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Sample records for inotropic effect binding

  1. Leukotriene C4 binds to receptors and has positive inotropic effects in bullfrog heart.

    PubMed

    Chiono, M; Heller, R S; Andazola, J J; Herman, C A

    1991-03-01

    Leukotriene (LT) C4, LTD4 and LTE4 have positive inotropic effects on contractility of the isolated perfused bullfrog heart. The effects of LTD4 and LTE4 but not LTC4 can be blocked by the mammalian antagonist L-649,923. Characterization of specific binding sites for [3H]LTC4 on membrane preparations from American bullfrog (Rana catesbeiana) ventricle was carried out. Binding assays were done in the presence of serine (5 mM) and borate (10 mM) for 30 min at 23 degrees C. Under these conditions, no metabolism of LTC4 to LTD4 occurred. Specific binding of [3H]LTC4 reached steady state within 10 min, remained constant for 60 min, and was reversible with the addition of 1000-fold excess unlabeled LTC4. Scatchard analysis of the binding data indicated a single class of binding sites with a Kd of 33.9 nM and maximal binding capacity of 51.6 pmol/mg of protein. Competition binding studies revealed an order of potency of LTC4 greater than LTD4 greater than LTE4 with Ki values of 47, 11766 and 32248 nM, respectively. Glutathione and hematin had Ki values of 50566 and 6014 nM, respectively, suggesting that the LTC4 receptor is not a site on glutathione transferase. Two mammalian LTD4 antagonists, L-649,923 and LY171883 failed to inhibit specific binding of [3H]LTC4, suggesting that the LTC4 receptor is distinct from the LTD4 receptor. Guanosine-5'-O-3-thiotriphosphate did not affect specific binding of [3H]LTC4 indicating that, like mammalian LTC4 receptors, a Gi protein is not involved in the transduction mechanism. LTC4 acts on bullfrog hearts through specific membrane receptors and is similar to its mammalian counterpart.

  2. Inotropic effect, binding properties, and calcium flux effects of the calcium channel agonist CGP 28392 in intact cultured embryonic chick ventricular cells

    SciTech Connect

    Laurent, S.; Kim, D.; Smith, T.W.; Marsh, J.D.

    1985-05-01

    CGP 28392 is a recently described dihydropyridine derivative with positive inotropic properties. To study the mechanism of action of this putative calcium channel agonist, we have related the effects of CGP 28392 on contraction (measured with an optical video system) and radioactive calcium uptake to ligand-binding studies in cultured, spontaneously beating chick embryo ventricular cells. CGP 28392 produced a concentration-dependent increase in amplitude and velocity of contraction (EC/sub 50/ = 2 x 10(-7) M; maximum contractile effect = 85% of the calcium 3.6 mM response). Nifedipine produced a shift to the right of the concentration-effect curve for CGP 28392 without decreasing the maximum contractile response, suggesting competitive antagonism (pA2 = 8.3). Computer analysis of displacement of (/sup 3/H)nitrendipine binding to intact heart cells by unlabeled CGP 28392 indicated a K /sub D/ = 2.2 +/- 0.95 x 10(-7) M, in good agreement with the EC/sub 50/ for the inotropic effect. CGP 28392 increased the rate of radioactive calcium influx (+39% at 10 seconds) without altering beating rate, while nifedipine decreased radioactive calcium influx and antagonized the CGP 28392-induced increase in calcium influx. Our results indicate that, in intact cultured myocytes, CGP 28392 acts as a calcium channel agonist and competes for the dihydropyridine-binding site of the slow calcium channel. In contrast to calcium channel blockers, CGP 28392 increases calcium influx and enhances the contractile state.

  3. Stimulation and inhibition of the sodium pump by cardioactive steroids in relation to their binding sites and their inotropic effect on guinea-pig isolated atria.

    PubMed

    Ghysel-Burton, J; Godfraind, T

    1979-06-01

    1 The actions of ouabain, ouabagenin and dihydroouabain on the contractility and on the ionic content have been investigated in left guinea-pig atria stimulated at 3.3 Hz. The specific binding of ouabain and its displacement by the other cardenolides have been determined. 2 The action of either ouabain or ouabagenin on Na and K content was qualitatively different according to the concentration employed. Low doses evoked a reduction of Nai whereas high doses produced an increase. Dihydroouabain evoked only a Nai gain. 3 The increase of KCl concentration from 2.7 to 12 mM decreased Nai in untreated atria and displaced ouabain dose-effect curves to the right. 4 ED50 values for the positive inotropic effect were lower than ED50 values for the inhibition of the pump and were not similarly affected by an increase in KCl concentration. 5 The specific binding of ouabain occurred at high and low affinity sites, related to Na pump stimulation and inhibition respectively. 6 The increase in KCl reduced the affinity of the two groups of sites for ouabain and increased the capacity of the high-affinity sites whereas the capacity of the other sites remained unchanged. 7 The results confirm the existence of two specific binding sites for ouabain in guinea-pig heart and suggest that the inhibition of the Na pump is not the only mechanism responsible for the positive inotropic effect of cardiac glycosides.

  4. Stimulation and inhibition of the sodium pump by cardioactive steroids in relation to their binding sites and their inotropic effect on guinea-pig isolated atria.

    PubMed Central

    Ghysel-Burton, J.; Godfraind, T.

    1979-01-01

    1 The actions of ouabain, ouabagenin and dihydroouabain on the contractility and on the ionic content have been investigated in left guinea-pig atria stimulated at 3.3 Hz. The specific binding of ouabain and its displacement by the other cardenolides have been determined. 2 The action of either ouabain or ouabagenin on Na and K content was qualitatively different according to the concentration employed. Low doses evoked a reduction of Nai whereas high doses produced an increase. Dihydroouabain evoked only a Nai gain. 3 The increase of KCl concentration from 2.7 to 12 mM decreased Nai in untreated atria and displaced ouabain dose-effect curves to the right. 4 ED50 values for the positive inotropic effect were lower than ED50 values for the inhibition of the pump and were not similarly affected by an increase in KCl concentration. 5 The specific binding of ouabain occurred at high and low affinity sites, related to Na pump stimulation and inhibition respectively. 6 The increase in KCl reduced the affinity of the two groups of sites for ouabain and increased the capacity of the high-affinity sites whereas the capacity of the other sites remained unchanged. 7 The results confirm the existence of two specific binding sites for ouabain in guinea-pig heart and suggest that the inhibition of the Na pump is not the only mechanism responsible for the positive inotropic effect of cardiac glycosides. PMID:465868

  5. Naloxone potentiates the inotropic effects of isoproterenol in vitro by a nonopiate receptor mechanism.

    PubMed

    Lechner, R B

    1992-11-01

    Naloxone potentiates the effects of adrenergic agonists when administered to hypovolemic dogs, and it has been assumed that this effect is due to naloxone's action at opiate receptors. To help determine the site and mechanism of this interaction, we administered naloxone and its "d" stereo-isomer (which does not bind to opiate receptors) to guinea pig papillary muscles in the presence and absence of pharmacologic (isoproterenol) and physiologic (treppe) inotropic stimulation. In control muscles and in rapidly paced muscles, naloxone was without significant inotropic effect. In the presence of isoproterenol, d- and l-naloxone exerted significant positive inotropic effects that were dose dependent. We conclude that, since both d- and l-naloxone potentiated the inotropic effects of isoproterenol and this was seen in the absence of opioids, naloxone may increase contractility by a nonopiate receptor-mediated mechanism.

  6. Importance of calcium in the inotropic effect of hyperosomotic agents, norepinephrine, paired electrical stimulation, and treppe.

    PubMed

    Willerson, J T; Crie, J S; Adcock, R C; Templeton, G H; Wildenthal, K

    1975-01-01

    The data obtained from these studies demonstrate that the inotropic effect of hyperosmolar mannitol and sucrose and of paired electrical stimulation is critically influenced by extracellular calcium concentration. The inotropic effect of norepinephrine is not prevented by maximal functional extracellular calcium concentrations. Inhibition of systolic calcium flux at the cell membrane by D600 does not prevent the inotropic effect of hyperosmolar mannitol or of paired electrical stimulation but it does prevent the inotropic effect of hyperosmolar intropic effect of treppe. Thus, intracellular calcium regulation appears to be of major importance in the inotropic effect in isolated cardiac muscle of mannitol and paired pacing while systolic calcium flux at the cell membrane appears to be of major importance in the inotropic effect of treppe.

  7. Evidence that the positive inotropic effects of the alkylxanthines are not due to adenosine receptor blockade.

    PubMed Central

    Collis, M. G.; Keddie, J. R.; Torr, S. R.

    1984-01-01

    We investigated the possibility that the positive inotropic effects of the alkylxanthines are due to adenosine receptor blockade. The potency of 8-phenyltheophylline, theophylline and enprofylline as adenosine antagonists was assessed in vitro, using the guinea-pig isolated atrium, and in vivo, using the anaesthetized dog. The order of potency of the alkylxanthines as antagonists of the negative inotropic response to 2-chloroadenosine in vitro, and of the hypotensive response to adenosine in vivo was 8-phenyltheophylline greater than theophylline greater than enprofylline. The order of potency of the alkylxanthines as positive inotropic and chronotropic agents in the anaesthetized dog was enprofylline greater than theophylline greater than 8-phenyltheophylline. The results of this study indicate that the inotropic effects of the alkylxanthines in the anaesthetized dog are not due to adenosine receptor blockade. PMID:6322898

  8. Relationship between theophylline uptake and inotropic effect in the guinea-pig heart

    PubMed Central

    Bellemann, P.; Scholz, H.

    1974-01-01

    positive inotropic effect of theophylline is partly due to an action of the drug on intracellular calcium binding or storage sites. However, the principal action of theophylline is assumed to be on the sarcolemma where it increases calcium influx from the extracellular space. This conclusion is based on the fact that the time courses of the increase in contractile force and of theophylline uptake into the cell were dissimilar. PMID:4451822

  9. Positive inotropic effect of Murraya koenigii (Linn.) Spreng extract on an isolated perfused frog heart.

    PubMed

    Shah, Kunal J; Juvekar, Archana R

    2006-06-01

    Ethanolic extract of fresh leaves of M. koenigii (MKEE) showed a dose dependent positive inotropic effect on isolated frog heart. The responses to MKEE (62.5-1000 microg) were not affected in either way by theophylline, imidazole, propranolol and sildenafil. The change in potassium and sodium concentration did not alter MKEE-induced positive inotropic effect. Lignocaine did not alter the responses to MKEE significantly. Responses to MKEE were significantly inhibited when calcium concentration was reduced to half (from 1.58 to 0.79 mM) and were significantly potentiated when calcium concentration was doubled (from 1.58 to 3.16 mM). Verapamil was found to inhibit the responses significantly. The results suggest that M. koenigii induced positive inotropic effect possibly by increasing availability of calcium from extra cellular sites.

  10. Induction by endogenous noradrenaline of an alpha 1-adrenoceptor-mediated positive inotropic effect in rabbit papillary muscles.

    PubMed Central

    Hattori, Y.; Takeda, Y.; Nakaya, H.; Kanno, M.

    1993-01-01

    1. The possible involvement of alpha 1-adrenoceptors in the inotropic and electrophysiological responses to endogenous noradrenaline released by tyramine was examined in rabbit papillary muscles. 2. A concentration-dependent positive inotropic effect was produced by tyramine. This effect of tyramine was not observed in muscles from rabbits pretreated with reserpine. 3. The positive inotropic effect of tyramine was greatly inhibited by propranolol, but not altered by prazosin. However, when beta-adrenoceptors were blocked by pretreatment with propranolol, tyramine still produced a positive inotropic effect, an effect which was antagonized by prazosin. 4. Tyramine caused a decrease in action potential duration (APD) and an increase in action potential amplitude in a concentration-dependent manner. Isoprenaline also produced the same electrophysiological effects. These electrophysiological effects of both agents were inhibited by propranolol. 5. When beta-adrenoceptors were blocked by propranolol, the observed prazosin-sensitive positive inotropic effect of tyramine was not accompanied by any change in APD. In contrast, APD was markedly prolonged by alpha 1-adrenoceptor stimulation with phenylephrine in the presence of propranolol, in association with the positive inotropic effect. 6. It is concluded that in rabbit papillary muscles, endogenous noradrenaline causes a positive inotropic effect predominantly mediated by beta-adrenoceptors, but can still evoke a positive inotropic effect through alpha 1-adrenoceptors when beta-adrenoceptor stimulation is eliminated. This suggests that the alpha 1-adrenoceptor-mediated positive intropic mechanism(s) may be masked by simultaneous activation of beta-adrenoceptors. In addition, this study indicates that APD prolongation is not involved in the alpha 1-adrenoceptor-mediated inotropic responses to endogenous noradrenaline. PMID:8401934

  11. Negative Inotropic Effects of Cytokines on the Heart Mediated by Nitric Oxide

    NASA Astrophysics Data System (ADS)

    Finkel, Mitchell S.; Oddis, Carmine V.; Jacob, Timothy D.; Watkins, Simon C.; Hattler, Brack G.; Simmons, Richard L.

    1992-07-01

    The direct effects of pro-inflammatory cytokines on the contractility of mammalian heart were studied. Tumor necrosis factor α, interleukin-6, and interleukin-2 inhibited contractility of isolated hamster papillary muscles in a concentration-dependent, reversible manner. The nitric oxide synthase inhibitor N^G-monomethyl-L-arginine (L-NMMA) blocked these negative inotropic effects. L-Arginine reversed the inhibition by L-NMMA. Removal of the endocardial endothelium did not alter these responses. These findings demonstrate that the direct negative inotropic effect of cytokines is mediated through a myocardial nitric oxide synthase. The regulation of pro-inflammatory cytokines and myocardial nitric oxide synthase may provide new therapeutic strategies for the treatment of cardiac disease.

  12. The negative inotropic effects of gaseous sulfur dioxide and its derivatives in the isolated perfused rat heart.

    PubMed

    Zhang, Quanxi; Meng, Ziqiang

    2012-03-01

    Epidemiological investigations have revealed that sulfur dioxide (SO(2) ) exposure is linked to cardiovascular diseases. The present study was designed to investigate the negative inotropic effects of gaseous SO(2) and its derivatives in the isolated perfused rat heart and the possible mechanisms involved in their effects. The results showed that both SO(2) and SO(2) derivatives elicited a negative inotropic effect in a dose-dependent manner, and SO(2) produced a higher negative effect than SO(2) derivatives. The mechanism of SO(2) -induced negative inotropic effects at low concentrations was different from that at high concentrations. At low concentrations, the mechanism of SO(2) -induced negative inotropic effects might occur through promoting the activities of protein kinase C (PKC), cycloxygenase, and cGMP, while the mechanism of SO(2) derivatives-induced effects might be related to the opening of ATP-sensitive K(+) (K(ATP) ) channel and the inhibition of Ca(2+) influx via L-type calcium-channel. At high concentrations, the mechanisms of SO(2) and SO(2) derivatives-induced negative inotropic effects were similar, which might be related to the K(ATP) channel and L-type calcium-channel as well as the possible alterations in PKC, cycloxygenase, and cGMP. Further work is needed to determine the relative contribution of each pathway in SO(2) -mediated inotropic effect.

  13. Resveratrol enhances the inotropic effect but inhibits the proarrhythmic effect of sympathomimetic agents in rat myocardium

    PubMed Central

    2017-01-01

    Background Resveratrol is a cardioprotective agent with known antiarrhythmic effects that has recently been shown to inhibit phosphodiesterase (PDE) enzyme activity. Thus, it is possible that resveratrol increases the inotropic effect of sympathomimetic agents, as PDE inhibitors do but, unlike other PDE inhibitors, its effect may not be accompanied by proarrhythmia due to its antiarrhythmic action. This work is aimed to test this hypothesis. Methods This is an “in vitro” concentration-response relationship study. The effects of noradrenaline, tyramine and isoproterenol, alone or in combination with either resveratrol or with the typical PDE inhibitor 3-isobutylmethylxantine (IBMX), were studied in electrically driven strips of right ventricle or in the spontaneously beating free wall of the right ventricle of rat heart in order to investigate inotropic or proarrhythmic effects respectively. Also, the effects of resveratrol or IBMX on the sinoatrial node rate were examined in the isolated right atria of rat heart. Results Resveratrol (10 µM and 100 µM) produces a leftward shift in the concentration-response curves for the contractile effects of noradrenaline, tyramine or isoproterenol and reduces the –log EC50 values of these three agents. IBMX produces similar effects. The spontaneous ventricular beating rate was increased by all three compounds, an effect that was further enhanced by the addition of IBMX. In contrast, resveratrol (100 µM) abolished the effects of these sympathomimetic agents on the ventricular rate. Resveratrol (1–100 µM) had no effect on the sinoatrial node rate, while IBMX produce a concentration dependent sinoatrial tachycardia. Discussion Taken together, the finding, indicate that resveratrol, like the PDE inhibitor IBMX enhances the contractile effects of sympathomimetic agents but, in contrast to IBMX, it does not enhance their proarrhythmic effect or produce sinoatrial tachycardia. This is most probably consequence of the

  14. Peptides from the N-terminal domain of chromogranin A (vasostatins) exert negative inotropic effects in the isolated frog heart.

    PubMed

    Tota, Bruno; Mazza, Rosa; Angelone, Tommaso; Nullans, Gerard; Metz-Boutigue, Marie-Hélène; Aunis, Dominique; Helle, Karen B

    2003-07-15

    The negative inotropic effects of synthetic peptides derived from the N-terminus of chromogranin A (CgA) were studied in an avascular model of the vertebrate myocardium, the isolated working frog heart (Rana esculenta). The peptides were frog and bovine CgA(4-16) and CgA(47-66), and bovine CgA(1-40) with (CgA(1-40SS)) and without an intact disulfide bridge (CgA(1-40SH)). Under basal cardiac conditions, four of the peptides caused a concentration-dependent negative inotropism that was comparable to the negative inotropy reported for human recombinant vasostatin I (CgA(1-78)) and bovine CgA(7-57). By comparison of the structural characteristics of the bovine and frog sequences with their minimally effective concentrations ranging from 68 to 125 nM of peptide, the results were consistent with the natural structure (CgA(17-38SS)) being essential for the negative inotropism. In addition, the partial sequences of the frog and bovine vasostatin I were effective in counteracting the characteristic positive inotropism exerted by isoproterenol (1 nM) at minimally effective concentrations ranging from 45 to 272 nM. Taken together, these results extend the first evidence for a cardiosuppressive role of the N-terminal domain of chromogranin A known for its co-storage with catecholamines in the sympathoadrenal system of vertebrates.

  15. Vasopressors and Inotropes in Sepsis.

    PubMed

    Stratton, Leeanne; Berlin, David A; Arbo, John E

    2017-02-01

    Vasopressor and inotropes are beneficial in shock states. Norepinephrine is considered the first-line vasopressor for patients with sepsis-associated hypotension. Dobutamine is considered the first-line inotrope in sepsis, and should be considered for patients with evidence of myocardial dysfunction or ongoing signs of hypoperfusion. Vasopressor and inotrope therapy has complex effects that are often difficult to predict; emergency providers should consider the physiology and clinical trial data. It is essential to continually reevaluate the patient to determine if the selected treatment is having the intended result.

  16. Diamide: positive inotropic effect in isolated atria and inhibition of Na+/Ca2+ exchange in cardiomyocytes.

    PubMed

    Antolini, M; Debetto, P; Trevisi, L; Luciani, S

    1991-02-01

    The influence of frequency of stimulation and external calcium on the positive inotropic response of guinea-pig left atria to diamide and the inhibitory action on Na+/Ca2+ exchange activity of rat cardiomyocytes by this oxidant of sulphhydryl groups have been investigated. Diamide (50-500 microM) induces a concentration-dependent positive inotropic effect which is more pronounced when atria are driven at 1.0 Hz rather than at 0.5 and 0.1 Hz, and are bathed in 2.72 mM rather than in 1.36 mM external calcium. A decrease in the positive inotropic effect at 35 degrees C with respect to 29 degrees C is also observed. In addition, diamide in positive inotropic concentrations (100-300 microM) significantly reduces Na+/Ca2+ exchange activity and cytoplasmic glutathione levels in adult rat cardiomyocytes. The thiol reducing agent dithiothreitol either reverses or prevents diamide effects both in isolated atria and cardiomyocytes, suggesting that the actions of diamide are correlated to its property to oxidize sulphhydryl groups to disulphides. In view of the functional importance of Na+/Ca2+ exchange in myocardial contractility, it is proposed that diamide may increase the heart force of contraction by an inhibition of the sarcolemmal Na+/Ca2+ exchange activity.

  17. Effects of nicardipine on coronary blood flow, left ventricular inotropic state and myocardial metabolism in patients with angina pectoris.

    PubMed

    Rousseau, M F; Vincent, M F; Cheron, P; van den Berghe, G; Charlier, A A; Pouleur, H

    1985-01-01

    The effects of intravenous nicardipine (2.5 mg) on the left ventricular (LV) inotropic state, LV metabolism, and coronary haemodynamics were analysed in 22 patients with angina pectoris. Measurements were made at fixed heart rate (atrial pacing), under basal state, and during a cold pressor test. After nicardipine, coronary blood flow and oxygen content in the coronary sinus increased significantly. The indices of inotropic state increased slightly, and the rate of isovolumic LV pressure fall improved. Myocardial oxygen consumption was unchanged despite the significant reduction in pressure-rate product, but LV lactate uptake increased, particularly during the cold pressor test. When nicardipine was administered after propranolol, the indices of inotropic state were unaffected. The lack of direct effect of nicardipine on LV inotropic state was further confirmed by intracoronary injection of 0.1 and 0.2 mg in a separate group of 10 patients. It is concluded that the nicardipine-induced coronary dilatation seems to improve perfusion and aerobic metabolism in areas with chronic ischaemia, resulting in reduced lactate production and augmented oxygen consumption.

  18. [Possible reasons for the variability of the inotropic insulin effect in papillary muscles of ground squirrel myocardium].

    PubMed

    Nakipova, O V; Chumaeva, L A; Andreeva, L A; Anufriev, A I; Kukushkin, N I

    2012-01-01

    The effects of insulin (0.1-50 nM) on isometric twitch force (0.1 to 1.0 Hz; 30 +/- 1 degree C; 1.8 mM Ca(2+)) were studied in right ventricular papillary muscles from active ground squirrels of different seasons (summer, n = 14; autumn, n = 16 and winter interbout, n = 16) in control conditions and after one-hour pretreatment of PM with 2 mkM nifedipine (an L-type Ca(2+)-channel inhibitor) and 1.0 mM orthovanadate (a tyrosine phosphatase inhibitor). In active animals of different seasonal periods insulin causes both positive and negative inotropic effects. At low frequencies (0.1-0.5 Hz), insulin of low concentrations (0.1-1.0 nM) induces a transient (within the first 20 min after application) positive effect (about 15-25%). Application of high hormone concentration (10 nM) in a low range of stimulation frequencies causes a biphasic effect (a small initial positive inotropic effect followed by a marked negative one). At frequencies above 0.5-Hz stimulation, insulin of 10 nM concentration causes presumably a negative inotropic effect. It was proposed that ICaL is possibly involved in the insulin-induced negative inotropy in ground squirrels hearts. Alteration of protein phosphorylation in tyrosine residues is known to be a major link in the mechanism of insulin action. We performed a study on orthovanadate action (a known inhibitor of tyrosine phosphatase) on the inotropic insulin effect. In the group of summer animals the pretreatment of papillary muscles with orthovanadate (100 mkM) does not change the negative inotropic effect of insulin in a low range of stimulation frequencies but almost completely removes this effect at stimulation frequencies above 0.3 Hz (n = 4). Nifedipine (1-1.5 hr pretreatment), a blocker of L-type calcium channel, reduces the inhibitory effect of insulin in autumn and winter animals, and on the contrary intensifies it in summer animals. This fact indicates that different mechanisms must be involved in insulin actions in animals of

  19. The negative inotropic effect of raised extracellular potassium and caesium ions on isolated frog atrial trabeculae.

    PubMed

    Chapman, R A; Rodrigo, G C

    1987-10-01

    The exposure of frog atrial trabeculae to Ringer solution containing an elevated K+ concentration, produces a depolarization of the membrane and a reduction of both the duration of the action potential and the strength of the heart beat. In voltage-clamped preparations, the effect of perfusion with K+-rich Ringer solution is threefold. First, a sustained inward current develops at the holding potential (-80 mV). Secondly, the contractions evoked by depolarizing clamp pulses are reduced: this effect which is greater upon the tonic phase of the contraction than the early phasic tension, is also seen to follow the addition of Cs+ ions to the bathing fluid; at equal concentrations K+ ions are the more effective. Thirdly, when measured with an ion-sensitive micro-electrode in ventricular trabeculae, the intracellular Na+ ion activity (aiNa) declines with a time course similar to the development of the negative inotropic effect. This suggests that the actions of raised [K+]o or [Cs+]o upon tension may be secondary to an effect on the movement of Na+ ions across the cell membrane, which by reducing aiNa may affect tension by way of the Na-Ca exchange.

  20. Amyloid beta peptide 22-35 induces a negative inotropic effect on isolated rat hearts

    PubMed Central

    Yousefirad, Neda; Kaygısız, Ziya; Aydın, Yasemin

    2016-01-01

    Evidences indicate that deposition of amyloid beta peptides (Aβs) plays an important role in the pathogenesis of Alzheimer disease. Aβs may influence cardiovascular system and ileum contractions. But the effect of amyloid beta peptide 22-35 (Aβ22-35) on cardiovascular functions and contractions of ileum has not been studied. Therefore, the present study aimed to investigate the possible effects of this peptide on isolated rat heart and ileum smooth muscle. Langendorff-perfused rat heart preparations were established. The hearts were perfused under constant pressure (60 mmHg) with modified Krebs-Henseleit solution. Aβ22-35 at doses of 1, 10 and 100 nM significantly decreased left ventricular developed pressure (LVDP; an index of cardiac contractility) and maximal rate of pressure development of left ventricle (+dP/dtmax; another index of cardiac contractility). This peptide at doses studied had no significant effect on heart rate, coronary flow, monophasic action potential amplitude (MAPamp), MAP duration at 90% repolarization (MAP90) and ileum contractions. We suggest that Aβ22-35 exerts a negative inotropism on isolated rat hearts with unchanged heart rate, coronary flow, MAPamp, MAP90 and smooth muscle contractility of ileum. PMID:28078053

  1. Mechanisms contributing to the cardiac inotropic effect of Na pump inhibition and reduction of extracellular Na

    PubMed Central

    1987-01-01

    Reduction of the transsarcolemmal [Na] gradient in rabbit cardiac muscle leads to an increase in the force of contraction. This has frequently been attributed to alteration of Ca movements via the sarcolemmal Na/Ca exchange system. However, the specific mechanisms that mediate the increased force at individual contractions have not been clearly established. In the present study, the [Na] gradient was decreased by reduction of extracellular [Na] or inhibition of the Na pump by either the cardioactive steroid acetylstrophanthidin or by reduction of extracellular [K]. Contractile performance and changes in extracellular Ca (sensed by double-barreled Ca-selective microelectrodes) were studied in order to elucidate the underlying basis for the increase in force. In the presence of agents that inhibit sarcoplasmic reticulum (SR) function (10 mM caffeine, 100-500 nM ryanodine), reduction of the [Na] gradient produced increases in contractile force similar to that observed in the absence of caffeine or ryanodine. It is concluded that an intact, functioning SR is not required for the inotropic effect of [Na] gradient reduction (at least in rabbit ventricle). However, this does not exclude a possible contribution of enhanced SR Ca release in the inotropic response to [Na] gradient reduction in the absence of caffeine or ryanodine. Acetylstrophanthidin (3-5 microM) usually leads to an increase in the magnitude of extracellular Ca depletions associated with individual contractions. However, acetylstrophanthidin can also increase extracellular Ca accumulation during the contraction, especially at potentiated contractions. This extracellular Ca accumulation can be suppressed by ryanodine and it is suggested that this apparent enhancement of Ca efflux is secondary to an enhanced release of Ca from the SR. Under conditions where Ca efflux during contractions is minimized (after a rest interval in the presence of ryanodine), acetylstrophanthidin increased both the rate and the

  2. Roselle Polyphenols Exert Potent Negative Inotropic Effects via Modulation of Intracellular Calcium Regulatory Channels in Isolated Rat Heart.

