transcription Factor Binding Sites from ChIP-seq Using Peak Shape Information

    Science.gov (United States)

    Wu, Hao; Ji, Hongkai

    2014-01-01

    ChIP-seq is a powerful technology for detecting genomic regions where a protein of interest interacts with DNA. ChIP-seq data for mapping transcription factor binding sites (TFBSs) have a characteristic pattern: around each binding site, sequence reads aligned to the forward and reverse strands of the reference genome form two separate peaks shifted away from each other, and the true binding site is located in between these two peaks. While it has been shown previously that the accuracy and resolution of binding site detection can be improved by modeling the pattern, efficient methods are unavailable to fully utilize that information in TFBS detection procedure. We present PolyaPeak, a new method to improve TFBS detection by incorporating the peak shape information. PolyaPeak describes peak shapes using a flexible Pólya model. The shapes are automatically learnt from the data using Minorization-Maximization (MM) algorithm, then integrated with the read count information via a hierarchical model to distinguish true binding sites from background noises. Extensive real data analyses show that PolyaPeak is capable of robustly improving TFBS detection compared with existing methods. An R package is freely available. PMID:24608116

  15. Effects of melatonin and prolactin in reproduction: review of literature.

    Science.gov (United States)

    Tenorio, Fernanda das Chagas Angelo Mendes; Simões, Manuel de Jesus; Teixeira, Valéria Wanderley; Teixeira, Álvaro Aguiar Coelho

    2015-01-01

    The pineal gland is responsible for producing a hormone called melatonin (MEL), and is accepted as the gland that regulates reproduction in mammals. Prolactin (PRL) also exhibits reproductive activity in animals in response to photoperiod. It is known that the concentrations of PRL are high in the summer and reduced during winter, the opposite of what is seen with melatonin in these seasons. In placental mammals, both prolactin and melatonin affect implantation, which is considered a critical point of pregnancy, since a successful pregnancy requires the development of a synchronous interaction between the endometrium and blastocyst for placental development. It is also known that PRL levels during pregnancy are essential for the maintenance of pregnancy, because this hormone induces the corpus luteum to produce progesterone, in addition to stimulating blastocyst implantation to maintain pregnancy and form the placenta. However, melatonin levels in plasma have also been shown to increase during pregnancy, peaking at the end of this period, which suggests that this hormone plays an important role in the maintenance of pregnancy. Thus, it is clear that treatment with prolactin or melatonin interferes with the processes responsible for the development and maintenance of pregnancy.

  16. Effects of melatonin and prolactin in reproduction: review of literature

    Directory of Open Access Journals (Sweden)

    Fernanda das Chagas Angelo Mendes Tenorio

    2015-06-01

    Full Text Available Summary The pineal gland is responsible for producing a hormone called melatonin (MEL, and is accepted as the gland that regulates reproduction in mammals. Prolactin (PRL also exhibits reproductive activity in animals in response to photoperiod. It is known that the concentrations of PRL are high in the summer and reduced during winter, the opposite of what is seen with melatonin in these seasons. In placental mammals, both prolactin and melatonin affect implantation, which is considered a critical point of pregnancy, since a successful pregnancy requires the development of a synchronous interaction between the endometrium and blastocyst for placental development. It is also known that PRL levels during pregnancy are essential for the maintenance of pregnancy, because this hormone induces the corpus luteum to produce progesterone, in addition to stimulating blastocyst implantation to maintain pregnancy and form the placenta. However, melatonin levels in plasma have also been shown to increase during pregnancy, peaking at the end of this period, which suggests that this hormone plays an important role in the maintenance of pregnancy. Thus, it is clear that treatment with prolactin or melatonin interferes with the processes responsible for the development and maintenance of pregnancy.

  17. Active site - a site of binding of affinity inhibitors in baker's yeast inorganic pyrophosphatase

    International Nuclear Information System (INIS)

    Svyato, I.E.; Sklyankina, V.A.; Avaeva, S.M.

    1986-01-01

    The interaction of the enzyme-substrate complex with methyl phosphate, O-phosphoethanolamine, O-phosphopropanolamine, N-acetylphosphoserine, and phosphoglyolic acid, as well as pyrophosphatase, modified by monoesters of phosphoric acid, with pyrophosphate and tripolyphosphate, was investigated. It was shown that the enzyme containing the substrate in the active site does not react with monophosphates, but modified pyrophosphatase entirely retains the ability to bind polyanions to the regulatory site. It is concluded that the inactivation of baker's yeast inorganic pyrophosphatase by monoesters of phosphoric acid, which are affinity inhibitors of it, is the result of modification of the active site of the enzyme

  18. Metabolic and stress-related roles of prolactin-releasing peptide.

    Science.gov (United States)

    Onaka, Tatsushi; Takayanagi, Yuki; Leng, Gareth

    2010-05-01

    In the modern world, improvements in human health can be offset by unhealthy lifestyle factors, including the deleterious consequences of stress and obesity. For energy homeostasis, humoral factors and neural afferents from the gastrointestinal tract, in combination with long-term nutritional signals, communicate information to the brain to regulate energy intake and expenditure. Energy homeostasis and stress interact with each other, and stress affects both food intake and energy expenditure. Prolactin-releasing peptide, synthesized in discrete neuronal populations in the hypothalamus and brainstem, plays an important role in integrating these responses. This review describes how prolactin-releasing peptide neurons receive information concerning both internal metabolic states and environmental conditions, and play a key role in energy homeostasis and stress responses. 2010 Elsevier Ltd. All rights reserved.

  19. Training increases the concentration of [3H]ouabain-binding sites in rat skeletal muscle

    DEFF Research Database (Denmark)

    Kjeldsen, K; Richter, Erik; Galbo, H

    1986-01-01

    ]ouabain-binding-site concentration in the diaphragm, but in the heart ventricles, the K+-dependent 3-O-methylfluorescein phosphatase activity increased by 20% (P less than 0.001). Muscle inactivity induced by denervation, plaster immobilisation or tenotomy reduced the [3H]ouabain-binding-site concentration by 20-30% (P less than 0...

  20. SP Transcription Factor Paralogs and DNA-Binding Sites Coevolve and Adaptively Converge in Mammals and Birds

    Science.gov (United States)

    Yokoyama, Ken Daigoro; Pollock, David D.

    2012-01-01

    Functional modification of regulatory proteins can affect hundreds of genes throughout the genome, and is therefore thought to be almost universally deleterious. This belief, however, has recently been challenged. A potential example comes from transcription factor SP1, for which statistical evidence indicates that motif preferences were altered in eutherian mammals. Here, we set out to discover possible structural and theoretical explanations, evaluate the role of selection in SP1 evolution, and discover effects on coregulatory proteins. We show that SP1 motif preferences were convergently altered in birds as well as mammals, inducing coevolutionary changes in over 800 regulatory regions. Structural and phylogenic evidence implicates a single causative amino acid replacement at the same SP1 position along both lineages. Furthermore, paralogs SP3 and SP4, which coregulate SP1 target genes through competitive binding to the same sites, have accumulated convergent replacements at the homologous position multiple times during eutherian and bird evolution, presumably to preserve competitive binding. To determine plausibility, we developed and implemented a simple model of transcription factor and binding site coevolution. This model predicts that, in contrast to prevailing beliefs, even small selective benefits per locus can drive concurrent fixation of transcription factor and binding site mutants under a broad range of conditions. Novel binding sites tend to arise de novo, rather than by mutation from ancestral sites, a prediction substantiated by SP1-binding site alignments. Thus, multiple lines of evidence indicate that selection has driven convergent evolution of transcription factors along with their binding sites and coregulatory proteins. PMID:23019068

  1. (/sup 3/H)Spiperone binding sites in brain: autoradiographic localization of multiple receptors

    Energy Technology Data Exchange (ETDEWEB)

    Palacios, J M; Niehoff, D L; Kuhar, M J [Johns Hopkins Univ., Baltimore, MD (USA). School of Medicine

    1981-01-01

    (/sup 3/H)Spiperone ((/sup 3/H)SP) binding sites were localized by light microscopic autoradiography, after in vitro labelling. The kinetic and pharmacological characteristics of these binding sites were studied in slide-mounted sections of rat forebrain, and optimal labeling conditions were defined. Autoradiograms were obtained by apposing emulsion-coated coverslips to labeled sections. Differential drug sensitivity allowed the selective displacement of (/sup 3/H)SP from dopamine receptors by ADTN, from serotonin receptors by cinanserin, from both by haloperidol and from unique spiperone sites by unlabeled spiperone. The various sites presented a differential anatomical localization. For example, only dopaminergic sites were found in the glomerular layer of the olfactory bulb; only serotonergic sites were found in lamina IV of the neocortex, and a high concentration of unique spiperone sites were found in parts of the hippocampus.

  2. Cell-type specificity of ChIP-predicted transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Håndstad Tony

    2012-08-01

    Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

  3. Evaluation of prolactin levels in patients with newly diagnosed pemphigus vulgaris and its correlation with pemphigus disease area index.

    Science.gov (United States)

    Lajevardi, Vahideh; Hallaji, Zahra; Daneshpazhooh, Maryam; Ghandi, Narges; Shekari, Peyman; Khani, Sepideh

    2016-06-01

    Prolactin is a hormone; in addition to it known roles, it has immunomodulatory effects on lymphocytes maturation and immunoglobulins production. Hyperprolactinemia has been demonstrated in various autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, type I diabetes mellitus, and Graves' disease. In view of the prolactin immunomodulatory roles, studying prolactin levels in pemphigus as an autoimmune blistering disease may introduce new ways of understanding disease etiology and developing treatment strategies. Our purpose was to determine the prolactin levels in patients with newly diagnosed pemphigus vulgaris and study its correlation with pemphigus disease area index. Our study was limited by the lack of a control group. In this cross-sectional study, prolactin and anti-desmoglein 1 and 3 autoantibodies levels were measured in 50 patients with newly diagnosed pemphigus vulgaris in Razi Dermatology Hospital. Pemphigus severity and extent was estimated using the Pemphigus Disease Area Index. Of the 50 patients, 18 were male and 32 were female with a mean age of 41.56 ± 13.66 years. Mean prolactin (PRL) level was 15.60 ± 11.72 ng/ml (10.68 in males and 18.37 in females). Mean anti-desmoglein 1 and 3 autoantibodies were 135.8 ± 119.8 and 245.8 ± 157.4 U/ml, respectively. Eleven out of 50 patients had a higher than normal prolactin range. No relation was found between prolactin level and disease activity ( p = .982). Also, correlation studies show no relation between prolactin and anti-desmoglein 1 and 3 autoantibodies levels (respectively, p = .771 and .738). In comparing the extent of the disease between the two groups with normal and high prolactin, paired t-test showed no significance ( p = .204). In our study, 22% of patients had hyperprolactinemia, which was greater among females. The highest PRL level was detected in mucocutaneous group. Although serum PRL levels were higher in patients with a greater Pemphigus Disease Area Index

  4. CTCF Binding Sites in the Herpes Simplex Virus 1 Genome Display Site-Specific CTCF Occupation, Protein Recruitment, and Insulator Function.

    Science.gov (United States)

    Washington, Shannan D; Musarrat, Farhana; Ertel, Monica K; Backes, Gregory L; Neumann, Donna M

    2018-04-15

    There are seven conserved CTCF binding domains in the herpes simplex virus 1 (HSV-1) genome. These binding sites individually flank the latency-associated transcript (LAT) and the immediate early (IE) gene regions, suggesting that CTCF insulators differentially control transcriptional domains in HSV-1 latency. In this work, we show that two CTCF binding motifs in HSV-1 display enhancer blocking in a cell-type-specific manner. We found that CTCF binding to the latent HSV-1 genome was LAT dependent and that the quantity of bound CTCF was site specific. Following reactivation, CTCF eviction was dynamic, suggesting that each CTCF site was independently regulated. We explored whether CTCF sites recruit the polycomb-repressive complex 2 (PRC2) to establish repressive domains through a CTCF-Suz12 interaction and found that Suz12 colocalized to the CTCF insulators flanking the ICP0 and ICP4 regions and, conversely, was removed at early times postreactivation. Collectively, these data support the idea that CTCF sites in HSV-1 are independently regulated and may contribute to lytic-latent HSV-1 control in a site-specific manner. IMPORTANCE The role of chromatin insulators in DNA viruses is an area of interest. It has been shown in several beta- and gammaherpesviruses that insulators likely control the lytic transcriptional profile through protein recruitment and through the formation of three-dimensional (3D) chromatin loops. The ability of insulators to regulate alphaherpesviruses has been understudied to date. The alphaherpesvirus HSV-1 has seven conserved insulator binding motifs that flank regions of the genome known to contribute to the establishment of latency. Our work presented here contributes to the understanding of how insulators control transcription of HSV-1. Copyright © 2018 American Society for Microbiology.

  5. PATTERN BASED DETECTION OF POTENTIALLY DRUGGABLE BINDING SITES BY LIGAND SCREENING

    Directory of Open Access Journals (Sweden)

    Uttam Pal

    2018-03-01

    Full Text Available This article describes an innovative way of finding the potentially druggable sites on a target protein, which can be used for orthosteric and allosteric lead detection in a single virtual screening setup. Druggability estimation for an alternate binding site other than the canonical ligand-binding pocket of an enzyme is rewarding for several inherent benefits. Allostery is a direct and efficient way of regulating biomacromolecule function. The allosteric modulators can fine-tune protein mechanics. Besides, allosteric sites are evolutionarily less conserved/more diverse even in very similarly related proteins, thus, provides high degree of specificity in targeting a particular protein. Therefore, targeting of allosteric sites is gaining attention as an emerging strategy in rational drug design. However, the experimental approaches provide a limited degree of characterization of new allosteric sites. Computational approaches are useful to analyze and select potential allosteric sites for drug discovery. Here, the use of molecular docking, which has become an integral part of the drug discovery process, has been discussed to predict the druggability of novel allosteric sites as well as the active site on target proteins by ligand screening. Genetic algorithm was used for docking and the whole protein was placed in the search space. For each ligand in the library of small molecules, the genetic algorithm was run for multiple times to populate all the druggable sites in the target protein, which was then translated into two dimensional density maps or “patterns”. High density clusters were observed for lead like molecules in these pattern diagrams. Each cluster in such a pattern diagram indicated a plausible binding site and the density gave its druggability score in terms of weighted probabilities. The patterns were filtered to find the leads for each of the druggable sites on the target protein. Such a novel pattern based analysis of the

  6. Druggable pockets and binding site centric chemical space: a paradigm shift in drug discovery.

    Science.gov (United States)

    Pérot, Stéphanie; Sperandio, Olivier; Miteva, Maria A; Camproux, Anne-Claude; Villoutreix, Bruno O

    2010-08-01

    Detection, comparison and analyses of binding pockets are pivotal to structure-based drug design endeavors, from hit identification, screening of exosites and de-orphanization of protein functions to the anticipation of specific and non-specific binding to off- and anti-targets. Here, we analyze protein-ligand complexes and discuss methods that assist binding site identification, prediction of druggability and binding site comparison. The full potential of pockets is yet to be harnessed, and we envision that better understanding of the pocket space will have far-reaching implications in the field of drug discovery, such as the design of pocket-specific compound libraries and scoring functions.

  7. Regulation of CCL2 expression by an upstream TALE homeodomain protein-binding site that synergizes with the site created by the A-2578G SNP.

    Science.gov (United States)

    Page, Stephen H; Wright, Edward K; Gama, Lucio; Clements, Janice E

    2011-01-01

    CC Chemokine Ligand 2 (CCL2) is a potent chemoattractant produced by macrophages and activated astrocytes during periods of inflammation within the central nervous system. Increased CCL2 expression is correlated with disease progression and severity, as observed in pulmonary tuberculosis, HCV-related liver disease, and HIV-associated dementia. The CCL2 distal promoter contains an A/G polymorphism at position -2578 and the homozygous -2578 G/G genotype is associated with increased CCL2 production and inflammation. However, the mechanisms that contribute to the phenotypic differences in CCL2 expression are poorly understood. We previously demonstrated that the -2578 G polymorphism creates a TALE homeodomain protein binding site (TALE binding site) for PREP1/PBX2 transcription factors. In this study, we identified the presence of an additional TALE binding site 22 bp upstream of the site created by the -2578 G polymorphism and demonstrated the synergistic effects of the two sites on the activation of the CCL2 promoter. Using chromatin immunoprecipitation (ChIP) assays, we demonstrated increased binding of the TALE proteins PREP1 and PBX2 to the -2578 G allele, and binding of IRF1 to both the A and G alleles. The presence of TALE binding sites that form inverted repeats within the -2578 G allele results in increased transcriptional activation of the CCL2 distal promoter while the presence of only the upstream TALE binding site within the -2578 A allele exerts repression of promoter activity.

  8. Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1

    Directory of Open Access Journals (Sweden)

    Wang Pu

    2010-10-01

    Full Text Available Abstract The Epstein-Barr Virus (EBV Nuclear Antigen 1 (EBNA1 protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP, regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP combined with massively parallel deep-sequencing (ChIP-Seq was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA

  9. Adenovirus-Mediated Delivery of Decoy Hyper Binding Sites Targeting Oncogenic HMGA1 Reduces Pancreatic and Liver Cancer Cell Viability.

    Science.gov (United States)

    Hassan, Faizule; Ni, Shuisong; Arnett, Tyler C; McKell, Melanie C; Kennedy, Michael A

    2018-03-30

    High mobility group AT-hook 1 (HMGA1) protein is an oncogenic architectural transcription factor that plays an essential role in early development, but it is also implicated in many human cancers. Elevated levels of HMGA1 in cancer cells cause misregulation of gene expression and are associated with increased cancer cell proliferation and increased chemotherapy resistance. We have devised a strategy of using engineered viruses to deliver decoy hyper binding sites for HMGA1 to the nucleus of cancer cells with the goal of sequestering excess HMGA1 at the decoy hyper binding sites due to binding competition. Sequestration of excess HMGA1 at the decoy binding sites is intended to reduce HMGA1 binding at the naturally occurring genomic HMGA1 binding sites, which should result in normalized gene expression and restored sensitivity to chemotherapy. As proof of principle, we engineered the replication defective adenovirus serotype 5 genome to contain hyper binding sites for HMGA1 composed of six copies of an individual HMGA1 binding site, referred to as HMGA-6. A 70%-80% reduction in cell viability and increased sensitivity to gemcitabine was observed in five different pancreatic and liver cancer cell lines 72 hr after infection with replication defective engineered adenovirus serotype 5 virus containing the HMGA-6 decoy hyper binding sites. The decoy hyper binding site strategy should be general for targeting overexpression of any double-stranded DNA-binding oncogenic transcription factor responsible for cancer cell proliferation.

  10. Effects of photoperiod and temperature on the release of prolactin from the pituitary gland of the goldfish, Carassius auratus L

    Energy Technology Data Exchange (ETDEWEB)

    McKeown, B.A. (Simon Fraser Univ., Burnaby, BC); Peter, R.E.

    1976-11-01

    A number of experiments were conducted to investigate the effects of photoperiod and temperature on prolactin release from the goldfish pituitary gland. Fish were acclimated to different photoperiods and temperatures, and also were subjected to a change in either of these two parameters after different acclimation conditions. Serum and pituitary samples were collected and analyzed by radioimmunoassay for prolactin levels. In other experiments samples for prolactin analysis were taken every 3 h intermittently over a period of 3 days from fish that were acclimated to different photoperiod and temperature conditions. Longer photoperiods and higher temperatures caused pituitary prolactin release. Serum prolactin changed on a circadian rhythm and the rhythm was modified depending on the length of the photoperiod.

  11. Polar bear hemoglobin and human Hb A0: same 2,3-diphosphoglycerate binding site but asymmetry of the binding?

    Science.gov (United States)

    Pomponi, Massimo; Bertonati, Claudia; Patamia, Maria; Marta, Maurizio; Derocher, Andrew E; Lydersen, Christian; Kovacs, Kit M; Wiig, Oystein; Bårdgard, Astrid J

    2002-11-01

    Polar bear (Ursus maritimus) hemoglobin (Hb) shows a low response to 2,3-diphosphoglycerate (2,3-DPG), compared to human Hb A0, even though these proteins have the same 2,3-DPG-binding site. In addition, polar bear Hb shows a high response to chloride and an alkaline Bohr effect (deltalog P50/deltapH) that is significantly greater than that of human Hb A0. The difference in sequence Pro (Hb A0)-->Gly (polar bear Hb) at position A2 in the A helix seems to be critical for reduced binding of 2,3-DPG. Our results also show that the A2 position may influence not only the flexibility of the A helix, but that differences in flexibility of the first turn of the A helix may affect the unloading of oxygen for the intrinsic ligand affinities of the alpha and beta chains. However, preferential binding to either chain can only take place if there is appreciable asymmetric binding of the phosphoric effector. Regarding this point, 31P NMR data suggest a loss of symmetry of the 2,3-DPG-binding site in the deoxyHb-2,3-DPG complex.

  12. BSSF: a fingerprint based ultrafast binding site similarity search and function analysis server

    Directory of Open Access Journals (Sweden)

    Jiang Hualiang

    2010-01-01

    Full Text Available Abstract Background Genome sequencing and post-genomics projects such as structural genomics are extending the frontier of the study of sequence-structure-function relationship of genes and their products. Although many sequence/structure-based methods have been devised with the aim of deciphering this delicate relationship, there still remain large gaps in this fundamental problem, which continuously drives researchers to develop novel methods to extract relevant information from sequences and structures and to infer the functions of newly identified genes by genomics technology. Results Here we present an ultrafast method, named BSSF(Binding Site Similarity & Function, which enables researchers to conduct similarity searches in a comprehensive three-dimensional binding site database extracted from PDB structures. This method utilizes a fingerprint representation of the binding site and a validated statistical Z-score function scheme to judge the similarity between the query and database items, even if their similarities are only constrained in a sub-pocket. This fingerprint based similarity measurement was also validated on a known binding site dataset by comparing with geometric hashing, which is a standard 3D similarity method. The comparison clearly demonstrated the utility of this ultrafast method. After conducting the database searching, the hit list is further analyzed to provide basic statistical information about the occurrences of Gene Ontology terms and Enzyme Commission numbers, which may benefit researchers by helping them to design further experiments to study the query proteins. Conclusions This ultrafast web-based system will not only help researchers interested in drug design and structural genomics to identify similar binding sites, but also assist them by providing further analysis of hit list from database searching.

  13. Binding of the mannose-specific lectin, griffithsin, to HIV-1 gp120 exposes the CD4-binding site

    CSIR Research Space (South Africa)

    Alexandre, Kabamba B

    2011-09-01

    Full Text Available of the lectin griffithsin (GRFT) with HIV-1 gp120 and its effects on exposure of the CD4-binding site (CD4bs). We found that GRFT enhanced the binding of HIV-1 onto plates coated with anti-CD4bs antibodies b12, b6 or the CD4 receptor mimetic, CD4-IgG2...

  14. Autoradiographic localization of calcitonin gene-related peptide (CGRP) binding sites in human and guinea pig lung

    International Nuclear Information System (INIS)

    Mak, J.C.; Barnes, P.J.

    1988-01-01

    125 I-Human calcitonin gene-related peptide (hCGRP) binding sites were localized in human and guinea pig lungs by an autoradiographic method. Scatchard analysis of saturation experiments from slide-mounted sections of guinea pig lung displayed specific 125 I-hCGRP binding sites with a dissociation constant (Kd) of 0.72 +/- 0.05 nM (mean +/- S.E.M., n = 3) and a maximal number of binding sites (Bmax) of 133.4 +/- 5.6 fmol/mg protein. In both human and guinea pig lung, autoradiography revealed that CGRP binding sites were widely distributed, with particularly dense labeling over bronchial and pulmonary blood vessels of all sizes and alveolar walls. Airway smooth muscle and epithelium of large airways was sparsely labeled but no labeling was found over submucosal glands. This localization corresponds well to the reported pattern of CGRP-like immunoreactive innervation. The findings of localization of CGRP binding sites on bronchial and pulmonary blood vessels indicate that CGRP may be important in the regulation of airway and pulmonary blood flow

  15. Effects of sodium on cell surface and intracellular 3H-naloxone binding sites

    International Nuclear Information System (INIS)

    Pollack, A.E.; Wooten, G.F.

    1987-01-01

    The binding of the opiate antagonist 3 H-naloxone was examined in rat whole brain homogenates and in crude subcellular fractions of these homogenates (nuclear, synaptosomal, and mitochondrial fractions) using buffers that approximated intra- (low sodium concentration) and extracellular (high sodium concentration) fluids. Saturation studies showed a two-fold decrease in the dissociation constant (Kd) in all subcellular fractions examined in extracellular buffer compared to intracellular buffer. In contrast, there was no significant effect of the buffers on the Bmax. Thus, 3 H-naloxone did not distinguish between binding sites present on cell surface and intracellular tissues in these two buffers. These results show that the sodium effect of opiate antagonist binding is probably not a function of altered selection of intra- and extracellular binding sites. 17 references, 2 tables

  16. Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

    Science.gov (United States)

    Denys, A; Allain, F; Carpentier, M; Spik, G

    1998-12-15

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB.

  17. Sugar-binding sites of the HA1 subcomponent of Clostridium botulinum type C progenitor toxin.

    Science.gov (United States)

    Nakamura, Toshio; Tonozuka, Takashi; Ide, Azusa; Yuzawa, Takayuki; Oguma, Keiji; Nishikawa, Atsushi

    2008-02-22

    Clostridium botulinum type C 16S progenitor toxin contains a hemagglutinin (HA) subcomponent, designated HA1, which appears to play an important role in the effective internalization of the toxin in gastrointestinal epithelial cells and in creating a broad specificity for the oligosaccharide structure that corresponds to various targets. In this study, using the recombinant protein fused to glutathione S-transferase, we investigated the binding specificity of the HA1 subcomponent to sugars and estimated the binding sites of HA1 based on X-ray crystallography and soaking experiments using various sugars. N-Acetylneuraminic acid, N-acetylgalactosamine, and galactose effectively inhibited the binding that occurs between glutathione S-transferase-HA1 and mucins, whereas N-acetylglucosamine and glucose did not inhibit it. The crystal structures of HA1 complex with N-acetylneuraminic acid, N-acetylgalactosamine, and galactose were also determined. There are two sugar-binding sites, sites I and II. Site I corresponds to the electron densities noted for all sugars and is located at the C-terminal beta-trefoil domain, while site II corresponds to the electron densities noted only for galactose. An aromatic amino acid residue, Trp176, at site I has a stacking interaction with the hexose ring of the sugars. On the other hand, there is no aromatic residue at site II; thus, the interaction with galactose seems to be poor. The double mutant W176A at site I and D271F at site II has no avidity for N-acetylneuraminic acid but has avidity for galactose. In this report, the binding specificity of botulinum C16S toxin HA1 to various sugars is demonstrated based on its structural features.