    PubMed

    Lim, Yi-Cheng; Budin, Siti Balkis; Othman, Faizah; Latip, Jalifah; Zainalabidin, Satirah

    2016-07-11

    Roselle (Hibiscus sabdariffa Linn.) calyces have demonstrated propitious cardioprotective effects in animal and clinical studies; however, little is known about its action on cardiac mechanical function. This study was undertaken to investigate direct action of roselle polyphenols (RP) on cardiac function in Langendorff-perfused rat hearts. We utilized RP extract which consists of 12 flavonoids and seven phenolic acids (as shown by HPLC profiling) and has a safe concentration range between 125 and 500 μg/ml in this study. Direct perfusion of RP in concentration-dependent manner lowered systolic function of the heart as shown by lowered LVDP and dP/dt max, suggesting a negative inotropic effect. RP also reduced heart rate (negative chronotropic action) while simultaneously increasing maximal velocity of relaxation (positive lusitropic action). Conversely, RP perfusion increased coronary pressure, an indicator for improvement in coronary blood flow. Inotropic responses elicited by pharmacological agonists for L-type Ca(2+) channel [(±)-Bay K 8644], ryanodine receptor (4-chloro-m-cresol), β-adrenergic receptor (isoproterenol) and SERCA blocker (thapsigargin) were all abolished by RP. In conclusion, RP elicits negative inotropic, negative chronotropic and positive lusitropic responses by possibly modulating calcium entry, release and reuptake in the heart. Our findings have shown the potential use of RP as a therapeutic agent to treat conditions like arrhythmia.

  3. The inotropic effect of nitric oxide on mammalian papillary muscle is dependent on the level of beta1-adrenergic stimulation.

    PubMed

    Reading, S A; Barclay, J K

    2002-06-01

    We tested the hypothesis that nitric oxide has a positive inotropic effect on mammalian cardiac muscle contractility and that this effect sums with the positive inotropic effect of beta1-adrenergic agonists when both are present. Feline right ventricular papillary muscles were stimulated to contract isometrically at 0.2 Hz in Krebs-Henseleit bicarbonate buffer (KREBS) gassed with 95% O2 and 5% CO2 (26 degrees C; pH 7.34). The nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine (SNAP, 10(-5) M), and the membrane permeable cGMP analog 8-bromoguanosine-3',5'-cyclophosphate sodium (Br-cGMP, 10(-5) M), significantly increased developed force by 13.3+/-1.5% (n = 11) and 7.8+/-2.8% (n = 7), respectively. SNAP, at 10(-5) M, significantly increased the force developed by papillary muscle treated with 10(-11) M or 10(-9) M dobutamine hydrochloride (a beta1-adrenergic agonist) (n = 25, 11.3+/-2.9% and 10.0+/-3.6%, respectively) when compared with the addition of KREBS (n = 27, 2.6+/-0.9% and 5.5+/-0.9%), but the increase was less than predicted by the sum of inotropic effects of SNAP and dobutamine. SNAP at 10(-5) M did not change developed force in muscles treated with 10(-7) M dobutamine but it significantly decreased developed force in muscles challenged with 10(-5) M dobutamine (n = 18, 29.3+/-5.0%) when compared with KREBS (n = 10, 41.5+/-6.8%). Similarly, 10(-4) M 8-bromo-adenosine cyclic 3',5'-hydrogen phosphate monosodium (a membrane permeable cAMP analog) increased developed force 14.9+/-3.3% and the addition of 10(-5) M Br-cGMP to those muscles significantly reduced developed force by 3.5%+/-1.1% (n = 7). Thus, the positive inotropic effect of NO decreased and ultimately became an attenuation as the level of beta1-adrenergic stimulation increased due at least in part, to an interaction between the cAMP and cGMP second messenger pathways.

  4. Ethanol extract of Lycopus lucidus elicits positive inotropic effect via activation of Ca2+ entry and Ca2+ release in beating rabbit atria.

    PubMed

    Cui, Hao Zhen; Oh, Hyun Cheol; Li, Xiang; Lee, Yun Jung; Cho, Kyung Woo; Kang, Dae Gill; Lee, Ho Sub

    2013-07-01

    Lycopus lucidus Turcz has been widely used as a traditional Oriental medicine (TOM) in Korea and China and prescribed for the enhancement of heart function. However, the precise effects have yet to be defined. The purpose of the present study was, therefore, to address whether the ethanol extract of Lycopus lucidus Turcz (ELT) has a positive inotropic effect. ELT-induced changes in atrial mechanical dynamics (pulse pressure, dp/dt, and stroke volume), and cAMP efflux were measured in perfused beating rabbit atria. Three active components, rosmarinic acid, betulinic acid, and oleanolic acid were identified in ELT by high performance liquid chromatography analysis. ELT increased atrial dynamics in a concentration-dependent manner without changes in atrial cAMP levels and cAMP efflux. The ELT-induced positive inotropic effect was blocked by inhibition of the L-type Ca(2+) channels and sarcoplasmic reticulum (SR). Inhibitors of β-adrenoceptors had no effect on the ELT-induced positive inotropic effect. The results suggest that ELT exerts a positive inotropic effect via activation of Ca(2+) entry through L-type Ca(2+) channel and Ca(2+) release from the SR in beating rabbit atria.

  5. Effects of inotropic stimulation on segmental left ventricular relaxation quantified by color kinesis.

    PubMed

    Carey, C F; Mor-Avi, V; Koch, R; Lang, R; Pérez, J E

    2000-06-15

    Although myocardial ischemia impairs left ventricular (LV) relaxation before contractile function, regional LV diastolic dysfunction is difficult to evaluate by conventional echocardiography. Because beta-adrenergic stimulation enhances myocardial relaxation, we sought to characterize segmental LV diastolic function (by color kinesis) during dobutamine stress echocardiography and compare it with independently assessed segmental systolic function. We studied 22 patients with suspected coronary artery disease with color kinesis by acquiring digital images with endocardial motion display throughout diastole. Quantification of LV segmental diastolic peak filling rate (SPFR, normalized to segmental end-diastolic area/s) was obtained at rest, low-dose, and peak dobutamine infusion in myocardial segments visualized from the short-axis and/or apical 4-chamber views. In patients with resting normal LV systolic function and a dobutamine-induced hypercontractile response (group I, n = 13 patients; 102 segments), progressive increases in SPFR (p <0.001) were seen in all segments. However, in LV segments with resting systolic wall motion abnormalities (group II, n = 9 patients; 74 segments) SPFR measured at rest was significantly lower than that in group I (p <0.005) and did not increase significantly in response to dobutamine. In both groups of patients, LV myocardial segments (n = 528; rest and after dobutamine)-systolic and quantitative diastolic function-were concordant in 84% and 77% as viewed from short-axis and apical views, respectively. Thus, segmental LV diastolic function can be measured with color kinesis at rest and after inotropic stimulation, allowing comparison with segmental systolic function during pharmacologic stress testing.

  6. Subcellular mechanisms underlying digitalis-induced arrhythmias: role of calcium/calmodulin-dependent kinase II (CaMKII) in the transition from an inotropic to an arrhythmogenic effect.

    PubMed

    Gonano, Luis Alberto; Petroff, Martín Vila

    2014-12-01

    Cardiotonic glycosides or digitalis are positive inotropes used in clinical practice for the treatment of heart failure, which also exist as endogenous ligands of the Na(+)/K(+) ATPase. An increase in the intracellular Ca2+ content mediates their positive inotropic effect, but has also been proposed as a trigger of life-threatening arrhythmias. Although the mechanisms involved in the positive inotropic effect of these compounds have been extensively studied, those underlying their arrhythmogenic action remain ill defined. Recent evidence has placed posttranslational modifications of the ryanodine receptor (RyR2), leading to arrhythmogenic Ca2+ release, in the centre of the storm. In this review we will examine, in depth, the mechanisms that generate the arrhythmogenic substrate, focussing on the role played by the RyR2 and how its CaMKII-dependent regulation may shift the balance from an inotropic to an arrhythmogenic Ca2+ release. Finally, we will provide evidence suggesting that stabilising RyR2 function could result in a potential new strategy to prevent cardiotonic glycoside-induced arrhythmias that could lead to a safer and more extensive use of these compounds.

  7. Inotropic changes in the left ventricle: the effect of changes in heart rate, aortic pressure and end-diastolic pressure

    PubMed Central

    Furnival, C. M.; Linden, R. J.; Snow, H. M.

    1970-01-01

    1. Under conditions where heart rate, mean aortic pressure and enddiastolic pressure in the left ventricle are held constant, the intravenous infusion of isoprenaline is accompanied by large changes in dP/dt max in the left ventricle. 2. Under similar conditions, during stepwise increments in the rate of infusion of isoprenaline the changes in dP/dt max (measured at a constant paced heart rate) were proportional to changes in the free (intrinsic) heart rate. It is concluded that dP/dt max is a quantitative index of inotropic changes in the left ventricle. 3. In comparison to dP/dt max, three other variables which have been used to indicate inotropic changes in the heart (peak pressure in the left ventricle, duration of systole and stroke work at constant end-diastolic pressure), were shown to be unreliable indices of inotropic changes. 4. Using dP/dt max to indicate inotropic changes, alteration in the heart rate while mean aortic pressure and end-diastolic pressure in the left ventricle were held constant, and in mean aortic pressure while heart rate ane end-diastolic pressure in the left ventricle were held constant, were each shown to be accompanied by small inotropic changes in the heart. 5. Under similar conditions, changes in end-diastolic pressure in the left ventricle alone were not accompanied by inotropic changes as indicated by dP/dt max. PMID:5501006

  8. Sensitivity Analysis of Vagus Nerve Stimulation Parameters on Acute Cardiac Autonomic Responses: Chronotropic, Inotropic and Dromotropic Effects

    PubMed Central

    Ojeda, David; Le Rolle, Virginie; Romero-Ugalde, Hector M.; Gallet, Clément; Bonnet, Jean-Luc; Henry, Christine; Bel, Alain; Mabo, Philippe; Carrault, Guy; Hernández, Alfredo I.

    2016-01-01

    Although the therapeutic effects of Vagus Nerve Stimulation (VNS) have been recognized in pre-clinical and pilot clinical studies, the effect of different stimulation configurations on the cardiovascular response is still an open question, especially in the case of VNS delivered synchronously with cardiac activity. In this paper, we propose a formal mathematical methodology to analyze the acute cardiac response to different VNS configurations, jointly considering the chronotropic, dromotropic and inotropic cardiac effects. A latin hypercube sampling method was chosen to design a uniform experimental plan, composed of 75 different VNS configurations, with different values for the main parameters (current amplitude, number of delivered pulses, pulse width, interpulse period and the delay between the detected cardiac event and VNS onset). These VNS configurations were applied to 6 healthy, anesthetized sheep, while acquiring the associated cardiovascular response. Unobserved VNS configurations were estimated using a Gaussian process regression (GPR) model. In order to quantitatively analyze the effect of each parameter and their combinations on the cardiac response, the Sobol sensitivity method was applied to the obtained GPR model and inter-individual sensitivity markers were estimated using a bootstrap approach. Results highlight the dominant effect of pulse current, pulse width and number of pulses, which explain respectively 49.4%, 19.7% and 6.0% of the mean global cardiovascular variability provoked by VNS. More interestingly, results also quantify the effect of the interactions between VNS parameters. In particular, the interactions between current and pulse width provoke higher cardiac effects than the changes on the number of pulses alone (between 6 and 25% of the variability). Although the sensitivity of individual VNS parameters seems similar for chronotropic, dromotropic and inotropic responses, the interacting effects of VNS parameters provoke

  9. The myosin activator omecamtiv mecarbil: a promising new inotropic agent.

    PubMed

    Nánási, Péter; Váczi, Krisztina; Papp, Zoltán

    2016-10-01

    Heart failure became a leading cause of mortality in the past few decades with a progressively increasing prevalence. Its current therapy is restricted largely to the suppression of the sympathetic activity and the renin-angiotensin system in combination with diuretics. This restrictive strategy is due to the potential long-term adverse effects of inotropic agents despite their effective influence on cardiac function when employed for short durations. Positive inotropes include inhibitors of the Na(+)/K(+) pump, β-receptor agonists, and phosphodiesterase inhibitors. Theoretically, Ca(2+) sensitizers may also increase cardiac contractility without resulting in Ca(2+) overload; nevertheless, their mechanism of action is frequently complicated by other pleiotropic effects. Recently, a new positive inotropic agent, the myosin activator omecamtiv mecarbil, has been developed. Omecamtiv mecarbil binds directly to β-myosin heavy chain and enhances cardiac contractility by increasing the number of the active force-generating cross-bridges, presumably without major off-target effects. This review focuses on recent in vivo and in vitro results obtained with omecamtiv mecarbil, and discusses its mechanism of action at a molecular level. Based on clinical data, omecamtiv mecarbil is a promising new tool in the treatment of systolic heart failure.

  10. The cardioprotective and inotropic components of the postconditioning effects of GLP-1 and GLP-1(9-36)a in an isolated rat heart.

    PubMed

    Ossum, Alvilde; van Deurs, Ulla; Engstrøm, Thomas; Jensen, Jan Skov; Treiman, Marek

    2009-11-01

    GLP-1 and its metabolite GLP-1(9-36)a have been shown to exert cardiotropic effects, and were demonstrated to be cardioprotective agents in isolated, postischemic rat or mouse hearts. An agent's total effect on myocardial performance in a postconditioning paradigm is a sum of its myocyte-preserving (cardioprotective) and contractility-affecting (negative or positive inotropic) action components. These components may not always be explicitly separated by the experimental protocol. We propose an analytical approach to identify and quantify the cardioprotective and inotropic components in a postconditioning protocol, as exemplified by use of GLP-1 and GLP-1(9-36)a following a global ischemia in isolated rat hearts. Peptides were administered during the first 15min of 120min reperfusion. GLP-1 0.3nM reduced infarct size from 23.2+/-2.4% to 14.1+/-2.3% of area-at-risk (n=15, P=0.0223), an effect abolished by the GLP-1 receptor antagonist, exendin(9-39) 5nM. GLP-1 showed only a small, non-significant tendency to increase mechanical performance (increase of LVDP by 26.7%, P=0.1621; RPP 33.5%, P=0.0858; dP/dt(max) 28.5%, P=0.1609). This could be accounted for by the cardioprotective component of GLP-1 action, rather than any true inotropic effect. In contrast, GLP-1(9-36)a did not reduce infarct size significantly, but acted as a strong negative inotrope in postischemic hearts, causing a contractility deficit (LVDP 58.8%, P=0.0004; RPP 58.2%, P=0.0007; dP/dt(max)=58.2%, P=0.0012), quantifiable by an analysis of infarct size-mechanical performance plots. These results help resolve certain apparent discrepancies between some of the published effects of GLP-1 and GLP-1(9-36)a.

  11. Negative inotropic effect of methylecgonidine, a major product of cocaine base pyrolysis, on ferret and human myocardium.

    PubMed

    Woolf, J H; Huang, L; Ishiguro, Y; Morgan, J P

    1997-09-01

    This study examined the physiological effects and potential mechanisms of action of methylecgonidine (MEG), the major pyrolysis product from smoking "crack cocaine," on cardiac function. Ferret right ventricular papillary muscles and human ventricular trabeculae were isolated and placed in a physiological solution at 30 degrees C containing 2.5 mM Ca2+ and stimulated at 0.33 Hz. MEG decreased peak tension and peak intracellular Ca2+ transients in a concentration-dependent manner (10 microM-1 mM). The negative inotropic effect (NIE) of MEG was reversible by atropine (1 microM). Atropine shifted the concentration-response curve of MEG rightward (pA2 = 9.17) similar to that of carbachol (pA2 = 8.70). With prior addition of histamine (1 microM) and Ca2+ (4.5 mM) in equiinotropic concentrations, MEG and carbachol decreased contractility to a greater extent in the histamine-stimulated muscles. To clarify whether the treatments altered responsiveness of the contractile elements to Ca2+, the effect of 2,3-butanedione monoxime (BDM), an agent that interferes with the interaction of actin and myosin, was tested after prior addition of histamine or increased Ca2+. No differential effect occurred. Moreover, the nitric oxide synthase inhibitor NG-nitro-L-arginine methylester (L-NAME; 0.1 mM), lessened the NIE of MEG compared with prior (pre-L-NAME) values. Furthermore, in human ventricular trabeculae (n = 7), MEG exhibited an NIE that was also reversible by atropine. We concluded that the NIE of MEG is caused by decreased calcium availability; the effect is not the result of a local anesthetic action but is mediated by stimulation of cholinergic receptors. This effect is potentiated by the nitric oxide pathway.

  12. Negative inotropic and chronotropic effects on the guinea pig atrium of extracts obtained from Averrhoa carambola L. leaves.

    PubMed

    Vasconcelos, C M L; Araújo, M S; Silva, B A; Conde-Garcia, E A

    2005-07-01

    It has been reported that star fruit can lead to a fatal outcome in uremic patients. The intoxication syndrome consists of hiccups, mental confusion, dizziness, and vomiting. On the other hand, folk medicine uses teas and infusions of carambola leaves to treat headache, vomiting, cough, insomnia, and diabetes. This motivated us to determine if Averrhoa carambola can act on the contractility and automaticity of the guinea pig heart. We measured the atrial isometric force in stimulated left atria and determined the chronotropic changes in spontaneously beating right atria. The carambola leaf extracts (1.5 mg/ml) abolished the contractile force in a concentration-dependent manner. Among the crude, methanolic, ethanolic, aqueous, and acetic extracts, the aqueous one was the most potent (EC50 = 520 +/- 94 microg/ml; flavonoids and tannins are the main constituents; Na+ and K+ contents in 1.0 mg/ml of aqueous extract were 0.12 +/- 0.016 and 1.19 +/- 0.15 mM, respectively). The aqueous extract abolished the positive Bowditch staircase phenomenon and reduced the inotropic response to CaCl2 (0.17-8.22 mM), events that are dependent on the cellular Ca2+ inward current. The adrenergic, muscarinic or opioid membrane receptors do not seem to participate in the mechanism of action of the cardioactive substance(s). In spontaneously beating atria, the aqueous extract promoted a negative chronotropic effect that was antagonized by 0.1 microM isoproterenol bitartrate. With this agonist, the EC50 of the aqueous extract increased from 133 +/- 58 to 650 +/- 100 microg/ml. These data regarding the effect of A. carambola on guinea pig atrial contractility and automaticity indicate an L-type Ca2+ channel blockade.

  13. Cardiovascular effects of a new positive inotropic agent, (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]-ethanol (TA-064) in the anesthetized dog and isolated guinea pig heart.

    PubMed

    Nagao, T; Ikeo, T; Murata, S; Sato, M; Nakajima, H

    1984-08-01

    The positive inotropic effect of TA-064, (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol, was studied in the anesthetized dog and isolated guinea pig heart. An intravenous administration of TA-064 dose-dependently increased the cardiac contractile force with little effect on blood pressure in dogs. The positive inotropic activity of TA-064 was 1/100 that of isoproterenol and similar to that of dobutamine. This effect of TA-064 was stereospecific, and it was blocked by practolol. Thus TA-064 has beta 1-adrenoceptor agonistic action. The positive inotropic effect of TA-064 was more pronounced than the positive chronotropic effect, compared with those of isoproterenol. Similar effect of TA-064 was observed in the reserpinized dog and in the isolated perfused heart of the guinea pig as well. TA-064 administered intraduodenally at a dose of 0.1 mg/kg increased contractile force by 120% of the control, and the effect lasted for more than 150 min. TA-064 given in the femoral artery demonstrated a very weak vasodilating effect on the artery. TA-064 is an orally active, positive inotropic agent. The selective positive inotropic action of TA-064 may result from its beta 1-adrenoceptor agonistic property.

  14. Effect of cephalic carbon dioxide tension on the cardiac inotropic response to carotid chemoreceptor stimulation in dogs.

    PubMed Central

    Hainsworth, R; Rankin, A J; Soladoye, A O

    1985-01-01

    Dogs were anaesthetized with chloralose and the cephalic circulation was perfused, through the brachiocephalic and left subclavian arteries, with blood equilibrated with various tensions of CO2. The vascularly isolated carotid bifurcations were perfused at a constant pressure with either arterial or venous blood. Inotropic responses were assessed by measuring the maximum rate of change of left ventricular pressure (dP/dt max) with heart rate and aortic pressure held constant. Stimulation of carotid chemoreceptors with venous blood, at all values of cephalic PCO2, always resulted in a decrease in dP/dt max. An increase in cephalic PCO2, during arterial perfusion of chemoreceptors, resulted in an increase in dP/dt max and the response to chemoreceptor stimulation was enhanced. Graded changes in cephalic PCO2 resulted in graded changes in dP/dt max during arterial perfusion of chemoreceptors. However, the value of dP/dt max during venous perfusion was not significantly affected by increases in cephalic PCO2 above normal but it did decrease significantly during cephalic hypocapnia. These results confirm that an increase in cephalic PCO2 and stimulation of carotid chemoreceptors result in opposite responses of the cardiac inotropic state. The responses to chemoreceptor stimulation were enhanced by cephalic hypercapnia but the responses to cephalic hypercapnia, although not to hypocapnia, were suppressed by chemoreceptor stimulation. PMID:3920387

  15. Roles of PKA, PI3K, and cPLA2 in the NO-mediated negative inotropic effect of beta2-adrenoceptor agonists in guinea pig right papillary muscles.

    PubMed

    Faucher, Fabien A; Gannier, François E; Lignon, Jacques M; Cosnay, Pierre; Malécot, Claire O

    2008-01-01

    Although beta(2)-adrenoceptors represent 15-25% of beta-adrenoceptors in the guinea pig heart, their functionality is controversial. We assessed the inotropic effects of beta(2)-adrenoceptor partial agonists in right papillary muscles. Salbutamol induced a small but significant concentration-dependent negative inotropic effect (NIE, -5% at 60 nM) followed by a moderate positive inotropic effect (+36% at 6 microM) due to activation of beta(1)-adrenoceptors. In the presence of 4 microM atenolol, the concentration-dependent NIE (-12% at 6 microM) was biphasic, best described by a double logistic equation with respective EC(50) values of 3 and approximately 420 nM, and was insensitive to SR59230A. In muscles from pertussis toxin-treated guinea pigs, the salbutamol-induced positive inotropic effect was sensitive to low concentrations of ICI-118551 in an unusual manner. Experiments in reserpinized animals revealed the importance of the phosphorylation-dephosphorylation processes. PKA inhibition reduced and suppressed the effects obtained at low and high concentrations, respectively, indicating that its activation was a prerequisite to the NIE. The effect occurring at nanomolar concentrations depended upon PKA/phosphatidylinositol 3-kinase/cytosolic phospholipase A(2) (cPLA(2)) activations leading to nitric oxide (NO) release via the arachidonic acid/cyclooxygenase pathway. NO release via PKA-dependent phosphorylation of the receptor was responsible for the inotropic effect observed at submicromolar concentrations, which is negatively controlled by cPLA(2). The possibility that these effects are due to an equilibrium between different affinity states of the receptor (G(s)/G(i) coupled and G(i) independent with different signaling pathways) that can be displaced by ICI-118551 is discussed. We conclude that beta(2)-adrenoceptors are functional in guinea pig heart and can modulate the inotropic state.

  16. The cardiovascular pharmacology of ICI 170777 ((6RS)-6-methyl-5-(pyrid-4-yl)-3H,6H-1,3,4- thiadiazin-2-one) a novel compound with positive inotropic and vasodilator effects.

    PubMed Central

    Collis, M. G.; Keddie, J. R.; Rouse, W.

    1989-01-01

    1. This paper describes the cardiovascular effects of ICI 170777, a novel compound which enhances cardiac contractility and causes arterial and venous dilatation. 2. The positive inotropic effects of ICI 170777 on the heart were demonstrated by an increase in left ventricular dP/dtmax in the anaesthetized and conscious dog, and by an increase in tension development in isolated papillary muscles from the cat. 3. In the anaesthetized dog, the positive inotropic effects of ICI 170777 and of isoprenaline were attenuated by atenolol (5 mg kg-1, i.v.). Atenolol displaced the dose-response curve to ICI 170777 to the right by 4 fold but displaced the isoprenaline dose-response curve to the right by 247 fold. In vitro, however, atenolol (10 microM) had no significant effect on the positive inotropic response to ICI 170777. In the ganglion-blocked anaesthetized dog, infusion of a low dose of ICI 170777 which had no effect on the basal left ventricular dP/dtmax, selectively potentiated the positive inotropic effects of isoprenaline. These results indicate that ICI 170777 has both a non-adrenoceptor-mediated positive inotropic effect on the heart and also facilitates the beta-adrenoceptor-mediated control of contractility. 4. In the denervated and perfused hind-limb of the dog, ICI 170777 reduced arterial perfusion pressure and increased limb circumference at a constant arterial flow and venous pressure. This indicates that ICI 170777 has direct dilator actions on both arterial and venous vessels. In this preparation, diazoxide exerted an arterial selective vasodilator effect and sodium nitroprusside was a relatively selective venous dilator.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2758224

  17. Negative inotropic action of denbufylline through interfering with the calcium channel independently of its PDE IV inhibitory activity in guinea pig ventricle papillary muscles.

    PubMed

    Sanae, F; Ohmae, S; Kobayashi, D; Takag, K; Miyamoto, K

    1996-04-01

    The inotropic actions of xanthine derivatives with long alkyl chains were investigated in guinea pig ventricular papillary muscle. A potent and nonselective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine, elicited a positive inotropy and inhibited the negative inotropic effects of calcium channel inhibitors, as did a selective PDE III inhibitor, amrinone, and these effects were canceled by a protein kinase inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89). However, 1,3-di-n-butyl-7-(2'oxopropyl)xanthine (denbufylline) and 1-n-butyl-3-n-propylxanthine (XT-044), which have potent and selective PDE IV-inhibitory activities, showed negative inotropic actions that became more potent in the presence of H-89. Denbufylline abolished the late restoration phase induced by ryanodine. This xanthine derivative attenuated the effects of both the calcium channel acting agents Bay K 8644 and verapamil, without interaction with caffeine and dihydropyridine calcium channel inhibitors, and denbufylline had little direct influence on the specific binding of [(3)H]azidopine and [(3)H]desmethoxyverapamil to cardiac membranes. A nonxanthine PDE IV inhibitor, Ro 20-1724, did not affect the inotropic actions of calcium channel inhibitors. The attenuation by denbufylline or XT-044 of the negative inotropic action of verapamil was not influenced by treatment with H-89. These results suggest that in the ventricular papillary muscle, these xanthine derivatives elicit negative inotropy by acting on a verapamil-sensitive site of the calcium channel without involving their PDE-inhibitory activity.

  18. Effects of a new positive inotropic agent, 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)- quinolinone (OPC-8212) and its solvent sulfolane on isolated heart preparations of the rat, guinea pig, and dog.

    PubMed

    Grupp, G; Grupp, I L; Newman, G; Schwartz, A

    1984-01-01

    3,4-Dihydro-6-[4-(3,4- dimethoxybenzoyl )-1-piperazinyl]-2(1H)- qu inolinone ( OPC -8212) has positive inotropic effects in guinea pig and dog isolated heart preparations. The rat heart ventricle does not respond to OPC -8212 but the atria do. OPC -8212 produces positive inotropic effects in the rat heart ventricle made hypodynamic by pretreatment with calcium entry blockers. Sulfolane , the solvent of OPC -8212, produced negative inotropic effects in most preparations. In isolated heart preparations of the rat and guinea pig, OPC -8212 produced increased coronary flow; sulfolane had only minor effects.

  19. Exenatide exerts a PKA-dependent positive inotropic effect in human atrial myocardium: GLP-1R mediated effects in human myocardium.

    PubMed

    Wallner, Markus; Kolesnik, Ewald; Ablasser, Klemens; Khafaga, Mounir; Wakula, Paulina; Ljubojevic, Senka; Thon-Gutschi, Eva Maria; Sourij, Harald; Kapl, Martin; Edmunds, Nicholas J; Kuzmiski, J Brent; Griffith, David A; Knez, Igor; Pieske, Burkert; von Lewinski, Dirk

    2015-12-01

    Glucagon-like peptide-1 receptor (GLP-1R) agonists are a rapidly growing class of drugs developed for treating type-2 diabetes mellitus. Patients with diabetes carry an up to 5-fold greater mortality risk compared to non-diabetic patients, mainly as a result of cardiovascular diseases. Although beneficial cardiovascular effects have been reported, exact mechanisms of GLP-1R-agonist action in the heart, especially in human myocardium, are poorly understood. The effects of GLP-1R-agonists (exenatide, GLP-1(7-36)NH2, PF-06446009, PF-06446667) on cardiac contractility were tested in non-failing atrial and ventricular trabeculae from 72 patients. The GLP-1(7-36)NH2 metabolite, GLP-1(9-36)NH2, was also examined. In electrically stimulated trabeculae, the effects of compounds on isometric force were measured in the absence and presence of pharmacological inhibitors of signal transduction pathways. The role of β-arrestin signaling was examined using a β-arrestin partial agonist, PF-06446667. Expression levels were tested by immunoblots. Translocation of GLP-1R downstream molecular targets, Epac2, GLUT-1 and GLUT-4, were assessed by fluorescence microscopy. All tested GLP-1R-agonists significantly increased developed force in human atrial trabeculae, whereas GLP-1(9-36)NH2 had no effect. Exendin(9-39)NH2, a GLP-1R-antagonist, and H-89 blunted the inotropic effect of exenatide. In addition, exenatide increased PKA-dependent phosphorylation of phospholamban (PLB), GLUT-1 and Epac2 translocation, but not GLUT-4 translocation. Exenatide failed to enhance contractility in ventricular myocardium. Quantitative real-time PCR (qRT-PCR) revealed a significant higher GLP-1R expression in the atrium compared to ventricle. Exenatide increased contractility in a dose-dependent manner via GLP-1R/cAMP/PKA pathway and induced GLUT-1 and Epac2 translocation in human atrial myocardium, but had no effect in ventricular myocardium. Therapeutic use of GLP-1R-agonists may therefore impart

  20. Intracellular cAMP increases during the positive inotropism induced by androgens in isolated left atrium of rat.

    PubMed

    Velasco, Lucía; Sánchez, Manuel; Rubín, José Manuel; Hidalgo, Agustín; Bordallo, Carmen; Cantabrana, Begoña

    2002-03-01

    Molecular interactions of androgens with the plasma membrane may produce rapid cardiovascular effects that cannot be explained by the classic genomic mechanisms. In this sense, 5 alpha- and 5 beta-dihydrotestosterone-induced an acute positive inotropic effect in isolated left atrium of rat, an effect which may be due to cAMP-dependent mechanisms. To prove this, intracellular levels of cAMP, after exposure to androgens in the organ bath, and binding to beta(1)-adrenoceptors were evaluated. After a 4-min exposure, 5 alpha- and 5 beta-dihydrotestosterone increased cAMP levels from 3.83+/-0.61 to 6.15+/-1.1 and 11.18+/-2.4 pmol cAMP/mg of protein, respectively. These increases were inhibited by atenolol and not modified by treatment of the rats with reserpine. The androgen-induced cAMP increase seems to be produced via an extracellular interaction, because positive inotropism and raised levels of cAMP were produced by 5 alpha-dihydrotestosterone conjugated with bovine serum albumin (BSA). In addition, it is independent of beta(1)-adrenoceptor activation, because neither androgen displaced [(3)H]dihydroalprenolol binding. Therefore, the androgens induced a positive inotropic effect via a postsynaptic effect that increases intracellular levels of cAMP. This effect is modulated by transcriptional mechanisms or by a protein with a short half-life.

  1. [Vasopressors and inotropes: use in pediatrics].

    PubMed

    García-Canales, Adrián; Peña-Juárez, Rocío Alejandra; Sandoval-Franco, Luz de María

    2017-03-20

    The cardiovascular system is a dynamic system whose function is to ensure adequate delivery of oxygen, nutrients and hormones to the tissues, necessary for cell metabolism, also synthesizes and modifies vasoactive components to regulate vascular tone and myocardial function. These components have become critical in the management of pediatric patients in critical condition with heart failure and shock which has proven its beneficial effects, however its use and abuse brings harmful effects, increase mortality, associated with arrhythmias and increase myocardial oxygen consumption favoring the presence of ischemia, therefore is necessary to know the mechanism of action and indications of these drugs to minimize the harmful effects. The purpose of this review is to describe the pharmacology and clinical applications of inotropic and vasopressor agents in the pediatric patient in critical condition.

  2. Crucial role of cytoskeleton reorganization in the negative inotropic effect of chromogranin A-derived peptides in eel and frog hearts.

    PubMed

    Mazza, Rosa; Mannarino, Cinzia; Imbrogno, Sandra; Barbieri, Sandra Francesca; Adamo, Cristina; Angelone, Tommaso; Corti, Angelo; Tota, Bruno

    2007-02-01

    Vasostatins (VSs), i.e. the main biologically active peptides generated by the proteolytic processing of chromogranin A (CGA) N-terminus, exert negative inotropism in vertebrate hearts. Here, using isolated working eel (Anguilla anguilla) and frog (Rana esculenta) heart preparations, we have studied the role of the cytoskeleton in the VSs-mediated inotropic response. In both eel and frog hearts, VSs-mediated-negative inotropy was abolished by treatment with inhibitors of cytoskeleton reorganization, such as cytochalasin-D (eel: 10 nM; frog: 1 nM), an inhibitor of actin polymerisation, wortmannin (0.01 nM), an inhibitor of PI3-kinase (PI3-K)/protein kinase B (Akt) signal-transduction cascade, butanedione 2-monoxime (BDM) (eel: 100 nM; frog: 10 nM), an antagonist of myosin ATPase, and N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide (W7) (eel: 100 nM; frog: 1 nM), a calcium-calmodulin antagonist. These results demonstrate that changes in cytoskeletal dynamics play a crucial role in the negative inotropic influence of VSs on eel and frog hearts.

  3. Assessing cardiac pumping capability by exercise testing and inotropic stimulation.

    PubMed Central

    Tan, L B; Bain, R J; Littler, W A

    1989-01-01

    In heart failure both functional capacity and prognosis are primarily determined by the degree of pump dysfunction. Although data on haemodynamic function at rest may indicate impaired cardiac function, they do not assess the capacity of the heart to respond to stress. Maximal bicycle ergometry and incremental intravenous inotropic stimulation in 31 patients with moderately severe heart failure were evaluated as methods of stressing the heart to determine cardiac pumping capability, which is defined as the cardiac power obtained during maximal stimulation. There was good agreement between the cardiac pumping capabilities assessed by these two methods. Maximal cardiac power output was better than maximal cardiac output and left ventricular stroke work index in representing cardiac pumping capability, because it was less dependent on the type of stimulation used during evaluation. Inotropic challenge is at least as effective as exercise testing in assessing cardiac pumping capability in heart failure, and may be a better method in patients who find physical exercise difficult. PMID:2757870

  4. Pharmacology and inotropic potential of forskolin in the human heart.

    PubMed Central

    Bristow, M R; Ginsburg, R; Strosberg, A; Montgomery, W; Minobe, W

    1984-01-01

    We evaluated the effects of the diterpene compound forskolin in human myocardial adenylate cyclase preparations, isolated trabeculae and papillary muscles derived from failing human hearts, and acutely instrumented dogs. Forskolin was a potent, powerful activator of human myocardial adenylate cyclase and produced maximal effects that were 4.82 (normally functioning left ventricle) and 6.13 (failing left ventricle) fold greater than isoproterenol. In contrast to isoproterenol, forskolin retained full activity in membrane preparations derived from failing hearts. In cyclase preparations, forskolin demonstrated unique substrate and Mg2+ kinetic properties that could be distinguished from hormone receptor-coupled agonists or fluoride ion. The adenylate cyclase stimulatory effect of forskolin was synergistic with isoproterenol, apparently due to the location of forskolin activation being beyond the level of hormone receptor-agonist in the receptor-cyclase complex. Forskolin was a potent positive inotrope in failing human myocardium, producing a stimulation of contraction that was similar to isoproterenol. Finally, in open chest dogs forskolin was a positive inotropic agent that reduced preload and afterload. We conclude that forskolin belongs to a class of agents that may have therapeutic potential in the treatment of congestive heart failure. Images PMID:6330174

  5. Use of inotropes and vasopressor agents in critically ill patients

    PubMed Central

    Bangash, Mansoor N; Kong, Ming-Li; Pearse, Rupert M

    2012-01-01

    Inotropes and vasopressors are biologically and clinically important compounds that originate from different pharmacological groups and act at some of the most fundamental receptor and signal transduction systems in the body. More than 20 such agents are in common clinical use, yet few reviews of their pharmacology exist outside of physiology and pharmacology textbooks. Despite widespread use in critically ill patients, understanding of the clinical effects of these drugs in pathological states is poor. The purpose of this article is to describe the pharmacology and clinical applications of inotropic and vasopressor agents in critically ill patients. LINKED ARTICLES This article is commented on by Bracht et al., pp. 2009–2011 and De Backer and Scolletta, pp. 2012–2014 of this issue. To view Bracht et al. visit http://dx.doi.org/10.1111/j.1476-5381.2011.01776.x and to view De Backer and Scolletta visit http://dx.doi.org/10.1111/j.1476-5381.2011.01746.x PMID:21740415

  6. Naloxone modifies the inotropic decrease induced by halothane on isolated left atria.

    PubMed

    Laorden, M L; Carceles, M D; Miralles, F S; Hernandez, J

    1991-01-01

    1. The present study evaluates the interaction between naloxone and halothane on the left atria. 2. Halothane produced significant decrease in auricular inotropism at concentrations ranging from 0.3 to 2.5 v/v%. 3. Naloxone modified the concentration response curve to halothane. The antagonism between both drugs is consistent with a competitive antagonism since the curve obtained had a slope which did not differ significantly from -1. 4. These results suggest that the negative inotropic effects induced by halothane could be mediated by opioid receptors.

  7. An analysis of the mechanism of the inotropic action of some milrinone analogues in guinea-pig isolated atria.

    PubMed Central

    Dorigo, P.; Gaion, R. M.; Belluco, P.; Mosti, L.; Borea, P. A.; Maragno, I.

    1991-01-01

    1. It has been reported previously that the milrinone analogues, ethyl 5-cyano-1,6-dihydro-2-methyl-6-oxo-3 pyridine carboxylate (I) and ethyl 5-cyano-1,6-dihydro-2-ethyl-6-oxo-3 pyridine carboxylate (II) exert a positive inotropic effect (EC50 = 15.6 +/- 0.2 microM and 40.3 +/- 0.1 microM) both on spontaneously beating and on electrically driven atria from reserpine-treated guinea-pigs. In the present study the mechanism of the inotropic action of these two agents was investigated. 2. In electrically driven left atrium from reserpine-treated guinea-pigs the EC50 values for inotropic activity for compounds (I) and (II) corresponded to that of milrinone (EC50 = 25 +/- 0.1 microM) but compound (I) induced a greater maximum effect. This corresponded to a percentage increase in developed tension over control of 63 +/- 0.3 whereas the maximum inotropic effect of milrinone was 48 +/- 0.3 and that of compound (II) was 47 +/- 0.2. 3. The inotropic activity of compounds (I) and (II) (10-100 microM) was resistant to propranolol (0.1 microM), thus excluding the involvement of beta-adrenoceptors. 4. Since the inotropism induced by compounds (I) and (II) was not reduced by carbachol (1 nM-0.5 microM), an action involving changes in adenosine 3':5'-cyclic monophosphate (cyclic AMP) can be excluded. 5. The inotropic action of compounds (I) and (II) was blocked selectively by 8-phenyltheophyline (10 microM) or adenosine deaminase (2 u ml-1). 6. Both (I) and (II) inhibited, in an apparently competitive manner, the negative inotropic effect induced by N6-(L-phenylisopropyl) adenosine (L-PIA), a stable adenosine agonist.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1810600

  8. Is inotropic therapy appropriate for patients with chronic congestive heart failure? Or is the digitalis leaf withering?

    PubMed Central

    Uretsky, B. F.

    1986-01-01

    The appropriateness of inotropic therapy in chronic heart failure was examined by critically reviewing five assumptions upon which this form of therapy has been justified. Only the first, that cardiac performance can be acutely improved by inotropic therapy, has been empirically proven. That such acute improvement is sustained appears to be true with non-catecholamine agents whereas the chronic haemodynamic efficacy of oral catecholamines remains in doubt. That any inotropic agent can improve exercise tolerance, make the patient feel better, or effect a change without deleteriously affecting the myocyte is very much in doubt. Thus, although the prospect of using powerful inotropic therapy in the patient with heart failure is theoretically appealing, its utility remains to be proven. PMID:2946034

  9. Use of Inotropic Agents in Treatment of Systolic Heart Failure.

    PubMed

    Tariq, Sohaib; Aronow, Wilbert S

    2015-12-04

    The most common use of inotropes is among hospitalized patients with acute decompensated heart failure, with reduced left ventricular ejection fraction and with signs of end-organ dysfunction in the setting of a low cardiac output. Inotropes can be used in patients with severe systolic heart failure awaiting heart transplant to maintain hemodynamic stability or as a bridge to decision. In cases where patients are unable to be weaned off inotropes, these agents can be used until a definite or escalated supportive therapy is planned, which can include coronary revascularization or mechanical circulatory support (intra-aortic balloon pump, extracorporeal membrane oxygenation, impella, left ventricular assist device, etc.). Use of inotropic drugs is associated with risks and adverse events. This review will discuss the use of the inotropes digoxin, dopamine, dobutamine, norepinephrine, milrinone, levosimendan, and omecamtiv mecarbil. Long-term inotropic therapy should be offered in selected patients. A detailed conversation with the patient and family shall be held, including a discussion on the risks and benefits of use of inotropes. Chronic heart failure patients awaiting heart transplants are candidates for intravenous inotropic support until the donor heart becomes available. This helps to maintain hemodynamic stability and keep the fluid status and pulmonary pressures optimized prior to the surgery. On the other hand, in patients with severe heart failure who are not candidates for advanced heart failure therapies, such as transplant and mechanical circulatory support, inotropic agents can be used for palliative therapy. Inotropes can help reduce frequency of hospitalizations and improve symptoms in these patients.

  10. Alteration of cardiac glycoside positive inotropic action by modulators of protein synthesis and degradation

    SciTech Connect

    Nosek, T.M.; Adams, R.J.

    1986-03-05

    Numerous membrane bound and cytoplasmic proteins participate in the cardiac expression of the positive inotropic action (PIA) of digitalis glycosides including the Na,K-ATPase (NKA). Exposure of the myocardium to an inhibitor of protein synthesis (cycloheximide, CYC) or of protein degradation (leupeptin, LEU) alters the PIA of ouabain in isolated, paced guinea pig papillary muscles (PM) in opposite ways. In vivo exposure to CYC for 3 hr resulted in a 30% depression of the in vitro PIA of ouabain at 1.7..mu..M compared to control. In vivo exposure to LEU for 1 hr resulted in a 47% enhancement of the in vitro PIA of 1.7..mu..M ouabain. Neither drug had an apparent effect on the ouabain PIA ED50. Neither CYC nor LEU exposure to PM in vitro affect resting or developed tension or the response of skinned PM to calcium. The mechanisms of the PIA alterations by CYC or LEU do not involve a direct effect on the digitalis receptor. Exposure of isolated cardiac sarcolemma enriched in NKA to 10-100..mu..M CYC or LEU did not affect NKA activity or /sup 3/H-ouabain binding. Although direct physicochemical effects of CYC or LEU may be involved in the alterations of the ouabain PIA, it is possible that modulation of the cellular levels or turnover rate of short-lived proteins may affect cardiac regulation of the digitalis PIA.

  11. Design and synthesis of new dihydrotestosterone derivative with positive inotropic activity.

    PubMed

    Lauro, Figueroa-Valverde; Francisco, Díaz-Cedillo; Elodia, García-Cervera; Eduardo, Pool-Gómez; Marcela, Rosas-Nexticapa; Lenin, Hau-Heredia; Betty, Sarabia Alcocer

    2015-03-01

    There are several reports which indicate that some steroid derivatives have inotropic activity; nevertheless, the cellular site and mechanism of action of steroid derivatives at cardiovascular level is very confusing. In order, to clarify these phenomena in this study, two dihydrotestosterone derivatives (compounds 5 and 10) were synthesized with the objective of to evaluate its biological activity on left ventricular pressure and characterize their molecular mechanism. In the first stage, the Langendorff technique was used to measure changes on perfusion pressure and coronary resistance in an isolated rat heart model in absence or presence of the steroid derivatives. Additionally, to characterize the molecular mechanism involved in the inotropic activity induced by the compound 5 was evaluated by measuring left ventricular pressure in absence or presence of following compounds; nifedipine, flutamide, indomethacin, prazosin, isoproterenol, propranolol and metoprolol. The results showed that the compound 5 significantly increased the perfusion pressure and coronary resistance in comparison with dihydrotestosterone, compound 10 and the control conditions. Other data indicate that 5 increase left ventricular pressure in a dose-dependent manner (0.001-100 nM); nevertheless, this phenomenon was significantly inhibited only by propranolol or metoprolol at a dose of 1 nM. These data suggest that positive inotropic activity induced by the compound 5 is through β1-adrenergic receptor however, this effect was independent of cAMP levels. This phenomenon is a particularly interesting because the positive inotropic activity induced by this steroid derivative involves a molecular mechanism different in comparison with other positive inotropic drugs.

  12. Inotropic action of the puberty hormone kisspeptin in rat, mouse and human: cardiovascular distribution and characteristics of the kisspeptin receptor.

    PubMed

    Maguire, Janet J; Kirby, Helen R; Mead, Emma J; Kuc, Rhoda E; d'Anglemont de Tassigny, Xavier; Colledge, William H; Davenport, Anthony P

    2011-01-01

    Kisspeptins, the ligands of the kisspeptin receptor known for its roles in reproduction and cancer, are also vasoconstrictor peptides in atherosclerosis-prone human aorta and coronary artery. The aim of this study was to further investigate the cardiovascular localisation and function of the kisspeptins and their receptor in human compared to rat and mouse heart. Immunohistochemistry and radioligand binding techniques were employed to investigate kisspeptin receptor localisation, density and pharmacological characteristics in cardiac tissues from all three species. Radioimmunoassay was used to detect kisspeptin peptide levels in human normal heart and to identify any pathological changes in myocardium from patients transplanted for cardiomyopathy or ischaemic heart disease. The cardiac function of kisspeptin receptor was studied in isolated human, rat and mouse paced atria, with a role for the receptor confirmed using mice with targeted disruption of Kiss1r. The data demonstrated that kisspeptin receptor-like immunoreactivity localised to endothelial and smooth muscle cells of intramyocardial blood vessels and to myocytes in human and rodent tissue. [(125)I]KP-14 bound saturably, with subnanomolar affinity to human and rodent myocardium (K(D) = 0.12 nM, human; K(D) = 0.44 nM, rat). Positive inotropic effects of kisspeptin were observed in rat, human and mouse. No response was observed in mice with targeted disruption of Kiss1r. In human heart a decrease in cardiac kisspeptin level was detected in ischaemic heart disease. Kisspeptin and its receptor are expressed in the human, rat and mouse heart and kisspeptins possess potent positive inotropic activity. The cardiovascular actions of the kisspeptins may contribute to the role of these peptides in pregnancy but the consequences of receptor activation must be considered if kisspeptin receptor agonists are developed for use in the treatment of reproductive disorders or cancer.

  13. Inotropic and coronary vasodilatory actions of the K-adenosine triphosphate channel modulator nicorandil in human tissue.

    PubMed

    Müller-Ehmsen, J; Brixius, K; Hoischen, S; Schwinger, R H

    1996-12-01

    The present study aimed to characterize the inotropic and vasodilatory properties of the K-ATP channel opener nicorandil (NIC) in isolated human cardiac tissue. For comparison, the Ca+2 channel blockers diltiazem (DIL) and nifedipine (NIF) have been studied. Concentration-dependent effects of NIC, DIL and NIF on the force of contraction (FOC) and the vascular tone have been studied on left ventricular papillary muscle strips (dilated cardiomyopathy, New York Heart Association Class IV, n = 20; nonfailing, donor hearts, n = 4), on right auricular trabeculae (nonfailing, n = 5) and on precontracted (prostaglandin F2 alpha: 0.3, 0.5 or 1 mumol/l) isolated human coronary artery rings (cardiac transplantation, n = 15). NIC, DIL and NIF concentration-dependently reduced the FOC of the papillary muscle preparations. However, the IC25 for the negative inotropic effect was significantly higher for NIC compared to DIL and NIF. The maximal negative inotropic effects of NIC, DIL and NIF (100 mumol/l) were -48.2 +/- 4.1, -92.9 +/- 0.9 and -93.4 +/- 1.4% of the basal FOC. The negative inotropic actions of NIC were similar in the human failing and the nonfailing ventricular and in the right atrial myocardium. Whereas pretreatment with methylene blue, an inhibitor of guanylyl cyclase, had no effect on the negative inotropic action of NIC, it was almost abolished by glibenclamide, a selective antagonist of the ATP-dependent K channels. NIC, DIL and NIF relaxed the coronary artery rings with 97.1 +/- 0.5, 90.7 +/- 0.9 and 96.4 +/- 0.7% of maximal relaxation (papaverine, 100 mumol/l). The rank order of vasodilatory potency was NIF > NIC > DIL. In conclusion, NIC is as effective as DIL and NIF in relaxing human coronary artery rings. However, NIC showed significantly lower negative inotropic effects when compared with the Ca+2 channel antagonists. The negative inotropic action of NIC is probably due to an interaction with the ATP-dependent K channels. In addition, activation of

  14. Celiprolol: a positive inotropic beta-adrenoceptor blocking agent in conscious dogs.

    PubMed Central

    Nganele, D. M.; DeLeonardis, V. M.; Hintze, T. H.

    1988-01-01

    1. beta-Adrenoceptor blocking agents are used to manage various cardiovascular disorders. A limiting factor in their use is the suppression of the cardiac contractile state. In our study, we examined the cardiac effects of celiprolol, a new beta-adrenoceptor blocking agent with reported positive inotropic effects. 2. Dogs were instrumented by use of sterile surgical techniques for the study of myocardial inotropic state, heart rate and internal left ventricular dimensions. Following complete recovery from surgery, experiments were conducted in the conscious state. 3. Intravenous injection of celiprolol (3 mg kg-1) in nine dogs, increased LV dP/dt by 13 +/- 2.6%, velocity of shortening (LV dD/dt) by 9.2 +/- 3.4%, and heart rate by 19 +/- 4.6% and decreased LV end-diastolic diameter by 1.8 +/- 0.8%, all significantly (P less than 0.05). Celiprolol blocked the inotropic actions of isoprenaline (0.5 micrograms kg-1) but only partially reduced its hypotensive effects. Propranolol, in contrast, reduced LV dP/dt by 17 +/- 3.3% and heart rate by 8.1 +/- 2.7% (P less than 0.05) while totally abolishing the hypotension, tachycardia and increase in LV dP/dt caused by isoprenaline. Following beta-adrenoceptor blockade with propranolol and with heart rate held constant by electrical pacing, celiprolol increased LV dP/dt by 16 +/- 4.0%, LV dD/dt by 12 +/- 3.0% and reduced LV end-diastolic diameter by 3.5 +/- 0.5% (P less than 0.05). 4. Thus, in conscious dogs, celiprolol increases inotropic state and reduces preload independently of beta 1-adrenoceptor mechanisms and the Bowditch phenomenon, while effectively blocking beta 1-receptors in the heart. These properties would make celiprolol useful in patients where a conventional beta-adrenoceptor blocking agent might lead to pump failure. PMID:2897217

  15. Vasostatins exert negative inotropism in the working heart of the frog.

    PubMed

    Corti, A; Mannarino, C; Mazza, R; Colombo, B; Longhi, R; Tota, B

    2002-10-01

    An in vitro isolated working frog heart (Rana esculenta) was used to study the effects of exogenous CGA(1-76) (vasostatin 1), CGA(1-113) (vasostatin 2), and the synthetic CGA(7-57) on cardiac performance. Under basal cardiac conditions, the dose-response curves of the three peptides from 10(-8) to 10(-7) M showed a significant calcium-dependent negative inotropism that involved neither the endocardial endothelium nor the adrenergic and muscarinic receptors. In addition, the CgA fragments clearly counteracted the typical positive inotropism of isoprenaline (10(-<9) M). Taken together, these results provide the first evidence for a cardio-suppressive role for the vasostatins.