  18. A model-based approach to identify binding sites in CLIP-Seq data.

    Directory of Open Access Journals (Sweden)

    Tao Wang

    Full Text Available Cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq has made it possible to identify the targeting sites of RNA-binding proteins in various cell culture systems and tissue types on a genome-wide scale. Here we present a novel model-based approach (MiClip to identify high-confidence protein-RNA binding sites from CLIP-seq datasets. This approach assigns a probability score for each potential binding site to help prioritize subsequent validation experiments. The MiClip algorithm has been tested in both HITS-CLIP and PAR-CLIP datasets. In the HITS-CLIP dataset, the signal/noise ratios of miRNA seed motif enrichment produced by the MiClip approach are between 17% and 301% higher than those by the ad hoc method for the top 10 most enriched miRNAs. In the PAR-CLIP dataset, the MiClip approach can identify ∼50% more validated binding targets than the original ad hoc method and two recently published methods. To facilitate the application of the algorithm, we have released an R package, MiClip (http://cran.r-project.org/web/packages/MiClip/index.html, and a public web-based graphical user interface software (http://galaxy.qbrc.org/tool_runner?tool_id=mi_clip for customized analysis.

  19. Effects of arachidonic acid and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine on prolactin secretion from anterior pituitary cells

    International Nuclear Information System (INIS)

    Camoratto, A.M.

    1988-01-01

    The role of two lipids, arachidonic acid and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, as modulators or prolactin secretion has been examined. Stimulators of phospholipase A 2 activity, melittin and mastoparan, were found to increase prolactin release. Melittin also caused release of previously incorporated 3 H-arachidonic acid and this effect was associated with loss of radiolabel from the phospholipid fraction. Exogenous arachidonic acid also stimulated prolactin secretion. Conversely, inhibitors of phospholipase A 2 activity, dibromoacetophenone and U10029A, decreased basal and stimulated prolactin release. Prolactin release could also be lowered by ETYA, BW755C and NDGA, inhibitors of arachidonic acid metabolism. In the second series of experiments the effects of the biologically active phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor, PAF) on prolactin release were examined. PAF is an ether-linked phospholipid known to stimulate granule release in a variety of cell types including both inflammatory and noninflammatory cells. PAF increased release of prolactin from dispersed rat anterior pituitary cells; stimulation was not due to cell lysis. PAF-induced prolactin release could be blocked by the dopaminergic agonists apomorphine and bromocriptine as well as by two PAF receptor antagonists, SRI 63-072 and L-652-731

  20. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

    Directory of Open Access Journals (Sweden)

    Gowda Veerabasappa T

    2007-12-01

    Full Text Available Abstract Background The snake venom group IIA secreted phospholipases A2 (SVPLA2, present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL at the membrane/water interface and by highly specific direct binding to: (i presynaptic membrane-bound or intracellular receptors; (ii natural PLA2-inhibitors from snake serum; and (iii coagulation factors present in human blood. Results Using surface plasmon resonance (SPR protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS. The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors.

  1. DNA Binding Drugs Targeting the Regulatory DNA Binding Site of the ETS Domain Family Transcription Factor Associated With Human Breast Cancer

    National Research Council Canada - National Science Library

    Wang, Yong-Dong

    1999-01-01

    .... The key approach is to prevent the binding of two transcription factors, ESX and AP-2, to the consensus DNA binding sites contained within the Her2/neu promoter resulting in inhibition of transcription factor function...

  2. Specificity of cellular DNA-binding sites of microbial populations in a Florida reservoir

    International Nuclear Information System (INIS)

    Paul, J.H.; Pichard, S.L.

    1989-01-01

    The substrate specificity of the DNA-binding mechanism(s) of bacteria in a Florida reservoir was investigated in short- and long-term uptake studies with radiolabeled DNA and unlabeled competitors. Thymine oligonucleotides ranging in size from 2 base pairs to 19 to 24 base pairs inhibited DNA binding in 20-min incubations by 43 to 77%. Deoxynucleoside monophosphates, thymidine, and thymine had little effect on short-term DNA binding, although several of these compounds inhibited the uptake of the radiolabel from DNA in 4-h incubations. Inorganic phosphate and glucose-1-phosphate inhibited neither short- nor long-term binding of [ 3 H]- or [ 32 P]DNA, indicating that DNA was not utilized as a phosphorous source in this reservoir. RNA inhibited both short- and long-term radiolabeled DNA uptake as effectively as unlabeled DNA. Collectively these results indicate that aquatic bacteria possess a generalized nuclei acid uptake/binding mechanism specific for compounds containing phosphodiester bonds and capable of recognizing oligonucleotides as short as dinucleotides. This binding site is distinct from nucleoside-, nucleotide-, phosphomonoester-, and inorganic phosphate-binding sites. Such a nucleic acid-binding mechanism may have evolved for the utilization of extracellular DNA (and perhaps RNA), which is abundant in many marine and freshwater environments

  3. Interaction of D-LSD with binding sites in brain: a study in vivo and in vitro

    International Nuclear Information System (INIS)

    Ebersole, B.L.J.

    1985-01-01

    The localization of [ 3 H]-d-lysergic acid diethylamide ([ 3 H]LSD) binding sites in the mouse brain was compared in vivo and in vitro. Radioautography of brain sections incubated with [ 3 H]LSD in vitro revealed substantial specific [ 3 H]LSD binding in cortical layers III-IV and areas CA1 and dentate gyrus in hippocampus. In contrast, in brain sections from animals that received [ 3 H]LSD in vivo, binding in hippocampus was scant and diffuse, although the pattern of labeling in cortex was similar to that seen in vitro. The low specific binding in hippocampus relative to cortex was confirmed by homogenate filtration studies of brain areas from mice that received injections of [ 3 H]LSD. Time-course studies established that peak specific binding at ten minutes was the same in cortex and hippocampus. At all times, binding in hippocampus was about one-third of that in cortex; in contrast, the concentration of free [ 3 H]LSD did not vary between regions. This finding was unexpected, because binding studies in vitro in membrane preparations indicated that the density and affinity of [ 3 H]LSD binding sites were similar in both brain regions. Saturation binding studies in vivo showed that the lower amount of [ 3 H]LSD binding in hippocampus was attributable to a lower density of sites labeled by [ 3 H]LSD. The pharmacological identify of [ 3 H]LSD binding sites in vivo may be relevant to the hallucinogenic properties of LSD and of other related hallucinogens

  4. Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

    Science.gov (United States)

    Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

    2011-09-08

    Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids.

  5. Characterisation of the human NMDA receptor subunit NR3A glycine binding site

    DEFF Research Database (Denmark)

    Nilsson, A; Duan, J; Mo-Boquist, L-L

    2007-01-01

    In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277...

  6. Defective distal regulatory element at the 5' upstream of rat prolactin gene of steroid-nonresponsive GH-subclone.

    Science.gov (United States)

    Kumar, V; Wong, D T; Pasion, S G; Biswas, D K

    1987-12-08

    The prolactin-nonproducing (PRL-) GH cell strains (rat pituitary tumor cells in culture). GH12C1 and F1BGH12C1, do not respond to steroid hormones estradiol or hydrocortisone (HC). However, the stimulatory effect of estradiol and the inhibitory effect of hydrocortisone on prolactin synthesis can be demonstrated in the prolactin-producing GH cell strain, GH4C1. In this investigation we have examined the 5' end flanking region of rat prolactin (rat PRL) gene of steroid-responsive, GH4C1 cells to identify the positive and negative regulatory elements and to verify the status of these elements in steroid-nonresponsive F1BGH12C1 cells. Results presented in this report demonstrate that the basel level expression of the co-transferred Neo gene (neomycin phosphoribosyl transferase) is modulated by the distal upstream regulatory elements of rat PRL gene in response to steroid hormones. The expression of adjacent Neo gene is inhibited by dexamethasone and is stimulated by estradiol in transfectants carrying distal regulatory elements (SRE) of steroid-responsive cells. These responses are not observed in transfectants with the rat PRL upstream sequences derived from steroid-nonresponsive cells. The basal level expression of the host cell alpha-2 tubulin gene is not affected by dexamethasone. We report here the identification of the distal steroid regulatory element (SRE) located between 3.8 and 7.8 kb upstream of the transcription initiation site of rat PRL gene. Both the positive and the negative effects of steroid hormones can be identified within this upstream sequence. This distal SRE appears to be nonfunctional in steroid-nonresponsive cells. Though the proximal SRE is functional, the defect in the distal SRE makes the GH substrain nonresponsive to steroid hormones. These results suggest that both the proximal and the distal SREs are essential for the mediation of action of steroid hormones in GH cells.

  7. DNA deformability changes of single base pair mutants within CDE binding sites in S. Cerevisiae centromere DNA correlate with measured chromosomal loss rates and CDE binding site symmetries

    Directory of Open Access Journals (Sweden)

    Marx Kenneth A

    2006-03-01

    Full Text Available Abstract Background The centromeres in yeast (S. cerevisiae are organized by short DNA sequences (125 bp on each chromosome consisting of 2 conserved elements: CDEI and CDEIII spaced by a CDEII region. CDEI and CDEIII are critical sequence specific protein binding sites necessary for correct centromere formation and following assembly with proteins, are positioned near each other on a specialized nucleosome. Hegemann et al. BioEssays 1993, 15: 451–460 reported single base DNA mutants within the critical CDEI and CDEIII binding sites on the centromere of chromosome 6 and quantitated centromere loss of function, which they measured as loss rates for the different chromosome 6 mutants during cell division. Olson et al. Proc Natl Acad Sci USA 1998, 95: 11163–11168 reported the use of protein-DNA crystallography data to produce a DNA dinucleotide protein deformability energetic scale (PD-scale that describes local DNA deformability by sequence specific binding proteins. We have used the PD-scale to investigate the DNA sequence dependence of the yeast chromosome 6 mutants' loss rate data. Each single base mutant changes 2 PD-scale values at that changed base position relative to the wild type. In this study, we have utilized these mutants to demonstrate a correlation between the change in DNA deformability of the CDEI and CDEIII core sites and the overall experimentally measured chromosome loss rates of the chromosome 6 mutants. Results In the CDE I and CDEIII core binding regions an increase in the magnitude of change in deformability of chromosome 6 single base mutants with respect to the wild type correlates to an increase in the measured chromosome loss rate. These correlations were found to be significant relative to 105 Monte Carlo randomizations of the dinucleotide PD-scale applied to the same calculation. A net loss of deformability also tends to increase the loss rate. Binding site position specific, 4 data-point correlations were also

  8. Phosphorus Binding Sites in Proteins: Structural Preorganization and Coordination

    DEFF Research Database (Denmark)

    Gruber, Mathias Felix; Greisen, Per Junior; Junker, Märta Caroline

    2014-01-01

    to individual structures that bind to phosphate groups; here, we investigate a total of 8307 structures obtained from the RCSB Protein Data Bank (PDB). An analysis of the binding site amino acid propensities reveals very characteristic first shell residue distributions, which are found to be influenced...... by the characteristics of the phosphorus compound and by the presence of cobound cations. The second shell, which supports the coordinating residues in the first shell, is found to consist mainly of protein backbone groups. Our results show how the second shell residue distribution is dictated mainly by the first shell...

  9. Serum Prolactin Levels in Patients with Major Depressive Disorder Receiving Selective Serotonin-Reuptake Inhibitor Monotherapy for 3 Months: A Prospective Study

    OpenAIRE

    Park, Young-Min

    2017-01-01

    Objective It is unclear whether selective serotonin-reuptake inhibitors (SSRIs) can significantly increase the prolactin level. The purpose of this study was to identify the relationship between the prolactin level and the administration of SSRIs such as escitalopram and sertraline. An additional purpose was to determine whether the elevation of prolactin differs between escitalopram and sertraline treatment. Methods Serum prolactin levels were measured at baseline and after 3 months in 23 pa...

  10. Interaction of Palmitic Acid with Metoprolol Succinate at the Binding Sites of Bovine Serum Albumin

    Directory of Open Access Journals (Sweden)

    Mashiur Rahman

    2014-12-01

    Full Text Available Purpose: The aim of this study was to characterize the binding profile as well as to notify the interaction of palmitic acid with metoprolol succinate at its binding site on albumin. Methods: The binding of metoprolol succinate to bovine serum albumin (BSA was studied by equilibrium dialysis method (ED at 27°C and pH 7.4, in order to have an insight in the binding chemistry of the drug to BSA in presence and absence of palmitic acid. The study was carried out using ranitidine as site-1 and diazepam as site-2 specific probe. Results: Different analysis of binding of metoprolol succinate to bovine serum albumin suggested two sets of association constants: high affinity association constant (k1 = 11.0 x 105 M-1 with low capacity (n1 = 2 and low affinity association (k2 = 4.0×105 M-1 constant with high capacity (n2 = 8 at pH 7.4 and 27°C. During concurrent administration of palmitic acid and metoprolol succinate in presence or absence of ranitidine or diazepam, it was found that palmitic acid displaced metoprolol succinate from its binding site on BSA resulting reduced binding of metoprolol succinate to BSA. The increment in free fraction of metoprolol succinate was from 26.27% to 55.08% upon the addition of increased concentration of palmitic acid at a concentration of 0×10-5 M to 16×10-5 M. In presence of ranitidine and diazepam, palmitic acid further increases the free fraction of metoprolol succinate from 33.05% to 66.95% and 40.68% to 72.88%, respectively. Conclusion: This data provided the evidence of interaction at higher concentration of palmitic acid at the binding sites on BSA, which might change the pharmacokinetic properties of metoprolol succinate.

  11. Effect of Sulpirid on blood serum prolactin- and TSH-levels

    International Nuclear Information System (INIS)

    Foldes, J.; Gyertyanfi, G.; Borvendeg, J.

    1979-01-01

    Euthyreoid and hyperthyreoid women were subjected to examinations investigating the effect of a dopamine-antagonist (Sulpirid) on serum TSH and prolactin (LTH)-levels. For measurements of serum concentrations the following kits were used: prolactine: CIS; TSH: Ria-mat-TSH (Byk-Mallinkrodt); thyroxine: Tiopac T 4 (Amersham); triiodothyronine: Ria-mat-T 3 (Byk-Mallinkrodt). Sulpirid increased both the LTH and the TSH-levels. In case of hyperthyreosis the effect of Sulpirid on LTH-levels was less pronounced and it had no effect on serum-TSH at all. Pre-treatment with a dopamine-agonist (Bromocryptin) impeded the effect of Sulpirid. It is concluded that dopamine-receptors do have a role in the regulation of TSH-secretion in the hypophysis. (L.E.)

  12. Genome-wide identification of estrogen receptor alpha-binding sites in mouse liver

    DEFF Research Database (Denmark)

    Gao, Hui; Fält, Susann; Sandelin, Albin

    2007-01-01

    We report the genome-wide identification of estrogen receptor alpha (ERalpha)-binding regions in mouse liver using a combination of chromatin immunoprecipitation and tiled microarrays that cover all nonrepetitive sequences in the mouse genome. This analysis identified 5568 ERalpha-binding regions...... genes. The majority of ERalpha-binding regions lie in regions that are evolutionarily conserved between human and mouse. Motif-finding algorithms identified the estrogen response element, and variants thereof, together with binding sites for activator protein 1, basic-helix-loop-helix proteins, ETS...... signaling in mouse liver, by characterizing the first step in this signaling cascade, the binding of ERalpha to DNA in intact chromatin....

  13. Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

    Energy Technology Data Exchange (ETDEWEB)

    Shyy, Y.J.; Tian, G.; Tsai, M.D.

    1987-10-06

    Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

  14. Identification of leukotriene D4 specific binding sites in the membrane preparation isolated from guinea pig lung

    International Nuclear Information System (INIS)

    Mong, S.; Wu, H.L.; Clark, M.A.; Stadel, J.M.; Gleason, J.G.; Crooke, S.T.

    1984-01-01

    A radioligand binding assay has been established to study leukotriene specific binding sites in the guinea pig and rabbit tissues. Using high specific activity [ 3 H]-leukotriene D4 [( 3 H]-LTD4), in the presence or absence of unlabeled LTD4, the diastereoisomer of LTD4 (5R,6S-LTD4), leukotriene E4 (LTE4) and the end-organ antagonist, FPL 55712, the authors have identified specific binding sites for [ 3 H]-LTD4 in the crude membrane fraction isolated from guinea pig lung. The time required for [ 3 H]-LTD4 binding to reach equilibrium was approximately 20 to 25 min at 37 degrees C in the presence of 10 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl. The binding of [ 3 H]-LTD4 to the specific sites was saturable, reversible and stereospecific. The maximal number of binding sites (Bmax), derived from Scatchard analysis, was approximately 320 +/- 200 fmol per mg of crude membrane protein. The dissociation constants, derived from kinetic and saturation analyses, were 9.7 nM and 5 +/- 4 nM, respectively. The specific binding sites could not be detected in the crude membrane fraction prepared from guinea pig ileum, brain and liver, or rabbit lung, trachea, ileum and uterus. In radioligand competition experiments, LTD4, FPL 55712 and 5R,6S-LTD4 competed with [ 3 H]-LTD4. The metabolic inhibitors of arachidonic acid and SKF 88046, an antagonist of the indirectly-mediated actions of LTD4, did not significantly compete with [ 3 H]-LTD4 at the specific binding sites. These correlations indicated that these specific binding sites may be the putative leukotriene receptors in the guinea-pig lung

  15. Neuropeptide Y binding sites in rat brain identified with purified neuropeptide Y-I125

    International Nuclear Information System (INIS)

    Walker, M.W.; Miller, R.J.

    1986-01-01

    Neuropeptide Y (NPY) is a widely distributed neuronally localized peptide with 36 amino acids, 5 of which are tyrosines. The authors wished to investigate the properties of specific receptors for NPY. They therefore labeled the tyrosines with I125 using chloramine T and then purified the peptide using HPLC. A single mono-iodinated species of NPY which yielded > 85% specific binding in rat forebrain synaptosomes was selected as the ligand for all subsequent experiments. A time course of binding showed that equilibrium conditions were reached in 60 minutes at 21 0 C. Scatchard plots revealed a single class of binding sites with a Kd and a Bmax of 3 x 10-10 M and 28 pmol/mg, respectively. Competition binding with unlabeled NPY showed 50% displacement of bound ligand at 1 x 10-10 M NPY. Competition binding with rat pancreatic polypeptide (RPP), a homologous peptide possessing little NPY-like activity, showed 50% displacement of bound ligand at 2 x 10 -7 M RPP. No binding was observed on F-11 or PC12 neuronal cell lines, or on HSWP fibroblast cells. They conclude that NPY-I125 purified to homogeneity with HPLC is a highly selective ligand for NPY receptor sites. They are currently investigating such sites in brain, gut, and other tissues

  16. Tuning the brain for motherhood: prolactin-like central signalling in virgin, pregnant, and lactating female mice.

    Science.gov (United States)

    Salais-López, Hugo; Lanuza, Enrique; Agustín-Pavón, Carmen; Martínez-García, Fernando

    2017-03-01

    Prolactin is fundamental for the expression of maternal behaviour. In virgin female rats, prolactin administered upon steroid hormone priming accelerates the onset of maternal care. By contrast, the role of prolactin in mice maternal behaviour remains unclear. This study aims at characterizing central prolactin activity patterns in female mice and their variation through pregnancy and lactation. This was revealed by immunoreactivity of phosphorylated (active) signal transducer and activator of transcription 5 (pSTAT5-ir), a key molecule in the signalling cascade of prolactin receptors. We also evaluated non-hypophyseal lactogenic activity during pregnancy by administering bromocriptine, which suppresses hypophyseal prolactin release. Late-pregnant and lactating females showed significantly increased pSTAT5-ir resulting in a widespread pattern of immunostaining with minor variations between pregnant and lactating animals, which comprises nuclei of the sociosexual and maternal brain, including telencephalic (septum, nucleus of the stria terminalis, and amygdala), hypothalamic (preoptic, paraventricular, supraoptic, and ventromedial), and midbrain (periaqueductal grey) regions. During late pregnancy, this pattern was not affected by the administration of bromocriptine, suggesting it to be elicited mostly by non-hypophyseal lactogenic agents, likely placental lactogens. Virgin females displayed, instead, a variable pattern of pSTAT5-ir restricted to a subset of the brain nuclei labelled in pregnant and lactating mice. A hormonal substitution experiment confirmed that estradiol and progesterone contribute to the variability found in virgin females. Our results reflect how the shaping of the maternal brain takes place prior to parturition and suggest that lactogenic agents are important candidates in the development of maternal behaviours already during pregnancy.

  17. Where's water? The many binding sites of hydantoin.

    Science.gov (United States)

    Gruet, Sébastien; Pérez, Cristóbal; Steber, Amanda L; Schnell, Melanie

    2018-02-21

    Prebiotic hydantoin and its complexes with one and two water molecules are investigated using high-resolution broadband rotational spectroscopy in the 2-8 GHz frequency range. The hyperfine structure due to the nuclear quadrupole coupling of the two 14 N atoms is analysed for the monomer and the complexes. This characteristic hyperfine structure will support a definitive assignment from low frequency radioastronomy data. Experiments with H 2 18 O provide accurate experimental information on the preferred binding sites of water, which are compared with quantum-chemically calculated coordinates. In the 2-water complexes, the water molecules bind to hydantoin as a dimer instead of individually, indicating the strong water-water interactions. This information provides first insight on how hydantoin interacts with water on the molecular level.

  18. Prolactin deficiency, obesity, and enlarged testes--a new syndrome?

    Science.gov (United States)

    Roitman, A; Assa, S; Kauli, R; Laron, Z

    1980-01-01

    A 4-year-old boy is described who was obese and slightly mentally retarded. His testes were enlarged. The only endocrine disorder present was a failure to increase plasma prolactin after stimulation. Images Figure PMID:7436524

  19. Alignment-independent comparison of binding sites based on DrugScore potential fields encoded by 3D Zernike descriptors.

    Science.gov (United States)

    Nisius, Britta; Gohlke, Holger

    2012-09-24

    Analyzing protein binding sites provides detailed insights into the biological processes proteins are involved in, e.g., into drug-target interactions, and so is of crucial importance in drug discovery. Herein, we present novel alignment-independent binding site descriptors based on DrugScore potential fields. The potential fields are transformed to a set of information-rich descriptors using a series expansion in 3D Zernike polynomials. The resulting Zernike descriptors show a promising performance in detecting similarities among proteins with low pairwise sequence identities that bind identical ligands, as well as within subfamilies of one target class. Furthermore, the Zernike descriptors are robust against structural variations among protein binding sites. Finally, the Zernike descriptors show a high data compression power, and computing similarities between binding sites based on these descriptors is highly efficient. Consequently, the Zernike descriptors are a useful tool for computational binding site analysis, e.g., to predict the function of novel proteins, off-targets for drug candidates, or novel targets for known drugs.

  20. Autoradiographic localization and characterization of atrial natriuretic peptide binding sites in the rat central nervous system and adrenal gland

    International Nuclear Information System (INIS)

    Gibson, T.R.; Wildey, G.M.; Manaker, S.; Glembotski, C.C.

    1986-01-01

    Atrial natriuretic peptides (ANP) have recently been identified in both heart and CNS. These peptides possess potent natriuretic, diuretic, and vasorelaxant activities, and are all apparently derived from a single prohormone. Specific ANP binding sites have been characterized in the adrenal zona glomerulosa and kidney cortex, and one study reported ANP binding sites in the CNS. However, a detailed examination of the localization of ANP binding sites throughout the brain has not been reported. In this study, quantitative autoradiography was employed to examine the distribution of ANP receptors in the rat CNS. The binding of (3- 125 I-iodotyrosyl28) rat ANP-28 to binding sites in the rat CNS was saturable, specific for ANP-related peptides, and displayed high affinity (Kd = 600 pM). When the relative concentrations of ANP binding sites were determined throughout the rat brain, the highest levels of ANP binding were localized to the circumventricular organs, including the area postrema and subfornical organ, and the olfactory apparatus. Moderate levels of ANP binding sites were present throughout the midbrain and brain stem, while low levels were found in the forebrain, diencephalon, basal ganglia, cortex, and cerebellum. The presence of ANP binding sites in the subfornical organ and the area postrema, regions considered to be outside the blood-brain barrier, suggests that peripheral ANP levels may regulate some aspects of CNS control of salt and water balance. The possible functions of ANP binding sites in other regions of the rat brain are not known, but, like many other peptides, ANP may act as a neurotransmitter or neuromodulator at these loci

  1. Engineering of specific uranyl-coordination sites in the calcium-binding motif of Calmodulin

    International Nuclear Information System (INIS)

    Beccia, M.; Pardoux, R.; Sauge-Merle, S.; Bremond, N.; Lemaire, D.; Berthomieu, C.; Delangle, P.; Guilbaud, P.

    2014-01-01

    Complete text of publication follows: Characterization of heavy metals interactions with proteins is fundamental for understanding the molecular factors and mechanisms governing ions toxicity and speciation in cells. This line of research will also help in developing new molecules able to selectively and efficiently bind toxic metal ions, which could find application for bio-detection or bioremediation purposes. We have used the regulatory calcium-binding protein Calmodulin (CaM) from A. thaliana as a structural model and, starting from it, we have designed various mutants by site-directed mutagenesis. We have analysed thermodynamics of uranyl ion binding to both sites I and II of CaM N-terminal domain and we have identified structural factors governing this interaction. Selectivity for uranyl ion has been tested by studying reactions of the investigated peptides with Ca 2+ , in the same conditions used for UO 2 2+ . Spectro-fluorimetric titrations and FTIR analysis have shown that the affinity for uranyl increases by phosphorylation of a threonine in site I, especially approaching the physiological pH, where the phospho-threonine side chain is deprotonated. Based on structural models obtained by Molecular Dynamics, we tested the effect of a two residues deletion on site I properties. We obtained an almost two orders of magnitude increase in affinity for uranyl, with a sub-nanomolar dissociation constant for the uranyl complex with the non phosphorylated peptide, and an improved uranyl/calcium selectivity. Allosteric effects depending on Ca 2+ and UO 2 2+ binding have been investigated by comparing thermodynamic parameters obtained for mutants having both sites I and II able to chelate metal ions with those of mutants consisting of just one active site

  2. Studies on the digitalis binding site in Na, K-ATPase

    International Nuclear Information System (INIS)

    Ahmed, K.; McParland, R.; Becker, R.; From, A.; Schimerlik, M.; Fullerton, D.S.