  16. Positive inotropic activity induced by a dehydroisoandrosterone derivative in isolated rat heart model.

    PubMed

    Figueroa-Valverde, L; Díaz-Cedillo, F; García-Cervera, E; Pool Gómez, E; López-Ramos, M; Rosas-Nexticapa, M; Martinez-Camacho, R

    2013-10-01

    Experimental studies indicate that some steroid derivatives have inotropic activity; nevertheless, there is scarce information about the effects of the dehydroisoandrosterone and its derivatives at cardiovascular level. In addition, to date the cellular site and mechanism of action of dehydroisoandrosterone at cardiovascular level is very confusing. In order, to clarify those phenomena in this study, a dehydroisoandrosterone derivative was synthesized with the objective of to evaluate its activity on perfusion pressure and coronary resistance and compare this phenomenon with the effect exerted by dehydroisoandrosterone. The Langendorff technique was used to measure changes on perfusion pressure and coronary resistance in an isolated rat heart model in absence or presence of dehydroisoandrosterone and its derivative. Additionally, to characterize the molecular mechanism involved in the inotropic activity induced by dehydroisoandrosterone derivative was evaluated by measuring left ventricular pressure in absence or presence of following compounds; flutamide, prazosin, metoprolol and nifedipine. The results showed that dehydroisoandrosterone derivative significantly increased the perfusion pressure and coronary resistance in comparison with the control conditions and dehydroisoandrosterone. Additionally, other data indicate that dehydroisoandrosterone derivative increase left ventricular pressure in a dose-dependent manner [1 × 10(-9)-1 × 10(-4) mmol]; nevertheless, this phenomenon was significantly inhibited by nifedipine at a dose of 1 × 10(-6) mmol. In conclusion, these data suggest that dehydroisoandrosterone derivative induces positive inotropic activity through of activation the L-type calcium channel.

  17. Nesfatin-1 as a new positive inotrope in the goldfish (Carassius auratus) heart.

    PubMed

    Mazza, R; Gattuso, A; Filice, M; Cantafio, P; Cerra, M C; Angelone, T; Imbrogno, S

    2015-12-01

    The hypothalamic neuropeptide Nesfatin-1 is present in both mammals and teleosts in which it elicits anorexigenic effects. In mammals, Nesfatin-1 acts on the heart by inducing negative inotropism and lusitropism, and cardioprotection against ischemic damages. We evaluated whether in teleosts, Nesfatin-1 also influences cardiac performance. In the goldfish (Carassius auratus), mature, fully processed Nesfatin-1 was detected in brain, gills, intestine and skeletal muscle, but not in the cardiac ventricle. However, on the isolated and perfused working goldfish heart, exogenous Nesfatin-1 induced a positive inotropic effect, revealed by a dose-dependent increase of stroke volume (SV) and stroke work (SW). Positive inotropism was abolished by inhibition of adenylate cyclase (AC; MDL123330A) and cAMP-dependent kinase (PKA; KT5720), suggesting a cAMP/PKA-mediated pathway. This was confirmed by the increased cAMP concentrations revealed by ELISA on Nesfatin-1-treated hearts. Perfusion with Diltiazem, Thapsigargin and PD98059 showed the involvement of L-type calcium channels, SERCA2a pumps and ERK1/2, respectively. The role of ERK1/2 and phospholamban in Nesfatin-1-induced cardiostimulation was supported by Western blotting analysis. In conclusion, this is the first report showing that in teleosts, Nesfatin-1 potentiates mechanical cardiac performance, strongly supporting the evolutionary importance of the peptide in the control of the cardiac function of vertebrates.

  18. Cardiac inotropic responses from changes in carbon dioxide tension in the cephalic circulation of anaesthetized dogs.

    PubMed Central

    Hainsworth, R; McGregor, K H; Rankin, A J; Soladoye, A O

    1984-01-01

    Experiments were performed on anaesthetized dogs to determine the effects of moderate changes in PCO2 in the cephalic circulation on the inotropic state of the heart and on the reflex inotropic responses from changes in carotid sinus pressure. The cephalic circulation was perfused, through the brachiocephalic and left subclavian arteries, with blood taken from the superior vena cava and equilibrated with various gas mixtures in a gas exchange unit. The carotid sinus regions were vascularly isolated and perfused with arterial blood at controlled pressures. Cardiac inotropic responses were assessed from the maximum rate of change of left ventricular pressure (dP/dtmax) with heart rate and mean aortic pressure held constant. An increase in cephalic blood PCO2 resulted in an increase in dP/dtmax and an increase in the unpaced heart rate. Small, graded changes in cephalic PCO2 resulted in graded responses of dP/dtmax. A change in carotid sinus pressure resulted in a significantly greater response of dP/dtmax when cephalic PCO2 was high. After interruption of the left cardiac sympathetic nerves, the responses of dP/dtmax to changes in cephalic PCO2 and carotid sinus pressure were nearly abolished. These results indicate that the tension of carbon dioxide in the cephalic circulation is likely to be of importance in the control of the inotropic state of the heart. They also imply that, in studies of cardiovascular reflex responses, it is important to control the carbon dioxide tension in the arterial blood. PMID:6439852

  19. Case Report: Exercise in a Patient with Acute Decompensated Heart Failure Receiving Positive Inotropic Therapy

    PubMed Central

    Camarda, Robert; Foley, Laura Little; Givertz, Michael M; Cahalin, Lawrence P

    2011-01-01

    Background and Purpose: The projected increase in persons with advanced heart failure and associated costs warrant the examination of exercise in patients receiving inotropic therapy. Literature supports the use of exercise and inotropic therapy in the treatment of patients with advanced heart failure. The purposes of this paper are to illustrate the use of exercise prescription and outcomes assessment with a 6-minute walk test in a patient with acute decompensated heart failure receiving tailored therapy with dobutamine and to discuss potential relationships resulting in observed improvements. Case Description: A 67-year old man was admitted to an acute care hospital with acute decompensated heart failure for tailored medical therapy including dobutamine. The patient received 14 days of tailored medical therapy, of which 12 days included exercise training by a physical therapist. Outcomes: Functional outcomes showed a clinically significant improvement in distance walked and improvement in the cardiorespiratory response. The improvement in estimated peak oxygen consumption was 7% greater than that predicted to be from tailored medical therapy. Discussion: Exercise was safely provided to a patient hospitalized with advanced heart failure on continuous inotropic therapy. The 6-minute walk test was effectively used to prescribe exercise and examine patient outcomes. PMID:21637393

  20. Evaluating an Educational Module on Home Inotrope Therapy.

    PubMed

    Lockman-Samkowiak, Jodie; Brenner, Phyllis S; Dunn, Deborah S; Qureshi, Elena

    2015-01-01

    Educating home health nurses presents significant challenges for nurse educators because of the vast geographical areas served and the types of patient cared for. The integration of technology into the home health care arena offers new and innovative opportunities to address the ongoing educational needs of nurses as required by accrediting bodies. This exploratory study evaluated a Web-based educational module on home inotrope therapy in regard to nurses' perceived knowledge and confidence.

  1. The mode of inotropic action of ciguatoxin on guinea-pig cardiac muscle.

    PubMed Central

    Seino, A.; Kobayashi, M.; Momose, K.; Yasumoto, T.; Ohizumi, Y.

    1988-01-01

    1. Ciguatoxin (CTX) caused a dose-dependent increase in the contractile force of the guinea-pig isolated left atria at concentrations ranging from 0.1 to 10 ng ml-1 with the ED50 value of 0.5 ng ml-1. 2. In the atria, tetrodotoxin (5 x 10(-7) M) inhibited markedly the inotropic action of CTX. The inotropic effect of CTX at low concentrations was abolished by practolol (10(-5) M) and reserpine (2 mg kg-1 daily, for 3 days), whereas that of CTX at high concentrations was partially inhibited by both drugs. 3. In single atrial cells, CTX (3 ng ml-1) produced a marked increase in the amplitude of longitudinal contractions. 4. CTX (3 ng ml-1) caused marked prolongation in the falling phase of action potentials of atrial strips without affecting the maximum rate of rise of action potentials and membrane resting potentials. The effect of CTX on action potentials was abolished by tetrodotoxin (10(-6) M). 5. The whole-cell patch-clamp experiments on myocytes revealed that CTX (20 ng ml-1) shifted the current-voltage curve of Na inward currents by 40 mV in the negative direction. CTX caused a small sustained Na inward current even at resting membrane potentials. 6. These results suggest that the inotropic action of lower concentrations of CTX is primarily due to an indirect action via noradrenaline release, whereas that of higher concentrations is caused not only by an indirect action but also by a direct action on voltage-dependent Na channels of cardiac muscle.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3207997

  2. Influence of calcium on the inotropic actions of hyperosmotic agents, norepinephrine, paired electrical stimulation, and treppe.

    PubMed

    Willerson, J T; Crie, J S; Adcock, R C; Templeton, G H; Wildenthal, K

    1974-10-01

    To analyze the interaction of calcium ion concentration with hypertonic agents and with other inotropic interventions, isolated right ventricular cat papillary muscles were studied under isometric conditions in Krebs-Ringer bicarbonate solution. Extracellular calcium concentrations were varied between 2.5 and 11.0 mM. Maximal inotropic effects occurred between 5 and 8.0 mM calcium and further elevation to 11.0 mM was without additional influence. The effect of hyperosmotic sucrose and mannitol on papillary muscle performance was compared with that of 10(-6) M norepinephrine at calcium concentrations of 2.5 and 10.0 mM and with paired electrical stimulation in 10.0 mM calcium. Both norepinephrine and the hyperosmotic agents produced significant increases in developed tension and in the maximal rate of tension rise (dT/dt) in Krebs-Ringer in 2.5 and 4.0 mM calcium. In 10 mM calcium norepinephrine increased developed tension and dT/dt, but sucrose and mannitol caused no change or small reductions in both. Paired electrical stimulation, like hyperosmolality, caused no increase in dT/dt in 10 mM calcium. The presence of a potent pharmacological inhibitor of systolic calcium transfer across the cell membrane (D600, 10(-6) M) reduced developed tension and dT/dt by 76+/-2.7 and 74+/-2.0%, respectively, and prevented and in fact reversed the expected increase in dT/dt associated with an increase in rate of stimulation (treppe). However, hypertonic mannitol and paired pacing persisted in causing marked increases in developed tension and dT/dt even in the presence of D600, suggesting that their inotropic effects are not dependent on increased intracellular transfer of calcium during systole through cell membrane channels in which D600 acts as a competitive inhibitor. The results of these studies suggest that apparent functional saturation of intracellular calcium receptor sites eliminates any additional inotropic effect of hyperosmolality or paired pacing. The data are

  3. Influence of Calcium on the Inotropic Actions of Hyperosmotic Agents, Norepinephrine, Paired Electrical Stimulation, and Treppe

    PubMed Central

    Willerson, James T.; Crie, J. Stanley; Adcock, Robert C.; Templeton, Gordon H.; Wildenthal, Kern

    1974-01-01

    To analyze the interaction of calcium ion concentration with hypertonic agents and with other inotropic interventions, isolated right ventricular cat papillary muscles were studied under isometric conditions in Krebs-Ringer bicarbonate solution. Extracellular calcium concentrations were varied between 2.5 and 11.0 mM. Maximal inotropic effects occurred between 5 and 8.0 mM calcium and further elevation to 11.0 mM was without additional influence. The effect of hyperosmotic sucrose and mannitol on papillary muscle performance was compared with that of 10-6 M norepinephrine at calcium concentrations of 2.5 and 10.0 mM and with paired electrical stimulation in 10.0 mM calcium. Both norepinephrine and the hyperosmotic agents produced significant increases in developed tension and in the maximal rate of tension rise (dT/dt) in Krebs-Ringer in 2.5 and 4.0 mM calcium. In 10 mM calcium norepinephrine increased developed tension and dT/dt, but sucrose and mannitol caused no change or small reductions in both. Paired electrical stimulation, like hyperosmolality, caused no increase in dT/dt in 10 mM calcium. The presence of a potent pharmacological inhibitor of systolic calcium transfer across the cell membrane (D600, 10-6 M) reduced developed tension and dT/dt by 76±2.7 and 74±2.0%, respectively, and prevented and in fact reversed the expected increase in dT/dt associated with an increase in rate of stimulation (treppe). However, hypertonic mannitol and paired pacing persisted in causing marked increases in developed tension and dT/dt even in the presence of D600, suggesting that their inotropic effects are not dependent on increased intracellular transfer of calcium during systole through cell membrane channels in which D600 acts as a competitive inhibitor. The results of these studies suggest that apparent functional saturation of intracellular calcium receptor sites eliminates any additional inotropic effect of hyperosmolality or paired pacing. The data are compatible

  4. Prevention by NMDA receptor antagonists of the centrally-evoked increases of cardiac inotropic responses in rabbits.

    PubMed Central

    Monassier, L.; Tibiriça, E.; Roegel, J. C.; Mettauer, B.; Feldman, J.; Bousquet, P.

    1994-01-01

    1. The purpose of this study was to investigate further the role of the excitatory amino acid (EAA) system of neurotransmission, particularly of the NMDA receptor, in the central regulation of cardiac function. 2. Electrical stimulation of the paraventricular nucleus of the hypothalamus (PVN) in pentobarbitone anaesthetized rabbits induced a cardiovascular response mainly characterized by a positive inotropic effect, hypertension and a marked increase in the myocardial oxygen demand index. 3. The intracerebroventricular (i.c.v.) or intravenous (i.v.) injection of different EAA antagonists acting on different sites of the NMDA receptor/channel complex dose-dependently blunted the excitatory cardiovascular effects of PVN stimulation. 4. 5,7 Dichlorokynurenic acid was used as a specific glycine site antagonist and 2-amino-5-phosphonovaleric acid was used to block the agonist recognition site; ketamine was used as a channel blocker site antagonist and ifenprodil as a blocker of the polyamine binding site. 5. 5,7 Dichlorokynurenic acid (125 and 250 micrograms kg-1, i.c.v.) virtually abolished the cardiovascular responses, inducing only haemodynamic depression at the highest dose used. 2-Amino-5-phosphonovaleric acid (0.1 to 1.0 mg kg-1, i.c.v.) elicited a reduction of the peak values observed during PVN stimulation which was accompanied by a decrease of the basal cardiovascular parameters. Ketamine (2.5 and 10 mg kg-1) and ifenprodil (1 mg kg-1), injected intravenously, blocked the haemodynamic response induced by PVN stimulation without marked reduction of the basal haemodynamics. 6. It is concluded that glutamate neurotransmission is not only involved in vasomotor tone control but also in the central control of cardiac function and can therefore modulate the myocardial oxygen demand. PMID:7913376

  5. Nucleotides of transcription factor binding sites exert interdependent effects on the binding affinities of transcription factors

    PubMed Central

    Bulyk, Martha L.; Johnson, Philip L. F.; Church, George M.

    2002-01-01

    We can determine the effects of many possible sequence variations in transcription factor binding sites using microarray binding experiments. Analysis of wild-type and mutant Zif268 (Egr1) zinc fingers bound to microarrays containing all possible central 3 bp triplet binding sites indicates that the nucleotides of transcription factor binding sites cannot be treated independently. This indicates that the current practice of characterizing transcription factor binding sites by mutating individual positions of binding sites one base pair at a time does not provide a true picture of the sequence specificity. Similarly, current bioinformatic practices using either just a consensus sequence, or even mononucleotide frequency weight matrices to provide more complete descriptions of transcription factor binding sites, are not accurate in depicting the true binding site specificities, since these methods rely upon the assumption that the nucleotides of binding sites exert independent effects on binding affinity. Our results stress the importance of complete reference tables of all possible binding sites for comparing protein binding preferences for various DNA sequences. We also show results suggesting that microarray binding data using particular subsets of all possible binding sites can be used to extrapolate the relative binding affinities of all possible full-length binding sites, given a known binding site for use as a starting sequence for site preference refinement. PMID:11861919

  6. Operative contractility: a functional concept of the inotropic state.

    PubMed

    Curiel, Roberto; Perez-Gonzalez, Juan; Torres, Edwar; Landaeta, Ruben; Cerrolaza, Miguel

    2005-10-01

    1. Initial unsuccessful attempts to evaluate ventricular function in terms of the 'heart as a pump' led to focusing on the 'heart as a muscle' and to the concept of myocardial contractility. However, no clinically ideal index exists to assess the contractile state. The aim of the present study was to develop a mathematical model to assess cardiac contractility. 2. A tri-axial system was conceived for preload (PL), afterload (AL) and contractility, where stroke volume (SV) was represented as the volume of the tetrahedron. Based on this model, 'operative' contractility ('OperCon') was calculated from the readily measured values of PL, AL and SV. The model was tested retrospectively under a variety of different experimental and clinical conditions, in 71 studies in humans and 29 studies in dogs. A prospective echocardiographic study was performed in 143 consecutive subjects to evaluate the ability of the model to assess contractility when SV and PL were measured volumetrically (mL) or dimensionally (cm). 3. With inotropic interventions, OperCon changes were comparable to those of ejection fraction (EF), velocity of shortening (Vcf) and dP/dt-max. Only with positive inotropic interventions did elastance (Ees) show significantly larger changes. With load manipulations, OperCon showed significantly smaller changes than EF and Ees and comparable changes to Vcf and dP/dt-max. Values of OperCon were similar when AL was represented by systolic blood pressure or wall stress and when volumetric or dimensional values were used. 4. Operative contractility is a reliable, simple and versatile method to assess cardiac contractility.

  7. Causal binding of actions to their effects.

    PubMed

    Buehner, Marc J; Humphreys, Gruffydd R

    2009-10-01

    According to widely held views in cognitive science harking back to David Hume, causality cannot be perceived directly, but instead is inferred from patterns of sensory experience, and the quality of these inferences is determined by perceivable quantities such as contingency and contiguity. We report results that suggest a reversal of Hume's conjecture: People's sense of time is warped by the experience of causality. In a stimulus-anticipation task, participants' response behavior reflected a shortened experience of time in the case of target stimuli participants themselves had generated, relative to equidistant, equally predictable stimuli they had not caused. These findings suggest that causality in the mind leads to temporal binding of cause and effect, and extend and generalize beyond earlier claims of intentional binding between action and outcome.

  8. Inotropes and inodilators for acute heart failure: sarcomere active drugs in focus.

    PubMed

    Nagy, László; Pollesello, Piero; Papp, Zoltán

    2014-09-01

    Acute heart failure (AHF) emerges as a major and growing epidemiological concern with high morbidity and mortality rates. Current therapies in patients with acute heart failure rely on different strategies. Patients with hypotension, hypoperfusion, or shock require inotropic support, whereas diuretics and vasodilators are recommended in patients with systemic or pulmonary congestion. Traditionally inotropic agents, referred to as Ca mobilizers load the cardiomyocyte with Ca and thereby increase oxygen consumption and risk for arrhythmias. These limitations of traditional inotropes may be avoided by sarcomere targeted agents. Direct activation of the cardiac sarcomere may be achieved by either sensitizing the cardiac myofilaments to Ca or activating directly the cardiac myosin. In this review, we focus on sarcomere targeted inotropic agents, emphasizing their mechanisms of action and overview the most relevant clinical considerations.

  9. Evaluation of activity inotropic of a new steroid derivative using an isolated rat heart model

    PubMed Central

    Lauro, Figueroa-Valverde; Francisco, Díaz-Cedillo; Elodia, García-Cervera; Eduardo, Pool-Gómez; Maria, López-Ramos; Marcela, Rosas-Nexticapa; Lenin, Hau-Heredia; Bety, Sarabia-Alcocer; Landy, Campos-Ramos

    2014-01-01

    There are studies which indicate that some steroid derivatives have inotropic activity; nevertheless, the cellular site and mechanism of action at cardiovascular level is very confusing. In order, to clarify these phenomena in this study, a new estradiol derivative was synthesized with the objective of to evaluate its biological activity on left ventricular pressure and characterize their molecular mechanism. The Langendorff technique was used to measure changes on perfusion pressure and coronary resistance in an isolated rat heart model in absence or presence of the estradiol derivative. Additionally, to characterize the molecular mechanism involved in the inotropic activity induced by the OTBDS-estradiol-hexanoic acid derivative was evaluated by measuring left ventricular pressure in absence or presence of following compounds; tamoxifen, prazosin, metoprolol, indomethacin and nifedipine. The results showed that the OTBDS-estradiol-hexanoic acid derivative significantly increased the perfusion pressure and coronary resistance in comparison with the control conditions. Additionally, other data indicate that OTBDS-estradiol-hexanoic acid derivative increase left ventricular pressure in a dose-dependent manner (0.001 to 100 nM); nevertheless, this phenomenon was significantly inhibited only by nifedipine at a dose of 1 nM. These data suggest that positive inotropic activity induced by the OTBDS-estradiol-hexanoic acid derivative is via activation of L-type calcium channel. This phenomenon is a particularly interesting because the positive inotropic activity induced by this steroid derivative involves a molecular mechanism different in comparison with other positive inotropic drugs. PMID:24995077

  10. Evaluation of activity inotropic of a new steroid derivative using an isolated rat heart model.

    PubMed

    Lauro, Figueroa-Valverde; Francisco, Díaz-Cedillo; Elodia, García-Cervera; Eduardo, Pool-Gómez; Maria, López-Ramos; Marcela, Rosas-Nexticapa; Lenin, Hau-Heredia; Bety, Sarabia-Alcocer; Landy, Campos-Ramos

    2014-01-01

    There are studies which indicate that some steroid derivatives have inotropic activity; nevertheless, the cellular site and mechanism of action at cardiovascular level is very confusing. In order, to clarify these phenomena in this study, a new estradiol derivative was synthesized with the objective of to evaluate its biological activity on left ventricular pressure and characterize their molecular mechanism. The Langendorff technique was used to measure changes on perfusion pressure and coronary resistance in an isolated rat heart model in absence or presence of the estradiol derivative. Additionally, to characterize the molecular mechanism involved in the inotropic activity induced by the OTBDS-estradiol-hexanoic acid derivative was evaluated by measuring left ventricular pressure in absence or presence of following compounds; tamoxifen, prazosin, metoprolol, indomethacin and nifedipine. The results showed that the OTBDS-estradiol-hexanoic acid derivative significantly increased the perfusion pressure and coronary resistance in comparison with the control conditions. Additionally, other data indicate that OTBDS-estradiol-hexanoic acid derivative increase left ventricular pressure in a dose-dependent manner (0.001 to 100 nM); nevertheless, this phenomenon was significantly inhibited only by nifedipine at a dose of 1 nM. These data suggest that positive inotropic activity induced by the OTBDS-estradiol-hexanoic acid derivative is via activation of L-type calcium channel. This phenomenon is a particularly interesting because the positive inotropic activity induced by this steroid derivative involves a molecular mechanism different in comparison with other positive inotropic drugs.

  11. A xanthine derivative denbufylline inhibits negative inotropic response to verapamil in guinea pig ventricular papillary muscles, independent of its phosphodiesterase inhibitory activity.

    PubMed

    Sanae, F; Ohmae, S; Takagi, K; Miyamoto, K

    1995-11-01

    A phosphodiesterase (PDE) III inhibitor, amrinone, inhibited both the negative inotropic actions of verapamil and nicardipine in guinea pig ventricular papillary muscle; this effect was canceled by the protein kinase A inhibitor H-89. The PDE IV inhibitor 1,3-di-n-butyl-7-(2'-oxopropyl)xanthine (denbufylline), which elicited a negative inotropic action by itself, attenuated the action of verapamil up to 10 microM, without any interaction with nicardipine. The attenuation by denbufylline was not influenced by H-89. This suggests that in the ventricular papillary muscle, denbufylline acts on some verapamil-sensitive site(s) in the membrane and interferes with the calcium channel function without involvement of its PDE inhibitory activity.

  12. Retroactivity effects dependency on the transcription factors binding mechanisms.

    PubMed

    Pantoja-Hernández, Libertad; Álvarez-Buylla, Elena; Aguilar-Ibáñez, Carlos F; Garay-Arroyo, Adriana; Soria-López, Alberto; Martínez-García, Juan Carlos

    2016-12-07

    Downstream connection effects on transcription are caused by retroactivity. When biomolecular dynamical systems interconnect retroactivity is a property that becomes important. The biological functional meaning of these effects is increasingly becoming an area of interest. Downstream targets, which are operator binding sites in transcriptional networks, may induce behaviors such as ultrasensitive responses or even represent an undesired issue in regulation. To the best of our knowledge, the role of the binding mechanisms of transcription factors in relation to minimizing - or enhancing - retroactivity effects has not been previously addressed. Our aim is to evaluate retroactivity effects considering how the binding mechanism impacts the number of free functional transcription factor (FFTF) molecules using a simple model via deterministic and stochastic simulations. We study four transcription factor binding mechanisms (BM): simple monomer binding (SMB), dimer binding (DB), cooperative sequential binding (CSB) and cooperative sequential binding with dimerization (CSB_D). We consider weak and strong binding regimes for each mechanism, where we contrast the cases when the FFTF is bound or unbound to the downstream loads. Upon interconnection, the number of FFTF molecules changed less for the SMB mechanism while for DB they changed the most. Our results show that for the chosen mechanisms (in terms of the corresponding described dynamics), retroactivity effects depend on transcription binding mechanisms. This contributes to the understanding of how the transcription factor regulatory function-such as decision making-and its dynamic needs for the response, may determine the nature of the selected binding mechanism.

  13. [Inotropic activity induced by carbamazepine-alkyne derivative in an isolated heart model and perfused to constant flow].

    PubMed

    Figueroa-Valverde, Lauro; Díaz-Cedillo, Francisco; López-Ramos, María; García-Cervera, Elodia; Quijano-Ascencio, Karen

    2011-06-01

    Inotropic activity induced by carbamazepine-alkyne derivative in an isolated heart model and perfused to constant flow Introduction. Few data exist with respect to the effects of carbamazepine and its derivatives at cardiovascular level; furthermore, the molecular mechanisms and cellular site of action are still unclear. Objective. The effects induced by carbamazepine-alquine derivative on perfusion pressure, vascular resistance and left ventricular pressure were evaluated. Materials and methods. The effects of carbamazepine and carbamazepine-alquine on the perfusion pressure, vascular resistance and left ventricular pressure were examined in isolated rat hearts (Langendorff model). Results. Four results were obtained: (1) The carbamazepine-alquine derivative 10-9 mM increased the perfusion pressure and vascular resistance in comparison with the carbamazepine 10-9 mM; (2) the effect of carbamazepine-alquine derivative 10-9-10-4 mM on left ventricular pressure not was inhibited by metoprolol or prazosin at a dose of 10-6 mM; (3) nifedipine 10-6 mM blocked the effects exerted by the carbamazepine-alquine derivative 10-9-10--4 mM on left ventricular pressure, and (4) the carbamazepine-alquine derivative at dose of 10-9 mM increased the concentration of intracellular calcium over a time period of 3-18 min; nevertheless, in presence of nifedipine 10-6 mM this effect was inhibited significantly (p=0.005). Conclusions. The activity exerted by carbamazepine-alquine derivative on perfusion pressure, vascular resistance and left ventricular pressure involved activation of calcium channel type-L, brought indirectly changes in the intracellular calcium levels and subsequently induced a positive inotropic effect.

  14. Effects of heparin on insulin binding and biological activity

    SciTech Connect

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

    1987-02-01

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

  15. Polycation binding to glomerular basement membrane. Effect of biochemical modification.

    PubMed

    Bertolatus, J A; Hunsicker, L G

    1987-02-01

    The polycation hexadimethrine (HDM) binds to anionic sites in the glomerular basement membrane (GBM) and causes heavy proteinuria when infused in vivo. An in vitro assay of 3H-HDM binding to isolated dog GBM was developed, to permit further analysis of the GBM components binding HDM. 3H-HDM binding to isolated GBM was saturable, reversible in dose-dependent fashion by competing polycations, and inhibited by increasing salt concentration and low pH. The pH dependence of binding suggested that most of the HDM binds to carboxyl groups rather than to the sulfate groups of proteoglycans. Removal of heparan sulfate by heparinase or purified heparatinase had no detectable effect on HDM binding. Treatment of GBM with neuraminidase, hyaluronidase, or chondroitinase reduced binding of HDM by a maximum of 20 to 38%. However, substitution of carboxyl anions with nonionizable glycine methyl ester residues resulted in complete elimination of HDM binding. Parallel results were obtained in studies of glomerular localization of cationized ferritin (CatF), pI 8.5. After carboxyl substitution, GBM did not bind CatF; heparinase-treated GBM bound CatF in a distribution not demonstrably different from normal. Cellulose acetate electrophoresis of glycosaminoglycan fractions prepared from treated GBM confirmed that carboxyl modification did not alter the content or charge of the heparan sulfate of GBM, but heparinase treatment removed at least 90% of heparan sulfate. The results indicate that carboxyl groups are quantitatively more important than heparan sulfate for binding of HDM in vitro. Since HDM causes proteinuria in vivo, carboxyl groups may be important for maintenance of normal permselectivity.

  16. Binding of endotoxin to macrophages: distinct effects of serum constituents.

    PubMed

    Tahri-Jouti, M A; Chaby, R

    1991-07-01

    The respective roles of serum lipoproteins, and of the complement component C3, in the binding of endotoxin (LPS) to macrophages were analyzed by an in vitro assay using [3H]LPS. The addition of an anti-C3 serum in the medium induced an apparent abolishment of the specific binding of LPS to mouse macrophages, but this effect appeared to be due to an actual increase of nonspecific binding. Isolated complexes of LPS with lipoproteins of high density (HDL3) and of very high density (VHDL) did not bind to macrophages. Furthermore, addition of HDL3 and VHDL in the incubation medium inhibited the specific binding of LPS to macrophages. These results suggest that C3 reduces nonspecific interactions between LPS and macrophages whereas associations between LPS and HDL3 or VHDL inhibit specific LPS-macrophage interactions.

  17. Long-term intravenous inotropes in low-output terminal heart failure?

    PubMed

    von Scheidt, Wolfgang; Pauschinger, Matthias; Ertl, Georg

    2016-06-01

    Intravenous inotropic therapy may be necessary to achieve short-term survival in end-stage heart failure patients with cardiogenic shock or extreme low output and severe organ hypoperfusion. However, mid- or long-term intravenous inotropic therapy is associated with an increased mortality in advanced stage D heart failure patients using β-adrenoceptor agonists (dobutamine) or PDE-3-inhibitors (milrinone). Intermittent levosimendan may evolve as a reasonable therapeutic option. Randomized trials or other meaningful scientific evidence addressing the optimal treatment of exclusively the most threatened subgroup of hospitalized patients with persistent severe organ hypoperfusion are missing, but urgently needed. Despite a lack of other beneficial pharmacological options, the use of long-term intravenous inotropic therapy as a treatment for refractory heart failure or as an obligatory criterion for high urgency (HU) listing of heart transplant candidates with a median waiting time of 66 days in Germany is not based on scientific evidence. In addition, it might create a disincentive to achieve the HU status as well as keeping it, thereby potentially exposing the patient to an unnecessary additional risk. Upcoming new allocation algorithms may possibly help to improve the inadequate present situation. There is need for both, a better definition and a better treatment of high risk terminal heart failure requiring high urgent transplant listing.

  18. GABAA receptor-mediated positive inotropism in guinea-pig isolated left atria: evidence for the involvement of capsaicin-sensitive nerves.

    PubMed

    Maggi, C A; Giuliani, S; Manzini, S; Meli, A

    1989-05-01

    1. Isolated left atria from reserpine-pretreated guinea-pigs, electrically driven (3 Hz) in the presence of atropine (1 microM), phentolamine (0.3 microM) and propranolol (1 microM), responded to a train of stimuli (10 Hz for 2.5s) with a delayed neurogenic positive inotropic response which was insensitive to hexamethonium (10 microM) but abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to calcitonin gene-related peptide (CGRP). 2. In these experimental conditions, gamma-aminobutyric acid (GABA) produced a concentration-related (10 microM-1 mM) positive inotropic response similar to that produced by electrical field stimulation. The effect of GABA was competitively antagonized by bicuculline methiodide (10 microM), a GABAA receptor antagonist. 3. The selective GABAA receptor agonists, muscimol and homotaurine mimicked the positive inotropic effect of GABA while baclofen, the selective GABAB receptor agonist, did not. 4. The action of GABA (1 mM) was abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to CGRP, while it was unaffected by hexamethonium. In contrast, the inotropic response to CGRP was unaffected by tetrodotoxin, omega-conotoxin, bicuculline methiodide, hexamethonium or in vitro capsaicin desensitization, but was abolished by CGRP desensitization. 5. In the spontaneously beating guinea-pig right atrium, GABA (1 microM) produced a small and transient positive chronotropic effect that was no longer observed after in vitro desensitization with capsaicin (1 microM). 6. In the guinea-pig isolated perfused heart from reserpine-pretreated animals (with atropine, phentolamine and propranolol in the perfusion medium), GABA (1 microM) produced a transient tachycardia and a small increase in coronary flow. Both capsaicin (1 microM) and CGRP (1 microM) produced marked tachycardias and increases in coronary flow

  19. Mg NMR in DNA solutions: Dominance of site binding effects.

    PubMed

    Rose, D M; Bleam, M L; Record, M T; Bryant, R G

    1980-11-01

    (25)Mg NMR spectroscopy is applied to a study of magnesium ion interactions with DNA, which is considered as a model for a linear polyelectrolyte. It is demonstrated that the magnesium ion spectrum is complicated by a non-Lorent-zian line shape and is dominated by the effects of chemical exchange with macromolecule binding sites. A distinction is made between specific-site interactions in which the magnesium ion loses a water molecule from the first coordination sphere on binding and those interactions, referred to as territorial binding, in which the ion maintains its first coordination sphere complement of solvent. The first type of site-binding interactions are shown to dominate the magnesium ion NMR spectrum, based on a consideration of the magnitudes of the observed (25)Mg relaxation rates compared with (23)Na relaxation rates, the clear contributions of chemical exchange-limited relaxation, and an ion displacement experiment employing sodium.

  20. Pharmacological profile of the novel inotropic agent (E,Z)-3-((2-aminoethoxy)imino)androstane-6,17-dione hydrochloride (PST2744).

    PubMed

    Micheletti, R; Mattera, G G; Rocchetti, M; Schiavone, A; Loi, M F; Zaza, A; Gagnol, R J P; De Munari, S; Melloni, P; Carminati, P; Bianchi, G; Ferrari, P

    2002-11-01

    The novel Na(+)/K(+)-ATPase inhibitor (E,Z)-3-((2-aminoethoxy)imino)androstane-6,17-dione hydrochloride (PST2744) was characterized for its inotropic and toxic properties. Inhibition potency on dog kidney Na(+)/K(+)-ATPase was comparable (0.43 microM) to that of digoxin (0.45 microM). PST2744 concentration-dependently increased force of contraction in guinea pig atria and twitch amplitude in isolated guinea pig myocytes; in the latter, aftercontractions developed significantly less than with digoxin. Intravenous infusion of 0.2 mg/kg/min PST2744 in anesthetized guinea pigs exerted an immediate and long-lasting inotropic effect (ED(80) of 1.89 +/- 0.37 mg/kg) without causing lethal arrhythmias up to a cumulative dose of 18 mg/kg. Conversely, an equieffective infusion of digoxin (0.016 mg/kg/min; ED(80) of 0.32 mg/kg) caused lethal arrhythmias at a cumulative dose of 0.81 mg/kg. At a higher rate (0.4 mg/kg/min), PST2744 induced lethal arrhythmias, with a lethal dose/ED(80) ratio significantly greater than digoxin (20.2 +/- 6.3 versus 3.23 +/- 0.55, p < 0.05). Decay of the inotropic effect (t(1/2), min) was significantly faster for PST2744 (6.0 +/- 0.39) than for digoxin (18.3 +/- 4.5, p < 0.05). In anesthetized dogs, PST2744 dose-dependently increased maximum velocity of pressure rise (+dP/dt(max)) in the range 32 to 500 microg/kg i.v. and was safer than digoxin. In conscious dogs with a healed myocardial infarction, PST2744 significantly increased resting values of +dP/dt(max), left ventricular pressure, and SPB, and increased +dP/dt(max) throughout treadmill exercise while reverting the increase in left ventricular end diastolic pressure seen in control animals. Digoxin significantly decreased basal heart rate, while not affecting the hemodynamic response to exercise. Thus, PST2744 represents a new class of Na(+)/K(+)-ATPase inhibitors endowed with inotropic activity comparable with that of digitalis but having greater safety.

  1. Transport effects on the kinetics of protein-surface binding.

    PubMed Central

    Balgi, G; Leckband, D E; Nitsche, J M

    1995-01-01

    A detailed model is presented for protein binding to active surfaces, with application to the binding of avidin molecules to a biotin-functionalized fiber optic sensor in experiments reported by S. Zhao and W. M. Reichert (American Chemical Society Symposium Series 493, 1992). Kinetic data for binding in solution are used to assign an intrinsic catalytic rate coefficient k to the biotin-avidin pair, deconvoluted from transport and electrostatic factors via application of coagulation theory. This intrinsic chemical constant is built into a reaction-diffusion analysis of surface binding where activity is restricted to localized sites (representing immobilized biotin molecules). The analysis leads to an effective catalytic rate coefficient keff characterizing the active surface. Thereafter, solution of the transport problem describing absorption of avidin molecules by the macroscopic sensor surface leads to predictions of the avidin flux, which are found to be in good agreement with the experimental data. The analysis suggests the following conclusions. 1) Translational diffusion limitations are negligible for avidin-biotin binding in solution owing to the small (kinetically limiting) value k = 0.00045 m/s. 2) The sparse distribution of biotin molecules and the presence of a repulsive hydration force produce an effective surface-average catalytic rate coefficient keff of order 10(-7) m/s, much smaller than k. 3) Avidin binding to the fiber optic sensor occurs in an intermediate regime where the rate is influenced by both kinetics and diffusion. Images FIGURE 1 FIGURE 3 PMID:7647232

  2. Effects of glycation on meloxicam binding to human serum albumin

    NASA Astrophysics Data System (ADS)

    Trynda-Lemiesz, Lilianna; Wiglusz, Katarzyna

    2011-05-01

    The current study reports a binding of meloxicam a pharmacologically important new generation, non-steroidal anti-inflammatory drug to glycated form of the human serum albumin (HSA). The interaction of the meloxicam with nonglycated and glycated albumin has been studied at pH 7.4 in 0.05 M sodium phosphate buffer with 0.1 M NaCl, using fluorescence quenching technique and circular dichroism spectroscopy. Results of the present study have shown that the meloxicam could bind both forms of albumin glycated and nonglycated at a site, which was close to the tryptophan residues. Similarly, how for native albumin glycated form has had one high affinity site for the drug with association constants of the order of 10 5 M -1. The glycation process of the HSA significantly has affected the impact of the meloxicam on the binding of other ligands such as warfarin and bilirubin. The affinity of the glycated albumin for bilirubin as for native albumin has been reduced by meloxicam but observed effect was weaker by half (about 20%) compared with nonglycated albumin. In contrast to the native albumin meloxicam binding to glycated form of the protein only slightly affected the binding of warfarin. It seemed possible that the effects on warfarin binding might be entirely attributable to the Lys 199 modification which was in site I.

  3. Beyond feature binding: interference from episodic context binding creates the bivalency effect in task-switching.

    PubMed

    Meier, Beat; Rey-Mermet, Alodie

    2012-01-01

    When switching between different tasks and bivalent stimuli occur only occasionally on one of them, performance is slowed on subsequent univalent trials even if they have no overlapping features with the bivalent stimulus. This phenomenon has been labeled the "bivalency effect." Recent evidence has revealed that this effect is robust, general, and enduring. Moreover, it challenges current theories of task-switching and cognitive control. Here, we review these theories and propose a new, episodic context binding account. According to this account, binding does not only occur between stimuli, responses, and tasks, but also for the more general context in which the stimuli occur. The result of this binding process is a complex representation that includes each of these components. When bivalent stimuli occur, the resulting conflict is associated with the general context, creating a new conflict-loaded representation. The reactivation of this representation causes interference on subsequent trials, that is, the bivalency effect. We evaluate this account in light of the empirical evidence.

  4. Polyclonal and monoclonal IgG binding on Protein A resins - Evidence of competitive binding effects.

    PubMed

    Weinberg, Justin; Zhang, Shaojie; Crews, Gillian; Healy, Edward; Carta, Giorgio; Przybycien, Todd

    2017-03-14

    Protein A (ProA) chromatography is used extensively in the biopharmaceutical industry for the selective capture of both polyclonal and monoclonal antibodies (mAbs). This work provides a comparison of the adsorptive behavior of a highly heterogeneous polyclonal hIgG versus that of a mAb as well as the behavior of their mixtures on representative ProA resins. Both pH gradient elution and frontal loading experiments using human polyclonal IgG (hIgG) reveal a distribution of IgG-ProA binding strengths likely associated with multiple IgG subclasses and the heterogeneity of the variable region. pH gradient analysis of fractions collected along the breakthrough curve demonstrate a clear progression from weaker binding (higher pH eluting) to stronger binding (lower pH eluting) IgG species leaving the column suggesting the possibility of stronger binding species displacing the weaker binding ones. Displacement is directly observed by visualizing the adsorption of fluorescently labeled mAb and hIgG using confocal laser scanning microscopy (CLSM). Here, the displacement of hIgG results in a broad adsorption front compared to the sharp, 'shrinking core' behavior typically observed with mAbs. Sequential CLSM adsorption experiments with a mAb and hIgG confirm that stronger or equivalent-binding hIgG species are able to displace and desorb bound mAb molecules. These phenomena are examined using a variety of ProA resins including CaptivA PriMAB, MabSelect, and MabSelect SuRe to understand the effect of different ligand properties on binding strength and competition among different IgG species. The results of these comparisons suggest that the competition kinetics are slower with ligands that have a single-point covalent attachment to the base matrix compared to a multi-point attachment. This article is protected by copyright. All rights reserved.

  5. Effect of clustered peptide binding on DNA condensation.

    PubMed

    Haley, Jennifer; Kabiru, Paul; Geng, Yan

    2010-01-01

    DNA condensation in-vitro has been studied as a model system to reveal common principles underlying gene packaging in biology, and as the critical first step towards the development of non-viral gene delivery vectors. In this study, we use a bio-inspired approach, where small DNA-binding peptides are controllably clustered by an amphiphilic block copolymer scaffold, to reveal the effect of clustered peptide binding on the energetics, size, shape and physical properties of DNA condensation in-vitro. This provides insights into the general architectural effect of gene-binding proteins on DNA condensation process. Moreover, the versatility afforded by regulating the clustering density and composition of peptides may provide a novel design platform for gene delivery applications in the future.

  6. Discriminatory effects in the optical binding of chiral nanoparticles

    NASA Astrophysics Data System (ADS)

    Forbes, Kayn A.; Bradshaw, David S.; Andrews, David L.

    2015-08-01

    The laser-induced intermolecular force that exists between two or more particles subjected to a moderately intense laser beam is termed `optical binding'. Completely distinct from the single-particle forces that give rise to optical trapping, the phenomenon of optical binding is a manifestation of the coupling between optically induced dipole moments in neutral particles. In conjunction with optical trapping, the optomechanical forces in optical binding afford means for the manipulation and fabrication of optically bound matter. The Casimir-Polder potential that is intrinsic to all matter can be overridden by the optical binding force in cases where the laser beam is of sufficient intensity. Chiral discrimination can arise when the laser input has a circular polarization, if the particles are themselves chiral. Then, it emerges that the interaction between particles with a particular handedness is responsive to the left- or right-handedness of the light. The present analysis, which expands upon previous studies of chiral discrimination in optical binding, identifies a novel mechanism that others have previously overlooked, signifying that the discriminatory effect is much more prominent than originally thought. The new theory leads to results for freely-tumbling chiral particles subjected to circularly polarized light. Rigorous conditions are established for the energy shifts to be non-zero and display discriminatory effects with respect to the handedness of the incident beam. Detailed calculations indicate that the energy shift is larger than those previously reported by three orders of magnitude.

  7. Effect of desipramine on dopamine receptor binding in vivo

    SciTech Connect

    Suhara, Tetsuya Jikei Univ., Tokyo ); Inoue, Osamu; Kobayasi, Kaoru )

    1990-01-01

    Effect of desipramine on the in vivo binding of {sup 3}H-SCH23390 and {sup 3}H-N-methylspiperone ({sup 3}H-NMSP) in mouse striatum was studied. The ratio of radioactivity in the striatum to that in the cerebellum at 15 min after i.v. injection of {sup 3}H-SCH23390 or 45 min after injection of {sup 3}H-NMSP were used as indices of dopamine D1 or D2 receptor binding in vivo, respectively. In vivo binding of D1 and D2 receptors was decreased in a dose-dependent manner by acute treatment with desipramine (DMI). A saturation experiment suggested that the DMI-induced reduction in the binding was mainly due to the decrease in the affinity of both receptors. No direct interactions between the dopamine receptors and DMI were observed in vitro by the addition of 1 mM of DMI into striatal homogenate. Other antidepressants such as imipramine, clomipramine, maprotiline and mianserin also decreased the binding of dopamine D1 and D2 receptors. The results indicated an important role of dopamine receptors in the pharmacological effect of antidepressants.

  8. Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides.

    PubMed Central

    Flitney, F W; Singh, J

    1980-01-01

    1. A study has been made of a well documented but poorly understood response of the isolated frog ventricle to treatment with exogenous adenosine 5' triphosphate (ATP). Measurements of membrane potential, isometric twitch tension and levels of endogenous 3',5'-cyclic nucleotides have been made at various times during the ATP-induced response. 2. ATP elicits a characteristic triphasic response, which comprises an initial, abrupt increase in contractility, rising to a maximum within a few beats (first phase); followed by a period when the twitch amplitude falls, sometimes to below the control level (second phase); and superceded by a more slowly developing and longer-lasting increase in contractile force (third phase). The response is unaffected by atropine, propranolol or phentolamine. However, the prostaglandin synthetase inhibitor indomethacin depresses the first phase and entirely suppresses the third phase. 3. The inotropic effects of ATP are accompanied by changes in the shape of the action potential. These effects are dose-related. The duration of the action potential (D-30mV) and its positive overshoot (O) are increased during all phases of the response, for [ATP]o's up to 10(-5) M. However, at higher [ATP]o's, D-30mV and O ar both reduced during the second phase (but not the first or third phase), when isometric twitch tension is also depressed. The relationship between action potential duration and twitch tension (P) for different [ATP]o's is linear for all three phases of the response, but the slopes of the curves (delta P/delta D) are markedly different, indicating that the sensitivity of the contractile system to membrane depolarization is not constant, but varies continuously throughout the response. 4. ATP has a potent stimulatory effect on the metabolism of endogenous 3',5'-cyclic nucleotides. The time courses of the changes in adenosine 3','5-cyclic monophosphate (3',5'-cyclic AMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cyclic GMP) are

  9. Effect of Ion Binding in Palmitoyl-Oleoyl Phosphatidylserine Monolayers

    NASA Astrophysics Data System (ADS)

    Eckler, Matthew; Matysiak, Silvina

    2013-03-01

    Molecular dynamics simulations of palmitoyl-oleoyl phosphatidylserine (POPS) monolayers at the air-water interface were performed with different ionic strengths with the aim of determining the specific organization and dynamics of counterion binding events. Na + ions penetrated the monolayers into both the ester carbonyl and carboxylate regions of the phospholipids. The binding events increase with the addition of salt. Differences in lipid order parameter, headgroup orientation, and prevalence of inter- and intramolecular hydrogen bonding events between the amine group of the lipid and oxygen groups are observed depending on whether the Na + is binding near the carboxylate or ester region of the lipid. The observed changes are explained in terms of the salting-out effect.

  10. Synthesis and positive inotropic evaluation of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine derivatives bearing substituted piperazine moieties.

    PubMed

    Ma, Long-Xu; Cui, Bai-Ri; Wu, Yan; Liu, Jia-Chun; Cui, Xun; Liu, Li-Ping; Piao, Hu-Ri

    2014-04-01

    Four series of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine derivatives bearing substituted piperazine moieties were synthesized and evaluated for their positive inotropic activity by measuring the left atrium stroke volume in isolated rabbit-heart preparations. Several compounds were developed and showed favorable activities compared to the standard drug milrinone, with (4-([1,2,4]triazolo[3,4-a]phthalazin-6-yl)piperazin-1-yl)(p-tolyl)methanone (5g) being identified as the most potent with an increased stroke volume of 19.15±0.22% (milrinone: 2.46±0.07%) at a concentration of 3×10(-5) M. A preliminary study of mechanism of action revealed that 5g displayed its positive inotropic effect may be related to the PDE-cAMP-PKA signaling pathway. Compounds exhibiting inotropic effects were also evaluated in terms of the chronotropic effects.

  11. Endogenous inotropic substance from heart tissue has digitalis-like properties

    SciTech Connect

    Khatter, J.C.; Agbanyo, M.; Navaratnam, S. )

    1991-01-01

    In the past few years, we developed an extraction procedure which we successfully used to isolate a crude fraction containing digitalis-like substance (DLS) from porcine left ventricular tissue. In this study, the crude fraction was found to cross-react with digoxin antibodies and showed immunoreactivity of 4.25 {plus minus} 0.6 ng digoxin equivalent/ml. On further purification of the crude fraction using silica gel G column chromatography, a fraction C was obtained, which was highly positive inotropic on canine trabeculae and it dose-dependently inhibited ouabain sensitive {sup 86}Rb{sup +} uptake in rate heart slices. A 50% inhibition of uptake was obtained by 25 ul of fraction C. Fraction C also inhibited canine kidney Na{sup +}, K{sup +}-ATPase dose-dependently and a 50% inhibition of this enzyme required 17 ul of fraction C. Ashing of the fraction C at 500{degree}C resulted in loss of inotropic and enzyme inhibitory activities, indicating an organic nature of the unknown digitalis-like substance.

  12. Upregulation of the alpha1-adrenoceptor-induced phosphoinositide and inotropic response in hypothyroid rat heart.

    PubMed

    Jalali, Shahrzad; Durston, Melanie; Panagia, Vincenzo; Mesaeli, Nasrin

    2006-02-01

    In this study, we examined changes in the biochemical and inotropic events of the alpha(1)-adrenoceptor signaling pathway in hypothyroid rat hearts. Hypothyroidism was induced by treating experimental animals with 0.05% 6-n-propyl-2-thiouracil (PTU) in drinking water for 7 weeks. A significant decrease of beta- and an increase in alpha(1)-adrenoceptor density as well as an increase in the basal activity of the phosphoinositide (4,5) bisphosphate hydrolyzing phospholipase C was observed in sarcolemmal membranes purified from hypothyroid hearts as compared to age-matched euthyroid controls. Following stimulation with 10 microM phenylephrine (in the presence of 10 microM atenolol), the increase of contractile parameters over baseline values was significantly higher in hypo- than euthyroid hearts, while the opposite occurred under beta-stimulation with 0.1 microM isoproterenol. Interestingly, the increase in phenylephrine-mediated positive inotropy was accompanied by a significant increase in the sarcolemmal phospholipase C activity and in the inositol 1,4,5-trisphosphate content in hypothyroid as compared to euthyroid controls. Our results suggest that cardiac alpha(1)-adrenoceptor and its associated phosphoinositide signaling pathway may act as a reserve for catecholamine inotropic response in hypothyroidism, where the beta-adrenoceptors are compromised.

  13. Nitrendipine: effects on vascular responses and myocardial binding.

    PubMed

    McBride, W; Mukherjee, A; Haghani, Z; Wheeler-Clark, E; Brady, J; Gandler, T; Bush, L; Buja, L M; Willerson, J T

    1984-11-01

    We have further defined the binding characteristics of [3H]nitrendipine to myocardial microsomal membranes of cats, dogs, rats, and rabbits and to canine coronary vasculature (1.5-3.0 mm OD), and we have studied nitrendipine's effect on contractile responses in isolated feline cardiac muscle and canine coronary arteries. [3H]nitrendipine binding is rapid, saturable, and reversible in all four species and in all of these tissues. Feline myocardium has a single binding site with a dissociation constant (KD) of 1.94 nM. Canine myocardium may have two classes of binding sites, with the high-affinity site having a KD of 0.17 nM. Nitrendipine depresses contractility in isolated feline cardiac muscle and canine coronary arteries in a dose-dependent manner [half-maximal dose (ED50) 0.20 microM in isolated feline cardiac muscle and 1.6-6.3 nM for potential dependent contractile responses in isolated canine coronary arteries] and severely blunts the contractile response to increases in extracellular calcium concentration in isolated feline papillary muscles. In contrast to verapamil and D 600, nitrendipine does not prevent the treppe phenomenon. In isolated feline cardiac muscle and large canine coronary arteries, the minimal nitrendipine concentration required for specific binding and for depression of contractile responses is similar. However, only in large canine coronary arteries is the ED50 for nifedipine's depression of contractility similar to the KD for [3H]nitrendipine binding in the respective tissue.

  14. Management of peripherally inserted central catheters (PICC) in pediatric heart failure patients receiving continuous inotropic support.