    1986-01-01

    Na, K-ATPase is believed to be the receptor for digitalis glycosides. The authors have previously documented that C17 side group of the cardenolide molecule is crucial to α subunit receptor binding. They have attempted to identify the structure of this binding site by labelling the enzyme with a 3 H-labelled photoactive probe localized in the C17 side group of the genin molecule. 3 H-α-subunit was purified and subjected to tryptic digestion. The digest was fractionated by gel filtration on Sephadex G-100. Fractions containing 3 H-labelled peptide were pooled and rechromatographed. The central peak fractions of 3 H-peptide were pooled, analyzed by SDS-PAGE, and subjected to amino acid sequence analysis. The tryptic peptide containing the 3 H-probe showed considerable sequence heterogeneity. Comparison of the sequence data with the published cDNA-based α-subunit sequence revealed that this peptide material was indeed a mixture of two tryptic peptides of nearly identical size containing the sequences from residue 68 through residue 146, and residues 263 through 342. The latter peptide contains the sequence ... glu tyr thr try leu glu ... speculated by Shull et al. as a possible ouabain binding site

  3. Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

    Science.gov (United States)

    Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

    2011-01-01

    In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

  4. Role of Mammary Prolactin in Carcinogenesis

    Science.gov (United States)

    1998-10-01

    lactating rats (167) and dairy kines tested, IL-4 was the most potent inhibitor of myome- cows (174) by bromocriptine did not lower immunoreactive trial...of prolactin in dairy cows during lactogenesis. J Dairy 151. Chapitis J, Riddick DH, Betz LM, Brumsted JR, Gibson M, Prior Sci 70:2241-2253 JC, Gout...factors, insulin (30) and EGF (31) stim- ing the presence of functional postreceptor signaling mech- ulate, whereas transforming growth factor-P

  5. Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

    Science.gov (United States)

    Kang, Hyeran; Bradley, Michael J.; McCullough, Brannon R.; Pierre, Anaëlle; Grintsevich, Elena E.; Reisler, Emil; De La Cruz, Enrique M.

    2012-01-01

    The assembly of actin monomers into filaments and networks plays vital roles throughout eukaryotic biology, including intracellular transport, cell motility, cell division, determining cellular shape, and providing cells with mechanical strength. The regulation of actin assembly and modulation of filament mechanical properties are critical for proper actin function. It is well established that physiological salt concentrations promote actin assembly and alter the overall bending mechanics of assembled filaments and networks. However, the molecular origins of these salt-dependent effects, particularly if they involve nonspecific ionic strength effects or specific ion-binding interactions, are unknown. Here, we demonstrate that specific cation binding at two discrete sites situated between adjacent subunits along the long-pitch helix drive actin polymerization and determine the filament bending rigidity. We classify the two sites as “polymerization” and “stiffness” sites based on the effects that mutations at the sites have on salt-dependent filament assembly and bending mechanics, respectively. These results establish the existence and location of the cation-binding sites that confer salt dependence to the assembly and mechanics of actin filaments. PMID:23027950

  6. Identification of a 3rd Na+ Binding Site of the Glycine Transporter, GlyT2.

    Directory of Open Access Journals (Sweden)

    Nandhitha Subramanian

    Full Text Available The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10.

  7. Mcm1p binding sites in ARG1 positively regulate Gcn4p binding and SWI/SNF recruitment

    OpenAIRE

    Yoon, Sungpil; Hinnebusch, Alan G.

    2009-01-01

    Transcription of the arginine biosynthetic gene ARG1 is activated by Gcn4p, a transcription factor induced by starvation for any amino acid. Previously we showed that Gcn4p binding stimulates the recruitment of Mcm1p and co-activator SWI/SNF to ARG1 in cells via Gcn4p induction through amino acid starvation. Here we report that Gcn4p binding is reduced by point mutations of the Mcm1p binding site and increased by overexpression of Mcm1p. This result suggests that Mcm1p plays a positive role i...

  8. Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin

    International Nuclear Information System (INIS)

    Hamilton, J.A.; Era, S.; Bhamidipati, S.P.; Reed, R.G.

    1991-01-01

    Binding of 13 C-enriched oleic acid to bovine serum albumin and to three large proteolytic fragments of albumin - two complementary fragments corresponding to the two halved of albumin and one fragment corresponding to the carboxyl-terminal domain - yielded unique patterns of NMR resonances (chemical shifts and relative intensities) that were used to identify the locations of binding of the first 5 mol of oleic acid to the multidomain albumin molecule. The first 3 mol of oleic acid added to intact albumin generated three distinct NMR resonances as a result of simultaneous binding of oleic acid to three heterogeneous sites (primary sites). This distribution suggests albumin to be a less symmetrical binding molecule than theoretical models predict. This work also demonstrates the power of NMR for the study of microenvironments of individual fatty acid binding sites in specific domain

  9. (/sup 3/H)diprenorphine binding to kappa-sites in guinea-pig and rat brain: Evidence for apparent heterogeneity

    Energy Technology Data Exchange (ETDEWEB)

    Wood, M.S.; Traynor, J.R.

    1989-07-01

    The binding of the unselective opioid antagonist (/sup 3/H)diprenorphine to homogenates prepared from rat brain and from guinea-pig brain and cerebellum has been studied in HEPES buffer containing 10 mM Mg2+ ions. Sequential displacement of bound (/sup 3/H)diprenorphine by ligands with selectivity for mu-, delta-, and kappa-opioid receptors uncovers the multiple components of binding. In the presence of cold ligands that occupy all mu-, delta-, and kappa-sites, opioid binding still remains. This binding represents 20% of total specific sites and is displaced by naloxone. The nature of these undefined opioid binding sites is discussed.

  10. Human Prolactin Point Mutations and Their Projected Effect on Vasoinhibin Generation and Vasoinhibin-Related Diseases

    Directory of Open Access Journals (Sweden)

    Jakob Triebel

    2017-11-01

    Full Text Available BackgroundA dysregulation of the generation of vasoinhibin hormones by proteolytic cleavage of prolactin (PRL has been brought into context with diabetic retinopathy, retinopathy of prematurity, preeclampsia, pregnancy-induced hypertension, and peripartum cardiomyopathy. Factors governing vasoinhibin generation are incompletely characterized, and the composition of vasoinhibin isoforms in human tissues or compartments, such as the circulation, is unknown. The aim of this study was to determine the possible contribution of PRL point mutations to the generation of vasoinhibins as well as to project their role in vasoinhibin-related diseases.MethodsProlactin sequences, point mutations, and substrate specificity information about the PRL cleaving enzymes cathepsin D, matrix metalloproteinases 8 and 13, and bone-morphogenetic protein 1 were retrieved from public databases. The consequences of point mutations in regard to their possible effect on vasoinhibin levels were projected on the basis of a score indicating the suitability of a particular sequence for enzymatic cleavage that result in vasoinhibin generation. The relative abundance and type of vasoinhibin isoforms were estimated by comparing the relative cleavage efficiency of vasoinhibin-generating enzymes.ResultsSix point mutations leading to amino acid substitutions in vasoinhibin-generating cleavage sites were found and projected to either facilitate or inhibit vasoinhibin generation. Four mutations affecting vasoinhibin generation in cancer tissues were found. The most likely composition of the relative abundance of vasoinhibin isoforms is projected to be 15 > 17.2 > 16.8 > 17.7 > 18 kDa vasoinhibin.ConclusionProlactin point mutations are likely to influence vasoinhibin levels by affecting the proteolysis efficiency of vasoinhibin-generating enzymes and should be monitored in patients with vasoinhibin-related diseases. Attempts to characterize vasoinhibin-related diseases

  11. Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux.

    Directory of Open Access Journals (Sweden)

    Soazig Le Lay

    Full Text Available Caveolin-1 (Cav1, a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs derived from wild type (WT or Cav1-deficient (Cav1(-/- animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/- cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/- MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/- cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.

  12. Comparison of Transcription Factor Binding Site Models

    KAUST Repository

    Bhuyan, Sharifulislam

    2012-05-01

    Modeling of transcription factor binding sites (TFBSs) and TFBS prediction on genomic sequences are important steps to elucidate transcription regulatory mechanism. Dependency of transcription regulation on a great number of factors such as chemical specificity, molecular structure, genomic and epigenetic characteristics, long distance interaction, makes this a challenging problem. Different experimental procedures generate evidence that DNA-binding domains of transcription factors show considerable DNA sequence specificity. Probabilistic modeling of TFBSs has been moderately successful in identifying patterns from a family of sequences. In this study, we compare performances of different probabilistic models and try to estimate their efficacy over experimental TFBSs data. We build a pipeline to calculate sensitivity and specificity from aligned TFBS sequences for several probabilistic models, such as Markov chains, hidden Markov models, Bayesian networks. Our work, containing relevant statistics and evaluation for the models, can help researchers to choose the most appropriate model for the problem at hand.

  13. Growth hormone and prolactin radioimmunoassay in early diagnosis of pituitary tumors

    International Nuclear Information System (INIS)

    Gembicki, M.; Kosowicz, J.

    1978-01-01

    Results of prolactin and HGH determination in basal conditions and following stimulation tests in the group of 68 patients with pituitary or suprasellar tumors are presented. In acromegaly elevated level of HGH in fasting state, lack of supression after glucose loading and parodoxical drop of HGH after L-dopa administration were observed. In pituitary tumors without acromegaly determinations of HGH during insulin induced hypoglycemia revealed lack of HGH response to such stimulation in 25 cases which indicated hypopituitarism. In 10 cases elevated prolactin levels (48 - 1000 ng/ml) were observed, this indicates that some of so-called inactive tumors are in fact hormonally active. (author)

  14. Prolactin, cortisol and thyroxine levels and the premature infant

    African Journals Online (AJOL)

    1983-04-16

    Apr 16, 1983 ... and the premature infant ... values in cord and maternal plasma to fetal age and weight and to the incidence of hyaline membrane disease (HMD) was .... thyroxine and prolactin values with an increase in weight has also.

  15. In vivo labelling in several rat tissues of 'peripheral type' benzodiazepine binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Benavides, J.; Guilloux, F.; Rufat, P.; Uzan, A.; Renault, C.; Dubroeucq, M.C.; Gueremy, C.; Le Fur, G. (Pharmuka Laboratoires, 92 - Gennevilliers (France))

    1984-03-16

    'Peripheral type' benzodiazepine binding sites in several rat tissues were labelled by intravenous injection of (/sup 3/H)PK 11195 and (/sup 3/H)RO5-4864. Binding was saturable in all tissues studied and regional distribution paralleled the in vitro binding. A similar potency order of displacing compounds was found in vivo and in vitro PK 11195 > PK 11211 > RO5-4864 > diazepam > dipyridamole > clonazepam. These results demonstrate the feasibility of using this technique to examine the effects of pharmacological manipulation on the binding sites in their native state. However, some properties (broader maximum during time course, higher percentage of particulate binding in the brain and independence of temperature) make (/sup 3/H)PK 11195 the most suitable ligand for this kind of studies.

  16. Assessing the model transferability for prediction of transcription factor binding sites based on chromatin accessibility.

    Science.gov (United States)

    Liu, Sheng; Zibetti, Cristina; Wan, Jun; Wang, Guohua; Blackshaw, Seth; Qian, Jiang

    2017-07-27

    Computational prediction of transcription factor (TF) binding sites in different cell types is challenging. Recent technology development allows us to determine the genome-wide chromatin accessibility in various cellular and developmental contexts. The chromatin accessibility profiles provide useful information in prediction of TF binding events in various physiological conditions. Furthermore, ChIP-Seq analysis was used to determine genome-wide binding sites for a range of different TFs in multiple cell types. Integration of these two types of genomic information can improve the prediction of TF binding events. We assessed to what extent a model built upon on other TFs and/or other cell types could be used to predict the binding sites of TFs of interest. A random forest model was built using a set of cell type-independent features such as specific sequences recognized by the TFs and evolutionary conservation, as well as cell type-specific features derived from chromatin accessibility data. Our analysis suggested that the models learned from other TFs and/or cell lines performed almost as well as the model learned from the target TF in the cell type of interest. Interestingly, models based on multiple TFs performed better than single-TF models. Finally, we proposed a universal model, BPAC, which was generated using ChIP-Seq data from multiple TFs in various cell types. Integrating chromatin accessibility information with sequence information improves prediction of TF binding.The prediction of TF binding is transferable across TFs and/or cell lines suggesting there are a set of universal "rules". A computational tool was developed to predict TF binding sites based on the universal "rules".

  17. Growth hormone (GH) binding and effects of GH analogs in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Bartke, A.; Steger, R.W. [Southern Illinois Univ., Carbondale, IL (United States); Turyn, D. [UBA-CONICET, Buenos Aires (Argentina)] [and others

    1994-12-31

    Overexpression of human (h) or bovine (b) growth hormone (GH) in transgenic mice is associated with marked (2- to 12-fold) and significant increase in hepatic binding of GH and prolactin (PRL). This is due to an increase in the number of GH and PRL receptors (GHR, PRLR) per mg of microsomal protein without changes in binding affinity. Comparison of results obtained in transgenic animals expressing bGH with a mouse metallothionein (MT) or a rat phosphoenolpyruvate carboxykinase (PEPCK) promoter suggests that effects of bGH on hepatic GHR and PRLR do not require GH overexpression during fetal life and, within the dose range tested, the effects on PRLR are not dose dependent. The increase in hepatic GHR was accompanied by significant increases in plasma GH-binding protein (GHBP) and in mean residence time of injected GH. Thus life-long elevation of peripheral GH levels alters the availability of both free GH and GHR. Site-directed in vitro mutagenesis was used to produce hGH and bGH analogs mutated within one of the sites involved in binding to GHR and PRLR. Mutating hGH to produce amino acid identity with bGH at Position 11, 18 (within Helix 1), 57, or 60 (within the loop between Helix 1 and 2) did not affect binding to GHR in vitro, or somatotropic activity in transgenic mice in vivo but reduced lactogenic activity in Nb{sub 2} cells by 22%-45%. Mutations of bGH designed to produce amino acid identity with hGH at one to four of the corresponding positions in the bGH molecule did not interfere with binding to GHR or somatotropic activity in vivo, and failed to produce significant binding to PRLR but resulted in alterations in the effects on the hypothalamic and anterior pituitary function in transgenic mice. Apparently region(s) outside the domains examined are essential for lactogenic activity of hGH, and different portions of the GH molecule are responsible for its diverse actions in vivo. 35 refs.

  18. Exploring the binding sites and binding mechanism for hydrotrope encapsulated griseofulvin drug on γ-tubulin protein.

    Directory of Open Access Journals (Sweden)

    Shubhadip Das

    Full Text Available The protein γ-tubulin plays an important role in centrosomal clustering and this makes it an attractive therapeutic target for treating cancers. Griseofulvin, an antifungal drug, has recently been used to inhibit proliferation of various types of cancer cells. It can also affect the microtubule dynamics by targeting the γ-tubulin protein. So far, the binding pockets of γ-tubulin protein are not properly identified and the exact mechanism by which the drug binds to it is an area of intense speculation and research. The aim of the present study is to investigate the binding mechanism and binding affinity of griseofulvin on γ-tubulin protein using classical molecular dynamics simulations. Since the drug griseofulvin is sparingly soluble in water, here we also present a promising approach for formulating and achieving delivery of hydrophobic griseofulvin drug via hydrotrope sodium cumene sulfonate (SCS cluster. We observe that the binding pockets of γ-tubulin protein are mainly formed by the H8, H9 helices and S7, S8, S14 strands and the hydrophobic interactions between the drug and γ-tubulin protein drive the binding process. The release of the drug griseofulvin from the SCS cluster is confirmed by the coordination number analysis. We also find hydrotrope-induced alteration of the binding sites of γ-tubulin protein and the weakening of the drug-protein interactions.

  19. ProBiS-ligands: a web server for prediction of ligands by examination of protein binding sites.

    Science.gov (United States)

    Konc, Janez; Janežič, Dušanka

    2014-07-01

    The ProBiS-ligands web server predicts binding of ligands to a protein structure. Starting with a protein structure or binding site, ProBiS-ligands first identifies template proteins in the Protein Data Bank that share similar binding sites. Based on the superimpositions of the query protein and the similar binding sites found, the server then transposes the ligand structures from those sites to the query protein. Such ligand prediction supports many activities, e.g. drug repurposing. The ProBiS-ligands web server, an extension of the ProBiS web server, is open and free to all users at http://probis.cmm.ki.si/ligands. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. In vivo binding of PRDM9 reveals interactions with noncanonical genomic sites

    DEFF Research Database (Denmark)

    Grey, Corinne; Clément, Julie A.J.; Buard, Jérôme

    2017-01-01

    In mouse and human meiosis, DNA double-strand breaks (DSBs) initiate homologous recombination and occur at specific sites called hotspots. The localization of these sites is determined by the sequence-specific DNA binding domain of the PRDM9 histone methyl transferase. Here, we performed...

  1. Rational design of a conformation-switchable Ca2+- and Tb3+-binding protein without the use of multiple coupled metal-binding sites.

    Science.gov (United States)

    Li, Shunyi; Yang, Wei; Maniccia, Anna W; Barrow, Doyle; Tjong, Harianto; Zhou, Huan-Xiang; Yang, Jenny J

    2008-10-01

    Ca2+, as a messenger of signal transduction, regulates numerous target molecules via Ca2+-induced conformational changes. Investigation into the determinants for Ca2+-induced conformational change is often impeded by cooperativity between multiple metal-binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca2+-dependent conformational changes, we report the design of a single-site Ca2+-binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2).CD2.trigger binds to Tb3+ and Ca2+ with dissociation constants of 0.3 +/- 0.1 and 90 +/- 25 microM, respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb3+ or Ca2+ binding results in folding of the native-like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca2+-dependent proteins and coupled metal-binding sites, represents an important step in the design of trigger proteins.

  2. Evaluation of the human prolactin of National Production for use in radioimmunoassay (RIA); Evaluacion de la prolactina humana de produccion nacional para su empleo en radioimmunoanalisis (RIA)

    Energy Technology Data Exchange (ETDEWEB)

    Caso, R [Centro de Isotopos, La Habana (Cuba); Arranz, C [Instituto Nacional de Endocrinologia, La Habana (Cuba)

    1996-07-01

    In this work was studied the possibility of using the Prolactin hormone as raw material to produce Kits-RIA of Prolactin. Was used the prolactin, which is obtained in Cuba by the Pharmaceutical Institute Mario Munoz. Was made the labbeling of Prolactin with I-125, was used the hormone as standard and were done the probes of quality control. The Prolactin Hormone had the necesary quality to produce Kits-RIA-Prolactin.

  3. Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

    DEFF Research Database (Denmark)

    Zweemer, Annelien J M; Bunnik, Julia; Veenhuizen, Margo

    2014-01-01

    be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor...

  4. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  5. Visualization of specific binding sites of benzodiazepine in human brain

    International Nuclear Information System (INIS)

    Shinotoh, H.; Yamasaki, T.; Inoue, O.; Itoh, T.; Suzuki, K.; Hashimoto, K.; Tateno, Y.; Ikehira, H.

    1986-01-01

    Using 11C-labeled Ro15-1788 and positron emission tomography, studies of benzodiazepine binding sites in the human brain were performed on four normal volunteers. Rapid and high accumulation of 11C activity was observed in the brain after i.v. injection of [11C]Ro15-1788, the maximum of which was within 12 min. Initial distribution of 11C activity in the brain was similar to the distribution of the normal cerebral blood flow. Ten minutes after injection, however, a high uptake of 11C activity was observed in the cerebral cortex and moderate uptake was seen in the cerebellar cortex, the basal ganglia, and the thalamus. The accumulation of 11C activity was low in the brain stem. This distribution of 11C activity was approximately parallel to the known distribution of benzodiazepine receptors. Saturation experiments were performed on four volunteers with oral administration of 0.3-1.8 mg/kg of cold Ro15-1788 prior to injection. Initial distribution of 11C activity following injection peaked within 2 min and then the accumulation of 11C activity decreased rapidly and remarkably throughout the brain. The results indicated that [11C] Ro15-1788 associates and dissociates to specific and nonspecific binding sites rapidly and has a high ratio of specific receptor binding to nonspecific binding in vivo. Carbon-11 Ro15-1788 is a suitable radioligand for the study of benzodiazepine receptors in vivo in humans

  6. Marked reduction in the number of platelet-tritiated imipramine binding sites in geriatric depression

    International Nuclear Information System (INIS)

    Nemeroff, C.B.; Knight, D.L.; Krishnan, R.R.; Slotkin, T.A.; Bissette, G.; Melville, M.L.; Blazer, D.G.

    1988-01-01

    The number (Bmax) and affinity (Kd) of platelet-tritiated imipramine binding sites was determined in young and middle-aged controls 50 years of age and younger (n = 25), elderly normal controls over 60 years of age (n = 18), patients who fulfilled DSM-III criteria for major depression who were under 50 years of age (n = 29), patients who fulfilled DSM-III criteria for major depression who were 60 years of age and older (n = 19), and patients who fulfilled both DSM-III criteria for primary degenerative dementia and National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria for probable Alzheimer's disease (n = 13). Both groups of depressed patients (under 50 and over 60 years of age) exhibited significant reductions (decreases 42%) in the number of platelet-tritiated imipramine binding sites with no change in affinity, when compared with their age-matched controls. There was little overlap in Bmax values between the elderly depressed patients and their controls. The patients with probable Alzheimer's disease showed no alteration in platelet-tritiated imipramine binding. There was no statistically significant relationship between postdexamethasone plasma cortisol concentrations and tritiated imipramine binding. These results indicate that platelet-tritiated imipramine binding may have potential utility as a diagnostic adjunct in geriatric depression, and moreover that the reduction in the number of platelet-tritiated imipramine binding sites is not due to hypercortisolemia

  7. A deeper look into transcription regulatory code by preferred pair distance templates for transcription factor binding sites

    KAUST Repository

    Kulakovskiy, Ivan V.

    2011-08-18

    Motivation: Modern experimental methods provide substantial information on protein-DNA recognition. Studying arrangements of transcription factor binding sites (TFBSs) of interacting transcription factors (TFs) advances understanding of the transcription regulatory code. Results: We constructed binding motifs for TFs forming a complex with HIF-1α at the erythropoietin 3\\'-enhancer. Corresponding TFBSs were predicted in the segments around transcription start sites (TSSs) of all human genes. Using the genome-wide set of regulatory regions, we observed several strongly preferred distances between hypoxia-responsive element (HRE) and binding sites of a particular cofactor protein. The set of preferred distances was called as a preferred pair distance template (PPDT). PPDT dramatically depended on the TF and orientation of its binding sites relative to HRE. PPDT evaluated from the genome-wide set of regulatory sequences was used to detect significant PPDT-consistent binding site pairs in regulatory regions of hypoxia-responsive genes. We believe PPDT can help to reveal the layout of eukaryotic regulatory segments. © The Author 2011. Published by Oxford University Press. All rights reserved.

  8. Time-dependent recovery of in vivo binding sites after drug dosing: A method for radiotracer evaluation

    International Nuclear Information System (INIS)

    Kilbourn, Michael R.

    1997-01-01

    The recovery of in vivo binding sites for (±)-α-[ 11 C]methoxytetrabenazine, a radioligand for the monoamine vesicular transporter (VMAT2), was determined in mouse brain at various times following a pharmacological dose of tetrabenazine. Concentrations of in vivo radioligand binding sites progressively increased and had reached control values by 8.5 h, and this recovery was consistent with the pharmacokinetics of the competing drug tetrabenazine and its active metabolite, dihydrotetrabenazine. This study demonstrates a simple experimental protocol of using a single dose of a reversible competing drug and time-dependent measurements of in vivo binding of a radioligand. This protocol is suitable for testing the sensitivity of an in vivo radiotracer for measurement of varying concentrations of in vivo binding sites

  9. Prolactin induces adrenal hypertrophy

    Directory of Open Access Journals (Sweden)

    E.J. Silva

    2004-02-01

    Full Text Available Although adrenocorticotropic hormone is generally considered to play a major role in the regulation of adrenal glucocorticoid secretion, several reports have suggested that other pituitary hormones (e.g., prolactin also play a significant role in the regulation of adrenal function. The aim of the present study was to measure the adrenocortical cell area and to determine the effects of the transition from the prepubertal to the postpubertal period on the hyperprolactinemic state induced by domperidone (4.0 mg kg-1 day-1, sc. In hyperprolactinemic adult and young rats, the adrenals were heavier, as determined at necropsy, than in the respective controls: adults (30 days: 0.16 ± 0.008 and 0.11 ± 0.007; 46 days: 0.17 ± 0.006 and 0.12 ± 0.008, and 61 days: 0.17 ± 0.008 and 0.10 ± 0.004 mg for treated and control animals, respectively; P < 0.05, and young rats (30 days: 0.19 ± 0.003 and 0.16 ± 0.007, and 60 days: 0.16 ± 0.006 and 0.13 ± 0.009 mg; P < 0.05. We selected randomly a circular area in which we counted the nuclei of adrenocortical cells. The area of zona fasciculata cells was increased in hyperprolactinemic adult and young rats compared to controls: adults: (61 days: 524.90 ± 47.85 and 244.84 ± 9.03 µm² for treated and control animals, respectively; P < 0.05, and young rats: (15 days: 462.30 ± 16.24 and 414.28 ± 18.19; 60 days: 640.51 ± 12.91 and 480.24 ± 22.79 µm²; P < 0.05. Based on these data we conclude that the increase in adrenal weight observed in the hyperprolactinemic animals may be due to prolactin-induced adrenocortical cell hypertrophy.

  10. Different serotonin receptor types participate in 5-hydroxytryptophan-induced gonadotropins and prolactin release in the female infantile rat.

    Science.gov (United States)

    Lacau-Mengido, I M; Libertun, C; Becú-Villalobos, D

    1996-05-01

    Serotonin (5-HT) receptors can be classified into at least three, possibly up to seven, classes of receptors. They comprise the 5-HT1, 5-HT2, and 5-HT3 classes, the "uncloned' 5-HT4 receptor and the recombinant receptors 5-ht5, 5-ht6 and 5-ht7. We investigated the role of different serotonin receptor types in a neuroendocrine response to the activation of the serotonergic system. Female immature rats were chosen as an experimental model as it has been shown that during the 3rd week of life, and not at later developmental stages, 5-hydroxytryptophan (5-HTP, a serotonin precursor) induces gonadotropin release in females and not in males. Besides, at this age, serotonin releases prolactin in both sexes. 5-HTP (50 mg/kg) released prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) as expected. Ketanserin (5-HT2A antagonist) and methysergide (5-HT2C antagonist) blocked 5-HTP-induced prolactin release, but did not block the LH or FSH responses. Ondansetron (5-HT3 receptor antagonist) did not modify prolactin response to 5-HTP, whereas it blocked 5-HTP-induced LH and FSH release. Propranolol (5-HT1 and beta-adrenergic antagonist) blocked prolactin, LH and FSH release induced by 5-HTP. The 5-HT2C agonist 1-(3-chlorophenyl)piperazine dihydrochloride released prolactin, without modifying LH or FSH release. Methyl-quipazine and phenylbiguanide (5-HT3 agonists) increased both LH and FSH levels, without altering prolactin secretion. The present experiments indicate that serotonin acting at the 5-HT3 receptor mediates LH and FSH release in infantile female rats, whereas 5-HT2C or 2A receptor types participate in the release of prolactin at this age. 5-HT1 receptor type may be involved in the release of the three hormones, though a beta-adrenergic component of the response cannot be discarded.

  11. Dangerous connections : on binding site models of infectious disease dynamics

    NARCIS (Netherlands)

    Leung, Ka Yin; Diekmann, Odo

    2017-01-01

    We formulate models for the spread of infection on networks that are amenable to analysis in the large population limit. We distinguish three different levels: (1) binding sites, (2) individuals, and (3) the population. In the tradition of physiologically structured population models, the

  12. The involvement of prolactin in avian molt: the effects of gender and breeding success on the timing of molt in Mute swans (Cygnus olor).

    Science.gov (United States)

    Dawson, A; Perrins, C M; Sharp, P J; Wheeler, D; Groves, S

    2009-04-01

    The aim of the study was to test the hypothesis that decreasing plasma prolactin stimulates or permits the initiation of avian molt. Changes in the concentration of plasma prolactin in Mute swans (Cygnus olor) were compared in non-breeding singletons and breeding pairs. In breeding swans, the onset of molt is delayed compared to non-breeders, and is delayed further in breeding males compared to their female partners. The seasonal decrease in prolactin in non-breeding birds of both sexes started at the end of May and was associated with the initiation of molt 4 weeks later. The decrease in plasma prolactin in incubating females was more pronounced, as a consequence of increased prolactin secretion associated with incubation behavior, but also started at end of May, and was associated the onset of molt 6 weeks later. In breeding males, plasma prolactin increased at the end of May when they started to care for their newly hatched cygnets. Correspondingly, prolactin began to decrease 3-5 weeks later in males than in females. These males started to molt in mid August, at least 4 weeks later than females. It is concluded that molt is related to decreasing plasma prolactin, and is inhibited when plasma prolactin is increasing or high.

  13. Selectivity of externally facing ion-binding sites in the Na/K pump to alkali metals and organic cations.

    Science.gov (United States)

    Ratheal, Ian M; Virgin, Gail K; Yu, Haibo; Roux, Benoît; Gatto, Craig; Artigas, Pablo

    2010-10-26

    The Na/K pump is a P-type ATPase that exchanges three intracellular Na(+) ions for two extracellular K(+) ions through the plasmalemma of nearly all animal cells. The mechanisms involved in cation selection by the pump's ion-binding sites (site I and site II bind either Na(+) or K(+); site III binds only Na(+)) are poorly understood. We studied cation selectivity by outward-facing sites (high K(+) affinity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. Guanidinium(+), methylguanidinium(+), and aminoguanidinium(+) produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly directed pump current at saturating K(+), and (ii) induction of pump-mediated, guanidinium-derivative-carried inward current at negative potentials without Na(+) and K(+). In contrast, formamidinium(+) and acetamidinium(+) induced K(+)-like outward currents. Measurement of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external K(+) congeners. Molecular dynamics simulations indicate that bound organic cations induce minor distortion of the binding sites. Among tested metals, only Li(+) induced Na(+)-like VDI, whereas all metals tested except Na(+) induced K(+)-like outward currents. Pump-mediated K(+)-like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by guanidinium(+) derivatives suggest that Na(+) binds to site III in a hydrated form and that the inward current observed without external Na(+) and K(+) represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites.

  14. Composite Structural Motifs of Binding Sites for Delineating Biological Functions of Proteins

    Science.gov (United States)

    Kinjo, Akira R.; Nakamura, Haruki

    2012-01-01

    Most biological processes are described as a series of interactions between proteins and other molecules, and interactions are in turn described in terms of atomic structures. To annotate protein functions as sets of interaction states at atomic resolution, and thereby to better understand the relation between protein interactions and biological functions, we conducted exhaustive all-against-all atomic structure comparisons of all known binding sites for ligands including small molecules, proteins and nucleic acids, and identified recurring elementary motifs. By integrating the elementary motifs associated with each subunit, we defined composite motifs that represent context-dependent combinations of elementary motifs. It is demonstrated that function similarity can be better inferred from composite motif similarity compared to the similarity of protein sequences or of individual binding sites. By integrating the composite motifs associated with each protein function, we define meta-composite motifs each of which is regarded as a time-independent diagrammatic representation of a biological process. It is shown that meta-composite motifs provide richer annotations of biological processes than sequence clusters. The present results serve as a basis for bridging atomic structures to higher-order biological phenomena by classification and integration of binding site structures. PMID:22347478

  15. Understanding the physical and chemical nature of the warfarin drug binding site in human serum albumin: experimental and theoretical studies.

    Science.gov (United States)

    Abou-Zied, Osama K

    2015-01-01

    Human serum albumin (HSA) is one of the major carrier proteins in the body and constitutes approximately half of the protein found in blood plasma. It plays an important role in lipid metabolism, and its ability to reversibly bind a large variety of pharmaceutical compounds makes it a crucial determinant of drug pharmacokinetics and pharmacodynamics. This review deals with one of the protein's major binding sites "Sudlow I" which includes a binding pocket for the drug warfarin (WAR). The binding nature of this important site can be characterized by measuring the spectroscopic changes when a ligand is bound. Using several drugs, including WAR, and other drug-like molecules as ligands, the results emphasize the nature of Sudlow I as a flexible binding site, capable of binding a variety of ligands by adapting its binding pockets. The high affinity of the WAR pocket for binding versatile molecular structures stems from the flexibility of the amino acids forming the pocket. The binding site is shown to have an ionization ability which is important to consider when using drugs that are known to bind in Sudlow I. Several studies point to the important role of water molecules trapped inside the binding site in molecular recognition and ligand binding. Water inside the protein's cavity is crucial in maintaining the balance between the hydrophobic and hydrophilic nature of the binding site. Upon the unfolding and refolding of HSA, more water molecules are trapped inside the binding site which cause some swelling that prevents a full recovery from the denatured state. Better understanding of the mechanism of binding in macromolecules such as HSA and other proteins can be achieved by combining experimental and theoretical studies which produce significant synergies in studying complex biochemical phenomena.

  16. Alcohol-Binding Sites in Distinct Brain Proteins: The Quest for Atomic Level Resolution

    Science.gov (United States)

    Howard, Rebecca J.; Slesinger, Paul A.; Davies, Daryl L.; Das, Joydip; Trudell, James R.; Harris, R. Adron

    2011-01-01

    Defining the sites of action of ethanol on brain proteins is a major prerequisite to understanding the molecular pharmacology of this drug. The main barrier to reaching an atomic-level understanding of alcohol action is the low potency of alcohols, ethanol in particular, which is a reflection of transient, low-affinity interactions with their targets. These mechanisms are difficult or impossible to study with traditional techniques such as radioligand binding or spectroscopy. However, there has been considerable recent progress in combining X-ray crystallography, structural modeling, and site-directed mutagenesis to define the sites and mechanisms of action of ethanol and related alcohols on key brain proteins. We review such insights for several diverse classes of proteins including inwardly rectifying potassium, transient receptor potential, and neurotransmit-ter-gated ion channels, as well as protein kinase C epsilon. Some common themes are beginning to emerge from these proteins, including hydrogen bonding of the hydroxyl group and van der Waals interactions of the methylene groups of ethanol with specific amino acid residues. The resulting binding energy is proposed to facilitate or stabilize low-energy state transitions in the bound proteins, allowing ethanol to act as a “molecular lubricant” for protein function. We discuss evidence for characteristic, discrete alcohol-binding sites on protein targets, as well as evidence that binding to some proteins is better characterized by an interaction region that can accommodate multiple molecules of ethanol. PMID:21676006

  17. Fabrication of supramolecular frameworks by tuning the binding site ...

    Indian Academy of Sciences (India)

    Administrator

    Fabrication of supramolecular frameworks by tuning the binding site of a tripodal ligand with d. 10 metal ions 803. Table 1. Crystal data and structure refinement parameters for 1 and 2. 1 .... e-mail: deposit@ccdc.cam.ac.uk web: http://www. ccdc. cam.ac.uk/deposit]. Supplementary figures and tables can be found in website ...

  18. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

    Science.gov (United States)

    Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

    1993-11-01

    Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

  19. Prolactin, thyrotropin, and growth hormone release during stress associated with parachute jumping.

    Science.gov (United States)

    Noel, G L; Dimond, R C; Earll, J M; Frantz, A G

    1976-05-01

    Prolactin, growth hormone, and thyrotropin (TSH) release during the stress of parachute jumping has been evaluated in 14 male subjects. Subjects were studied at several times before and immediately after their first military parachute jump. All three hormones had risen significantly 1 to 14 min after the jump, compared to mean levels measured immediately beforehand. Earlier studies of physical exercise by ourselves and others would suggest that emotional stress played a role in producing changes of this magnitude. We conclude that prolactin, TSH, and growth hormone are released in physiologically significant amounts in association with the stress of parachute jumping.

  20. Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor

    International Nuclear Information System (INIS)

    Smith, L.I.

    1989-01-01

    The primary goal of these studies was to localize the steroid-binding and phosphorylated sites of the glucocorticoid receptor. The synthetic steroid, dexamethasone 21-mesylate (DM) forms a covalent thioether bond via the sulfhydryl group of a cysteine residue in the receptor. To determine the covalent site of attachment of this ligand, receptors in WEHI-7 mouse thymoma cells were labeled with [ 3 H]DM and purified with a monoclonal antibody. The receptor was completely digested with trypsin and a single peptide covalently labeled with steroid identified by reversed-phase HPLC. This peptide was analyzed by automated Edman degradation to determine the location of the steroid-labeled residue. A similar analysis was performed on an overlapping peptide produced by Staphylococcus aureus protease digestion. Analysis of tryptic peptides from receptors labeled with both [ 3 H]DM and L-[ 35 S]methionine indicated that this peptide contained methionine. These analyses, coupled with the published amino acid sequence of the receptor, identified Cysteine-644 in the steroid-binding domain of the mouse glucocorticoid receptor as the residue involved in covalent steroid-binding. A synthetic peptide representing amino acids 640-650 of the mouse receptor was prepared and analyzed to confirm the identification. These biochemical studies represent a direct demonstration of an amino acid important in receptor function. It has been proposed that the receptor functions through a phosphorylation-dephosphorylation cycle to explain the dependence of hormone binding capacity upon cellular ATP. The glucocorticoid receptor has been shown to be a phosphoprotein. As an initial step to identifying a role of phosphorylation in receptor action, phosphorylated sites within the functional domains of the protein were identified

  1. Local production of donkey anti-rabbit's sera for human prolactin radioimmunoassay

    International Nuclear Information System (INIS)

    Hassan, Ammar Mohamed Elamin

    2001-11-01

    Pure Rabbit's IgG was used in this study to raise donkey anti rabbit's sera to be used as separating agent in radioimmunoassay. Two healthy donkeys have been immunized. The anti rabbit's sera have been titrated as (i) crude, (ii) purified and dialysed coupled to magnetic particles. Then this antibody was used as separating agent in a radioimmunoassay for measurement of human prolactin (PRL). Coupled Sudanese donkey and rabbit's sera (Sud-DARS) was used as 1/8 titre using chelsea RIA kit for human prolactin while 1/200 of liquid Sud-DARS was found to be the best titre using the Chinese kit. The best condition for estimation of the prolactin were optimized by determining the suitable incubation time and temperature. The assay can be done at room temperature but it should be incubated for 6 hours as recommended by the Chinese kit. Validity tests were done. The regression coefficients were 0.994 and 0.999 for linearity and recovery tests respectively. Measurement of human PRL wa found to be reproducible using Sud-DARS as separating agent since the coefficient of variation (C.V. %) was found to be less than 15% for both within batch and between assays. Comparing Sud D ARS to the Chinese kit, separating agent as reference agent, regression coefficient was found to be 0.977 which indicate that Sud-DARS can be used as separating agent. Prolactin in Sudanese subject was determined using the Chinese kit the Sud-DARS as separating agent. The ranges were 74-398 mIU/L in males and 102-414 mI/L in the preovulatory phase for the females while in the post ovulatory phase it was 114-442 mIU/L. Ovulation was confirmed by measurement of progesterone level 7 days before the next suspected mensuration. (Author)

  2. Autologous peptides constitutively occupy the antigen binding site on Ia

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1988-01-01

    Low molecular weight material associated with affinity-purified class II major histocompatibility complex (MHC) molecules of mouse (Ia) had the expected properties of peptides bound to the antigen binding site of Ia. Thus, the low molecular weight material derived from the I-Ad isotype...

  3. Ontogeny of basic fibroblast growth factor binding sites in mouse ocular tissues

    International Nuclear Information System (INIS)

    Fayein, N.A.; Courtois, Y.; Jeanny, J.C.

    1990-01-01

    Basic fibroblast growth factor (bFGF) binding to ocular tissues has been studied by autoradiographical and biochemical approaches directly performed on sections during mouse embryonic and postnatal development. Frozen sections of embryos (9 to 18 days), newborns, and adults (1 day to 6 months) were incubated with iodinated bFGF. One specific FGF binding site (KD = 2.5 nM) is colocalized with heparan sulfate proteoglycans of the basement membranes and is heparitinase sensitive. It first appears at Day 9 around the neural tube, the optic vesicles, and below the head ectoderm and by Day 14 of embryonic development is found in all basement membranes of the eye. At Day 16, very intensely labeled patches appear, corresponding to mast cells which have been characterized by metachromatic staining of their heparin-rich granulations with toluidine blue. In addition to the latter binding, we have also observed a general diffuse distribution of silver grains on all tissues and preferentially in the ecto- and neuroectodermic tissues. From Days 17-18, there is heterogeneous labeling inside the retina, localized in the pigmented epithelium and in three different layers colocalized with the inner and outer plexiform layers and with the inner segments of the photoreceptors. This binding is heparitinase resistant but N-glycanase sensitive and may represent a second specific binding site corresponding to cellular FGF receptors (KD = 280 pM). Both types of binding patterns observed suggest a significant role for bFGF in eye development and physiology

  4. Cortisol and prolactin concentrations during repeated blood sample collection from freely moving, mouse-sized mammals (Phodopus spp.).

    Science.gov (United States)

    Reburn, C J; Wynne-Edwards, K E

    2000-04-01

    Validation of a method for obtaining blood samples that does not change cortisol or prolactin concentrations yet allows serial blood samples to be collected from animals under anesthesia, without prior handling, from freely interacting social groups of small mammals. Results from five experiments are reported. Male dwarf hamsters (Phodopus spp.) were housed in modified home cages under continuous flow of compressed air that could be switched to isoflurane in O2 vehicle without approaching the cages. Dwarf hamsters respond to manual restraint with behavioral distress and increase in the concentration of the dominant glucocorticoid, cortisol, and decrease in prolactin concentration. Both effects are evident within one minute. In contrast, when this new method was used, neither cortisol nor prolactin changed in response to repeated sample collection (up to 8 successive samples at 2 hour intervals), prolonged isoflurane exposure, or substantial blood volume reduction (30%). Prolactin concentration was suppressed and cortisol concentration was increased in response to stimuli from other hamsters tested without anesthesia. Suppression of prolactin concentration was graded in response to the degree of stress and equaled the pharmacologic reduction caused by bromocryptine mesylate (50 microg of CB154 x 3 days). The technique is superior to alternatives for studies of behavioral endocrinology of freely interacting small mammals.

  5. Three-dimensional binding sites volume assessment during cardiac pacing lead extraction

    Directory of Open Access Journals (Sweden)

    Bich Lien Nguyen

    2015-07-01

    Conclusions: Real-time 3D binding sites assessment is feasible and improves transvenous lead extraction outcomes. Its role as a complementary information requires extensive validation, and might be beneficial for a tailored strategy.

  6. Tentative identification of the second substrate binding site in Arabidopsis phytochelatin synthase.

    Directory of Open Access Journals (Sweden)

    Ju-Chen Chia

    Full Text Available Phytochelatin synthase (PCS uses the substrates glutathione (GSH, γGlu-Cys-Gly and a cadmium (Cd-bound GSH (Cd∙GS2 to produce the shortest phytochelatin product (PC2, (γGlu-Cys2-Gly through a ping-pong mechanism. The binding of the 2 substrates to the active site, particularly the second substrate binding site, is not well-understood. In this study, we generated a structural model of the catalytic domain of Arabidopsis AtPCS1 (residues 12-218 by using the crystal structure of the γGlu-Cys acyl-enzyme complex of the PCS of the cyanobacterium Nostoc (NsPCS as a template. The modeled AtPCS1 revealed a cavity in proximity to the first substrate binding site, consisting of 3 loops containing several conserved amino acids including Arg152, Lys185, and Tyr55. Substitutions of these amino acids (R152K, K185R, or double mutation resulted in the abrogation of enzyme activity, indicating that the arrangement of these 2 positive charges is crucial for the binding of the second substrate. Recombinant AtPCS1s with mutations at Tyr55 showed lower catalytic activities because of reduced affinity (3-fold for Y55W for the Cd∙GS2, further suggesting the role of the cation-π interaction in recognition of the second substrate. Our study results indicate the mechanism for second substrate recognition in PCS. The integrated catalytic mechanism of PCS is further discussed.

  7. Circular dichroism study of the interaction between mutagens and bilirubin bound to different binding sites of serum albumins

    Science.gov (United States)

    Orlov, Sergey; Goncharova, Iryna; Urbanová, Marie

    Although recent investigations have shown that bilirubin not only has a negative role in the organism but also exhibits significant antimutagenic properties, the mechanisms of interactions between bilirubin and mutagens are not clear. In this study, interaction between bilirubin bound to different binding sites of mammalian serum albumins with structural analogues of the mutagens 2-aminofluorene, 2,7-diaminofluorene and mutagen 2,4,7-trinitrofluorenone were investigated by circular dichroism and absorption spectroscopy. Homological human and bovine serum albumins were used as chiral matrices, which preferentially bind different conformers of bilirubin in the primary binding sites and make it observable by circular dichroism. These molecular systems approximated a real system for the study of mutagens in blood serum. Differences between the interaction of bilirubin bound to primary and to secondary binding sites of serum albumins with mutagens were shown. For bilirubin bound to secondary binding sites with low affinity, partial displacement and the formation of self-associates were observed in all studied mutagens. The associates of bilirubin bound to primary binding sites of serum albumins are formed with 2-aminofluorene and 2,4,7-trinitrofluorenone. It was proposed that 2,7-diaminofluorene does not interact with bilirubin bound to primary sites of human and bovine serum albumins due to the spatial hindrance of the albumins binding domains. The spatial arrangement of the bilirubin bound to serum albumin along with the studied mutagens was modelled using ligand docking, which revealed a possibility of an arrangement of the both bilirubin and 2-aminofluorene and 2,4,7-trinitrofluorenone in the primary binding site of human serum albumin.

  8. Crystal structure of equine serum albumin in complex with cetirizine reveals a novel drug-binding site

    OpenAIRE

    Handing, Katarzyna B.; Shabalin, Ivan G.; Szlachta, Karol; Majorek, Karolina A.; Minor, Wladek

    2016-01-01

    Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1 ?. Cetirizine is bound in two sites ? a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizi...

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

    Science.gov (United States)

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

    2017-06-02

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

  10. Mapping the Binding Site for Escitalopram and Paroxetine in the Human Serotonin Transporter Using Genetically Encoded Photo-Cross-Linkers

    DEFF Research Database (Denmark)

    Rannversson, Hafsteinn; Andersen, Jacob; Bang-Andersen, Benny

    2017-01-01

    amber codon suppression in hSERT to encode the photo-cross-linking unnatural amino acid p-azido-l-phenylalanine into the suggested high- and low-affinity binding sites. We then employ UV-induced cross-linking with azF to map the binding site of escitalopram and paroxetine, two prototypical selective...... serotonin reuptake inhibitors (SSRIs). We find that the two antidepressant drugs exclusively cross-link to azF incorporated at the high-affinity binding site of hSERT, while cross-linking is not observed at the low-affinity binding site. Combined with previous homology models and recent structural data on h...

  11. Urea, Uric Acid, Prolactin and fT4 Concentrations in Aqueous Humor of Keratoconus Patients.

    Science.gov (United States)

    Stachon, Tanja; Stachon, Axel; Hartmann, Ulrike; Seitz, Berthold; Langenbucher, Achim; Szentmáry, Nóra

    2017-06-01

    Keratoconus is a noninflammatory disease of the cornea associated with progressive thinning and conical shape. Metabolic alterations in the urea cycle, with changes in collagen fibril stability, oxidative stress, thyroid hormones and prolactin with regulatory effect on biosynthesis and biomechanical stability of corneal stroma, may all play a role in keratoconus etiology. Our purpose was to determine urea, uric acid, prolactin and free thyroxin (fT4) concentrations in human aqueous humor (hAH) of keratoconus and cataract patients. hAH was collected from 100 keratoconus (penetrating keratoplasty) (41.9 ± 14.9 years, 69 males) and 100 cataract patients (cataract surgery) (71.2 ± 12.4 years, 58 males). Urea, uric acid, prolactin and fT4 concentrations were measured by Siemens clinical chemistry or immunoassay system. For statistical analysis, a generalized linear model (GLM) was used. Urea concentration was 11.88 ± 3.03 mg/dl in keratoconus and 16.44 ± 6.40 mg/dl in cataract patients, uric acid 2.04 ± 0.59 mg/dl in keratoconus and 2.18 ± 0.73 mg/dl in cataract groups. Prolactin concentration was 3.18 ± 0.34 ng/ml in keratoconus and 3.33 ± 0.32 ng/ml in cataract patients, fT4 20.57 ± 4.76 pmol/l in KC and 19.06 ± 3.86 pmol/l in cataract group. Urea concentration was effected through gender (p = 0.039), age (p = 0.001) and diagnosis (p = 0.025). Uric acid concentration was not effected through any of the analyzed parameters (p > 0.056). Prolactin and fT4 concentration were effected only through diagnosis (p = 0.009 and p = 0.006). Urea and prolactin concentrations are decreased, fT4 concentration is increased in aqueous humor of keratoconus patients, and uric acid concentration remains unchanged. Urea concentration in aqueous humor is also increased in older and male patients. Therefore, metabolic disorder and hormonal balance may both have an impact on keratoconus development. Further studies are necessary to assess the specific impact.

  12. Radioligands for PET studies of central benzodiazepine receptors and PK (peripheral benzodiazepine) binding sites -current status

    International Nuclear Information System (INIS)

    Pike, V.W.; Osman, S.; Shah, F.; Turton, D.R.; Waters, S.L.; Crouzel, C.; Nutt, D.J.

    1993-01-01

    The status of the radiochemical development and biological evaluation of radioligands for PET studies of central benzodiazepine (BZ) receptors and the so-called peripheral benzodiazepine binding sites, here discriminated and referred to as PK binding sites, is reviewed against current pharmacological knowledge, indicating those agents with present value and those with future potential. Practical recommendations are given for the preparation of two useful radioligands for PET studies, [N-methyl- 11 C]flumazenil for central BZ receptors, and [N-methyl- 11 C]PK 11195 for PK binding sites. Quality assurance and plasma metabolite analysis are also reviewed for these radioligands and practical recommendations are given on methodology for their performance. (Author)

  13. Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

    Science.gov (United States)

    Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

    2018-06-01

    Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Increased Prolactin Levels Are Associated with Impaired Processing Speed in Subjects with Early Psychosis

    OpenAIRE

    Montalvo, Itziar; Gutiérrez-Zotes, Alfonso; Creus, Marta; Monseny, Rosa; Ortega, Laura; Franch, Joan; Lawrie, Stephen M.; Reynolds, Rebecca M.; Vilella, Elisabet; Labad, Javier

    2014-01-01

    Hyperprolactinaemia, a common side effect of some antipsychotic drugs, is also present in drug-naïve psychotic patients and subjects at risk for psychosis. Recent studies in non-psychiatric populations suggest that increased prolactin may have negative effects on cognition. The aim of our study was to explore whether high plasma prolactin levels are associated with poorer cognitive functioning in subjects with early psychoses. We studied 107 participants: 29 healthy subjects and 78 subjects w...

  15. An homologous human prolactin (hPRL) radioimmunoassay with an antibody against ''little'' hPRL

    International Nuclear Information System (INIS)

    Werder, K. von; Felixberger, F.; Gottsmann, M.; Kerner, W.; Gloeckner, B.

    1978-01-01

    Since it is tedious to prepare prolactin (PRL) from human pituitaries that is sufficiently pure for immunization, the authors have used the serum of a male patient with complete panhypopituitarism, a PRL-producing pituitary tumour and excessively high hPRL-levels (18-20μg per ml) as a source of the antigen. Ten millilitres of serum were passed through 3cm x 110cm Sephadex G-75 columns. The ''big'' hPRL (20% of the total immunoreactivity) was discarded and the ''little'' hPRL (80%) from two chromatographic runs was lyophilized (approximately 50μg hPRL) and injected into a rabbit together with 1ml of Freund's adjuvant. Though the polyacrylamide gel electrophoresis of the preparation showed a marked protein heterogeneity, labelling of this material with 125 I and subsequent Sephadex G-50 and G-75 chromatography led to an elution pattern comparable to that of 125 I-VLS-hPRL. Specific hPRL antibodies could be demonstrated after three injections. After nine injections the binding (B 0 ) of 125 I-hPRL at a final antibody dilution of 1:100,000 was 22.5%. This dilution was suitable for a highly specific prolactin radioimmunoassay (hPRL RIA) with a lower limit of detection (B 0 minus 3SD) below 0.1ng of VLS-hPRL and a maximal inhibition of tracer binding when 10ng of unlabelled hPRL were added. No cross-reaction with hGH, hPL, hFSH, hLH or hTSH was found. Dilution curves of galactorrhea serum, pregnancy serum, and ''big'' and ''little'' hPRL preparations from serum were shown to run parallel to the standard curve. For routine measurements, pooled pregnancy serum was calibrated with the MRC standard A-71/222 and used as standard in the RIA (1ng of VLS-hPRL equals 20μU of 71/222hPRL). These findings show that serum of a patient hyperprolactinaemia and panhypopituitarism can be an ideal source of the hPRL immunogen since, in contrast to pituitary extracts, no separation from other contaminating anterior pituitary hormones is needed. (author)

  16. Change in Prolactin Levels in Pediatric Patients Given Antipsychotics for Schizophrenia and Schizophrenia Spectrum Disorders: A Network Meta-Analysis

    Directory of Open Access Journals (Sweden)

    Chakrapani Balijepalli

    2018-01-01

    Full Text Available Background. Treatment of schizophrenia with first- and second-generation antipsychotics has been associated with elevated prolactin levels, which may increase the risk for prolactin-related adverse events. Methods. Randomized controlled trials (RCTs included in a recent systematic review were considered for this analysis. A Bayesian network meta-analysis was used to compare changes in prolactin levels in pediatric patients diagnosed with schizophrenia or schizophrenia spectrum disorders treated with second-generation antipsychotics (SGAs. Results. Five RCTs, including 989 patients combined, have evaluated the changes in prolactin for pediatric patients after 6 weeks of treatment with risperidone, quetiapine, aripiprazole, olanzapine, and paliperidone. In the overall study population, treatment with risperidone was associated with the highest increase in mean prolactin levels compared to other SGAs. Patients treated with risperidone 4–6 mg/day were found to experience the greatest increases (55.06 ng/ml [95% CrI: 40.53–69.58] in prolactin levels, followed by risperidone 1–3 mg/day, paliperidone 3–6 mg/day, and paliperidone 6–12 mg/day. Conclusions. This study shows that there are differences in SGAs ability to cause hyperprolactinemia. Further, there is clear evidence of safety concerns with risperidone and paliperidone treatment in adolescent schizophrenia patients. Registration. PROSPERO CRD42014009506.