    PubMed

    Giangregorio, Maeve; Mott, Sandra; Tong, Elizabeth; Handa, Sonia; Gauvreau, Kimberlee; Connor, Jean Anne

    2014-01-01

    The study aim was to evaluate present practice of maintaining PICC line patency in pediatric heart failure patients receiving continuous inotropes by comparing one cohort receiving low dose continuous heparin with one receiving no heparin. A case control retrospective chart review compared the two cohorts on duration of patency (measured in days) and need for thrombolytic agents. Median duration of patency for the heparin group was 24 days versus 16 days for the no heparin group (p=0.07). Use of thrombolytic agents was 28% in the heparin group compared to 50% in the no heparin group (p=0.08). Although not statistically significant, findings were clinically significant and supportive of current practice.

  15. Effects of N-acetylimidazole on oxytocin binding in bovine mammary tissue

    SciTech Connect

    Zhao, X.; Gorewit, R.C.; Currie, W.B. )

    1990-01-01

    The effects of N-acetylimidazole on specific binding of oxytocin to microsomal fractions of bovine mammary gland were studied. N-acetylimidazole suppressed oxytocin binding, with time and concentration dependence. Decreased oxytocin binding activity appeared to be due to decreased affinity of the hormone for its receptor. Acetylation of oxytocin, rather than of oxytocin receptors, seemed to be responsible for the decreased binding.

  16. The Ca2+ -antagonist and binding properties of the phenylalkylamine, anipamil.

    PubMed Central

    Dillon, J. S.; Nayler, W. G.

    1988-01-01

    1. Isolated, Langendorff-perfused rat hearts, isolated membranes, and pharmacological and receptor binding techniques were used to study the properties of the newly developed verapamil derivative, anipamil. 2. When added acutely to isolated, spontaneously beating or electrically paced hearts, anipamil (0.01-0.15 microM) exerted a dose-dependent negative inotropic effect which developed slowly and persisted after 60 min washout. 3. When added acutely (0.05-0.1 microM) to isolated hearts, or when given intravenously (2 mg kg-1 body weight 1 h before the animals were killed), anipamil displaced the dose-response curves for the positive inotropic effect of (0.10-3.0 mM) Ca2+ and (10-50 nM) Bay K 8644 to the right. 4. When added to freshly isolated cardiac membranes, 0.1 microM anipamil increased the dissociation constant (KD) of the phenylalkylamine (-)-[3H]-desmethoxyverapamil ((-)-[3H]-D888) from 1.22 +/- 0.2 to 2.91 +/- 0.46 nM, without any significant change in density (Bmax; control: 163 +/- 17; anipamil: 117 +/- 20 fmol mg-1 protein). Bound (-)-[3H]-D888 was displaceable by (-)-D888 (Ki 1.7 +/- 0.4 nM) greater than (-)-D600 (Ki 12 +/- 0.5 nM) greater than verapamil (Ki 55 +/- 11 nM) greater than (+)-D600 (Ki 108 +/- 12.2) greater than anipamil (Ki 471 +/- 52 nM). 5. In cardiac membranes isolated from rats pretreated with anipamil (2 mg kg-1 i.v.) 1h before they were killed, the KD of (-)-[3H]-D888 binding was increased (P less than 0.05) from 1.59 +/- 0.18 to 3.28 +/-0.65 nM with no significant change in density, compared to the placebo-treated (control) rats. 6. These results establish that anipamil interacts in a competitive manner with the phenylalkylamine binding sites in cardiac membranes, and that it resembles other Ca2+ antagonists in displacing the dose-response curve for the positive inotropic effect of Ca2+ to the right. The results also show that although anipamil binds tightly to the cardiac membranes, it binds to the (-)-[3H]-D888 recognition sites

  17. Pharmacokinetics and a simulation model of colforsin daropate, new forskolin derivative inotropic vasodilator, in patients undergoing coronary artery bypass grafting.

    PubMed

    Kikura, Mutsuhito; Morita, Koji; Sato, Shigehito

    2004-03-01

    Colforsin daropate, a water-soluble forskolin derivative, is an adenyl cyclase activator with positive inotropic and vasodilatory effects that are useful in the treatment of ventricular dysfunction. We investigated the pharmacokinetics of colforsin daropate in cardiac surgery patients and performed simulations to determine the dosage necessary to maintain an effective plasma concentration following cardiopulmonary bypass. In six patients undergoing coronary artery bypass graft, colforsin daropate (0.01mgkg(-1)) was administered immediately after separation from cardiopulmonary bypass. Arterial blood was sampled over the next 16h and plasma concentrations of colforsin daropate and its initial active metabolite were determined by gas-chromatography. Extended nonlinear least-squares regression was used to fit a three-compartment model to each patient's data. Distribution half-life (t(1/2alpha)) was 3.9+/-1.1min, metabolic half-life (t(1/2beta)) was 1.9+/-0.7h, and elimination half-life (t(1/2gamma)) was 95.3+/-15.2h. Central-compartment volume was 591.0+/-42.8mlkg(-1), volume distribution was 2689.2+/-450.6mlkg(-1), and elimination clearance was 27.7+/-14.7mlkg(-1)min(-1). In the pharmacokinetic simulation model, 0.5, 0.75, and 1.0microgkg(-1)min(-1) continuous infusion of colforsin daropate produce effective concentration (5-10ngml(-1)) within 30, 20, and 10min, respectively following administration. An initial active metabolite of decreased rapidly to less than 1.0ngml(-1) within the first 10min.A colforsin daropate infusion of 0.7-1.0microgkg(-1)min(-1) for 10-20min followed by 0.5microgkg(-1)min(-1) continuous infusion is recommended to produce an effective concentration (5-10ngml(-1)) within 10-20min and to maintain a therapeutic concentration throughout the administration period after cardiopulmonary bypass.

  18. Chemical Binding Effects in Neutron Resonance Scattering and Absorption.

    NASA Astrophysics Data System (ADS)

    Shamaoun, Adib Iskandar

    The Doppler broadening of neutron absorption and scattering resonances is an effect of considerable importance in calculating reactor parameters. This broadening is known to depend upon the state of the atom from which the scattering of the neutron occurs. This dependence is called the chemical binding effect. A key assumption in the usual computations of Doppler broadening is to ignore the dependence of the total resonance width on the chemical binding state of the compound nucleus. This is an excellent approximation for the gamma line. We derive an expression for the neutron line width as a function of the energy of the compound nucleus for an ideal gas. The influence of energy on the width with energy is examined at two different temperatures 4K and 1000K. It is found that these effects are very small, of the order of 10^{4-} . The assumption of constancy of the resonance width is thus shown to be a good approximation for the neutron line width. Also we examine the influence of the crystalline binding on the 6.67 eV resonance energy of U-238 line shape in uranium carbide and uranium dioxide. This model treats the crystal as a gas with an effective temperature and an effective mass determined by harmonic crystal phonon spectrum developed by Koppel and Houston. Based on this model, the line shape of U-238 is Gaussian plus a recoilless part. We also compute the broadening using a harmonic crystal model. As the temperature of U-238 target is decreased, disagreement between the two models becomes pronounced. However the results agree in the limiting case of high temperature. As the nucleus becomes more tightly bound, shifts in the resonance peak to lower energies are also observed. A general formula for the differential scattering cross section is developed starting from the transition probability (T-matrix). The formalism is applied to the gas and harmonic crystal models to determine the chemical binding effect. Although the resonance broadening is determined in

  19. Angiotensin receptor binding and pressor effects in cat subretrofacial nucleus

    SciTech Connect

    Allen, A.M.; Dampney, R.A.L.; Mendelsohn, F.A.O. Univ. of Sydney )

    1988-11-01

    Central administration of angiotensin II (ANG II) increases arterial blood pressure via increased sympathetic activity. The authors have examined the possibility that one site of action of ANG II is the subretrofacial (SRF) nucleus in the rostral ventrolateral medulla, since this nucleus is known to play a critical role in the tonic and phasic control of arterial pressure. In vitro autoradiography, employing {sup 125}I-labeled (Sar{sup 1}, Ile{sup 8})ANG II as radioligand, was used to localize binding sites for ANG-II in the cat ventrolateral medulla. A high density of ANG II-receptor binding sites was found confined to the SRF nucleus. In a second group of experiments in anesthetized cats, microinjections of ANG II, in doses ranging from 10 to 50 pmol, were made into histologically identified sites within and outside the SRF nucleus. Microinjections into the nucleus resulted in a dose-dependent increase in arterial pressure, which was abolished by systemic administration of the ganglion-blocking drug hexamethonium bromide. In contrast, microinjections just outside the SRF nucleus had no effect on arterial pressure. It is concluded that activation of ANG II-receptor binding sites within the SRF nucleus leads to an increase in arterial pressure via increased sympathetic efferent activity.

  20. Half-logistic time constants as inotropic and lusitropic indices for four sequential phases of isometric tension curves in isolated rabbit and mouse papillary muscles.

    PubMed

    Mizuno, Ju; Morita, Shigeho; Otsuji, Mikiya; Arita, Hideko; Hanaoka, Kazuo; Akins, Robert E; Hirano, Shuta; Kusakari, Yoichiro; Kurihara, Satoshi

    2009-05-01

    The waveforms of myocardial tension and left ventricular (LV) pressure curves are useful for evaluating myocardial and LV performance, and especially for inotropism and lusitropism. Recently, we found that half-logistic (h-L) functions provide better fits for the two partial rising and two partial falling phases of the isovolumic LV pressure curve compared to mono-exponential (m-E) functions, and that the h-L time constants for the four sequential phases are superior inotropic and lusitropic indices compared to the m-E time constants. In the present study, we tested the hypothesis that the four sequential phases of the isometric tension curves in mammalian cardiac muscles could be curve-fitted accurately using h-L functions. The h-L and m-E curve-fits were compared for the four phases of the isometric twitch tension curves in 7 isolated rabbit right ventricular and 15 isolated mouse LV papillary muscles. The isometric tension curves were evaluated in the four temporal phases: from the beginning of twitch stimulation to the maximum of the first order time derivative of tension (dF/dt(max)) (Phase I), from dF/dt(max) to the peak tension (Phase II), from the peak tension to the minimum of the first order time derivative of tension (dF/dt(min)) (Phase III), and from dF/dt(min) to the resting tension (Phase IV). The mean h-L correlation coefficients (r) of 0.9958, 0.9996, 0.9995, and 0.9999 in rabbit and 0.9950, 0.9996, 0.9994, and 0.9997 in mouse for Phases I, II, III, and IV, respectively, were higher than the respective m-E r-values (P < 0.001). The h-L function quantifies the amplitudes and time courses of the two partial rising and two partial falling phases of the isometric tension curve, and the h-L time constants for the four partial phases serve as accurate and useful indices for estimation of inotropic and lusitropic effects.

  1. Effective Hamiltonians for fastly driven tight-binding chains

    NASA Astrophysics Data System (ADS)

    Itin, A. P.; Neishtadt, A. I.

    2014-02-01

    We consider a single particle tunnelling in a tight-binding model with nearest-neighbour couplings, in the presence of a periodic high-frequency force. An effective Hamiltonian for the particle is derived using an averaging method resembling classical canonical perturbation theory. Three cases are considered: uniform lattice with periodic and open boundary conditions, and lattice with a parabolic potential. We find that in the latter case, interplay of the potential and driving leads to appearance of the effective next-nearest neighbour couplings. In the uniform case with periodic boundary conditions the second- and third-order corrections to the averaged Hamiltonian are completely absent, while in the case with open boundary conditions they have a very simple form, found before in some particular cases by S. Longhi (2008) [10]. These general results may found applications in designing effective Hamiltonian models in experiments with ultracold atoms in optical lattices, e.g. for simulating solid-state phenomena.

  2. Protein kinase C epsilon induces systolic cardiac failure marked by exhausted inotropic reserve and intact Frank-Starling mechanism.

    PubMed

    Montgomery, David E; Rundell, Veronica L M; Goldspink, Paul H; Urboniene, Dalia; Geenen, David L; de Tombe, Pieter P; Buttrick, Peter M

    2005-11-01

    Myofilament dysfunction is a common point of convergence for many forms of heart failure. Recently, we showed that cardiac overexpression of PKC epsilon initially depresses myofilament activity and then leads to a progression of changes characteristic of human heart failure. Here, we examined the effects of PKC epsilon on contractile reserve, Starling mechanism, and myofilament activation in this model of end-stage dilated cardiomyopathy. Pressure-volume loop analysis and echocardiography showed that the PKC epsilon mice have markedly compromised systolic function and increased end-diastolic volumes. Dobutamine challenge resulted in a small increase in contractility in PKC epsilon mice but failed to enhance cardiac output. The PKC epsilon mice showed a normal length-dependent tension development in skinned cardiac muscle preparations, although Frank-Starling mechanism appeared to be compromised in the intact animal. Simultaneous measurement of tension and ATPase demonstrated that the maximum tension and ATPase were markedly lower in the PKC epsilon mice at any length or Ca2+ concentration. However, the tension cost was also lower indicating less energy expenditure. We conclude 1) that prolonged overexpression of PKC epsilon ultimately leads to a dilated cardiomyopathy marked by exhausted contractile reserve, 2) that PKC epsilon does not compromise the Frank-Starling mechanism at the myofilament level, and 3) that the Starling curve excursion is limited by the inotropic state of the heart. These results reflect the significance of the primary myofilament contractilopathy induced by phosphorylation and imply a role for PKC epsilon-mediated phosphorylation in myofilament physiology and the pathophysiology of decompensated cardiac failure.

  3. Contribution of beta 1- and beta 2-adrenoceptors of human atrium and ventricle to the effects of noradrenaline and adrenaline as assessed with (-)-atenolol.

    PubMed Central

    Lemoine, H.; Schönell, H.; Kaumann, A. J.

    1988-01-01

    1. (-)-Atenolol was used as a tool to assess the function of beta 1- and beta 2-adrenoceptors in human heart. Right atrial and left ventricular preparations from patients undergoing open heart surgery were set up to contract isometrically. Membrane particles were prepared for beta-adrenoceptor labelling with [3H]-(-)-bupranolol and adenylate cyclase assays. 2. The positive inotropic effects of (-)-noradrenaline were antagonized to a similar extent by (-)-atenolol in atrial and ventricular preparations. (-)-Atenolol consistently antagonized the effects of (-)-adrenaline to a lesser extent than those of (-)-noradrenaline in atrial preparations. In ventricular preparations (-)-atenolol antagonized the effects of low concentrations of (-)-adrenaline to a lesser extent than those of high concentrations. 3. pKB values (M) of (-)-atenolol, estimated with non-linear analysis from the blockade of the positive inotropic effects of the catecholamines, were 7.4 for beta 1-adrenoceptors and 6.0 for beta 2-adrenoceptors. 4. (-)-Atenolol inhibited the binding of [3H]-(-)-bupranolol to ventricular beta 1-adrenoceptors with a pKD (M) of 5.9 and to ventricular beta 2-adrenoceptors with a pKD of 4.6. 5. (-)-Atenolol inhibited the catecholamine-induced adenylate cyclase stimulation in the atrium and ventricle with pKB values of 5.8-6.4 for beta 1- and pKB values of 4.7-5.7 for beta 2-adrenoceptors. The binding and cyclase assays suggest a partial affinity loss for (-)-atenolol inherent to membrane preparations. 6. beta 1-Adrenoceptors mediate the maximum positive inotropic effects of (-)-noradrenaline in both the atrium and ventricle of man. beta 2-Adrenoceptors appear to be capable of mediating maximal positive inotropic effects of (-)-adrenaline in atrium. In contrast, ventricular beta 2-adrenoceptors mediated only submaximal effects of (-)-adrenaline. PMID:2851354

  4. Magnesium ion is an effective inhibitor of the binding of Escherichia coli to heparin

    PubMed Central

    Kang, Jonghoon; Lee, Myung Soog; Gorenstein, David G.

    2007-01-01

    We investigated the effects of ions and temperature on the binding of E. coli to heparin using a chemiluminescence electrophoretic mobility shift assay. We found that magnesium ion is an effective inhibitor of the binding. The method can be readily applied to discover agents that can block the binding. PMID:17967492

  5. The effectiveness of ski bindings and their professional adjustment for preventing alpine skiing injuries.

    PubMed

    Finch, C F; Kelsall, H L

    1998-06-01

    This article presents a critical review of the extent to which alpine ski bindings and their adjustment have been formally demonstrated to prevent injuries. It considers a range of evidence, from anecdotal evidence and informed opinion to biomechanical studies, testing of equipment, epidemiological studies and controlled field evaluations. A total of 15 published studies examining the effectiveness of bindings and their adjustment were identified. All of these included anecdotal or informed opinion, and all but one focused on equipment design. Seven studies involved the testing of bindings or binding prototypes, 2 studies presented biomechanical models of the forces involved in binding operation, 6 reported an epidemiological evaluation of ski bindings and 2 considered skiers' behaviours towards binding adjustment. Some of the reviewed articles relate to the study of the biomechanics of ski bindings and their release in response to various loads and loading patterns. Other studies examined the contribution of bindings and binding-release to lower extremity, equipment-related injuries, the effect of various methods of binding adjustment on injury risk and the determinants of skiers' behaviour relating to professional binding adjustment. Most of the evidence suggests that currently used bindings are insufficient for the multidirectional release required to reduce the risk of injury to the lower limb, especially at the knee. This evidence suggests that further technical developments and innovations are required. The standard of the manufacture of bindings and boots also needs to be considered. The optimal adjustment of bindings using a testing device has been shown to be associated with a reduced risk of lower extremity injury. Generally, however, the adjustment of bindings has been shown to be inadequate, especially for children's bindings. Recommendations for further research, development and implementation with respect to ski binding and their adjustment are given

  6. Four limb amputations due to peripheral gangrene from inotrope use – Case report and review of the literature

    PubMed Central

    Ang, Chuan Han; Koo, Oon Thien; Howe, Tet Sen

    2015-01-01

    INTRODUCTION We present a rare case of 4 limb amputations due to peripheral gangrene which resulted from the use of inotropes for septic shock. PRESENTATION OF CASE A 72-year-old woman with no past medical history presented with fever and pain in bilateral big toes. She was diagnosed with Streptococcal pneumoniae septicaemia and was started on broad spectrum antibiotics, dopamine and noradrenaline in the medical intensive care unit. She developed peripheral gangrene of all 4 extremities due to microvascular spasm from inotrope use and 4 limb amputations were performed electively in a single stage. DISCUSSION The gangrene was contributed by the presence of disseminated intravascular coagulation and septic shock. There was no evidence of an autoimmune disorder or vasculitis on laboratory investigations and tissue histology. CONCLUSION Microvascular spasm is a rare complication of inotrope use which may lead to extensive peripheral gangrene. Anecdotal reports of reversal agents have been discussed. Four limb amputations are a reasonable option especially if done in an elective setting after the gangrene has demarcated itself. Rehabilitation with prosthesis after 4 limb amputations can result in good functional outcome. PMID:26232740

  7. Binding Energies, Effective Masses and Screenings Effects of Fröhlich Bipolarons

    NASA Astrophysics Data System (ADS)

    Cataudella, V.; Iadonisi, G.; Ninno, D.

    1991-01-01

    The bipolaron ground state binding energy and the effective masses are calculated self-consistently in a scheme where the electron-phonon interaction is described by the Fröhlich interaction. We explicitly use the total linear momentum conservation and both two-and three-dimensional systems are considered. We review results for binding energies and show that the bipolaron effective mass increases with the electron-phonon coupling constant α more rapidly than two free polaron masses. As expected, the increase is greater in two than in three dimensions. We estimate the screening effects due to an electronic or hole density n in a range of values such that nR2b ll 1 (here Rb is the bipolaron radius). We find that the bipolaron binding energy decreases with n and eventually becomes positive indicating the existence of a metastable bipolaron state. Finally we discuss the possible connections between our results and high Tc superconductivity.

  8. Autoradiographic analysis of tritiated imipramine binding in the human brain post mortem: effects of suicide

    SciTech Connect

    Gross-Isseroff, R.; Israeli, M.; Biegon, A.

    1989-03-01

    In vitro quantitative autoradiography of high-affinity tritiated imipramine binding sites was performed on brains of 12 suicide victims and 12 matched controls. Region-specific differences in imipramine binding were found between the two groups. Thus, the pyramidal and molecular layers of the cornu ammoni hippocampal fields and the hilus of the dentate gyrus exhibited 80%, 60%, and 90% increases in binding in the suicide group, respectively. The postcentral cortical gyrus, insular cortex, and claustrum had 45%, 28%, and 75% decreases in binding in the suicide group, respectively. No difference in imipramine binding was observed in prefrontal cortical regions, in the basal ganglia, and in mesencephalic nuclei. No sex and postmortem delay effects on imipramine binding were found. Imipramine binding was positively correlated with age, the effect of age being most pronounced in portions of the basal ganglia and temporal cortex.

  9. Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-Binding Module

    SciTech Connect

    Taylor, C. B.; Talib, M. F.; McCabe, C.; Bu, L.; Adney, W. S.; Himmel, M. E.; Crowley, M. F.; Beckham, G. T.

    2012-01-27

    Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3-6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function.

  10. Binding isotope effects as a tool for distinguishing hydrophobic and hydrophilic binding sites of HIV-1 RT.

    PubMed

    Krzemińska, Agnieszka; Paneth, Piotr; Moliner, Vicent; Świderek, Katarzyna

    2015-01-22

    The current treatment for HIV-1 infected patients consists of a cocktail of inhibitors, in an attempt to improve the potency of the drugs by adding the possible effects of each supplied compound. In this contribution, nine different inhibitors of HIV-1 RT, one of the three key proteins responsible for the virus replication, have been selected to develop and test a computational protocol that allows getting a deep insight into the inhibitors' binding mechanism. The interaction between the inhibitors and the protein have been quantified by computing binding free energies through FEP calculations, while a more detailed characterization of the kind of inhibitor-protein interactions is based on frequency analysis of the ligands in the initial and final state, i.e. in solution and binding the protein. QM/MM calculation of heavy atoms ((13)C, (15)N, and (18)O) binding isotope effects (BIE) have been used to identify the binding sites of the different inhibitors. Specific interactions between the isotopically labeled atoms of the inhibitors and polar residues and magnesium cations on the hydrophilic pocket of the protein are responsible for the frequencies shifting that can be detected when comparing the IR spectra of the compounds in solution and in the protein. On the contrary, it seems that changes in vdW interactions from solution to the final state when the ligand is interacting with residues of the hydrophobic cavity, does not influence frequency modes and then no BIE are observed. Our results suggest that a proper computational protocol can be a valuable tool which in turn can be used to increase the efficiency of anti AIDS drugs.

  11. Chelate effects in sulfate binding by amide/urea-based ligands.

    PubMed

    Jia, Chuandong; Wang, Qi-Qiang; Begum, Rowshan Ara; Day, Victor W; Bowman-James, Kristin

    2015-07-07

    The influence of chelate and mini-chelate effects on sulfate binding was explored for six amide-, amide/amine-, urea-, and urea/amine-based ligands. Two of the urea-based hosts were selective for SO4(2-) in water-mixed DMSO-d6 systems. Results indicated that the mini-chelate effect provided by a single urea group with two NH binding sites appears to provide enhanced binding over two amide groups. Furthermore, additional urea binding sites incorporated into the host framework appeared to overcome to some extent competing hydration effects with increasing water content.

  12. Actions of Ethanol on Voltage-Sensitive Sodium Channels: Effects on Neurotoxin Binding

    DTIC Science & Technology

    1987-01-01

    Exprnmantal Trherpeutics Ped in I.SA. Actions of Ethanol on Voltage-Sensitive Sodium Channels: Effects on Neurotoxin Binding1 MICHAEL J. MULLIN 2 and... sodium channels. This indirect allosteric mechanism for inhibition of [H]BTX-B binding. effect orethanol was concentration-dependent and was affected...ethanol increased the equilibrium binding constant without af- that ethanol can affect the voltage-sensitive sodium channels in fecting the apparent

  13. Effect of FGF-binding Protein 3 on Vascular Permeability*

    PubMed Central

    Zhang, Wentao; Chen, Yifan; Swift, Matthew R.; Tassi, Elena; Stylianou, Dora C.; Gibby, Krissa A.; Riegel, Anna T.; Wellstein, Anton

    2008-01-01

    Fibroblast growth factor-binding protein 1 (FGF-BP1 is BP1) is involved in the regulation of embryonic development, tumor growth, and angiogenesis by mobilizing endogenous FGFs from their extracellular matrix storage. Here we describe a new member of the FGF-BP family, human BP3. We show that the hBP3 protein is secreted from cells, binds to FGF2 in vitro and in intact cells, and inhibits FGF2 binding to heparin. To determine the function of hBP3 in vivo, hBP3 was transiently expressed in chicken embryos and resulted in >50% lethality within 24 h because of vascular leakage. The onset of vascular permeability was monitored by recording the extravasation kinetics of FITC-labeled 40-kDa dextran microperfused into the vitelline vein of 3-day-old embryos. Vascular permeability increased as early as 8 h after expression of hBP3. The increased vascular permeability caused by hBP3 was prevented by treatment of embryos with PD173074, a selective FGFR kinase inhibitor. Interestingly, a C-terminal 66-amino acid fragment (C66) of hBP3, which contains the predicted FGF binding domain, still inhibited binding of FGF2 to heparin similar to full-length hBP3. However, expression of the C66 fragment did not increase vascular permeability on its own, but required the administration of exogenous FGF2 protein. We conclude that the FGF binding domain and the heparin binding domain are necessary for the hBP3 interaction with endogenous FGF and the activation of FGFR signaling in vivo. PMID:18669637

  14. Binding energy effects in cascade evolution and sputtering

    SciTech Connect

    Robinson, M.T.

    1995-06-01

    The MARLOWE model was extended to include a binding energy dependent on the local crystalline order, so that atoms are bound less strongly to their lattice sites near surfaces or associated damage. Sputtering and cascade evolution were studied on the examples of self-ion irradiations of Cu and Au monocrystals. In cascades, the mean binding energy is reduced {approximately}8% in Cu with little dependence on the initial recoil energy; in Au, it is reduced {approximately}9% at 1 keV and {approximately}15% at 100 keV. In sputtering, the mean binding energy is reduced {approximately}8% in Cu and {approximately}15% in Au with little energy dependence; the yields are increased about half as much. Most sites from which sputtered atoms originate are isolated in both metals. Small clusters of such sites occur in Cu, but there are some large clusters in Au, especially in [111] targets. There are always more large clusters with damage-dependent binding than with a constant binding energy, but only a few clusters are compact enough to be regarded as pits.

  15. 43 CFR 1810.3 - Effect of laches; authority to bind government.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 43 Public Lands: Interior 2 2014-10-01 2014-10-01 false Effect of laches; authority to bind... GUIDANCE General Rules § 1810.3 Effect of laches; authority to bind government. (a) The authority of the... agents when they enter into an arrangement or agreement to do or cause to be done what the law does...

  16. On the effect of nuclear motion on chemical binding

    NASA Astrophysics Data System (ADS)

    Dohnert, A.