  17. Subnucleosomes and their relationships to the arrangement of histone binding sites along nucleosome deoxyribonucleic acid

    International Nuclear Information System (INIS)

    Nelson, D.A.; Mencke, A.J.; Chambers, S.A.; Oosterhof, D.K.; Rill, R.L.

    1982-01-01

    Micrococcal nuclease cleaves within nucleosomes at sites spaced about 10.4 base pairs (bp) apart. Cleavages at sites equivalent to 30-35 bp from the ends of 146-bp cores cause spontaneous loss of an H2a-H2b pair associated with 30-40 bp length DNA. Cleavages at certain other sites do not affect the nucleosome integrity unless a solvent perturbant such as urea is added. Chromatin moderately digested with micrococcal nuclease, when fractionated by sedimentation or electrophoresis in the presence of 3 M urea, yielded four previously unobserved subnucleosomes with the following histone/DNA compositions: (H3) 2 (H4) 2 (H2a)(H2b)/95-115 bp; (H3)(H4)/70-80 bp DNA; (H2a)(H2b)/50-60 bp DNA; and (H1)/60-70 bp DNA. All but the latter subnucleosome were also obtained upon DNase I digestion of purified nucleosome cores labeled on the 5' ends with 32 P. Only subnucleosomes that retained H2a and H2b also retained labeled ends. These results show that H2a and H2b are paired on the terminal 30-40 bp of core DNA, as suggested from analyses of histone-DNA cross-link products by Mirzabekov and coworkers. Considerations of the orgins and compositions of subnucleosomes and of cross-linking data suggest an expanded model for the locations of histone binding sites along nucleosome core DNA. The principal features of this model are (i) strong electrostatic binding sites of H2a and H2b occur at positions approximately 20-30 bp from the core ends, (ii) strong electrostatic binding sites of H3 and H4 occur primarily on the central 40 bp of core DNA, (iii) strong nonelectrostatic, urea-sensitive binding sites of H3 and H4 occur at positions approximately 30-50 bp from the core ends, and (iv) urea-sensitive binding sites of H2a or H2b may occur on the terminal 10-20 bp of core DNA

  18. Peptide microarrays to probe for competition for binding sites in a protein interaction network

    NARCIS (Netherlands)

    Sinzinger, M.D.S.; Ruttekolk, I.R.R.; Gloerich, J.; Wessels, H.; Chung, Y.D.; Adjobo-Hermans, M.J.W.; Brock, R.E.

    2013-01-01

    Cellular protein interaction networks are a result of the binding preferences of a particular protein and the entirety of interactors that mutually compete for binding sites. Therefore, the reconstruction of interaction networks by the accumulation of interaction networks for individual proteins

  19. Iodine 125-lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells

    International Nuclear Information System (INIS)

    Yagaloff, K.A.; Hartig, P.R.

    1985-01-01

    125 I-Lysergic acid diethylamide ( 125 I-LSD) binds with high affinity to serotonergic sites on rat choroid plexus. These sites were localized to choroid plexus epithelial cells by use of a novel high resolution stripping film technique for light microscopic autoradiography. In membrane preparations from rat choroid plexus, the serotonergic site density was 3100 fmol/mg of protein, which is 10-fold higher than the density of any other serotonergic site in brain homogenates. The choroid plexus site exhibits a novel pharmacology that does not match the properties of 5-hydroxytryptamine-1a (5-HT1a), 5-HT1b, or 5-HT2 serotonergic sites. 125 I-LSD binding to the choroid plexus site is potently inhibited by mianserin, serotonin, and (+)-LSD. Other serotonergic, dopaminergic, and adrenergic agonists and antagonists exhibit moderate to weak affinities for this site. The rat choroid plexus 125 I-LSD binding site appears to represent a new type of serotonergic site which is located on non-neuronal cells in this tissue

  20. Evolutionary aspects of growth hormones and prolactins and their receptors

    International Nuclear Information System (INIS)

    Tarpey, J.F.

    1986-01-01

    The interactions of GH's, PRL's and PL's with receptors for GH and PRL were examined from a comparative and evolutionary viewpoint. The binding of 125 I-bGH to membrane preparations from liver of representatives of the major classes of non-mammalian vertebrates was also studied. Only hepatic membranes from sturgeon and Gillichthys had significant bGH binding and were further characterized and compared with male rabbit liver membranes in terms of time, temperature, pH, and membrane concentration to optimize binding conditions. The binding of several members of the GH, PRL, PL family of hormones to GH receptors from liver of sturgeon, Gillichthys, rabbit, mouse and rat was investigated. in terms of hormonal specificity, the mammalian receptors and the sturgeon binding sites were similar, while Gillichthys receptors had a different pattern of hormonal specificity. The binding of 125 I-oPRL to renal membranes of the turtle, Pseudemys scripta elegans, was characterized and compared to PRL binding sites of kidney membranes of the bullfrog, Rana catesbeiana, and the tiger salamander, Ambystoma tigrinum

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

    International Nuclear Information System (INIS)

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

    1986-01-01

    This study examined the binding of purified 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 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 0 C. Binding was inversely related to the temperature of incubation. A total of 80% of the labeled toxin binding at 4 0 C dissociated from MVM when the temperature was raised to 37 0 C, but reassociated when the temperature was again brought to 4 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

  2. Follicle-stimulating hormone (FSH) unmasks specific high affinity FSH-binding sites in cell-free membrane preparations of porcine granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ford, K.A.; LaBarbera, A.R.

    1988-11-01

    The purpose of these studies was to determine whether changes in FSH receptors correlated with FSH-induced attenuation of FSH-responsive adenylyl cyclase in immature porcine granulosa cells. Cells were incubated with FSH (1-1000 ng/ml) for up to 24 h, treated with acidified medium (pH 3.5) to remove FSH bound to cells, and incubated with (125I)iodo-porcine FSH to quantify FSH-binding sites. FSH increased binding of FSH in a time-, temperature-, and FSH concentration-dependent manner. FSH (200 ng/ml) increased binding approximately 4-fold within 16 h. Analysis of equilibrium saturation binding data indicated that the increase in binding sites reflected a 2.3-fold increase in receptor number and a 5.4-fold increase in apparent affinity. The increase in binding did not appear to be due to 1) a decrease in receptor turnover, since the basal rate of turnover appeared to be very slow; 2) an increase in receptor synthesis, since agents that inhibit protein synthesis and glycosylation did not block the increase in binding; or 3) an increase in intracellular receptors, since agents that inhibit cytoskeletal components had no effect. Agents that increase intracellular cAMP did not affect FSH binding. The increase in binding appeared to result from unmasking of cryptic FSH-binding sites, since FSH increased binding in cell-free membrane preparations to the same extent as in cells. Unmasking of cryptic sites was hormone specific, and the sites bound FSH specifically. Unmasking of sites was reversible in a time- and temperature-dependent manner after removal of bound FSH. The similarity between the FSH dose-response relationships for unmasking of FSH-binding sites and attenuation of FSH-responsive cAMP production suggests that the two processes are functionally linked.

  3. Multi-binding site model-based curve-fitting program for the computation of RIA data

    International Nuclear Information System (INIS)

    Malan, P.G.; Ekins, R.P.; Cox, M.G.; Long, E.M.R.

    1977-01-01

    In this paper, a comparison will be made of model-based and empirical curve-fitting procedures. The implementation of a multiple binding-site curve-fitting model which will successfully fit a wide range of assay data, and which can be run on a mini-computer is described. The latter sophisticated model also provides estimates of binding site concentrations and the values of the respective equilibrium constants present: the latter have been used for refining assay conditions using computer optimisation techniques. (orig./AJ) [de

  4. Benzodiazepines: rat pinealocyte binding sites and augmentation of norepinephrine-stimulated N-acetyltransferase activity

    Energy Technology Data Exchange (ETDEWEB)

    Matthew, E.; Parfitt, A.G.; Sugden, D.; Engelhardt, D.L.; Zimmerman, E.A.; Klein, D.C.

    1984-02-01

    Studies of (/sup 3/H)diazepam binding to intact rat pineal cells were carried out in tissue culture preparations. The binding was saturable, reversible and proportional to the number of cells used. Scatchard analysis resulted in a linear plot (Kd . 23 nM, maximum binding sites (Bmax) . 1.56 pmol/mg of protein for cells in monolayer culture; Kd . 7 nM, Bmax . 1.3 pmol/mg of protein for cells in suspension culture). Inhibition constants (Ki) for clonazepam (500 nM), flunitrazepam (38 nM) and Ro-5-4864 (5 nM) indicated that the binding sites were probably of the ''peripheral'' type. In addition, the effects of diazepam on norepinephrine-stimulated N-acetyltransferase (NAT) activity were studied in organ culture and dissociated cell culture. Diazepam (10-50 microM) both prolonged and increased the magnitude of the norepinephrine-induced increase in NAT activity but did not affect the initial rate of rise of enzyme activity. The effect was dose-dependent and was also seen with clonazepam, flunitrazepam and Ro-5-4864, but not with Ro-15-1788. Diazepam, by itself, at these concentrations, had no effect on NAT, but enzyme activity was increased by higher concentrations (0.1-1 mM). Although a relationship between the (/sup 3/H)diazepam binding sites described here and the effect of benzodiazepines on NAT cannot be established from these studies, the data suggest that the benzodiazepines may alter melatonin levels through their action on NAT.

  5. Prolactin-secreting pituitary adenoma in a man with gigantism: a case report.

    Science.gov (United States)

    Peillon, F; Philippon, J; Brandi, A M; Fohanno, D; Laplane, D; Dubois, M P; Decourt, J

    1979-12-01

    A prolactin-secreting pituitary adenoma was removed trans-sphenoidally from a 37 years old man with gigantism (218 cm). Serum levels of prolactin (PRL) were elevated pre-operatively and decreased after administration of L-Dopa with no increase after TRH as is usually observed in PRL-secreting adenomas. Growth hormone (GH) and somatomedin serum levels were normal with no modification of GH after insulin hypoglycemia, oral glucose loading or L-Dopa. Morphological examination of the tumour demonstrated the presence of lactotrophs by light and electron microscopy and by immunofluorescense staining. No somatotrophs were found. In this unique case, the relationship between a PRL-secreting adenoma and gigantism is discussed.

  6. Polymorphism of the prolactin gene (PRL) and its relationship with ...

    African Journals Online (AJOL)

    enoh

    2012-04-10

    Apr 10, 2012 ... prolactin gene (PRL), associated with milk production and quality (Brymet et al., 2005; ... synthesis and secretion of proteins, lactose, lipids, and other important ..... genotype AB in Black Pied cattle affected milk, fat and protein ...

  7. Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

    International Nuclear Information System (INIS)

    Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R

    2002-01-01

    The clostridial neurotoxins include the closely related tetanus (TeNT) and botulinum (BoNT) toxins. Botulinum toxin is used to treat severe muscle disorders and as a cosmetic wrinkle reducer. Large quantities of botulinum toxin have also been produced by terrorists for use as a biological weapon. Because there are no known antidotes for these toxins, they thus pose a potential threat to human health whether by an accidental overdose or by a hostile deployment. Thus, the discovery of high specificity and affinity compounds that can inhibit their binding to neural cells can be used as antidotes or in the design of chemical detectors. Using the crystal structure of the C fragment of the tetanus toxin (TetC), which is the cell recognition and cell surface binding domain, and the computational program DOCK, sets of small molecules have been predicted to bind to two different sites located on the surface of this protein. While Site-1 is common to the TeNT and BoNTs, Site-2 is unique to TeNT. Pairs of these molecules from each site can then be linked together synthetically to thereby increase the specificity and affinity for this toxin. Electrospray ionization mass spectroscopy was used to experimentally screen each compound for binding. Mixtures containing binders were further screened for activity under biologically relevant conditions using nuclear magnetic resonance (NMR) methods. The screening of mixtures of compounds offers increased efficiency and throughput as compared to testing single compounds and can also evaluate how possible structural changes induced by the binding of one ligand can influence the binding of the second ligand. In addition, competitive binding experiments with mixtures containing ligands predicted to bind the same site could identify the best binder for that site. NMR transfer nuclear Overhauser effect (trNOE) confirm that TetC binds doxorubicin but that this molecule is displaced by N-acetylneuraminic acid (sialic acid) in a mixture that

  8. Comparative conventional- and quantum dot-labelling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

    Directory of Open Access Journals (Sweden)

    Londiwe Siphephise Mgcina

    2015-05-01

    Full Text Available Lipopolysaccharide (LPS from Gram-negative bacteria is recognized as a microbe-associated molecular pattern (MAMP and not only induces an innate immune response in plants, but also stimulates the development of characteristic defense responses. However, identification and characterization of a cell surface LPS-receptor/binding site, as described in mammals, remains elusive in plants. As an amphiphilic, macromolecular lipoglycan, intact LPS potentially contains three MAMP-active regions, represented by the O-polysaccharide chain, the core and the lipid A. Binding site studies with intact labelled LPS were conducted in Arabidopsis thaliana protoplasts and quantified using flow cytometry fluorescence changes. Qdots, which allow non-covalent, hydrophobic labelling were used as a novel strategy in this study and compared to covalent, hydrophilic labelling with Alexa 488. Affinity for LPS-binding sites was clearly demonstrated by concentration-, temperature- and time-dependent increases in protoplast fluorescence following treatment with the labelled LPS. Moreover, this induced fluorescence increase was convincingly reduced following pre-treatment with excess unlabeled LPS, thereby indicating reversibility of LPS binding. Inhibition of the binding process is also reported using endo- and exocytosis inhibitors. Here, we present evidence for the anticipated presence of LPS-specific binding sites in Arabidopsis protoplasts, and furthermore propose Qdots as a more sensitive LPS-labelling strategy in comparison to the conventional Alexa 488 hydrazide label for binding studies.

  9. Exploration of N-arylpiperazine Binding Sites of D2 Dopaminergic Receptor.

    Science.gov (United States)

    Soskic, Vukic; Sukalovic, Vladimir; Kostic-Rajacic, Sladjana

    2015-01-01

    The crystal structures of the D3 dopamine receptor and several other G-protein coupled receptors (GPCRs) were published in recent times. Those 3D structures are used by us and other scientists as a template for the homology modeling and ligand docking analysis of related GPCRs. Our main scientific interest lies in the field of pharmacologically active N-arylpiperazines that exhibit antipsychotic and/or antidepressant properties, and as such are dopaminergic and serotonergic receptor ligands. In this short review article we are presenting synthesis and biological data on the new N-arylpipereazine as well our results on molecular modeling of the interactions of those N-arylpiperazines with the model of D2 dopamine receptors. To obtain that model the crystal structure of the D3 dopamine receptor was used. Our results show that the N-arylpiperazines binding site consists of two pockets: one is the orthosteric binding site where the N-arylpiperazine part of the ligand is docked and the second is a non-canonical accessory binding site for N-arylpipereazine that is formed by a second extracellular loop (ecl2) of the receptor. Until now, the structure of this receptor region was unresolved in crystal structure analyses of the D3 dopamine receptor. To get a more complete picture of the ligand - receptor interaction, DFT quantum mechanical calculations on N-arylpiperazine were performed and the obtained models were used to examine those interactions.

  10. Characterization of the 3,3',5-triiodo-L-thyronine-binding site on plasma membranes from human placenta

    International Nuclear Information System (INIS)

    Alderson, R.; Pastan, I.; Cheng, S.

    1985-01-01

    The binding of [ 125 I]T3 to sites on human placenta plasma membranes was characterized, and the binding site was solubilized after affinity labeling with N-bromoacetyl-[ 125 I]T3 (BrAc[ 125 I]T3). Two classes of T3-binding sites were detected. One class has a high affinity (K /sub d/ = 2.0nM) and a low capacity (approximately 320 fmol/mg protein); the other has a low affinity (K /sub k/ = 18.5 microM) and a high capacity (approximately 2.2 pmol/mg protein). The binding sites were found to be specific for T3 in that other thyroid hormone analogs (D-T3, rT3, D-T4, and L-T4) were less effective or ineffective in displacing the bound [ 125 I]T3. The affinity labeling ligand BrAc[ 125 I]T3 was found to specifically label a protein with an apparent mol wt of 65,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The BrAc[ 125 I]T3-labeled protein was solubilized with 2 mM 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate. The apparent mol wt of the labeled protein was between 140,000 and 150,000 by Sephadex-G-200 gel filtration. These data demonstrate that a high affinity binding site specific for T3 is present on plasma membranes from human placenta and that the binding site is a protein, most likely a dimer, with a native mol wt between 140,000 and 150,000

  11. Suppressed serum prolactin in sinoaortic-denervated rats

    International Nuclear Information System (INIS)

    Alexander, N.; Melmed, S.; Morris, M.

    1987-01-01

    The authors investigated the effect of arterial baroreceptor deafferentation on serum and pituitary prolactin (PRL) and on catecholamines in median eminence (ME) and anterior and posterior pituitaries. Male Wistar rats were sinoaortic denervated (SAD) or sham operated (SO). Three days after surgery serum prolactin, measured by radioimmunoassay, was suppressed in SAD rats, and dopamine (DA) and norepinephrine (NE) concentrations, measured by radioenzymatic or high-performance liquid chromatography electron capture methods, were significantly reduced in ME of SAD rats. Simultaneously, anterior pituitary of SAD rats had significant increases in both catecholamines, whereas posterior pituitary showed no changes. Four hours after surgery serum PRL was also reduced in SAD rats, but no changes in ME catecholamines were found. Mean arterial pressure (MAP) and heart rate were measured before and after injection of bromocriptine in SAD and SO rats 3 days after surgery. Bromocriptine markedly suppressed serum PRL in both groups and reduced MAP from 144 +/- 10 to 84 +/- 5 and from 116 +/- 2 to 99 +/- 3 in SAD and SO rats, respectively; heart rate was reduced in SAD rats. They conclude that the SAD rat is a model of hypertension with suppressed serum PRL and that interruption of arterial baroreceptor nerves suppresses PRL secretion probably by modulating tuberoinfundibular turnover of catecholamines

  12. A Unified Model of the GABA(A) Receptor Comprising Agonist and Benzodiazepine Binding Sites

    DEFF Research Database (Denmark)

    Kongsbak, Kristine Grønning; Bergmann, Rikke; Sørensen, Pernille Louise

    2013-01-01

    We present a full-length a1b2c2 GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate......-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode...... of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines a1T206 and c2T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important a1H101 and the N-methyl group near a1Y159, a1T206, and a1Y209. We present a binding mode...

  13. Human platelet ( sup 125 I)R-DOI binding sites. Characterization by in vitro autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Himeno, A.; Saavedra, J.M. (National Institute of Mental Health, Bethesda, MD (USA))

    1990-02-01

    We quantified binding sites for 2,5-dimethoxy-4-iodo-phenylisopropylamine (DOI), a 5-HT2 agonist and hallucinogen, in human platelets. We incubated sections from human platelet pellets with ({sup 125}I)R-DOI with or without 1 mumol/L ketanserin, followed by autoradiography and computerized microdensitometry. We corrected the values of binding density by the protein content of each section with a densitometric protein assay. The present method revealed a single class of high affinity binding sites for ({sup 125}I)R-DOI, with a Kd of 6.4 +/- 0.7 nmol/L and a Bmax of 100 +/- 10 fmol/mg protein. Kd and Bmax for ({sup 125}I)R-DOI determined by the classical membrane binding assay, were 2.7 +/- 0.4 nmol/L and 100 +/- 10 fmol/mg protein, respectively. The present method is precise, very sensitive, and allows the characterization of ({sup 125}I)R-DOI binding in sections obtained from as little as 3 ml of blood. Standardization is possible after correction by the protein content of each individual section.

  14. Antipsychotic Medication in Children and Adolescents : A Descriptive Review of the Effects on Prolactin Level and Associated Side Effects

    NARCIS (Netherlands)

    Roke, Yvette; van Harten, Peter N.; Boot, Annemieke M.; Buitelaar, Jan K.

    Objective: This review reports the incidence of hyperprolactinemia, its relationship with genotype, and prolactin-related side effects in children and adolescents treated with antipsychotics. Method: Data on prolactin levels were available for haloperidol, pimozide, risperidone, olanzapine,

  15. Antipsychotic medication in children and adolescents: a descriptive review of the effects on prolactin level and associated side effects.

    NARCIS (Netherlands)

    Roke, Y.; Harten, P.N. van; Boot, A.M.; Buitelaar, J.K.

    2009-01-01

    OBJECTIVE: This review reports the incidence of hyperprolactinemia, its relationship with genotype, and prolactin-related side effects in children and adolescents treated with antipsychotics. METHOD: Data on prolactin levels were available for haloperidol, pimozide, risperidone, olanzapine,

  16. Prolactin-releasing peptide: a new tool for obesity treatment

    Czech Academy of Sciences Publication Activity Database

    Kuneš, Jaroslav; Pražienková, V.; Popelová, A.; Mikulášková, Barbora; Zemenová, J.; Maletínská, L.

    2016-01-01

    Roč. 230, č. 2 (2016), R51-R58 ISSN 0022-0795 R&D Projects: GA ČR(CZ) GA15-08679S Institutional support: RVO:67985823 Keywords : prolactin-releasing peptide * lipidization * obesity * GPR10 * anorexigenic * mice Subject RIV: ED - Physiology Impact factor: 4.706, year: 2016

  17. Calculation of Relative Binding Free Energy in the Water-Filled Active Site of Oligopeptide-Binding Protein A.

    Science.gov (United States)

    Maurer, Manuela; de Beer, Stephanie B A; Oostenbrink, Chris

    2016-04-15

    The periplasmic oligopeptide binding protein A (OppA) represents a well-known example of water-mediated protein-ligand interactions. Here, we perform free-energy calculations for three different ligands binding to OppA, using a thermodynamic integration approach. The tripeptide ligands share a high structural similarity (all have the sequence KXK), but their experimentally-determined binding free energies differ remarkably. Thermodynamic cycles were constructed for the ligands, and simulations conducted in the bound and (freely solvated) unbound states. In the unbound state, it was observed that the difference in conformational freedom between alanine and glycine leads to a surprisingly slow convergence, despite their chemical similarity. This could be overcome by increasing the softness parameter during alchemical transformations. Discrepancies remained in the bound state however, when comparing independent simulations of the three ligands. These difficulties could be traced to a slow relaxation of the water network within the active site. Fluctuations in the number of water molecules residing in the binding cavity occur mostly on a timescale larger than the simulation time along the alchemical path. After extensive simulations, relative binding free energies that were converged to within thermal noise could be obtained, which agree well with available experimental data.

  18. Carotenoid-binding sites of the major light-harvesting complex II of higher plants

    NARCIS (Netherlands)

    Croce, Roberta; Weiss, Saskia; Bassi, Roberto

    1999-01-01

    Recombinant light-harvesting complex II (LHCII) proteins with modified carotenoid composition have been obtained by in vitro reconstitution of the Lhcb1 protein overexpressed in bacteria. The monomeric protein possesses three xanthophyll-binding sites. The L1 and L2 sites, localized by electron

  19. Distribution of basic fibroblast growth factor binding sites in various tissue membrane preparations from adult guinea pig

    International Nuclear Information System (INIS)

    Ledoux, D.; Mereau, A.; Dauchel, M.C.; Barritault, D.; Courty, J.

    1989-01-01

    In order to localize a rich source of basic FGF receptor, we examined the distribution of basic FGF binding sites in brain, stomach, lung, spleen, kidney, liver and intestine membrane preparations from adult guinea pig. Comparative binding studies using iodinated basic FGF showed that a specific binding was detected in all the membrane preparations tested. Scatchard plots from iodinated basic FGF competition experiment with native basic FGF in various membrane preparations, suggested the presence of one class of binding sites in some tissues such as liver, kidney, spleen, lung, stomach, and intestine with an apparent dissociation constant (appKD) value ranging from 4 to 7.5 nM and the existence of a second class of higher affinity sites in brain membranes with appKD value of 15 pM. Characterization of these basic FGF high affinity interaction sites was performed using a cross-linking reagent. These results show for the first time that specific interaction sites for basic FGF are widely distributed, suggesting that this growth factor might play a role in the physiological functions of a number of adult organs

  20. Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus.

    Science.gov (United States)

    Bae, Ji-Eun; Hwang, Kwang Yeon; Nam, Ki Hyun

    2018-06-16

    Glucose isomerase (GI) catalyzes the reversible enzymatic isomerization of d-glucose and d-xylose to d-fructose and d-xylulose, respectively. This is one of the most important enzymes in the production of high-fructose corn syrup (HFCS) and biofuel. We recently determined the crystal structure of GI from S. rubiginosus (SruGI) complexed with a xylitol inhibitor in one metal binding mode. Although we assessed inhibitor binding at the M1 site, the metal binding at the M2 site and the substrate recognition mechanism for SruGI remains the unclear. Here, we report the crystal structure of the two metal binding modes of SruGI and its complex with glucose. This study provides a snapshot of metal binding at the SruGI M2 site in the presence of Mn 2+ , but not in the presence of Mg 2+ . Metal binding at the M2 site elicits a configuration change at the M1 site. Glucose molecule can only bind to the M1 site in presence of Mn 2+ at the M2 site. Glucose and Mn 2+ at the M2 site were bridged by water molecules using a hydrogen bonding network. The metal binding geometry of the M2 site indicates a distorted octahedral coordination with an angle of 55-110°, whereas the M1 site has a relatively stable octahedral coordination with an angle of 85-95°. We suggest a two-step sequential process for SruGI substrate recognition, in Mn 2+ binding mode, at the M2 site. Our results provide a better understanding of the molecular role of the M2 site in GI substrate recognition. Copyright © 2018. Published by Elsevier Inc.

  1. Osteopontin: A uranium phosphorylated binding-site characterization

    International Nuclear Information System (INIS)

    Safi, Samir; Jeanson, Aurelie; Roques, Jerome; Simoni, Eric; Creff, Gaelle; Qi, Lei; Basset, Christian; Vidaud, Claude; Solari, Pier Lorenzo; Den Auwer, Christophe

    2013-01-01

    Herein, we describe the structural investigation of one possible uranyl binding site inside a non structured protein. This approach couples spectroscopy, thermodynamics, and theoretical calculations (DFT) and studies the interaction of uranyl ions with a phospho-peptide, thus mimicking a possible osteopontin (OPN) hydroxyapatite growth-inhibition site. Although thermodynamical aspects were investigated by using time-resolved laser fluorescence spectroscopy (TRLFS) and isothermal titration calorimetry (ITC), structural characterization was performed by extended X-ray absorption fine structure (EXAFS) at the U L(III)-edge combined with attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. From the vibrational and fluorescence spectra, several structural models of a UO 2 2+ /peptide complex were developed and subsequently refined by using theoretical calculations to fit the experimental EXAFS obtained. The structural effect of the pH value was also considered under acidic to moderately acidic conditions (pH 1.5-5.5). Most importantly, the uranyl/peptide coordination environment was similar to that of the native protein. (authors)

  2. Status of the substrate binding sites of ribulose bisphosphate carboxylase as determined with 2-C-carboxyarabinitol 1,5-bisphosphate. [Spinacia oleracea

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Genhai; Jensen, R.G. (Univ. of Arizona, Tucson (USA))

    1990-05-01

    The properties of the tight and specific binding of 2-C-carboxy-D-arabinitol 1,5-bisphosphate (CABP), which occurs only to reaction sites of ribulose 1,5-bisphosphate carboxylase (Rubisco) that are activated by CO{sub 2} and Mg{sup 2+}, were studied. With fully active purified spinach (Spinacia oleracea) Rubisco the rate of tight binding of ({sup 14}C)CABP fit a multiple exponential rate equation with half of the sites binding with a rate constant of 40 per minute and the second half of the sites binding at 3.2 per minute. This suggests that after CABP binds to one site of a dimer of Rubisco large subunits, binding to the second site is considerably slower, indicating negative cooperativity as previously reported. The rate of CABP binding to partially activated Rubisco was complete within 2 to 5 minutes, with slower binding to inactive sites as they formed the carbamate and bound Mg{sup 2+}. Addition of ({sup 14}C)CABP and EDTA stopped binding of Mg{sup 2+} and allowed tight binding of the radiolabel only to sites which were CO{sub 2}/Mg{sup 2+}-activated at that moment. The rate of CO{sub 2} fixation was proportional to the CO{sub 2}/Mg{sup 2+}-activated sites. During light-dependent CO{sub 2} fixation with isolated spinach chloroplasts, the amount of carbamylation was proportional to Rubisco activity either initially upon lysis of the plastids or following total activation with Mg{sup 2+} and CO{sub 2}. Lysis of chloroplasts in media with ({sup 14}C)CABP plus EDTA estimated those carbamylated sites having Mg{sup 2+}. The loss of Rubisco activation during illumination was partially due to the lack of Mg{sup 2+} to stabilize the carbamylated sites.

  3. Further investigations on the inorganic phosphate binding site of beef heart mitochondrial F1-ATPase

    International Nuclear Information System (INIS)

    Pougeois, R.; Lauquin, G.J.

    1985-01-01

    The possibility that 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (P /sub i/ ), could mimic ATP was investigated. ANPP was hydrolyzed in the dark by sarcoplasmic reticulum Ca 2+ -ATPase in the presence of Ca 2+ but not in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANPP was not hydrolyzed by purified mitochondrial F1-ATPase; however, ADP and ATP protected F1-ATPase against ANPP photoinactivation. On the other hand, the trinitrophenyl nucleotide analogues (TNP-ADP, TNP-ATP, and TNP-AMP-PNP), which bind specifically at the two catalytic sites of F1-ATPase, abolished P /sub i/ binding on F1-ATPase; they do not protect F1-ATPase against ANPP photoinactivation. Furthermore, ANPP-photoinactivated F1-ATPase binds the TNP analogues in the same way as the native enzyme. The Pi binding site of F1-ATPase, which is shown to be photolabeled by ANPP, does not appear to be at the gamma-phosphate position of the catalytic sites

  4. Interpretation of Ocular Melanin Drug Binding Assays. Alternatives to the Model of Multiple Classes of Independent Sites.

    Science.gov (United States)

    Manzanares, José A; Rimpelä, Anna-Kaisa; Urtti, Arto

    2016-04-04

    Melanin has a high binding affinity for a wide range of drugs. The determination of the melanin binding capacity and its binding affinity are important, e.g., in the determination of the ocular drug distribution, the prediction of drug effects in the eye, and the trans-scleral drug delivery. The binding parameters estimated from a given data set vary significantly when using different isotherms or different nonlinear fitting methods. In this work, the commonly used bi-Langmuir isotherm, which assumes two classes of independent sites, is confronted with the Sips isotherm. Direct, log-log, and Scatchard plots are used, and the interpretation of the binding curves in the latter is critically analyzed. In addition to the goodness of fit, the emphasis is placed on the physical meaning of the binding parameters. The bi-Langmuir model imposes a bimodal distribution of binding energies for the sites on the melanin granules, but the actual distribution is most likely continuous and unimodal, as assumed by the Sips isotherm. Hence, the latter describes more accurately the distribution of binding energies and also the experimental results of melanin binding to drugs and metal ions. Simulations are used to show that the existence of two classes of sites cannot be confirmed on the sole basis of the shape of the binding curve in the Scatchard plot, and that serious doubts may appear on the meaning of the binding parameters of the bi-Langmuir model. Experimental results of melanin binding to chloroquine and metoprolol are used to illustrate the importance of the choice of the binding isotherm and of the method used to evaluate the binding parameters.

  5. Thermodynamic Characterization of Hydration Sites from Integral Equation-Derived Free Energy Densities: Application to Protein Binding Sites and Ligand Series.

    Science.gov (United States)

    Güssregen, Stefan; Matter, Hans; Hessler, Gerhard; Lionta, Evanthia; Heil, Jochen; Kast, Stefan M

    2017-07-24

    Water molecules play an essential role for mediating interactions between ligands and protein binding sites. Displacement of specific water molecules can favorably modulate the free energy of binding of protein-ligand complexes. Here, the nature of water interactions in protein binding sites is investigated by 3D RISM (three-dimensional reference interaction site model) integral equation theory to understand and exploit local thermodynamic features of water molecules by ranking their possible displacement in structure-based design. Unlike molecular dynamics-based approaches, 3D RISM theory allows for fast and noise-free calculations using the same detailed level of solute-solvent interaction description. Here we correlate molecular water entities instead of mere site density maxima with local contributions to the solvation free energy using novel algorithms. Distinct water molecules and hydration sites are investigated in multiple protein-ligand X-ray structures, namely streptavidin, factor Xa, and factor VIIa, based on 3D RISM-derived free energy density fields. Our approach allows the semiquantitative assessment of whether a given structural water molecule can potentially be targeted for replacement in structure-based design. Finally, PLS-based regression models from free energy density fields used within a 3D-QSAR approach (CARMa - comparative analysis of 3D RISM Maps) are shown to be able to extract relevant information for the interpretation of structure-activity relationship (SAR) trends, as demonstrated for a series of serine protease inhibitors.

  6. Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

    Science.gov (United States)

    Long, Katherine S.

    2012-01-01

    Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms. PMID:22143525

  7. In vitro and in vivo characterisation of [{sup 3}H]ANSTO-14 binding to the {sigma}{sub 1} binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Vu H. E-mail: V.H.Nguyen@ansto.gov.au; Mardon, Karine; Kassiou, Michael; Christie, MacDonald J

    1999-02-01

    ABSTRACT. N-(4-phenylbutyl)-3-hydroxy-4-azahexacyclo[5.4.1.0{sup 2,6}.0{sup 3,10}.0{sup 5,9}.0{sup 8}= {sup ,11}]dodecane (ANSTO-14) showed the highest activity for the {sigma}{sub 1} site ( K{sub i}=9.4 nM) and 19-fold {sigma}{sub 1}/{sigma}{sub 2} selectivity. The present study showed that [{sup 3}H]ANSTO-14 binds to a single high-affinity site in guinea pig brain membranes with an equilibrium K{sub d} of 8.0 {+-} 0.3 nM, in good agreement with the kinetic studies ( K{sub d}=13.3{+-}5.4 nM, n=4), and a B{sub max} of 3,199 {+-} 105 fmol/mg protein ( n=4). The in vivo biodistribution of [{sup 3}H]ANSTO-14 showed a high uptake in the diencephalon. Pretreatment of rats with {sigma} ligands including (+)-pentazocine ({sigma}{sub 1}), ANSTO-14 ({sigma}{sub 1}), and DTG ({sigma}{sub 1} and {sigma}{sub 2}) did not significantly reduce radiotracer uptake in the brain, but did in the spleen. A labelled metabolite was found in the liver and brain. Due to its insensitivity to {sigma} ligands, the accumulation of [{sup 3}H]ANSTO-14 in the brain indicates high nonspecific binding. Therefore, [{sup 3}H]ANSTO-14 is a suitable ligand for labelling {sigma}{sub 1} sites in vitro but is not suitable for brain imaging of {sigma} binding sites in vivo.

  8. The hormone prolactin is a novel, endogenous trophic factor able to regulate reactive glia and to limit retinal degeneration.

    Science.gov (United States)

    Arnold, Edith; Thebault, Stéphanie; Baeza-Cruz, German; Arredondo Zamarripa, David; Adán, Norma; Quintanar-Stéphano, Andrés; Condés-Lara, Miguel; Rojas-Piloni, Gerardo; Binart, Nadine; Martínez de la Escalera, Gonzalo; Clapp, Carmen

    2014-01-29

    Retinal degeneration is characterized by the progressive destruction of retinal cells, causing the deterioration and eventual loss of vision. We explored whether the hormone prolactin provides trophic support to retinal cells, thus protecting the retina from degenerative pressure. Inducing hyperprolactinemia limited photoreceptor apoptosis, gliosis, and changes in neurotrophin expression, and it preserved the photoresponse in the phototoxicity model of retinal degeneration, in which continuous exposure of rats to bright light leads to retinal cell death and retinal dysfunction. In this model, the expression levels of prolactin receptors in the retina were upregulated. Moreover, retinas from prolactin receptor-deficient mice exhibited photoresponsive dysfunction and gliosis that correlated with decreased levels of retinal bFGF, GDNF, and BDNF. Collectively, these data unveiled prolactin as a retinal trophic factor that may regulate glial-neuronal cell interactions and is a potential therapeutic molecule against retinal degeneration.

  9. An effective approach for identification of in vivo protein-DNA binding sites from paired-end ChIP-Seq data

    Directory of Open Access Journals (Sweden)

    Wilson Zoe A

    2010-02-01

    Full Text Available Abstract Background ChIP-Seq, which combines chromatin immunoprecipitation (ChIP with high-throughput massively parallel sequencing, is increasingly being used for identification of protein-DNA interactions in vivo in the genome. However, to maximize the effectiveness of data analysis of such sequences requires the development of new algorithms that are able to accurately predict DNA-protein binding sites. Results Here, we present SIPeS (Site Identification from Paired-end Sequencing, a novel algorithm for precise identification of binding sites from short reads generated by paired-end solexa ChIP-Seq technology. In this paper we used ChIP-Seq data from the Arabidopsis basic helix-loop-helix transcription factor ABORTED MICROSPORES (AMS, which is expressed within the anther during pollen development, the results show that SIPeS has better resolution for binding site identification compared to two existing ChIP-Seq peak detection algorithms, Cisgenome and MACS. Conclusions When compared to Cisgenome and MACS, SIPeS shows better resolution for binding site discovery. Moreover, SIPeS is designed to calculate the mappable genome length accurately with the fragment length based on the paired-end reads. Dynamic baselines are also employed to effectively discriminate closely adjacent binding sites, for effective binding sites discovery, which is of particular value when working with high-density genomes.

  10. Prolactin-releasing peptide: a new tool for obesity treatment

    Czech Academy of Sciences Publication Activity Database

    Kuneš, Jaroslav; Pražienková, Veronika; Popelová, Andrea; Mikulášková, Barbora; Zemenová, Jana; Maletínská, Lenka

    2016-01-01

    Roč. 230, č. 2 (2016), R51-R58 ISSN 0022-0795 R&D Projects: GA ČR(CZ) GA15-08679S Institutional support: RVO:61388963 Keywords : prolactin-releasing peptide * lipidization * obesity * GPR10 * anorexigenic * mice Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.706, year: 2016

  11. Cortisol rapidly reduces prolactin release and cAMP and 45Ca2+ accumulation in the cichlid fish pituitary in vitro

    International Nuclear Information System (INIS)

    Borski, R.J.; Helms, L.M.H.; Richman, N.H. III; Grau, E.G.

    1991-01-01

    During in vitro incubation, prolactin release is inhibited in a dose-related manner by cortisol. This action is mimicked by the synthetic glucocorticoid agonist dexamethasone but not by other steroids tested. Perifusion studies indicate that the inhibition of [ 3 H]prolactin release by cortisol occurs within 20 min. Cortisol (50 nM) also inhibits cAMP accumulation and reduces 45 Ca 2+ accumulation in the tilapia rostral pars distalis within 15 min. Cortisol's action on prolactin release is blocked in the presence of either the Ca 2+ ionophore A23187 or a combination of dibutyryl cAMP and 3-isobutyl-1-methylxanthine, which increase intracellular Ca 2+ and cAMP, respectively. Taken together, these findings suggest that cortisol may play a physiologically relevant role in the rapid modulation of prolactin secretion in vivo. These studies also suggest that the inhibition of prolactin release by cortisol is a specific glucocorticoid action that may be mediated, in part, through cortisol's ability to inhibit intracellular cAMP and Ca 2+ metabolism

  12. Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

    Science.gov (United States)

    Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

    2017-09-08

    Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

  13. Localization of substance P binding sites in submucous plexus of guinea pig ileum, using whole-mount autoradiography

    International Nuclear Information System (INIS)

    Burcher, E.; Bornstein, J.C.

    1988-01-01

    Whole mounts of guinea pig ileum submucosa were incubated with radiolabeled tachykinins, and binding sites were visualized using autoradiography. Very dense specific binding for [ 125 I]-Bolton-Hunter substance P (BHSP) was observed over ganglia of the submucous plexus, with weaker binding over internodal strands. Dense specific binding was also seen over occasional strands of circular muscle, with weak binding over clumps of mucosa. Although very weak binding was seen over some large blood vessels, no binding was associated with smaller blood vessels. Localization of binding was absent in whole-mounts coincubated with 1 microM substance P, used to define nonspecific binding. Localization of BHSP-specific binding was also abolished in whole-mounts coincubated with 1 nM substance P, but not with 1 nM neurokinin B, suggesting that binding was probably to an NK-1 tachykinin receptor. In whole-mounts incubated in [ 125 I]-iodohistidyl neurokinin A (INKA) or [ 125 I]-Bolton-Hunter neurokinin B (BHNKB), no specific binding over ganglia was observed. These binding sites for BHSP are probably identical with the neuronal substance P receptors mediating mucosal ion transport

  14. Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.

    Directory of Open Access Journals (Sweden)

    Yaron Orenstein

    Full Text Available The new technology of protein binding microarrays (PBMs allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.

  15. Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site.

    Science.gov (United States)

    Abe, Yukiko; Nakagawa, Osamu; Yamaguchi, Rie; Sasaki, Shigeki

    2012-06-01

    DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Characterisation of the effect of ion channel modulators on I1-imidazoline binding sites in bovine adrenal medulla

    International Nuclear Information System (INIS)

    Musgrave, I.F.; Kotsopoulos, D.; Hughes, R.A.

    1998-01-01

    Full text: The structure of I 1 -imidazoline binding sites is still unknown and we have proposed that they represent ion channels (i). In these experiments we characterised the effects of the known ion channel modulators methyltriphenylphosphonium (MTPP), 4-aminopyridine (4-AP) and tetraethyl ammonium (TEA) on [ 3 H] clonidine binding in bovine adrenal medullary membranes as these membranes have a relatively well defined I 1 -imidazoline binding site (Molderings et al, 1993). Membranes from bovine adrenal medulla's were prepared by a minor modification of the method of Rapier et al. [ 3 H] Clonidine binding was performed by the method of Ernsberger et al (3), with [ 3 H] clonidine (62 Ci/mmol) used at a final concentration of 5 nM. [ 3 H] Clonidine binding was displaced from bovine adrenal medullary membranes by adrenergic drugs with the order of potency being oxymetazoline > clonidine > moxonidine = idazoxan >> yonimbine. This order of potency is consistent with previous studies of I 1 -imidazoline binding sites (4). Non-linear curve fitting to this data was consistent with a single site model. Both TEA and 4-AP displaced [ H] clonidine with similar potency to its effect on ion channels, TEA having a EC>> of 54 ± 0.3 μM (n=3). The displacement of [ 3 H] clonidine produced by both TEA and 4-AP also fitted to a single site model. Displacement of [ 3 H] clonidine by MTPP fitted a two site model (p 1 -imidazoline binding sites defined with [ 3 H] clonidine may represent ion channels. We have used this data to perform molecular modelling and have determined a common conformation of I 1 -prefering ligands which will aid in the development of I 1 -selective ligands in the future. Copyright (1998) Australian Neuroscience Society

  17. Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

    OpenAIRE

    Denys, A; Allain, F; Carpentier, M; Spik, G

    1998-01-01

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes...

  18. Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction

    International Nuclear Information System (INIS)

    Rebelo, Tânia S.C.R.; Santos, C.; Costa-Rodrigues, J.; Fernandes, M.H.; Noronha, João P.; Sales, M. Goreti F.

    2014-01-01

    Graphical abstract: EF13-201, Novel Prostate Specific Antigen plastic antibody designed with charged binding sites for an improved protein binding and its application in a biosensor of potentiometric transduction. - Abstract: This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomers around the binding site enhances protein binding. These charged receptor sites are placed over a neutral polymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed by preparing control materials with neutral monomers and also with non-imprinted template. This concept has been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate cancer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associated cancer. Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surface imprinting over graphene layers to which the protein was first covalently attached. Vinylbenzyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labelling the binding site and were allowed to self-organize around the protein. The subsequent polymerization was made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without oriented charges and non imprinted materials (NIM) obtained without template were used as controls. These materials were used to develop simple and inexpensive potentiometric sensor for PSA. They were included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liquid conductive contacts, made of conductive carbon over a syringe or of inner reference solution over micropipette tips. The electrodes with charged monomers showed a more stable and sensitive response, with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10 −11 mol/L (2 ng/mL). The corresponding non-imprinted sensors showed lower

  19. A Parzen window-based approach for the detection of locally enriched transcription factor binding sites.

    Science.gov (United States)

    Vandenbon, Alexis; Kumagai, Yutaro; Teraguchi, Shunsuke; Amada, Karlou Mar; Akira, Shizuo; Standley, Daron M

    2013-01-21

    Identification of cis- and trans-acting factors regulating gene expression remains an important problem in biology. Bioinformatics analyses of regulatory regions are hampered by several difficulties. One is that binding sites for regulatory proteins are often not significantly over-represented in the set of DNA sequences of interest, because of high levels of false positive predictions, and because of positional restrictions on functional binding sites with regard to the transcription start site. We have developed a novel method for the detection of regulatory motifs based on their local over-representation in sets of regulatory regions. The method makes use of a Parzen window-based approach for scoring local enrichment, and during evaluation of significance it takes into account GC content of sequences. We show that the accuracy of our method compares favourably to that of other methods, and that our method is capable of detecting not only generally over-represented regulatory motifs, but also locally over-represented motifs that are often missed by standard motif detection approaches. Using a number of examples we illustrate the validity of our approach and suggest applications, such as the analysis of weaker binding sites. Our approach can be used to suggest testable hypotheses for wet-lab experiments. It has potential for future analyses, such as the prediction of weaker binding sites. An online application of our approach, called LocaMo Finder (Local Motif Finder), is available at http://sysimm.ifrec.osaka-u.ac.jp/tfbs/locamo/.

  20. Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

    Science.gov (United States)

    Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

    2009-01-01

    A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

  1. The Antinociceptive Agent SBFI-26 Binds to Anandamide Transporters FABP5 and FABP7 at Two Different Sites

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Hao-Chi [Cryo-EM Structural; Tong, Simon [Department of Chemistry, Stony Brook University, Stony Brook, New York 11794, United States; Zhou, Yuchen [Department of Applied Mathematics; Elmes, Matthew W. [Department of Biochemistry and; Yan, Su [Department of Chemistry, Stony Brook University, Stony Brook, New York 11794, United States; Kaczocha, Martin [Department of Biochemistry and; Department of Anesthesiology, Stony Brook University, Stony; Deutsch, Dale G. [Department of Biochemistry and; Institute of Chemical Biology and; Rizzo, Robert C. [Department of Applied Mathematics; Institute of Chemical Biology and; Laufer; Ojima, Iwao [Department of Chemistry, Stony Brook University, Stony Brook, New York 11794, United States; Institute of Chemical Biology and; Li, Huilin [Cryo-EM Structural; Institute of Chemical Biology and

    2017-06-28

    Human FABP5 and FABP7 are intracellular endocannabinoid transporters. SBFI-26 is an α-truxillic acid 1-naphthyl monoester that competitively inhibits the activities of FABP5 and FABP7 and produces antinociceptive and anti-inflammatory effects in mice. The synthesis of SBFI-26 yields several stereoisomers, and it is not known how the inhibitor binds the transporters. Here we report co-crystal structures of SBFI-26 in complex with human FABP5 and FABP7 at 2.2 and 1.9 Å resolution, respectively. We found that only (S)-SBFI-26 was present in the crystal structures. The inhibitor largely mimics the fatty acid binding pattern, but it also has several unique interactions. Notably, the FABP7 complex corroborates key aspects of the ligand binding pose at the canonical site previously predicted by virtual screening. In FABP5, SBFI-26 was unexpectedly found to bind at the substrate entry portal region in addition to binding at the canonical ligand-binding pocket. Our structural and binding energy analyses indicate that both R and S forms appear to bind the transporter equally well. We suggest that the S enantiomer observed in the crystal structures may be a result of the crystallization process selectively incorporating the (S)-SBFI-26–FABP complexes into the growing lattice, or that the S enantiomer may bind to the portal site more rapidly than to the canonical site, leading to an increased local concentration of the S enantiomer for binding to the canonical site. Our work reveals two binding poses of SBFI-26 in its target transporters. This knowledge will guide the development of more potent FABP inhibitors based upon the SBFI-26 scaffold.

  2. A Flexible Binding Site Architecture Provides New Insights into CcpA Global Regulation in Gram-Positive Bacteria.

    Science.gov (United States)

    Yang, Yunpeng; Zhang, Lu; Huang, He; Yang, Chen; Yang, Sheng; Gu, Yang; Jiang, Weihong

    2017-01-24

    Catabolite control protein A (CcpA) is the master regulator in Gram-positive bacteria that mediates carbon catabolite repression (CCR) and carbon catabolite activation (CCA), two fundamental regulatory mechanisms that enable competitive advantages in carbon catabolism. It is generally regarded that CcpA exerts its regulatory role by binding to a typical 14- to 16-nucleotide (nt) consensus site that is called a catabolite response element (cre) within the target regions. However, here we report a previously unknown noncanonical flexible architecture of the CcpA-binding site in solventogenic clostridia, providing new mechanistic insights into catabolite regulation. This novel CcpA-binding site, named cre var , has a unique architecture that consists of two inverted repeats and an intervening spacer, all of which are variable in nucleotide composition and length, except for a 6-bp core palindromic sequence (TGTAAA/TTTACA). It was found that the length of the intervening spacer of cre var can affect CcpA binding affinity, and moreover, the core palindromic sequence of cre var is the key structure for regulation. Such a variable architecture of cre var shows potential importance for CcpA's diverse and fine regulation. A total of 103 potential cre var sites were discovered in solventogenic Clostridium acetobutylicum, of which 42 sites were picked out for electrophoretic mobility shift assays (EMSAs), and 30 sites were confirmed to be bound by CcpA. These 30 cre var sites are associated with 27 genes involved in many important pathways. Also of significance, the cre var sites are found to be widespread and function in a great number of taxonomically different Gram-positive bacteria, including pathogens, suggesting their global role in Gram-positive bacteria. In Gram-positive bacteria, the global regulator CcpA controls a large number of important physiological and metabolic processes. Although a typical consensus CcpA-binding site, cre, has been identified, it remains

  3. Integrin activation dynamics between the RGD-binding site and the headpiece hinge.

    Science.gov (United States)

    Puklin-Faucher, Eileen; Vogel, Viola

    2009-12-25

    Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII(10)-bound alpha(V)beta(3) integrin headpiece how the binding pocket and interdomain betaA/hybrid domain hinge on the distal end of the betaA domain are allosterically linked via a hydrophobic T-junction between the middle of the alpha1 helix and top of the alpha7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca(2+) in place of Mg(2+) at the site adjacent to the metal ion-dependent adhesion site ("ADMIDAS"). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca(2+) at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated.

  4. Validation of binding of SE-75 labeled sucralfate to sites of gastrointestinal ulceration

    Energy Technology Data Exchange (ETDEWEB)

    Maurer, A.H.; Knight, L.C.; Kollman, M.; Krevsky, B.; Pleet, D.; D' Ercole, F.; Siegel, J.A.; Fisher, R.S.; Malmud, L.S.

    1985-05-01

    This study was performed to determine if and for how long sucralfate (SU) binds selectively to sites of gastro-intestinal (GI) ulceration. Se-Su was prepared by sulfating sucrose with tracer Se-75 and precipitating it as the basic Al salt. All patients (pts) had endoscopy to confirm the presence of either: esophagitis (n=5), gastritis (GA) (n=5), gastric ulcers (GU) (n=5), duodenal ulcers (DU) (n=5), or no ulceration (NU) (n=5). Following an overnight fast the pts swallowed 1 gm with 100 ..mu..Ci of Se-SU and were imaged continuously over 24 hours or until no activity remained in the upper GI tract. Pts with GU visually demonstrated focal SU binding at the ulcers for an average of 3.9 +- 1.1 hrs. with a mean GET of 68 +- 25 min. Mean GET for pts with DU was prolonged, 171 +- 63 min, however focal binding at duodenal ulcers was not seen. All pts with GA had diffuse retention of SU in the stomach with a mean GET of 118 +- 34 min. Focal binding of SU at all sites of esophagitis was seen with a T-1/2 of 65 +- 32 min at the ulcerations. In conclusion these data support the theory that the mechanism of ulcer healing with SU is related to its ability to adhere to the ulcer site forming a protective barrier. In addition Se-SU is a potential ulcer imaging agent which can be used to noninvasively assess healing.

  5. Validation of binding of SE-75 labeled sucralfate to sites of gastrointestinal ulceration

    International Nuclear Information System (INIS)

    Maurer, A.H.; Knight, L.C.; Kollman, M.; Krevsky, B.; Pleet, D.; D'Ercole, F.; Siegel, J.A.; Fisher, R.S.; Malmud, L.S.