    1985-05-01

    The surfaces defined by Berlin (1951) to characterize the role of intramolecular forces in chemical binding are obtained analytically for the case in which the atomic nuclei are in motion. The generalized equation for the force function is derived from the hypervirial theorem rather than by the method of coordinate scaling as reported by Garcia-Sucre et al. (1984). Numerical results for the lowest vibrational level of the ground state of the H2 molecule are presented in a graph, and the inclusion of nuclear motion is shown to reduce the volume of the spatial 'binding region' and hence to localize the bond and reduce the degree of binding (relative to the fixed-nucleus model).

  17. Electronic and Steric Effects in Binding of Deep Cavitands

    PubMed Central

    Hooley, Richard J.; Shenoy, Siddhartha R.; Rebek, Julius

    2009-01-01

    A deep, self-folding cavitand responds to minor electronic differences between suitably sized adamantane guests. Binding constants range from <0.5 to 4000 M-1 for guests as similar as 1-bromoadamantane and 1-cyanoadamantane. The barriers to guest exchange also vary up to 3 kcal mol-1. PMID:18989966

  18. The effect of chronic ethanol on glutamate binding in human and rat brain

    SciTech Connect

    Cummins, J.T.; Sack, M.; von Hungen, K. Univ. of California School of Medicine, Los Angeles )

    1990-01-01

    Quantitative autoradiographic techniques demonstrate that chronic alcohol administration causes a decrease in ({sup 3}H)-glutamate binding to hippocampal N-methyl-D-aspartate (NMDA) receptors. A 14% decrease in ({sup 3}H)-glutamate binding in the hippocampal CA{sub 1} region is seen both in the rat after five days of ethanol administration and in postmortem hippocampal tissues from alcoholics. In the rat, 24 hr ethanol withdrawal values are intermediate between control and alcohol binding levels. There was no significant effect of ethanol on ({sup 3}H)-glutamate binding in the cortex or caudate.

  19. Bose polaron problem: Effect of mass imbalance on binding energy

    NASA Astrophysics Data System (ADS)

    Ardila, L. A. Peña; Giorgini, S.

    2016-12-01

    By means of quantum Monte Carlo methods we calculate the binding energy of an impurity immersed in a Bose-Einstein condensate at T =0 . The focus is on the attractive branch of the Bose polaron and on the role played by the mass imbalance between the impurity and the surrounding particles. For an impurity resonantly coupled to the bath, we investigate the dependence of the binding energy on the mass ratio and on the interaction strength within the medium. In particular, we determine the equation of state in the case of a static (infinite mass) impurity, where three-body correlations are irrelevant and the result is expected to be a universal function of the gas parameter. For the mass ratio corresponding to 40K impurities in a gas of 87Rb atoms, we provide an explicit comparison with the experimental findings of a recent study carried out at JILA.

  20. A role of ionic strength on the inotropic effects of osmolarity change in frog atrium.

    PubMed

    Ohba, M

    1984-01-01

    The influence of varying the ionic strength of the bathing solution on the contraction of chemically skinned frog atrial muscle fibers was studied. The rate of tension development activated by calcium slows as the ionic strength is elevated. The size of caffeine contracture, however, was larger in the fiber preloaded with calcium at a higher ionic strength. There was a decrease in the maintained tension at 10(-6) M Ca when a fiber was bathed in a high ionic strength solution. Returning to a normal ionic strength solution caused a transient tension increase. When the fiber was bathed in a low ionic strength solution, the maintained tension increased transiently to a high value and then declined to reach a plateau. The response was also observed in a solution of pCa 8. In the caffeine-treated fiber or in the fiber bathed in ATP free solution, although the maintained tension level was changed corresponding to the altered ionic strength, the transient responses were blocked. These responses were not much influenced by the kinds of salts used to change the ionic strength. When osmolarity of the medium was altered by sucrose, transient responses were not induced. The results could qualitatively explain the isometric tension change of an intact fiber of frog atrium bathed in a hyper- or hypotonic solution.

  1. Binding of Polycarboxylic Acids to Cationic Mixed Micelles: Effects of Polymer Counterion Binding and Polyion Charge Distribution.

    PubMed

    Yoshida; Sokhakian; Dubin

    1998-09-15

    Mixed micelles of cetyltrimethylammonium chloride (CTAC) and n-dodecyl hexaoxyethylene glycol monoether (C12E8) bind to polyanions when the mole fraction of the cationic surfactant exceeds a critical value (Yc). Yc corresponds to a critical micelle surface charge density at which polyelectrolyte will bind to this colloidal particle. Turbidimetric titrations were used to determine Yc for such cationic-nonionic micelles in the presence of acrylic acid and acrylamido-2-methylpropane sulfonate homopolymers (PAA and PAMPS, respectively) and their copolymers with acrylamide, as function of pH, ionic strength, and polyelectrolyte counterion. In 0.20 M NaCl, Yc for PAA is found to be remarkably insensitive to pH, i.e., virtually independent of the apparent polymer charge density xiapp. On the other hand, the expected inverse relationship between Yc and xiapp is observed either for PAA when NaCl is replaced by TMACl (tetramethylammonium chloride), or when xiapp is manipulated using acrylic acid/acrylamide copolymers at high pH. The effective charge density of PAA is thus seen to be suppressed by specific sodium ion binding, indicating that the influence of salts on the interaction of polycarboxylic acids with colloidal particles may differ qualitatively from their effect on the analogous behavior of strong polyanions. Comparisons between homo- and copolymers of acrylic acid were carried out also to test the hypothesis that the "mobility" of charges on PAA at moderate pH (degree of ionization less than unity) could make this "annealed" polymer exhibit the behavior of a more highly charged one. The results, while consistent with this expectation, were obscured by the likely effect of copolymer sequence distributions. Copyright 1998 Academic Press.

  2. A Quantitative Study of the Effects of Guest Flexibility on Binding Inside a Coordination Cage Host.

    PubMed

    Taylor, Christopher G P; Cullen, William; Collier, Olivia M; Ward, Michael D

    2017-01-01

    We have performed a systematic investigation of the effects of guest flexibility on their ability to bind in the cavity of a coordination cage host in water, using two sets of isomeric aliphatic ketones that differ only in the branching patterns of their alkyl chains. Apart from the expected increase in binding strength for C9 over C7 ketones associated with their greater hydrophobic surface area, within each isomeric set there is a clear inverse correlation between binding free energy and guest flexibility, associated with loss of conformational entropy. This can be parameterized by the number of rotatable C-C bonds in the guest, with each additional rotatable bond resulting in a penalty of around 2 kJ mol(-1) in the binding free energy, in good agreement with values obtained from protein/ligand binding studies. We used the binding data for the new flexible guests to improve the scoring function that we had previously developed that allowed us to predict binding constants of relatively rigid guests in the cage cavity using the molecular docking programme GOLD (Genetic Optimisation of Ligand Docking). This improved scoring function resulted in a significant improvement in the ability of GOLD to predict binding constants for flexible guests, without any detriment to its ability to predict binding for more rigid guests.

  3. Effects of water models on binding affinity: evidence from all-atom simulation of binding of tamiflu to A/H5N1 neuraminidase.

    PubMed

    Nguyen, Trang Truc; Viet, Man Hoang; Li, Mai Suan

    2014-01-01

    The influence of water models SPC, SPC/E, TIP3P, and TIP4P on ligand binding affinity is examined by calculating the binding free energy ΔG(bind) of oseltamivir carboxylate (Tamiflu) to the wild type of glycoprotein neuraminidase from the pandemic A/H5N1 virus. ΔG(bind) is estimated by the Molecular Mechanic-Poisson Boltzmann Surface Area method and all-atom simulations with different combinations of these aqueous models and four force fields AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L. It is shown that there is no correlation between the binding free energy and the water density in the binding pocket in CHARMM. However, for three remaining force fields ΔG(bind) decays with increase of water density. SPC/E provides the lowest binding free energy for any force field, while the water effect is the most pronounced in CHARMM. In agreement with the popular GROMACS recommendation, the binding score obtained by combinations of AMBER-TIP3P, OPLS-TIP4P, and GROMOS-SPC is the most relevant to the experiments. For wild-type neuraminidase we have found that SPC is more suitable for CHARMM than TIP3P recommended by GROMACS for studying ligand binding. However, our study for three of its mutants reveals that TIP3P is presumably the best choice for CHARMM.

  4. Effects of vitamin B-6 nutrition on benzodiazepine (BDZ) receptor binding in the developing rat brain

    SciTech Connect

    Borek, J.P.; Guilarte, T.R. )

    1990-02-26

    A dietary deficiency of vitamin B-6 promotes seizure activity in neonatal animals and human infants. Previous studied have shown that neonatal vitamin B-6 deprivation results in reduced levels of brain gamma-aminobutyric acid (GABA) and increased binding at the GABA site of the GABA/BDZ receptor complex. Since the GABA and BDZ receptors are allosterically linked, this study was undertaken to determine if vitamin B-6 deprivation had an effect on BDZ receptor binding. Benzodiazepine receptor binding isotherms using {sup 3}H-flunitrazepam as ligand were performed in the presence and absence of 10 {mu}M GABA. The results indicate a significant increase in the binding affinity (Kd) in the presence of GABA in cerebellar membranes from deficient rat pups at 14 days of age with no effect on receptor number (Bmax). By 28 days of age, the increase in Kd was no longer present. No change in Kd or Bmax was observed in cortical tissue from deficient animals at 14 or 28 days of age. Preliminary studies of GABA-enhancement of {sup 3}H-flunitrazepam binding indicate that vitamin B-6 deficiency also induces alterations in the ability of GABA to enhance BZD receptor binding. In summary, these results indicate that the effects of vitamin B-6 deprivation on BDZ receptor binding are region specific and age related.

  5. Concentration-dependent effect of fibrinogen on IgG-specific antigen binding and phagocytosis.

    PubMed

    Boehm, Tobias Konrad; Sojar, Hakimuddin; Denardin, Ernesto

    2010-01-01

    In this paper, we aim to characterize fibrinogen-IgG interactions, and explore how fibrinogen alters IgG-mediated phagocytosis. Using enzyme-linked binding assays, we found that fibrinogen binding to IgG is optimized for surfaces coated with high levels of IgG. Using a similar method, we have shown that for an antigen unable to specifically bind fibrinogen, fibrinogen enhances binding of antibodies towards that antigen. For binding of IgG antibodies to cells expressing Fc receptors, we found a bimodal binding response, where low levels of fibrinogen enhance binding of antibody to Fc receptors and high levels reduce it. This corresponds to a bimodal effect on phagocytosis of IgG-coated particles, which is inhibited in the presence of excess IgG during coating of the particles with antibodies and fibrinogen. We conclude that fibrinogen can modulate phagocytosis of IgG-coated particles in vitro by changing IgG binding behavior, and that high fibrinogen levels could negatively affect phagocytosis.

  6. Identification of procollagen promoter DNA-binding proteins: effects of dexamethasone

    SciTech Connect

    Sweeney, C.; Cutroneo, K.R.

    1987-05-01

    Glucocorticoids selectively decrease procollagen synthesis by decreasing procollagen mRNA transcription. Dexamethasone coordinately decreased total cellular type I and type III procollagen mRNAs in mouse embryonic skin fibroblasts. Since sequence specific DNA-binding proteins are known to modulate eukaryotic gene expression the authors identified in mouse fibroblasts nuclear proteins which bind to types I and III procollagen promoter DNAs. Nuclear proteins were electrophoresed, blotted onto nitrocellulose and probed with /sup 32/P-end-labeled type I and type III procollagen promoter DNAs in the presence of equimolar amounts of /sup 32/P-end-labeled vector DNA. Differences in total DNA binding were noted by the densitometric scans of the nuclear proteins. Dexamethasone treatment enhanced total DNA binding. Increasing the NaCl concentration decreased the number of promoter DNA-binding proteins without altering the relative specificity for the promoter DNAs. Promoter DNA binding to nuclear proteins was also inhibited by increasing concentrations of E. coli DNA. The number of DNA-binding proteins was greater for type III procollagen promoter DNA. The effect of dexamethasone treatment on promoter DNA binding to nuclear proteins was determined.

  7. The effect of vesicle shape, line tension, and lateral tension on membrane-binding proteins

    NASA Astrophysics Data System (ADS)

    Hutchison, Jaime B.

    Model membranes allow for the exploration of complex biological phenomena with simple, controllable components. In this thesis we employ model membranes to determine the effect of vesicle properties such as line tension, lateral tension, and shape on membrane-binding proteins. We find that line tension at the boundary between domains in a phase separated vesicle can accumulate model membrane-binding proteins (green fluorescent protein with a histidine tag), and that those proteins can, in turn, alter vesicle shape. These results suggest that domains in biological membranes may enhance the local concentration of membrane-bound proteins and thus alter protein function. We also explore how membrane mechanical and chemical properties alter the function of the N-BAR domain of amphiphysin, a membrane-binding protein implicated in endocytosis. We find that negatively charged lipids are necessary for N-BAR binding to membranes at detectable levels, and that, at least for some lipid species, binding may be cooperative. Measurements of N-BAR binding as a function of vesicle tension reveal that modest membrane tension of around 2 mN/m, corresponding to a strain of around 1%, strongly increases N-BAR binding. We attribute this increase in binding with tension to the insertion of N-BAR's N-terminal amphipathic helix into the membrane which increases the membrane area. We propose that N-BAR, which was previously described as being able to sense membrane curvature, may be sensing strain instead. Measurements of membrane deformation by N-BAR as a function of membrane tension reveal that tension can hinder membrane deformation. Thus, tension may favor N-BAR binding yet suppress membrane deformation/tubulation, which requires work against tension. These results suggest that membrane tension, a parameter that is often not controlled in model membranes but is tightly controlled in biological cells, may be important in regulating protein binding and assembly and, hence, protein

  8. Effects of sodium on cell surface and intracellular TH-naloxone binding sites

    SciTech Connect

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

    1987-07-27

    The binding of the opiate antagonist TH-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, TH-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.

  9. The effect of saturation of ACE binding sites on the pharmacokinetics of enalaprilat in man.

    PubMed Central

    Wade, J R; Meredith, P A; Hughes, D M; Elliott, H L

    1992-01-01

    1. Eight healthy male volunteers received oral enalapril, 10 mg, in the presence and absence of pretreatment with captopril, 50 mg, twice daily for 5 days. 2. Enalaprilat pharmacokinetics were characterised after both doses of enalapril to investigate the effect of saturating ACE binding sites by pretreatment with captopril. 3. The pharmacokinetics of enalaprilat were best described by a one compartment model with zero order input incorporating saturable binding to plasma and tissue ACE. 4. Values of AUC (0.72 h) for enalaprilat were 419 +/- 97 and 450 +/- 87 ng ml-1 h in the presence and absence of captopril, respectively. The difference was not statistically significant nor were there any other differences in model parameters. 5. Induction of ACE by captopril resulting in an increase in the number of ACE binding sites, may have obscured any effect of captopril on the occupancy of ACE binding sites by enalapril. PMID:1312853

  10. Effects of the central potassium ions on the G-quadruplex and stabilizer binding.

    PubMed

    Wang, Zhiguo; Liu, Jun-Ping

    2017-03-01

    Human telomeres undertake the structure of intra-molecular parallel G-quadruplex in the presence of K(+) in eukaryotic cell. Stabilization of the telomere G-quadruplex represents a potential strategy to prevent telomere lengthening by telomerase in cancer therapy. Current work demonstrates that the binding of central K(+) with the parallel G-quadruplex is a coordinated water directed step-wise process. The K(+) above the top G-tetrad is prone to leak into environment and the 5'-adenine quickly flips over the top G-tetrad, leading to the bottom gate of G-tetrads as the only viable pathway of K(+) binding. Present molecular dynamics studies on the two most potent stabilizers RHPS4 and BRACO-19 reveal that the central K(+) has little influence on the binding conformations of the bound stabilizers. But without the central K(+), either RHPS4 or BRACO-19 cannot stabilize the structure of G-quadruplex. The binding strength of stabilizers evaluated by the MM-PBSA method follows the order of BRACO-19> RHPS4, which agrees with the experimental results. The difference in binding affinities between RHPS4 and BRACO-19 is probably related to the ability to form intramolecular hydrogen bonds and favorable van del Waals interactions with G-quadruplex. In the models that have one central K(+) located at the upper/lower binding site, the corresponding top/bottom stacked stabilizers show more favorable binding affinities, indicating the apparent promoting effect of central K(+) on the stabilizer binding. Our findings provide further insights into the regulatory effect of K(+) on the G-quadruplex targeted binding, which is meaningful to the development of G-quadruplex stabilizers.

  11. Modulatory effects of unsaturated fatty acids on the binding of glucocorticoids to rat liver glucocorticoid receptors.

    PubMed

    Vallette, G; Vanet, A; Sumida, C; Nunez, E A

    1991-09-01

    Binding of the synthetic glucocorticoid dexamethasone to the rat liver cytosol glucocorticoid receptor was inhibited by physiological concentrations of nonesterified fatty acids as a function of increasing dose, degree of unsaturation, and chain length of the fatty acid. Polyunsaturated fatty acids were the most potent inhibitors. Scatchard analysis and Line-weaver-Burk plots of the binding data revealed that both the association constants and number of binding sites decreased and that polyunsaturated fatty acids inhibition was of a mixed non-competitive type. The dissociation rate constant of [3H]dexamethasone from glucocorticoid receptors was increased by up to 10 times in the presence of docosahexaenoic acid, whereas a competitive inhibitor like the glucocorticoid antagonist RU 38486 had no effect. Moreover, sucrose density gradient analysis showed that docosahexaenoic acid inhibited the binding of [3H] dexamethasone to both the 8.8S and 4S forms. The results strongly suggest that unsaturated fatty acids are interacting at a site on the receptor different from the hormone binding site and the heat shock protein and that by binding to a second site unsaturated fatty acids greatly change the conformation of the hormone binding site to reduce its affinity for the hormone, either partially or completely depending on the concentration and the class of the fatty acid.

  12. Disconfirming (Double Bind) Effects of Incongruent Multi-Channel Messages.

    ERIC Educational Resources Information Center

    Rummel, Lynda; And Others

    In an experiment designed to explore the immediate disconfirming effects of strong and mild incongruent messages and the impact of certain contextual constraints on disconfirming effects, 192 male undergraduates evaluated videotaped examples of strong and mild sarcasm and joking and the complementary congruent forms of straightforward positive and…

  13. Synthesis and biological evaluation of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine derivatives bearing substituted benzylpiperazine moieties as positive inotropic agents.

    PubMed

    Wu, Yan; Sun, Liang-Peng; Ma, Long-Xu; Che, Jian; Song, Ming-Xia; Cui, Xun; Piao, Hu-Ri

    2013-05-01

    Two series of [1,2,4]triazolo[3,4-a]phthalazine and tetrazolo[5,1-a]phthalazine derivatives bearing substituted benzylpiperazine moieties have been synthesized and evaluated for their positive inotropic activity by measuring left atrium stroke volume on isolated rabbit heart preparations. The majority of the derivatives exhibited better in vitro activity than the existing drug, milrinone, and 6-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)tetrazolo[5,1-a]phthalazine. 8 m in particular was identified as the most potent with an increased stroke volume of 12.02 ± 0.20% (milrinone: 2.46 ± 0.07%) at a concentration of 3 × 10(-5)  m. The chronotropic effects of the compounds that exhibited good potency were also evaluated.

  14. The effect of gamma-enhancing binaural beats on the control of feature bindings.

    PubMed

    Colzato, Lorenza S; Steenbergen, Laura; Sellaro, Roberta

    2017-04-13

    Binaural beats represent the auditory experience of an oscillating sound that occurs when two sounds with neighboring frequencies are presented to one's left and right ear separately. Binaural beats have been shown to impact information processing via their putative role in increasing neural synchronization. Recent studies of feature-repetition effects demonstrated interactions between perceptual features and action-related features: repeating only some, but not all features of a perception-action episode hinders performance. These partial-repetition (or binding) costs point to the existence of temporary episodic bindings (event files) that are automatically retrieved by repeating at least one of their features. Given that neural synchronization in the gamma band has been associated with visual feature bindings, we investigated whether the impact of binaural beats extends to the top-down control of feature bindings. Healthy adults listened to gamma-frequency (40 Hz) binaural beats or to a constant tone of 340 Hz (control condition) for ten minutes before and during a feature-repetition task. While the size of visuomotor binding costs (indicating the binding of visual and action features) was unaffected by the binaural beats, the size of visual feature binding costs (which refer to the binding between the two visual features) was considerably smaller during gamma-frequency binaural beats exposure than during the control condition. Our results suggest that binaural beats enhance selectivity in updating episodic memory traces and further strengthen the hypothesis that neural activity in the gamma band is critically associated with the control of feature binding.

  15. Inversion of the Bohr effect upon oxygen binding to 24-meric tarantula hemocyanin.

    PubMed Central

    Sterner, R; Decker, H

    1994-01-01

    The Bohr effect describes the usually negative coupling between the binding of oxygen and the binding of protons to respiratory proteins. It was first described for hemoglobin and provides for an optimal oxygen supply of the organism under changing physiological conditions. Our measurements of both oxygen and proton binding to the 24-meric tarantula hemocyanin establish the unusual case where a respiratory protein binds protons at low degrees of oxygenation but releases protons at high degrees of oxygenation. In contrast to what is observed with hemoglobin and other respiratory proteins, this phenomenon amounts to the inversion of the Bohr effect in the course of an oxygen-binding curve at a given pH value. Therefore, protons in spider blood can act either as allosteric activators or as allosteric inhibitors of oxygen binding, depending on the degree of oxygenation of hemocyanin. These functional properties of tarantula hemocyanin, which cannot be explained by classical allosteric models, require at least four different conformational states of the subunits. Inspection of the known x-ray structures of closely related hemocyanins suggests that salt bridges between completely conserved histidine and glutamate residues located at particular intersubunit interfaces are responsible for the observed phenomena. Images PMID:8197143

  16. Probing the Functional Heterogeneity of Surface Binding Sites by Analysis of Experimental Binding Traces and the Effect of Mass Transport Limitation

    PubMed Central

    Svitel, Juraj; Boukari, Hacène; Van Ryk, Donald; Willson, Richard C.; Schuck, Peter

    2007-01-01

    Many techniques rely on the binding activity of surface-immobilized proteins, including antibody-based affinity biosensors for the detection of analytes, immunoassays, protein arrays, and surface plasmon resonance biosensors for the study of thermodynamic and kinetic aspects of protein interactions. To study the functional homogeneity of the surface sites and to characterize their binding properties, we have recently proposed a computational tool to determine the distribution of affinity and kinetic rate constants from surface binding progress curves. It is based on modeling the experimentally measured binding signal as a superposition of signals from binding to sites spanning a range of rate and equilibrium constants, with regularization providing the most parsimonious distribution consistent with the data. In the present work, we have expanded the scope of this approach to include a compartment-like transport step, which can describe competitive binding to different surface sites in a zone of depleted analyte close to the sensor surface. This approach addresses a major difficulty in the analysis of surface binding where both transport limitation as well as unknown surface site heterogeneity may be present. In addition to the kinetic binding parameters of the ensemble of surface sites, it can provide estimates for effective transport rate constants. Using antibody-antigen interactions as experimental model systems, we studied the effects of the immobilization matrix and of the analyte flow-rate on the effective transport rate constant. Both were experimentally observed to influence mass transport. The approximate description of mass transport by a compartment model becomes critical when applied to strongly transport-controlled data, and we examined the limitations of this model. In the presence of only moderate mass transport limitation the compartment model provides a good description, but this approximation breaks down for strongly transport-limited surface

  17. Alpha-Amylase Starch Binding Domains: Cooperative Effects of Binding to Starch Granules of Multiple Tandemly Arranged Domains▿

    PubMed Central

    Guillén, D.; Santiago, M.; Linares, L.; Pérez, R.; Morlon, J.; Ruiz, B.; Sánchez, S.; Rodríguez-Sanoja, R.

    2007-01-01

    The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch. PMID:17468268

  18. Alpha-amylase starch binding domains: cooperative effects of binding to starch granules of multiple tandemly arranged domains.

    PubMed

    Guillén, D; Santiago, M; Linares, L; Pérez, R; Morlon, J; Ruiz, B; Sánchez, S; Rodríguez-Sanoja, R

    2007-06-01

    The Lactobacillus amylovorus alpha-amylase starch binding domain (SBD) is a functional domain responsible for binding to insoluble starch. Structurally, this domain is dissimilar from other reported SBDs because it is composed of five identical tandem modules of 91 amino acids each. To understand adsorption phenomena specific to this SBD, the importance of their modular arrangement in relationship to binding ability was investigated. Peptides corresponding to one, two, three, four, or five modules were expressed as His-tagged proteins. Protein binding assays showed an increased capacity of adsorption as a function of the number of modules, suggesting that each unit of the SBD may act in an additive or synergic way to optimize binding to raw starch.

  19. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    PubMed

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms.

  20. MODELING THE EFFECTS OF FLEXIBILITY ON THE BINDING OF ENVIRONMENTAL ESTROGENS TO THE ESTROGEN RECEPTOR

    EPA Science Inventory

    Modeling the effects of flexibility on the binding of environmental estrogens to the estrogen receptor
    There are many reports of environmental endocrine disruption in the literature, yet it has been difficult to identify the specific chemicals responsible for these effects. ...

  1. Maltose-binding protein effectively stabilizes the partially closed conformation of the ATP-binding cassette transporter MalFGK2.

    PubMed

    Weng, Jingwei; Gu, Shuo; Gao, Xin; Huang, Xuhui; Wang, Wenning

    2017-04-05

    Maltose transporter MalFGK2 is a type-I importer in the ATP-binding cassette (ABC) transporter superfamily. Upon the binding of its periplasmic binding protein, MalE, the ATPase activity of MalFGK2 can be greatly enhanced. Crystal structures of the MalFGK2-MalE-maltose complex in a so-called "pretranslocation" ("pre-T") state with a partially closed conformation suggest that the formation of this MalE-stabilized intermediate state is a key step leading to the outward-facing catalytic state. On the contrary, crosslinking and fluorescence studies suggest that ATP binding alone is sufficient to promote the outward-facing catalytic state, thereby doubting the role of MalE binding. To clarify the role of MalE binding and to gain deeper understanding of the molecular mechanisms of MalFGK2, we calculated the free energy surfaces (FESs) related to the lateral motion in the presence and absence of MalE using atomistic metadynamics simulations. The results showed that, in the absence of MalE, laterally closing motion was energetically forbidden but, upon MalE binding, more closed conformations similar to the pre-T state become more stable. The significant effect of MalE binding on the free energy landscapes was in agreement with crystallographic studies and confirmed the important role of MalE in stabilizing the pre-T state. Our simulations also revealed that the allosteric effect of MalE stimulation originates from the MalE-binding-promoted vertical motion between MalF and MalG cores, which was further supported by MD simulation of the MalE-independent mutant MalF500.