    1985-01-01

    This study was performed to determine if and for how long sucralfate (SU) binds selectively to sites of gastro-intestinal (GI) ulceration. Se-Su was prepared by sulfating sucrose with tracer Se-75 and precipitating it as the basic Al salt. All patients (pts) had endoscopy to confirm the presence of either: esophagitis (n=5), gastritis (GA) (n=5), gastric ulcers (GU) (n=5), duodenal ulcers (DU) (n=5), or no ulceration (NU) (n=5). Following an overnight fast the pts swallowed 1 gm with 100 μCi of Se-SU and were imaged continuously over 24 hours or until no activity remained in the upper GI tract. Pts with GU visually demonstrated focal SU binding at the ulcers for an average of 3.9 +- 1.1 hrs. with a mean GET of 68 +- 25 min. Mean GET for pts with DU was prolonged, 171 +- 63 min, however focal binding at duodenal ulcers was not seen. All pts with GA had diffuse retention of SU in the stomach with a mean GET of 118 +- 34 min. Focal binding of SU at all sites of esophagitis was seen with a T-1/2 of 65 +- 32 min at the ulcerations. In conclusion these data support the theory that the mechanism of ulcer healing with SU is related to its ability to adhere to the ulcer site forming a protective barrier. In addition Se-SU is a potential ulcer imaging agent which can be used to noninvasively assess healing

  6. Simultaneous radioimmunoassay for luteinizing hormone and prolactin

    International Nuclear Information System (INIS)

    Steele, M.K.; Deschepper, C.F.

    1985-01-01

    A combined radioimmunoassay (RIA) for the measurement of the anterior pituitary proteins luteinizing hormone (LH) and prolactin (PRL) is described and compared with individual RIAs for these hormones. The standard curves and the sample values for LH and PRL were identical when determined in a combined or in an individual RIA. This technique may prove useful to a number of laboratories where it is desirable to determine levels of more than one hormone in limited sample volumes

  7. Localization of 125I-insulin binding sites in the rat hypothalamus by quantitative autoradiography

    International Nuclear Information System (INIS)

    Corp, E.S.; Woods, S.C.; Figlewicz, D.P.; Porte, D. Jr.; Baskin, D.G.; Dorsa, D.M.

    1986-01-01

    In vitro autoradiography and computer video densitometry were used to localize and quantify binding of 125 I-insulin in the hypothalamus of the rat brain. Highest specific binding was found in the arculate, dorsomedial, suprachiasmatic, paraventricular and periventricular regions. Significantly lower binding was present in the ventromedial nucleus and median eminence. The results are consistent with the hypothesis that insulin modulates the neural regulation of feeding by acting at sites in the hypothalamus. (author)

  8. Low nucleosome occupancy is encoded around functional human transcription factor binding sites

    Directory of Open Access Journals (Sweden)

    Daenen Floris

    2008-07-01

    Full Text Available Abstract Background Transcriptional regulation of genes in eukaryotes is achieved by the interactions of multiple transcription factors with arrays of transcription factor binding sites (TFBSs on DNA and with each other. Identification of these TFBSs is an essential step in our understanding of gene regulatory networks, but computational prediction of TFBSs with either consensus or commonly used stochastic models such as Position-Specific Scoring Matrices (PSSMs results in an unacceptably high number of hits consisting of a few true functional binding sites and numerous false non-functional binding sites. This is due to the inability of the models to incorporate higher order properties of sequences including sequences surrounding TFBSs and influencing the positioning of nucleosomes and/or the interactions that might occur between transcription factors. Results Significant improvement can be expected through the development of a new framework for the modeling and prediction of TFBSs that considers explicitly these higher order sequence properties. It would be particularly interesting to include in the new modeling framework the information present in the nucleosome positioning sequences (NPSs surrounding TFBSs, as it can be hypothesized that genomes use this information to encode the formation of stable nucleosomes over non-functional sites, while functional sites have a more open chromatin configuration. In this report we evaluate the usefulness of the latter feature by comparing the nucleosome occupancy probabilities around experimentally verified human TFBSs with the nucleosome occupancy probabilities around false positive TFBSs and in random sequences. Conclusion We present evidence that nucleosome occupancy is remarkably lower around true functional human TFBSs as compared to non-functional human TFBSs, which supports the use of this feature to improve current TFBS prediction approaches in higher eukaryotes.

  9. Competitive binding of Chlorin p6 and Dansyl-L-Proline to Sudlow's site II of human serum albumin

    Science.gov (United States)

    Patel, Sunita; Sharma, Kaushal Kishor; Datta, Anindya

    2015-03-01

    The binding of chlorin p6, a model photosensitizer for photodynamic therapy (PDT), to the Sudlow's site II of Human Serum Albumin (HSA) has been monitored by different spectroscopic methods. Displacement of Dansyl-L-Proline (DP) from its conjugate with HSA is manifested in the spectral shift and decrease in its fluorescence intensity as well as the emergence of component with lifetime of 2-3 ns, which is characteristic of free DP. As DP is known to bind specifically to the Sudlow's site II of human serum albumin, its displacement by chlorin p6 indicates the residence of the photosensitizer in the same site, in addition to Sudlow's site I. The binding constants for Sudlow's site II, determined by the stopped-flow technique, are found to be two orders of magnitude smaller than that for Sudlow's site I.

  10. Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (. mu. ) and enkephalin (delta) binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Kazmi, S.M.I.; Mishra, R.K.

    1986-06-13

    Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of ..mu.. and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of (/sup 3/H)-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin (DADLE) in the presence of 10/sup -5/M D-Pro/sup 4/-morphiceptin (to block the ..mu.. receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of (/sup 3/H)-dihydromorphine, together with the higher potency of morphine analogues to displace (/sup 3/H)-naloxone binding established the presence of ..mu.. sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of (/sup 3/H)-DADLE binding.

  11. Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

    International Nuclear Information System (INIS)

    Chabot, J.G.; Walker, P.; Pelletier, G.

    1986-01-01

    The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of [ 125 I]EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of [ 125 I]EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of [ 125 I]EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland

  12. Prolactin, cortisol and thyroxine levels and the premature infant ...

    African Journals Online (AJOL)

    The relationship of prolactin, cortisol and thyroxine values in cord and maternal plasma to fetal age and weight and to the incidence of hyaline membrane disease (HMD) was investigated in 80 neonates of whom 40 were born at more than 37 weeks' gestation. Of the 40 born at less than 36 weeks 11 developed HMD.

  13. high doses of prolactin inhibit testosterone secretion in rat leydig cells

    African Journals Online (AJOL)

    Femi Olaleye

    1 The effect of prolactin on dispersed rat Leydig cells was investigated. Leydig cells from adult rat testes of proven fertility were isolated via collagenase digestion and dispersion. About 100,000 Leydig .... Hormones, Drugs and Reagents.

  14. Na,K-ATPase binding sites in human erythrocytes in cirrhosis of the liver

    International Nuclear Information System (INIS)

    Schober, O.; Oetting, G.; Bossaller, C.

    1985-01-01

    The number of red blood cell ouabain binding sites, total-body potassium (TBK), serum potassium, exchangeable sodium, and serum sodium was studied in 24 patients with cirrhosis of the liver. The number of red cell ouabain binding sites, measured by equilibrium binding of 3 H-ouabain, showed a significant increase in the number of Na,K pumps in patients with cirrhosis of the liver (447+-99) as compared with a control group (281+-50, n=36). TBK was measured by counting the endogenous K-40 in a whole-body counter. TBK was 76+-10% in cirrhosis. This significant reduction in TBK was accompanied by normal serum potassium levels, and slightly decreased serum sodium levels in cirrhosis, however exchangeable sodium (Na-24) was increased in cirrhosis of the liver (55+-13 mmol/kg) compared with controls (40+-7 mmol/kg). These results support the suggestion that changes of sodium-potassium concentration at the cell membrane may regulate the synthesis of Na,K-pump molecules. (orig.) [de

  15. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Directory of Open Access Journals (Sweden)

    Marlet Martinez-Archundia

    2012-01-01

    Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

  16. Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

    Science.gov (United States)

    André, S; Ortega, P J; Perez, M A; Roy, R; Gabius, H J

    1999-11-01

    Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential

  17. Characterization of dFOXO binding sites upstream of the Insulin Receptor P2 promoter across the Drosophila phylogeny.

    Directory of Open Access Journals (Sweden)

    Dorcas J Orengo

    Full Text Available The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. Here we have focused on the Insulin Receptor (InR expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO. We have finely characterized this transcription factor binding sites in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, we have tested the effect of mutations in the characterized dFOXO sites of D. melanogaster in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription.

  18. In vitro studies of immunoglobulin heavy-chain binding protein (BiP, GRP78). Interactions of BiP with newly synthesized proteins and adenine nucleotides

    International Nuclear Information System (INIS)

    Kassenbrock, C.K.

    1988-01-01

    Here we examine the interaction of BiP with newly synthesized polypeptides in an in vitro protein translations-translocation system. We find that BiP forms tight complexes with nonglycosylated yeast invertase and incorrectly disulfide-bonded prolactin but not with glycosylated invertase or correctly disulfide-bonded prolactin. Moreover, BiP associates detectably only with completed chains of prolactin, not with chains undergoing synthesis. We conclude that BiP recognizes and binds with high affinity to aberrantly folded or aberrantly glycosylated polypeptides in vitro, but not to all nascent chains as they are folding. BiP also binds APT and can be purified by APT affinity chromatography. We show that submicromolar levels of ATP or ADP decrease the rate of absorption of 125 I-BiP to nitrocellulose filters coated with protein or nonionic detergents. ATP and ADP also protect portions of BiP from proteolytic degradation. In contrast, micromolar levels of AMP increase the rate of adsorption and the rate of proteolytic degradation of BiP. We also show that an ATPase activity co-purifies with BiP, but its slow turnover number suggests a regulatory, rather than a functional role. The BiP-associated ATPase shares several properties with the related cytoplasmic protein, HSC70/clathrin uncoating ATPase

  19. Autoradiographic localization of (125I-Tyr4)bombesin-binding sites in rat brain

    International Nuclear Information System (INIS)

    Zarbin, M.A.; Kuhar, M.J.; O'Donohue, T.L.; Wolf, S.S.; Moody, T.W.

    1985-01-01

    The binding of ( 125 I-Tyr 4 )bombesin to rat brain slices was investigated. Radiolabeled (Tyr 4 )bombesin bound with high affinity (K/sub d/ . 4 nM) to a single class of sites (B/sub max/ . 130 fmol/mg of protein); the ratio of specific to nonspecific binding was 6/1. Also, pharmacology studies indicated that the C-terminal of bombesin was important for the high affinity binding activity. Autoradiographic studies indicated that the ( 125 I-Tyr4)bombesin-binding sites were discretely distributed in certain gray but not white matter regions of rat brain. Highest grain densities were present in the olfactory bulb and tubercle, nucleus accumbens, suprachiasmatic and periventricular nuclei of the hypothalamus, central medial thalamic nucleus, medial amygdaloid nucleus, hippocampus, dentate gyrus, subiculum, nucleus of the solitary tract, and substantia gelatinosa. Moderate grain densities were present in the parietal cortex, deep layers of the neocortex, rhinal cortex, caudate putamen, stria terminalis, locus ceruleus, parabrachial nucleus, and facial nucleus. Low grain densities were present in the globus pallidus, lateral thalamus, and midbrain. Negligible grain densities were present in the cerebellum, corpus callosum, and all regions treated with 1 microM unlabeled bombesin. The discrete regional distribution of binding suggests that endogenous bombesin-like peptides may function as important regulatory agents in certain brain loci

  20. Conversion of the low affinity ouabain-binding site of non-gastric H,K-ATPase into a high affinity binding site by substitution of only five amino acids.

    NARCIS (Netherlands)

    Qiu, L.Y.; Swarts, H.G.P.; Tonk, E.C.; Willems, P.H.G.M.; Koenderink, J.B.; Pont, J.J.H.H.M. de

    2006-01-01

    P-type ATPases of the IIC subfamily exhibit large differences in sensitivity toward ouabain. This allows a strategy in which ouabain-insensitive members of this subfamily are used as template for mutational elucidation of the ouabain-binding site. With this strategy, we recently identified seven

  1. Differential alterations of cortical glutamatergic binding sites in senile dementia of the Alzheimer type

    International Nuclear Information System (INIS)

    Chalmers, D.T.; Dewar, D.; Graham, D.I.; Brooks, D.N.; McCulloch, J.

    1990-01-01

    Involvement of cortical glutamatergic mechanisms in senile dementia of the Alzheimer type (SDAT) has been investigated with quantitative ligand-binding autoradiography. The distribution and density of Na(+)-dependent glutamate uptake sites and glutamate receptor subtypes--kainate, quisqualate, and N-methyl-D-aspartate--were measured in adjacent sections of frontal cortex obtained postmortem from six patients with SDAT and six age-matched controls. The number of senile plaques was determined in the same brain region. Binding of D-[3H]aspartate to Na(+)-dependent uptake sites was reduced by approximately 40% throughout SDAT frontal cortex relative to controls, indicating a general loss of glutamatergic presynaptic terminals. [3H]Kainate receptor binding was significantly increased by approximately 70% in deep layers of SDAT frontal cortex compared with controls, whereas this binding was unaltered in superficial laminae. There was a positive correlation (r = 0.914) between kainate binding and senile plaque number in deep cortical layers. Quisqualate receptors, as assessed by 2-amino-3-hydroxy-5-[3H]methylisoxazole-4-propionic acid binding, were unaltered in SDAT frontal cortex compared with controls. There was a small reduction (25%) in N-methyl-D-aspartate-sensitive [3H]glutamate binding only in superficial cortical layers of SDAT brains relative to control subjects. [3H]Glutamate binding in SDAT subjects was unrelated to senile plaque number in superficial cortical layers (r = 0.104). These results indicate that in the presence of cortical glutamatergic terminal loss in SDAT plastic alterations occur in some glutamate receptor subtypes but not in others

  2. Serum prolactin, leptin, lipids and lipoproteins levels during antipsychotics treatment in Parkinson's disease and related psychosis.

    Science.gov (United States)

    Rustembegovic, Avdo; Sofic, Emin; Wichart, Ildiko

    2006-01-01

    Weight gain is a common adverse effect associated with the use of most typical and atypical antipsychotic. Aim of this study was to investigate serum prolactin, leptin, cholesterol, triglyceride, lipoproteins, such high density lipoprotein (HDL), and low density lipoprotein (LDL) levels in patients with Parkinson's disease (PD)-related psychosis during long-term medication with atypical antipsychotic. The study population comprised 40 patients, who were divided into 4 groups: olanzapine (n=10), risperidone (n=10), seroquel (n=10) monotherapy, a group of 10 patients receiving only antiparkinson drugs and a control group of 8 healthy persons. The patients were evaluated at baseline and at the sixth and twelfth week according to the Positive and Negative Syndrome Scale (PANSS), body mass index (BMI), and fasting serum prolactin, leptin, lipids and lipoproteins levels. Treatment of patients with olanzapine caused marked increase of serum LDL, cholesterol, triglyceride, and leptin levels (prelationship between serum leptin, lipid levels and BMI. However, treatment of patients with seroquel did not cause changes in serum prolactin, leptin, lipids, and lipoproteins levels. Our results suggest that treatment of patients with PD-related psychosis with seroquel appears to have minimal influence on serum leptin, prolactin, lipids, lipoproteins and BMI compared with olanzapine and risperidone.

  3. Investigation of the Copper Binding Site And the Role of Histidine As a Ligand in Riboflavin Binding Protein

    Energy Technology Data Exchange (ETDEWEB)

    Smith, S.R.; Bencze, K.Z.; Russ, K.A.; Wasiukanis, K.; Benore-Parsons, M.; Stemmler, T.L.

    2009-05-26

    Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 {angstrom}, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O{sub 3}N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.

  4. DISTINCT ROLES OF β1 MIDAS, ADMIDAS AND LIMBS CATION-BINDING SITES IN LIGAND RECOGNITION BY INTEGRIN α2β1*

    Science.gov (United States)

    Valdramidou, Dimitra; Humphries, Martin J.; Mould, A. Paul

    2012-01-01

    Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as α2β1, ligand recognition takes place exclusively at the α subunit I domain. However, activation of the αI domain depends on its interaction with a structurally similar domain in the β subunit known as the I-like or βI domain. The top face of the βI domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS) and LIMBS (ligand-associated metal binding site). The role of these sites in controlling ligand binding to the αI domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to α2β1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcome by the activating mAb TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between αI and βI whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of βI. An activating mutation in the α2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca2+, Mg2+ and Mn2+ on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn2+ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2+-binding site, occupancy of which increases the affinity of Mg2+ for the MIDAS. PMID:18820259

  5. Prediction of protein binding sites using physical and chemical descriptors and the support vector machine regression method

    International Nuclear Information System (INIS)

    Sun Zhong-Hua; Jiang Fan

    2010-01-01

    In this paper a new continuous variable called core-ratio is defined to describe the probability for a residue to be in a binding site, thereby replacing the previous binary description of the interface residue using 0 and 1. So we can use the support vector machine regression method to fit the core-ratio value and predict the protein binding sites. We also design a new group of physical and chemical descriptors to characterize the binding sites. The new descriptors are more effective, with an averaging procedure used. Our test shows that much better prediction results can be obtained by the support vector regression (SVR) method than by the support vector classification method. (rapid communication)

  6. Radioimmunoassay of human homologous prolactin in serum with commercially available reagents. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Kao, P.C.; Jiang, N.S.; Abboud, C.F.

    1977-09-01

    A clinically useful and reproducible radioimmunoassay for human homologous prolactin, established with commercially available reagents, was studied and validated. We present detailed conditions for iodination and purification of labeled prolactin and the optimal conditions for the assay. By the method, we found values (..mu..g/liter) as follows for serum prolactin: normal men, 8.9 +- 5.2 (mean +- SD); normal women, 11.8 +- 5.5; normal women taking contraceptive pills, 9.2 +- 5.0; pregnant women in the third trimester, 188 +- 69.5; patients with various diseases other than of the hypothalamic-pituitary axis, 9.3 +- 6.3; in some patients with amenorrhea and galactorrhea of diverse origin, 78.2 +- 87.4; and in some patients with surgically proven pituitary tumor, 1414 +- 1980. Results under provocative testing are also presented for a patient with normal hypothalamic-pituitary function.

  7. Structure-based stabilization of HIV-1 gp120 enhances humoral immune responses to the induced co-receptor binding site.

    Directory of Open Access Journals (Sweden)

    Barna Dey

    2009-05-01

    Full Text Available The human immunodeficiency virus type 1 (HIV-1 exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.

  8. Comparison of Plasma Neurosteroid and Prolactin Levels in Patients with Schizophrenia and Healthy Individuals

    Directory of Open Access Journals (Sweden)

    Forough Riahi

    2016-01-01

    Full Text Available Background. The present study aimed to compare plasma levels of cortisol, testosterone, dehydroepiandrosterone (DHEA, and prolactin in patients with schizophrenia and healthy individuals. Method. A total of 100 patients with schizophrenia disorder (69 men and 31 women and 190 healthy individuals (94 men and 96 women participated in this cross-sectional study. They were tested for hormone levels and completed demographic questionnaires. Data were analyzed using multivariate analysis of variance (MANOVA and one-way analysis of variance. Results. Serum testosterone level was significantly higher in men with schizophrenia than in healthy men. Women with schizophrenia had a significantly higher level of testosterone and lower level of prolactin compared to healthy women. There were no significant differences in hormone levels across various subtypes of schizophrenia. No significant differences also were observed in hormones levels in patients with first-episode schizophrenia disorder compared to those in patients with recurrent episodes. Conclusion. This study indicated that abnormal testosterone and prolactin levels might be associated with pathophysiology of schizophrenia disorder.

  9. Manipulation of EphB2 regulatory motifs and SH2 binding sites switches MAPK signaling and biological activity.

    Science.gov (United States)

    Tong, Jiefei; Elowe, Sabine; Nash, Piers; Pawson, Tony

    2003-02-21

    Signaling by the Eph family of receptor tyrosine kinases (RTKs) is complex, because they can interact with a variety of intracellular targets, and can potentially induce distinct responses in different cell types. In NG108 neuronal cells, activated EphB2 recruits p120RasGAP, in a fashion that is associated with down-regulation of the Ras-Erk mitogen-activated kinase (MAPK) pathway and neurite retraction. To pursue the role of the Ras-MAPK pathway in EphB2-mediated growth cone collapse, and to explore the biochemical and biological functions of Eph receptors, we sought to re-engineer the signaling properties of EphB2 by manipulating its regulatory motifs and SH2 binding sites. An EphB2 mutant that retained juxtamembrane (JM) RasGAP binding sites but incorporated a Grb2 binding motif at an alternate RasGAP binding site within the kinase domain had little effect on basal Erk MAPK activation. In contrast, elimination of all RasGAP binding sites, accompanied by the addition of a Grb2 binding site within the kinase domain, led to an increase in phospho-Erk levels in NG108 cells following ephrin-B1 stimulation. Functional assays indicated a correlation between neurite retraction and the ability of the EphB2 mutants to down-regulate Ras-Erk MAPK signaling. These data suggest that EphB2 can be designed to repress, stabilize, or activate the Ras-Erk MAPK pathway by the manipulation of RasGAP and Grb2 SH2 domain binding sites and support the notion that Erk MAPK regulation plays a significant role in axon guidance. The behavior of EphB2 variants with mutations in the JM region and kinase domains suggests an intricate pattern of regulation and target recognition by Eph receptors.

  10. The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites

    International Nuclear Information System (INIS)

    Zeng Zhizhen; Chen, T.-B.; Miller, Patricia J.; Dean, Dennis; Tang, Y.S.; Sur, Cyrille; Williams, David L.

    2006-01-01

    We have characterized the interaction of the serotonin transporter ligand [ 3 H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [ 3 H]-DASB, a tritiated version of the widely used [ 11 C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K d =0.20±0.04 nM). The serotonin transporter density (B max ) obtained for rhesus frontal cortex was found to be 66±8 fmol/mg protein using [ 3 H]-DASB, similar to the B max value obtained using the reference radioligand [ 3 H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83±22 fmol/mg protein). Specific binding sites of both [ 3 H]-DASB and [ 3 H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [ 3 H]-citalopram binding in a competition autoradiographic study, with K i values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [ 3 H]-DASB and [ 3 H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [ 11 C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates

  11. Response and binding elements for ligand-dependent positive transcription factors integrate positive and negative regulation of gene expression

    International Nuclear Information System (INIS)

    Rosenfeld, M.G.; Glass, C.K.; Adler, S.; Crenshaw, E.B. III; He, X.; Lira, S.A.; Elsholtz, H.P.; Mangalam, H.J.; Holloway, J.M.; Nelson, C.; Albert, V.R.; Ingraham, H.A.

    1988-01-01

    Accurate, regulated initiation of mRNA transcription by RNA polymerase II is dependent on the actions of a variety of positive and negative trans-acting factors that bind cis-acting promoter and enhancer elements. These transcription factors may exert their actions in a tissue-specific manner or function under control of plasma membrane or intracellular ligand-dependent receptors. A major goal in the authors' laboratory has been to identify the molecular mechanisms responsible for the serial activation of hormone-encoding genes in the pituitary during development and the positive and negative regulation of their transcription. The anterior pituitary gland contains phenotypically distinct cell types, each of which expresses unique trophic hormones: adrenocorticotropic hormone, thyroid-stimulating hormone, prolactin, growth hormone, and follicle-stimulating hormone/luteinizing hormone. The structurally related prolactin and growth hormone genes are expressed in lactotrophs and somatotrophs, respectively, with their expression virtually limited to the pituitary gland. The reported transient coexpression of these two structurally related neuroendocrine genes raises the possibility that the prolactin and growth hormone genes are developmentally controlled by a common factor(s)

  12. [In vitro evaluation of the gels properties prepared on Pluronic F-127 as vehicles for administration prolactin by injection].

    Science.gov (United States)

    Karolewicz, Bozena; Pawlik-Gałczyńska, Anna; Pluta, Janusz; Ryszka, Florian

    2011-01-01

    The aim of this study was to prepare a thermoresponsive formulations, which are a carrier for proteins--prolactin administered directly into solid tumor and which obtain sol-gel transitions at physiological ranges of temperature. Prolactin (PRL) is a hormone that in vivo and in vitro exhibits antiangiogenic properties. Application of this protein in the proposed formulations can be particularly advantageous because of its relatively low stability and limited ability to transmembrane penetration. The paper prepared thermoresponsive carriers, based on nonionic polymer Pluronic F-127 with selected excipients such as dextran 7000, PEG 400, Tween 20 and Tween 80. The sol-gel transition temperature of the formulations was investigated and their physicochemical properties such as pH, density, osmotic pressure were studied. In the remainder of the work carried out tests of prolactin release from the proposed media. The results obtained indicate that a significant influence on the theological parameters obtained carriers and the availability of pharmaceutical composition of prolactin was developed formulation.

  13. Osmoregulatory effects of hypophysectomy and homologous prolactin replacement in hybrid striped bass

    DEFF Research Database (Denmark)

    Jackson, Leslie F; McCormick, Stephen D; Madsen, Steffen S

    2005-01-01

    The effects of ovine prolactin (oPRL) and striped bass prolactin (sbPRL; Morone saxatilis) on plasma osmolality, electrolyte balance, and gill Na+,K+-ATPase activity were investigated in hypophysectomized (Hx), freshwater (FW)-acclimated, hybrid striped bass (M. saxatilis x Morone chrysops...... or 100 ng/g), or hormone vehicle (0.9% NaCl) at 48-h intervals (days 0, 2, 4, and 6) in FW and then sampled for blood plasma 24 h after the fourth injection (day 7). In Hx fish, oPRL (5 and 20 microg/g) and sbPRL (10 and 100 ng/g) were effective in maintaining plasma osmolality and levels of Na+, Cl...... balance in FW-adapted hybrid striped bass, and that this may involve downregulation of branchial Na+,K+-ATPase activity....

  14. Cytochrome c1 exhibits two binding sites for cytochrome c in plants.

    Science.gov (United States)

    Moreno-Beltrán, Blas; Díaz-Quintana, Antonio; González-Arzola, Katiuska; Velázquez-Campoy, Adrián; De la Rosa, Miguel A; Díaz-Moreno, Irene

    2014-10-01

    In plants, channeling of cytochrome c molecules between complexes III and IV has been purported to shuttle electrons within the supercomplexes instead of carrying electrons by random diffusion across the intermembrane bulk phase. However, the mode plant cytochrome c behaves inside a supercomplex such as the respirasome, formed by complexes I, III and IV, remains obscure from a structural point of view. Here, we report ab-initio Brownian dynamics calculations and nuclear magnetic resonance-driven docking computations showing two binding sites for plant cytochrome c at the head soluble domain of plant cytochrome c1, namely a non-productive (or distal) site with a long heme-to-heme distance and a functional (or proximal) site with the two heme groups close enough as to allow electron transfer. As inferred from isothermal titration calorimetry experiments, the two binding sites exhibit different equilibrium dissociation constants, for both reduced and oxidized species, that are all within the micromolar range, thus revealing the transient nature of such a respiratory complex. Although the docking of cytochrome c at the distal site occurs at the interface between cytochrome c1 and the Rieske subunit, it is fully compatible with the complex III structure. In our model, the extra distal site in complex III could indeed facilitate the functional cytochrome c channeling towards complex IV by building a "floating boat bridge" of cytochrome c molecules (between complexes III and IV) in plant respirasome. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Hyperprolactinemia with normal serum prolactin: Its clinical significance

    Directory of Open Access Journals (Sweden)

    Manika Agarwal

    2010-01-01

    Full Text Available Amenorrhea and infertility with an added feature of galactorrhea makes a provisional diagnosis of hyperprolactinemia. But again, normal serum prolactin with all clinical features of hyperprolactinemia might question the diagnosis and further management. The answer lies in the heterogeneity of the peptide hormone - the immunoactive and the bioactive forms. This has been further illustrated with the help of a case which had been treated with cabergoline.

  16. Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

    Energy Technology Data Exchange (ETDEWEB)

    Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita; Karkashon, Shay; Bonanno, Jeffrey B.; Poulos, Thomas L.; Yeh, Syun-Ru (Einstein); (UCI)

    2017-11-22

    Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

  17. Plasminogen fragments K 1-3 and K 5 bind to different sites in fibrin fragment DD.

    Science.gov (United States)

    Grinenko, T V; Kapustianenko, L G; Yatsenko, T A; Yusova, O I; Rybachuk, V N

    2016-01-01

    Specific plasminogen-binding sites of fibrin molecule are located in Аα148-160 regions of C-terminal domains. Plasminogen interaction with these sites initiates the activation process of proenzyme and subsequent fibrin lysis. In this study we investigated the binding of plasminogen fragments K 1-3 and K 5 with fibrin fragment DD and their effect on Glu-plasminogen interaction with DD. It was shown that the level of Glu-plasminogen binding to fibrin fragment DD is decreased by 50-60% in the presence of K 1-3 and K 5. Fragments K 1-3 and K 5 have high affinity to fibrin fragment DD (Kd is 0.02 for K 1-3 and 0.054 μМ for K 5). K 5 interaction is independent and K 1-3 is partly dependent on C-terminal lysine residues. K 1-3 interacts with complex of fragment DD-immobilized K 5 as well as K 5 with complex of fragment DD-immobilized K 1-3. The plasminogen fragments do not displace each other from binding sites located in fibrin fragment DD, but can compete for the interaction. The results indicate that fibrin fragment DD contains different binding sites for plasminogen kringle fragments K 1-3 and K 5, which can be located close to each other. The role of amino acid residues of fibrin molecule Аα148-160 region in interaction with fragments K 1-3 and K 5 is discussed.

  18. Chromatin immunoprecipitation to analyze DNA binding sites of HMGA2.

    Directory of Open Access Journals (Sweden)

    Nina Winter

    Full Text Available BACKGROUND: HMGA2 is an architectonic transcription factor abundantly expressed during embryonic and fetal development and it is associated with the progression of malignant tumors. The protein harbours three basically charged DNA binding domains and an acidic protein binding C-terminal domain. DNA binding induces changes of DNA conformation and hence results in global overall change of gene expression patterns. Recently, using a PCR-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment procedure two consensus sequences for HMGA2 binding have been identified. METHODOLOGY/PRINCIPAL FINDINGS: In this investigation chromatin immunoprecipitation (ChIP experiments and bioinformatic methods were used to analyze if these binding sequences can be verified on chromatin of living cells as well. CONCLUSION: After quantification of HMGA2 protein in different cell lines the colon cancer derived cell line HCT116 was chosen for further ChIP experiments because of its 3.4-fold higher HMGA2 protein level. 49 DNA fragments were obtained by ChIP. These fragments containing HMGA2 binding sites have been analyzed for their AT-content, location in the human genome and similarities to sequences generated by a SELEX study. The sequences show a significantly higher AT-content than the average of the human genome. The artificially generated SELEX sequences and short BLAST alignments (11 and 12 bp of the ChIP fragments from living cells show similarities in their organization. The flanking regions are AT-rich, whereas a lower conservation is present in the center of the sequences.

  19. Integrin Activation Dynamics between the RGD-binding Site and the Headpiece Hinge*

    Science.gov (United States)

    Puklin-Faucher, Eileen; Vogel, Viola

    2009-01-01

    Integrins form mechanical links between the extracellular matrix and the cytoskeleton. Although integrin activation is known to be regulated by an allosteric conformational change, which can be induced from the extracellular or intracellular end of the molecule, little is known regarding the sequence of structural events by which signals propagate between distant sites. Here, we reveal with molecular dynamics simulations of the FnIII10-bound αVβ3 integrin headpiece how the binding pocket and interdomain βA/hybrid domain hinge on the distal end of the βA domain are allosterically linked via a hydrophobic T-junction between the middle of the α1 helix and top of the α7 helix. The key results of this study are: 1) that this T-junction is induced by ligand binding and hinge opening, and thus displays bidirectionality; 2) that formation of this junction can be accelerated by ligand-mediated force; and 3) how formation of this junction is inhibited by Ca2+ in place of Mg2+ at the site adjacent to the metal ion-dependent adhesion site (“ADMIDAS”). Together with recent experimental evidence that integrin complexes can form catch bonds (i.e. become strengthened under force), as well as earlier evidence that Ca2+ at the ADMIDAS results in lower binding affinity, these simulations provide a common structural model for the dynamic process by which integrins become activated. PMID:19762919

  20. CONREAL web server: identification and visualization of conserved transcription factor binding sites

    NARCIS (Netherlands)

    Berezikov, E.; Guryev, V.; Cuppen, E.

    2005-01-01

    The use of orthologous sequences and phylogenetic footprinting approaches have become popular for the recognition of conserved and potentially functional sequences. Several algorithms have been developed for the identification of conserved transcription factor binding sites (TFBSs), which are

  1. Serum prolactin revisited: parametric reference intervals and cross platform evaluation of polyethylene glycol precipitation-based methods for discrimination between hyperprolactinemia and macroprolactinemia.

    Science.gov (United States)

    Overgaard, Martin; Pedersen, Susanne Møller

    2017-10-26

    Hyperprolactinemia diagnosis and treatment is often compromised by the presence of biologically inactive and clinically irrelevant higher-molecular-weight complexes of prolactin, macroprolactin. The objective of this study was to evaluate the performance of two macroprolactin screening regimes across commonly used automated immunoassay platforms. Parametric total and monomeric gender-specific reference intervals were determined for six immunoassay methods using female (n=96) and male sera (n=127) from healthy donors. The reference intervals were validated using 27 hyperprolactinemic and macroprolactinemic sera, whose presence of monomeric and macroforms of prolactin were determined using gel filtration chromatography (GFC). Normative data for six prolactin assays included the range of values (2.5th-97.5th percentiles). Validation sera (hyperprolactinemic and macroprolactinemic; n=27) showed higher discordant classification [mean=2.8; 95% confidence interval (CI) 1.2-4.4] for the monomer reference interval method compared to the post-polyethylene glycol (PEG) recovery cutoff method (mean=1.8; 95% CI 0.8-2.8). The two monomer/macroprolactin discrimination methods did not differ significantly (p=0.089). Among macroprolactinemic sera evaluated by both discrimination methods, the Cobas and Architect/Kryptor prolactin assays showed the lowest and the highest number of misclassifications, respectively. Current automated immunoassays for prolactin testing require macroprolactin screening methods based on PEG precipitation in order to discriminate truly from falsely elevated serum prolactin. While the recovery cutoff and monomeric reference interval macroprolactin screening methods demonstrate similar discriminative ability, the latter method also provides the clinician with an easy interpretable monomeric prolactin concentration along with a monomeric reference interval.

  2. msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

    Directory of Open Access Journals (Sweden)

    Anil Raj

    Full Text Available Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

  3. msCentipede: Modeling Heterogeneity across Genomic Sites and Replicates Improves Accuracy in the Inference of Transcription Factor Binding.

    Science.gov (United States)

    Raj, Anil; Shim, Heejung; Gilad, Yoav; Pritchard, Jonathan K; Stephens, Matthew

    2015-01-01

    Understanding global gene regulation depends critically on accurate annotation of regulatory elements that are functional in a given cell type. CENTIPEDE, a powerful, probabilistic framework for identifying transcription factor binding sites from tissue-specific DNase I cleavage patterns and genomic sequence content, leverages the hypersensitivity of factor-bound chromatin and the information in the DNase I spatial cleavage profile characteristic of each DNA binding protein to accurately infer functional factor binding sites. However, the model for the spatial profile in this framework fails to account for the substantial variation in the DNase I cleavage profiles across different binding sites. Neither does it account for variation in the profiles at the same binding site across multiple replicate DNase I experiments, which are increasingly available. In this work, we introduce new methods, based on multi-scale models for inhomogeneous Poisson processes, to account for such variation in DNase I cleavage patterns both within and across binding sites. These models account for the spatial structure in the heterogeneity in DNase I cleavage patterns for each factor. Using DNase-seq measurements assayed in a lymphoblastoid cell line, we demonstrate the improved performance of this model for several transcription factors by comparing against the Chip-seq peaks for those factors. Finally, we explore the effects of DNase I sequence bias on inference of factor binding using a simple extension to our framework that allows for a more flexible background model. The proposed model can also be easily applied to paired-end ATAC-seq and DNase-seq data. msCentipede, a Python implementation of our algorithm, is available at http://rajanil.github.io/msCentipede.

  4. An allosteric binding site at the human serotonin transporter mediates the inhibition of escitalopram by R-citalopram: kinetic binding studies with the ALI/VFL-SI/TT mutant.

    Science.gov (United States)

    Zhong, Huailing; Hansen, Kasper B; Boyle, Noel J; Han, Kiho; Muske, Galina; Huang, Xinyan; Egebjerg, Jan; Sánchez, Connie

    2009-10-25

    The human serotonin transporter (hSERT) has primary and allosteric binding sites for escitalopram and R-citalopram. Previous studies have established that the interaction of these two compounds at a low affinity allosteric binding site of hSERT can affect the dissociation of [(3)H]escitalopram from hSERT. The allosteric binding site involves a series of residues in the 10th, 11th, and 12th trans-membrane domains of hSERT. The low affinity allosteric activities of escitalopram and R-citalopram are essentially eliminated in a mutant hSERT with changes in some of these residues, namely A505V, L506F, I507L, S574T, I575T, as measured in dissociation binding studies. We confirm that in association binding experiments, R-citalopram at clinically relevant concentrations reduces the association rate of [(3)H]escitalopram as a ligand to wild type hSERT. We demonstrate that the ability of R-citalopram to reduce the association rate of escitalopram is also abolished in the mutant hSERT (A505V, L506F, I507L, S574T, I575T), along with the expected disruption the low affinity allosteric function on dissociation binding. This suggests that the allosteric binding site mediates both the low affinity and higher affinity interactions between R-citalopram, escitalopram, and hSERT. Our data add an additional structural basis for the different efficacies of escitalopram compared to racemic citalopram reported in animal studies and clinical trials, and substantiate the hypothesis that hSERT has complex allosteric mechanisms underlying the unexplained in vivo activities of its inhibitors.

  5. Substrate binding in the active site of cytochrome P450cam

    NARCIS (Netherlands)

    Swart, M.; Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K.

    2005-01-01

    We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calculations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of the reaction taking place. The DFT

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  7. Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes.

    Science.gov (United States)

    Denys, A; Allain, F; Foxwell, B; Spik, G

    1997-08-01

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway and released in biological fluids. We have recently demonstrated that both free CyPB and CyPB-CsA complex specifically bind to peripheral blood T lymphocytes and are internalized. These results suggest that CyPB might promote the targeting of the drug into sensitive cells. Peripheral blood lymphocytes are subdivided in several populations according to their biological functions and sensitivity to CsA. We have investigated the binding of CyPB to these different subsets using a CyPB derivatized by fluorescein through its single cysteine which retains its binding properties. We have confirmed that only T cells were involved in the interaction with CyPB. The ligand binding was found to be heterogeneously distributed on the different T-cell subsets and surface-bound CyPB was mainly associated with the CD4-positive cells. No significant difference was noted between the CD45RA and CD45RO subsets, demonstrating that CyPB-binding sites were equally distributed between native and memory T cells. CD3 stimulation of T lymphocytes led to a decrease in the CyPB-binding capacity, that may be explained by a down-regulation of the CyPB-receptor expression upon T-cell activation. Finally, we demonstrated that CyPB-receptor-positive cells, isolated on CyPB sulphydryl-coupled affinity matrices, are more sensitive to CyPB-complexed CsA than mixed peripheral blood lymphocytes, suggesting that CyPB potentiates CsA activity through the binding of the complex. Taken together, our results demonstrate that CyPB-binding sites are mainly associated with resting cells of the helper T lymphocyte, and that CyPB might modulate the distribution of CsA through the drug targeting to sensitive cells.

  8. CavityPlus: a web server for protein cavity detection with pharmacophore modelling, allosteric site identification and covalent ligand binding ability prediction.

    Science.gov (United States)

    Xu, Youjun; Wang, Shiwei; Hu, Qiwan; Gao, Shuaishi; Ma, Xiaomin; Zhang, Weilin; Shen, Yihang; Chen, Fangjin; Lai, Luhua; Pei, Jianfeng

    2018-05-10

    CavityPlus is a web server that offers protein cavity detection and various functional analyses. Using protein three-dimensional structural information as the input, CavityPlus applies CAVITY to detect potential binding sites on the surface of a given protein structure and rank them based on ligandability and druggability scores. These potential binding sites can be further analysed using three submodules, CavPharmer, CorrSite, and CovCys. CavPharmer uses a receptor-based pharmacophore modelling program, Pocket, to automatically extract pharmacophore features within cavities. CorrSite identifies potential allosteric ligand-binding sites based on motion correlation analyses between cavities. CovCys automatically detects druggable cysteine residues, which is especially useful to identify novel binding sites for designing covalent allosteric ligands. Overall, CavityPlus provides an integrated platform for analysing comprehensive properties of protein binding cavities. Such analyses are useful for many aspects of drug design and discovery, including target selection and identification, virtual screening, de novo drug design, and allosteric and covalent-binding drug design. The CavityPlus web server is freely available at http://repharma.pku.edu.cn/cavityplus or http://www.pkumdl.cn/cavityplus.

  9. Cadmium mimics estrogen-driven cell proliferation and prolactin secretion from anterior pituitary cells.

    Directory of Open Access Journals (Sweden)

    Sonia A Ronchetti

    Full Text Available Cadmium (Cd is a heavy metal of considerable occupational and environmental concern affecting wildlife and human health. Recent studies indicate that Cd, like other heavy metals, can mimic effects of 17β-estradiol (E2 involving E2 receptor (ER activation. Lactotrophs, the most abundant cell type in anterior pituitary gland, are the main target of E2, which stimulates cell proliferation and increases prolactin secretion through ERα. The aim of this work was to examine whether Cd at nanomolar concentrations can induce cell proliferation and prolactin release in anterior pituitary cells in culture and whether these effects are mediated through ERs. Here we show that 10 nM Cd was able to stimulate lactotroph proliferation in anterior pituitary cell cultures from female Wistar rats and also in GH3 lactosomatotroph cell line. Proliferation of somatotrophs and gonadotrophs were not affected by Cd exposure. Cd promoted cell cycle progression by increasing cyclins D1, D3 and c-fos expression. Cd enhanced prolactin synthesis and secretion. Cd E2-like effects were blocked by the pure ERs antagonist ICI 182,780 supporting that Cd acts through ERs. Further, both Cd and E2 augmented full-length ERαexpression and its 46 kDa-splicing variant. In addition, when co-incubated Cd was shown to interact with E2 by inducing ERα mRNA expression which indicates an additive effect between them. This study shows for the first time that Cd at nanomolar concentration displays xenoestrogenic activities by inducing cell growth and stimulating prolactin secretion from anterior pituitary cells in an ERs-dependent manner. Cd acting as a potent xenoestrogen can play a key role in the aetiology of different pathologies of the anterior pituitary and in estrogen-responsive tissues which represent considerable risk to human health.

  10. Functional analysis of a potential regulatory K+-binding site in the Na+, K+-ATPase

    DEFF Research Database (Denmark)

    Schack, Vivien Rodacker; Vilsen, Bente

    The Na+, K+-ATPase functions by actively transporting 3 Na+ ions out of and 2 K+ ions into the cell, thereby creating ion gradients crucial for many physiological processes. Recently, a combined structural and functional study of the closely related Ca2+-ATPase indicated the presence...... of a regulatory K+-binding site in the P-domain of the enzyme, identifying E732 as being of particular importance (Sorensen, Clausen et al. 2004). In addition, P709 is thought to play a significant role in the structural organization of this site. Both E732 and P709 are highly conserved among P-type ATPases (E732...... is present as either glutamic acid or aspartic acid), which supports their importance and additionally raises the question whether this site may play a general role among P-type ATPases. In Na+, K+-ATPase, K+ functions directly as a substrate for membrane binding sites, however, an additional regulatory...

  11. Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches

    DEFF Research Database (Denmark)

    Møller, Charlotte; Sprenger, Richard R.; Stürup, Stefan

    2011-01-01

    Using insulin as a model protein for binding of oxaliplatin to proteins, various mass spectrometric approaches and techniques were compared. Several different platinum adducts were observed, e.g. addition of one or two diaminocyclohexane platinum(II) (Pt(dach)) molecules. By top-down analysis...... and fragmentation of the intact insulin-oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges...... were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin-oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached...

  12. Prolactin-secreting adenoma as part of the multiple endocrine neoplasia--type I (MEN-I) syndrome.

    Science.gov (United States)

    Levine, J H; Sagel, J; Rosebrock, G; Gonzalez, J J; Nair, R; Rawe, S; Powers, J M

    1979-06-01

    Two patients presented with the galactorrhea-amenorrhea syndrome. One patient had previously had parathyroid hyperplasia and the other an insulinoma. Preoperative evaluation of each patient revealed hyperprolactinemia and radiological evidence of an abnormal sella turcica. Pituitary adenomas were identified and removed at surgery. Immunostaining techniques confirmed the presence of prolactin-containing cells in both tumors. We propose that prolactin-secreting tumors be considered as part of the MEN-I syndrome, and that patients presenting with the galactorrhea-amenorrhea syndrome be screened and followed sequentially for evidence of other endocrine neoplasia.

  13. The serotonin transporter in rhesus monkey brain: comparison of DASB and citalopram binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Zeng Zhizhen [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)]. E-mail: zhizhen_zeng@merck.com; Chen, T.-B. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Miller, Patricia J. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Dean, Dennis [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Tang, Y.S. [Labeled Compound Synthesis Group, Drug Metabolism, Merck Research Laboratories, Rahway, NJ 07065-0900 (United States); Sur, Cyrille [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States); Williams, David L. [Imaging Department, Merck Research Laboratories, West Point, PA 19486 (United States)

    2006-05-15

    We have characterized the interaction of the serotonin transporter ligand [{sup 3}H]-N,N-dimethyl-2-(2-amino-4-cyanophenylthio)-benzylamine (DASB) with rhesus monkey brain in vitro using tissue homogenate binding and autoradiographic mapping. [{sup 3}H]-DASB, a tritiated version of the widely used [{sup 11}C] positron emission tomography tracer, was found to selectively bind to a single population of sites with high affinity (K {sub d}=0.20{+-}0.04 nM). The serotonin transporter density (B {sub max}) obtained for rhesus frontal cortex was found to be 66{+-}8 fmol/mg protein using [{sup 3}H]-DASB, similar to the B {sub max} value obtained using the reference radioligand [{sup 3}H]-citalopram, a well-characterized and highly selective serotonin reuptake inhibitor (83{+-}22 fmol/mg protein). Specific binding sites of both [{sup 3}H]-DASB and [{sup 3}H]-citalopram were similarly and nonuniformly distributed throughout the rhesus central nervous system, in a pattern consistent with serotonin transporter localization reported for human brain. Regional serotonin transporter densities, estimated from optical densities of the autoradiographic images, were well correlated between the two radioligands. Finally, DASB and fluoxetine showed dose-dependent full inhibition of [{sup 3}H]-citalopram binding in a competition autoradiographic study, with K {sub i} values in close agreement with those obtained from rhesus brain homogenates. This side-by-side comparison of [{sup 3}H]-DASB and [{sup 3}H]-citalopram binding sites in rhesus tissue homogenates and in adjacent rhesus brain slices provides additional support for the use of [{sup 11}C]-DASB to assess the availability and distribution of serotonin transporters in nonhuman primates.

  14. Study on the binding sites of radiosensitivity associated transcription factor in the promoter region of Ier5 gene

    International Nuclear Information System (INIS)

    Cui Wei; Yin Lingling; Dong Lingyue

    2012-01-01

    Objective: To clarify the mechanism of immediate early response gene 5 (Ier5) transcription induced by radiation. Methods: Deletant construction, site-specific mutagenesis,electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) were used to forecast the promoter region, binding sites and transcription factors of Ier5 gene in HeLa cells. Results: The promoter region of Ier5 gene might be in the region of Ier5 -8 deletant (-408 - -238 bp). The Ier5 gene had two transcription factors of GCF and NFI, and GCF had two binding sites located in the region of -388 - -382 bp and -274 - -270 bp of Ier5 promoter. The binding site of NFI was located in -362 - -357 bp of Ier5 promoter. GCF could inhibit the expression of Ier5 gene and this inhibition was diminished when the radiation dose increased. In contrast, NFI increased the expression of Ier5. Conclusions: The most possible region of Ier5 promoter is from -408 to -238 bp which has two binding sites for the radiosensitivity transcription factors of GCF and NFI that could negatively and positively regulate the expression of Ier5 respectively. (authors)

  15. Tannic acid and chromic chloride-induced binding of protein to red cells: a preliminary study of possible binding sites and reaction mechanisms.

    Science.gov (United States)

    Hunt, A F; Reed, M I

    1990-07-01

    The binding mechanisms and binding sites involved in the tannic acid and chromic chloride-induced binding of protein to red cells were investigated using the binding of IgA paraprotein to red cells as model systems. Inhibition studies of these model systems using amino acid homopolymers and compounds (common as red cell membrane constituents) suggest that the mechanisms involved are similar to those proposed for the conversion of hide or skin collagen to leather, as in commercial tanning. These studies also suggest that tannic acid-induced binding of IgA paraprotein to red cells involves the amino acid residues of L-arginine, L-lysine, L-histidine, and L-proline analogous to tanning with phenolic plant extracts. The amino acid residues of L-aspartate, L-glutamate and L-asparagine are involved in a similar manner in chronic chloride-induced binding of protein to red cells.

  16. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    International Nuclear Information System (INIS)

    Leff, S.E.; Creese, I.

    1985-01-01

    The interactions of dopaminergic agonists and antagonists with 3 H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [ 3 H]dopamine and [ 3 H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/[ 3 H]dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [ 3 H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [ 3 H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [ 3 H]dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/[ 3 H]flupentixol competition curves. Both D3 specific [ 3 H] dopamine binding and the high affinity agonist-binding component of dopamine/[ 3 H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor

  17. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

    Science.gov (United States)

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  18. EFFECT OF MORINGA OLEIFERA ON LEVEL OF PROLACTIN AND BREAST MILK PRODUCTION IN POSTPARTUM MOTHERS

    Directory of Open Access Journals (Sweden)

    Yuni Sulistiawati

    2017-04-01

    Full Text Available Background: Breastfeeding among postpartum mothers has been a problem due to low milk supply. As a result, mothers often decide to give formula milk or other additional foods, which might affect to the infant’s growth and development. Objective: This study aims to investigate the effect of Moringa Oliefera on the levels of prolactin and breast milk production (baby’s weight and sleep duration in postpartum mothers. Methods: Quasi-Experimental study with Non Equivalent control group design. There were 30 respondents recruited by purposive sampling, consisted of 15 respondents in intervention group and 15 respondents in the control group. This study was conducted from November until December 2016 in Four Midwive Independent Practice (BPM in the working area of the Health Center of Tlogosari wetan Semarang. Data were analyzed using Independent t-test. Results: Findings showed that there was a mean difference of prolactin level in the intervention group (231.72 ng / ml, and the control group (152.75 ng / ml; and a significant effect on increasing the levels of prolactin (p = 0.002. The mean of baby’s weight in the intervention group was 3783.33 grams, and in the control group was 3599.00 grams. However, there was no significant effect of moringa oleifera on baby’s weight (p = 0.313> 0.05. While the mean difference on sleep duration was 128.20 minutes in the intervention group and 108.80 minutes in the control group. There was a significant effect on baby’s sleep duration (p= 0.000. Conclusion: There were significant effects of moringa oleifera on mother’s prolactin and sleep duration of the baby. However, there was no significant effect on baby’s weight. Thus, it can be suggested that moringa oleifera can be used as an alternative treatment to increase breast milk production and prolactin hormones. Midwives should promote the benefits of moringa leaves as one of alternative supplements.

  19. Possible role of serum testosterone, gonadotropins and prolactin in patients with premature ejaculation.

    Science.gov (United States)

    Abu El-Hamd, M; Farah, A

    2018-02-01

    Premature ejaculation (PE) is the most common male sexual dysfunction. This study aimed to investigate the role of serum testosterone, gonadotropins and prolactin in patients with PE. In a prospective a case-controlled study, it was conducted on 90 male patients with PE and 90 male healthy participants as controls. Patients were evaluated by Premature Ejaculation Diagnostic Tool (PEDT) and intravaginal ejaculatory latency time (IELT). Patients with mean IELT values ≤60 s and PEDT total scores ≥11 were considered to have PE. Serum levels of total testosterone (TT), free testosterone (FT), follicle-stimulating hormone (FSH), luteinising hormone (LH) and prolactin (PL) were investigated in patients with PE and controls. There was no statistically significant difference between patients with PE and controls regarding the serum levels of TT, FT, FSH, LH and PL (p value ˃.05). There was no significant correlation between the sex hormones levels (TT, FT, FSH, LH and PL) and (age, body mass index (BMI), IELTS and total PEDT scores of the patients; p value ˃.05). This study concluded that there was no disturbance in serum levels of testosterone, gonadotropins and prolactin in patients with PE and controls. These hormones could not relate to pathogenesis of PE. © 2017 Blackwell Verlag GmbH.

  20. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Conroy, W.G.

    1988-01-01

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG 3 k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [ 3 H]naloxone. The antibody which did not inhibit the binding of [ 3 H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG 3 k antibody that blocked the binding of [ 3 H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form