  2. Mapping the Effect of Gly Mutations in Collagen on α2β1 Integrin Binding*

    PubMed Central

    Yigit, Sezin; Yu, Hongtao; An, Bo; Hamaia, Samir; Farndale, Richard W.; Kaplan, David L.; Lin, Yu-Shan; Brodsky, Barbara

    2016-01-01

    The replacement of one Gly in the essential repeating tripeptide sequence of the type I collagen triple helix results in the dominant hereditary bone disorder osteogenesis imperfecta. The mechanism leading to pathology likely involves misfolding and autophagy, although it has been hypothesized that some mutations interfere with known collagen interactions. Here, the effect of Gly replacements within and nearby the integrin binding GFPGER sequence was investigated using a recombinant bacterial collagen system. When a six-triplet human type I collagen sequence containing GFPGER was introduced into a bacterial collagen-like protein, this chimeric protein bound to integrin. Constructs with Gly to Ser substitutions within and nearby the inserted human sequence still formed a trypsin-resistant triple helix, suggesting a small local conformational perturbation. Gly to Ser mutations within the two Gly residues in the essential GFPGER sequence prevented integrin binding and cell attachment as predicted from molecular dynamics studies of the complex. Replacement of Gly residues C-terminal to GFPGER did not affect integrin binding. In contrast, Gly replacements N-terminal to the GFPGER sequence, up to four triplets away, decreased integrin binding and cell adhesion. This pattern suggests either an involvement of the triplets N-terminal to GFPGER in initial binding or a propagation of the perturbation of the triple helix C-terminal to a mutation site. The asymmetry in biological consequences relative to the mutation site may relate to the observed pattern of osteogenesis imperfecta mutations near the integrin binding site. PMID:27432884

  3. Effect of DNA binding on geminate CO recombination kinetics in CooA

    NASA Astrophysics Data System (ADS)

    Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan; Poulos, Thomas; Champion, Paul

    2012-02-01

    CooA proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding to RrCooA. The effects of DNA binding and the truncation of the DNA binding domain on the CO geminate recombination kinetics were investigated. The CO rebinding kinetics in these CooA complexes takes place on ultrafast timescales but remains non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the timescale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in RrCooA relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Analysis of our data reveals that the uncomplexed and inherently flexible DNA binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases and the distribution of the conformations available in the heme domain becomes restricted.

  4. Effects of Water Models on Binding Affinity: Evidence from All-Atom Simulation of Binding of Tamiflu to A/H5N1 Neuraminidase

    PubMed Central

    Nguyen, Trang Truc; Viet, Man Hoang

    2014-01-01

    The influence of water models SPC, SPC/E, TIP3P, and TIP4P on ligand binding affinity is examined by calculating the binding free energy ΔGbind of oseltamivir carboxylate (Tamiflu) to the wild type of glycoprotein neuraminidase from the pandemic A/H5N1 virus. ΔGbind is estimated by the Molecular Mechanic-Poisson Boltzmann Surface Area method and all-atom simulations with different combinations of these aqueous models and four force fields AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L. It is shown that there is no correlation between the binding free energy and the water density in the binding pocket in CHARMM. However, for three remaining force fields ΔGbind decays with increase of water density. SPC/E provides the lowest binding free energy for any force field, while the water effect is the most pronounced in CHARMM. In agreement with the popular GROMACS recommendation, the binding score obtained by combinations of AMBER-TIP3P, OPLS-TIP4P, and GROMOS-SPC is the most relevant to the experiments. For wild-type neuraminidase we have found that SPC is more suitable for CHARMM than TIP3P recommended by GROMACS for studying ligand binding. However, our study for three of its mutants reveals that TIP3P is presumably the best choice for CHARMM. PMID:24672329

  5. Effect of protein aggregates on characterization of FcRn binding of Fc-fusion therapeutics.

    PubMed

    Bajardi-Taccioli, Adriana; Blum, Andrew; Xu, Chongfeng; Sosic, Zoran; Bergelson, Svetlana; Feschenko, Marina

    2015-10-01

    Recycling of antibodies and Fc containing therapeutic proteins by the neonatal Fc receptor (FcRn) is known to prolong their persistence in the bloodstream. Fusion of Fc fragment of IgG1 to other proteins is one of the strategies to improve their pharmacokinetic properties. Accurate measurement of Fc-FcRn binding provides information about the strength of this interaction, which in most cases correlates with serum half-life of the protein. It can also offer insight into functional integrity of Fc region. We investigated FcRn binding activity of a large set of Fc-fusion samples after thermal stress by the method based on AlphaScreen technology. An unexpected significant increase in FcR binding was found to correlate with formation of aggregates in these samples. Monomer purified from a thermally-stressed sample had normal FcRn binding, confirming that its Fc portion was intact. Experiments with aggregates spiked into a sample with low initial aggregation level, demonstrated strong correlation between the level of aggregates and FcRn binding. This correlation varied significantly in different methods. By introducing modifications to the assay format we were able to minimize the effects of aggregated species on FcRn binding, which should prevent masking functional changes of Fc-fusion protein. Biolayer interferometry (BLI) was used as an alternative method to measure FcRn binding. Both optimized AlphaScreen- and BLI-based assays were sensitive to structural changes in Fc portion of the molecule, such as oxidation of methionines 252 and 428, and therefore suitable for characterization of FcRn binding.

  6. Uncoupling of gamma-aminobutyric acid B receptors from GTP-binding proteins by N-ethylmaleimide: effect of N-ethylmaleimide on purified GTP-binding proteins

    SciTech Connect

    Asano, T.; Ogasawara, N.

    1986-03-01

    Treatment of membranes from bovine cerebral cortex with N-ethylmaleimide (NEM) resulted in inhibition of gamma-aminobutyric acid (GABA) binding to GABAB receptors. The binding curve for increasing concentrations of agonist was shifted to the right by NEM treatment. Guanine nucleotide had little effect on the binding of GABA to NEM-treated membranes. The addition of purified GTP-binding proteins, which were the substrates of islet-activating protein (IAP), pertussis toxin, to the NEM-treated membranes caused a shift of the binding curve to the left, suggesting modification of GTP-binding proteins rather than receptors by NEM. The effect of NEM on two purified GTP-binding proteins, Gi (composed of three subunits with molecular weight of alpha, 41,000; beta, 35,000; gamma, 10,000) and Go (alpha, 39,000; beta, 35,000; gamma, 10,000) was studied. NEM did not significantly change guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding and GTPase activity of these two proteins. NEM-treated Gi and Go were not ADP-ribosylated by IAP and did not increase GABA binding to NEM-treated membranes. When alpha and beta gamma subunits were treated with NEM and then mixed with nontreated alpha and beta gamma to form Gi or Go, respectively, both oligomers with NEM-treated alpha-subunits lost their abilities to be IAP substrates and to couple to receptors. Results indicate that NEM uncoupled GTP-binding proteins from receptors by modifying alpha-subunits of GTP-binding proteins, and the site seemed to be on or near the site of ADP-ribosylation by IAP. When alpha and beta gamma subunits were treated with NEM and then mixed to form Gi or Go, GTP gamma S binding in the absence of Mg2+ and GTPase activity were changed, although they were not affected when oligomers were treated with NEM. Results suggest the existence of another sulfhydryl group which is protected from NEM by the association of subunits.

  7. Bivalent cation binding effect on formation of the peptide bond

    NASA Astrophysics Data System (ADS)

    Remko, Milan; Rode, Bernd Michael

    2000-01-01

    The reactions between formic acid (or glycine) and ammonia, without and with Mg 2+, Ni 2+ and Cu 2+ cations as catalysts, have been studied as model reactions for peptide bond formation using the Becke3LYP functional and 6-311+G(d,p) basis set of DFT theory. Enthalpies and free energies for the stationary points of each reaction have been calculated to determine the thermodynamics of reactions investigated. A substantial decrease in reaction enthalpies and free energies was found for formic acid-ammonia and glycine-ammonia reactions catalysed by Mg 2+, Ni 2+ and Cu 2+ ions compared with those of the uncatalysed amide bond formation. The catalytic effect of the transition metal ions Ni 2+ and Cu 2+ is of similar strength and more pronounced than that of the Mg 2+ cation.

  8. Effect of chain length on binding of fatty acids to Pluronics in microemulsions.

    PubMed

    James-Smith, Monica A; Shekhawat, Dushyant; Cheung, Sally; Moudgil, Brij M; Shah, Dinesh O

    2008-03-15

    We investigated the effect of fatty acid chain length on the binding capacity of drug and fatty acid to Pluronic F127-based microemulsions. This was accomplished by using turbidity experiments. Pluronic-based oil-in-water microemulsions of various compositions were synthesized and titrated to turbidity with concentrated Amitriptyline, an antidepressant drug. Sodium salts of C(8), C(10), or C(12) fatty acid were used in preparation of the microemulsion and the corresponding binding capacities were observed. It has been previously determined that, for microemulsions prepared with sodium caprylate (C(8) fatty acid soap), a maximum of 11 fatty acid molecules bind to the microemulsion per 1 molecule of Pluronic F127 and a maximum of 12 molecules of Amitriptyline bind per molecule of F127. We have found that with increasing the chain length of the fatty acid salt component of the microemulsion, the binding capacity of both the fatty acid and the Amitriptyline to the microemulsion decreases. For sodium salts of C(8), C(10) and C(12) fatty acids, respectively, a maximum of approximately 11, 8.4 and 8.3 molecules of fatty acid molecules bind to 1 Pluronic F127 molecule. We propose that this is due to the decreasing number of free monomers with increasing chain length. As chain length increases, the critical micelle concentration (cmc) decreases, thus leading to fewer monomers. Pluronics are symmetric tri-block copolymers consisting of propylene oxide (PO) and ethylene oxide (EO). The polypropylene oxide block, PPO is sandwiched between two polyethylene oxide (PEO) blocks. The PEO blocks are hydrophilic while PPO is hydrophobic portion in the Pluronic molecule. Due to this structure, we propose that the fatty acid molecules that are in monomeric form most effectively diffuse between the PEO "tails" and bind to the hydrophobic PPO groups.

  9. Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies.

    PubMed

    Bittoun, P; Bagheri-Yarmand, R; Chaubet, F; Crépin, M; Jozefonvicz, J; Fermandjian, S

    1999-06-15

    CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

  10. Structures of apo IRF-3 and IRF-7 DNA binding domains: effect of loop L1 on DNA binding

    SciTech Connect

    De Ioannes, Pablo; Escalante, Carlos R.; Aggarwal, Aneel K.

    2013-11-20

    Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-{beta} (IFN-{beta}) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-{beta} promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures of IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.

  11. Lomofungin and dilomofungin: inhibitors of MBNL1-CUG RNA binding with distinct cellular effects

    PubMed Central

    Hoskins, Jason W.; Ofori, Leslie O.; Chen, Catherine Z.; Kumar, Amit; Sobczak, Krzysztof; Nakamori, Masayuki; Southall, Noel; Patnaik, Samarjit; Marugan, Juan J.; Zheng, Wei; Austin, Christopher P.; Disney, Matthew D.; Miller, Benjamin L.; Thornton, Charles A.

    2014-01-01

    Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disorder resulting from expression of RNA containing an expanded CUG repeat (CUGexp). The pathogenic RNA is retained in nuclear foci. Poly-(CUG) binding proteins in the Muscleblind-like (MBNL) family are sequestered in foci, causing misregulated alternative splicing of specific pre-mRNAs. Inhibitors of MBNL1-CUGexp binding have been shown to restore splicing regulation and correct phenotypes in DM1 models. We therefore conducted a high-throughput screen to identify novel inhibitors of MBNL1-(CUG)12 binding. The most active compound was lomofungin, a natural antimicrobial agent. We found that lomofungin undergoes spontaneous dimerization in DMSO, producing dilomofungin, whose inhibition of MBNL1–(CUG)12 binding was 17-fold more potent than lomofungin itself. However, while dilomofungin displayed the desired binding characteristics in vitro, when applied to cells it produced a large increase of CUGexp RNA in nuclear foci, owing to reduced turnover of the CUGexp transcript. By comparison, the monomer did not induce CUGexp accumulation in cells and was more effective at rescuing a CUGexp-induced splicing defect. These results support the feasibility of high-throughput screens to identify compounds targeting toxic RNA, but also demonstrate that ligands for repetitive sequences may have unexpected effects on RNA decay. PMID:24799433

  12. Large-scale molecular dynamics simulation: Effect of polarization on thrombin-ligand binding energy

    PubMed Central

    Duan, Li L.; Feng, Guo Q.; Zhang, Qing G.

    2016-01-01

    Molecular dynamics (MD) simulations lasting 500 ns were performed in explicit water to investigate the effect of polarization on the binding of ligands to human α-thrombin based on the standard nonpolarizable AMBER force field and the quantum-derived polarized protein-specific charge (PPC). The PPC includes the electronic polarization effect of the thrombin-ligand complex, which is absent in the standard force field. A detailed analysis and comparison of the results of the MD simulation with experimental data provided strong evidence that intra-protein, protein-ligand hydrogen bonds and the root-mean-square deviation of backbone atoms were significantly stabilized through electronic polarization. Specifically, two critical hydrogen bonds between thrombin and the ligand were broken at approximately 190 ns when AMBER force field was used and the number of intra-protein backbone hydrogen bonds was higher under PPC than under AMBER. The thrombin-ligand binding energy was computed using the molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) method, and the results were consistent with the experimental value obtained using PPC. Because hydrogen bonds were unstable, it was failed to predict the binding affinity under the AMBER force field. Furthermore, the results of the present study revealed that differences in the binding free energy between AMBER and PPC almost comes from the electrostatic interaction. Thus, this study provides evidence that protein polarization is critical to accurately describe protein-ligand binding. PMID:27507430

  13. Trimer Enhancement Mutation Effects on HIV-1 Matrix Protein Binding Activities

    PubMed Central

    Alfadhli, Ayna; Mack, Andrew; Ritchie, Christopher; Cylinder, Isabel; Harper, Logan; Tedbury, Philip R.; Freed, Eric O.

    2016-01-01

    ABSTRACT The HIV-1 matrix (MA) protein is the amino-terminal domain of the HIV-1 precursor Gag (Pr55Gag) protein. MA binds to membranes and RNAs, helps transport Pr55Gag proteins to virus assembly sites at the plasma membranes of infected cells, and facilitates the incorporation of HIV-1 envelope (Env) proteins into virions by virtue of an interaction with the Env protein cytoplasmic tails (CTs). MA has been shown to crystallize as a trimer and to organize on membranes in hexamer lattices. MA mutations that localize to residues near the ends of trimer spokes have been observed to impair Env protein assembly into virus particles, and several of these are suppressed by the 62QR mutation at the hubs of trimer interfaces. We have examined the binding activities of wild-type (WT) MA and 62QR MA variants and found that the 62QR mutation stabilized MA trimers but did not alter the way MA proteins organized on membranes. Relative to WT MA, the 62QR protein showed small effects on membrane and RNA binding. However, 62QR proteins bound significantly better to Env CTs than their WT counterparts, and CT binding efficiencies correlated with trimerization efficiencies. Our data suggest a model in which multivalent binding of trimeric HIV-1 Env proteins to MA trimers contributes to the process of Env virion incorporation. IMPORTANCE The HIV-1 Env proteins assemble as trimers, and incorporation of the proteins into virus particles requires an interaction of Env CT domains with the MA domains of the viral precursor Gag proteins. Despite this knowledge, little is known about the mechanisms by which MA facilitates the virion incorporation of Env proteins. To help elucidate this process, we examined the binding activities of an MA mutant that stabilizes MA trimers. We found that the mutant proteins organized similarly to WT proteins on membranes, and that mutant and WT proteins revealed only slight differences in their binding to RNAs or lipids. However, the mutant proteins showed

  14. Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors

    PubMed Central

    2009-01-01

    Background Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity. Results At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 μM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [3H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [35S]GTPγS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [35S]GTPγS binding at M2 and M4 receptors. In contrast, 0.1 μM rapacuronium significantly increased ACh potency at M1, M3, and M5 receptors. Kinetic measurements at M3 receptors showed acceleration of the rate of ACh-induced [35S]GTPγS binding by rapacuronium. Conclusions Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 μM) but not at high concentrations (10 μM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M3 receptor-mediated bronchoconstriction. PMID:20038295

  15. Quantum confined Stark effect in Gaussian quantum wells: A tight-binding study

    SciTech Connect

    Ramírez-Morales, A.; Martínez-Orozco, J. C.; Rodríguez-Vargas, I.

    2014-05-15

    The main characteristics of the quantum confined Stark effect (QCSE) are studied theoretically in quantum wells of Gaussian profile. The semi-empirical tight-binding model and the Green function formalism are applied in the numerical calculations. A comparison of the QCSE in quantum wells with different kinds of confining potential is presented.

  16. Effect of d-amino acids on IgE binding to peanut allergens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    D-amino acids are formed when L-amino acids are exposed to heat. The objective was to determine the existence of D-amino acids in roasted peanut and their effect on IgE binding. Raw and roasted peanut protein extracts were hydrolyzed in 6 N HCL under vacuum. The hydrolysates were then analyzed for D...

  17. Catalytic and ligand binding properties of the FK506 binding protein FKBP12: effects of the single amino acid substitution of Tyr82 to Leu.

    PubMed Central

    Bossard, M J; Bergsma, D J; Brandt, M; Livi, G P; Eng, W K; Johnson, R K; Levy, M A

    1994-01-01

    The binding of FK506 and rapamycin to their cytosolic receptor FKBP12 is an intermediate step in the paths leading to their potent immunosuppressive properties. One of the amino acids defining the hydrophobic binding cleft for the macrocycles is Tyr82, which is thought to form a hydrogen bond with the amide oxygens of the common pipecolyl structural element within the two macrolides. To understand better the influence of this amino acid residue in catalytic activity (cis-trans peptidyl prolyl isomerization) and ligand binding properties, a Tyr82 to Leu site-specific modification of FKBP12 was prepared, purified and characterized. Kinetic experiments have demonstrated that the Tyr82 to Leu modification has a greater effect on catalytic properties than on ligand binding affinities, a result which indicates that these inhibitors may not be binding as true transition-state analogues. In an additional test for cellular function, expression of both wild-type and mutant human FKBP12 in a strain of Saccharomyces cerevisiae rendered resistant to rapamycin by deletion of the gene encoding a cytosolic rapamycin binding protein (RPB1), the yeast homologue of FKBP12, restored wild-type drug sensitivity. PMID:7507662

  18. The effect of hypobaric hypoxia on misonidazole binding in normal and tumour-bearing mice.

    PubMed Central

    MacManus, M. P.; Maxwell, A. P.; Abram, W. P.; Bridges, J. M.

    1989-01-01

    The effect of hypobaric hypoxia on the in vivo binding of misonidazole was investigated in normal mice and mice bearing T50/80 or CA NT mammary carcinomas. After the intraperitoneal injection of radiolabelled misonidazole, mice were randomised to breathe either room air or air at 0.5 atmospheres. The distribution of misonidazole in liver, kidney, heart, spleen and tumour tissue, 24 h later, was studied by scintillation counting and by autoradiography. Significantly higher misonidazole binding occurred in the livers (x2.5), kidneys (x2.4), spleens (x2.9) and hearts (x1.8) of hypoxic mice compared to controls. Hypobaric hypoxia was associated with a greater than four-fold increase in misonidazole binding within T50/80 tumours. However, significantly higher binding was not demonstrated within CA NT tumours after exposure of tumour-bearing animals to hypoxic conditions. In autoradiographs of hypoxic liver, labelling was intense in regions near to hepatic veins but sparse in areas surrounding portal tracts. This pattern was striking and consistent. In hypoxic kidney, labelling was most intense over tubular cells, less intense over glomeruli and sparse in the renal medulla. It is likely that the hepatic and renal cortical distributions of misonidazole binding reflect local oxygen gradients. Images Figure 4 Figure 5 PMID:2930698

  19. Effect of terbium(III) on the binding of aromatic guests with sodium taurocholate aggregates.

    PubMed

    Pace, Tamara C S; Souza, Sergio P; Zhang, Hui Ting; Bohne, Cornelia

    2011-10-01

    The effect of binding Tb(3+) to sodium taurocholate aggregates containing polyaromatic hydrocarbon guests was examined using pyrene and 1-ethylnaphthalene as guests that bind to the primary aggregate, and 1-naphthyl-1-ethanol as a secondary aggregate guest. Time-resolved fluorescence quenching studies were used to study the binding site properties, while laser flash photolysis quenching studies provided information on the dynamics of the guest-aggregate system. Both the primary and secondary aggregate binding sites became more compact in the presence of bound Tb(3+), while only the primary aggregate became more accessible to anionic molecules. The binding dynamics for the guest-primary aggregate system became faster when Tb(3+) was bound to the aggregate. In contrast, for the guest-secondary aggregate the presence of Tb(3+) resulted in a small decrease in the dissociation rate constant. The influence of bound Tb(3+) on the primary and secondary bile salt aggregates is significantly different, which affects how these aggregates can be used as supramolecular host systems to modify guest reactivity.

  20. Beyond the detergent effect: a binding site for sodium dodecyl sulfate (SDS) in mammalian apoferritin

    SciTech Connect

    Liu, Renyu Bu, Weiming; Xi, Jin; Mortazavi, Shirin R.; Cheung-Lau, Jasmina C.; Dmochowski, Ivan J.; Loll, Patrick J.

    2012-05-01

    Using X-ray crystallography and isothermal titration calorimetry, we show that sodium dodecyl sulfate (SDS) binds specifically to a pre-formed internal cavity in horse-spleen apoferritin. Although sodium dodecyl sulfate (SDS) is widely used as an anionic detergent, it can also exert specific pharmacological effects that are independent of the surfactant properties of the molecule. However, structural details of how proteins recognize SDS are scarce. Here, it is demonstrated that SDS binds specifically to a naturally occurring four-helix bundle protein: horse apoferritin. The X-ray crystal structure of the apoferritin–SDS complex was determined at a resolution of 1.9 Å and revealed that the SDS binds in an internal cavity that has previously been shown to recognize various general anesthetics. A dissociation constant of 24 ± 9 µM at 293 K was determined by isothermal titration calorimetry. SDS binds in this cavity by bending its alkyl tail into a horseshoe shape; the charged SDS head group lies in the opening of the cavity at the protein surface. This crystal structure provides insights into the protein–SDS interactions that give rise to binding and may prove useful in the design of novel SDS-like ligands for some proteins.

  1. Effect of limited enzymatic hydrolysis on linoleic acid binding properties of β-lactoglobulin.

    PubMed

    Sponton, Osvaldo E; Perez, Adrián A; Carrara, Carlos; Santiago, Liliana G

    2014-03-01

    β-Lactoglobulin (BLG) is a member of lipocalin family, proteins with ability to bind small hydrophobic ligands, such as retinol, vitamins and fatty acids. Moreover, BLG is susceptible to protease action producing a wide range of polypeptides depending on the hydrolysis degree (HD). In the present work, the effect of limited enzymatic hydrolysis on fatty acid binding properties of BLG was studied. Linoleic acid (LA) was used as a model fatty acid. Limited enzymatic hydrolysis was performed using α-chymotrypsin immobilised on agarose microparticles. BLG hydrolysates were produced at HD: 1%, 3% and 5%. In order to determine the influence of HD on BLG molecular weight SDS-PAGE was used. BLG structural modification and LA binding properties were monitored by means of fluorescence spectroscopic techniques. The increase in HD produced: (i) a BLG degradation and a molecular weight distribution of BLG hydrolysates and (ii) an increased exposition of buried hydrophobic residues, however it was observed a decrease in surface hydrophobicity possibly due to a deterioration of hydrophobic protein domains. It was observed that enzymatic hydrolysis treatment produced a decrease in BLG ability for binding LA. It was concluded that limited enzymatic hydrolysis could deteriorate the specific site on BLG structure necessary for binding LA.

  2. Effects of oxymorphazone in frogs: long lasting antinociception in vivo, and apparently irreversible binding in vitro

    SciTech Connect

    Benyhe, S.; Hoffman, G.; Varga, E.; Hosztafi, S.; Toth, G.; Borsodi, A.; Wollemann, M.

    1989-01-01

    Oxymorphazone was found to be a relatively weak antinociceptive drug in intact frog (Rana esculenta) when acetic acid was used as pain stimulus. Frogs remained analgesic for at least 48 hrs following oxymorphazone administration. The ligand increased the latency of wiping reflex in spinal frogs too. There effects were blocked by naloxone. In equilibrium binding studies (/sup 3/H)oxymorphazone had high affinity to the opioid receptors of frog brain and spinal cord as well. Kinetic experiments show that only 25% of the bound (/sup 3/H)oxymorphazone is readily dissociable. Preincubation of the membranes with labeled oxymorphazone results in a washing resistant inhibition of the opioid binding sites. At least 70% of the (/sup 3/H)oxymorphazone specific binding is apparently irreversible after reaction at 5 nM ligand concentration, and this can be enhanced by a higher concentration of tritiated ligand.

  3. Potent enhancement of (/sup 3/H)nitrendipine binding in rat cerebral cortical and cardiac homogenates: a putative mechanism for the action of MDL 12,330A

    SciTech Connect

    Lee, H.R.; Jaros, J.A.; Roeske, W.R.; Wiech, N.L.; Ursillo, R.; Yamamura, H.I.

    1985-06-01

    (/sup 3/H)Nitrendipine ((/sup 3/H)NTD), a specific high-affinity calcium channel antagonist, was used to label dihydropyridine binding sites associated with calcium channels in rat cerebral cortical and cardiac homogenates. A novel lactamimide compound, MDL 12,330A, has been shown previously to have negative inotropic and chronotropic effects in isolated working guinea-pig hearts and these effects are reversed by the administration of calcium. MDL 12,330A is potent in enhancing (/sup 3/H)NTD binding in membranes prepared from the cerebral cortex and the heart, with EC50 values of 6.1 X 10(-8) and 3.4 X 10(-8) M, respectively, at 37 degrees C. This allosteric effect by MDL 12,330A is similar to that produced by a known calcium channel antagonist, d-cis diltiazem, which has been shown previously to enhance (/sup 3/H)NTD binding at 37 degrees C. The extent of enhancement by MDL 12,330A depends on incubation temperature (37 degrees C greater than 25 degrees C greater than 0 degrees C). The mechanism of this enhancement by MDL 12,330A is due to a decrease in the dissociation rate constant of the dihydropyridine-calcium channel supramolecular complex. MDL 12,330A is the most potent drug thus far examined which demonstrates the enhancement of (/sup 3/H)NTD binding.

  4. Metal loading effect on rare earth element binding to humic acid: Experimental and modelling evidence

    NASA Astrophysics Data System (ADS)

    Marsac, Rémi; Davranche, Mélanie; Gruau, Gérard; Dia, Aline

    2010-03-01

    The effect of metal loading on the binding of rare earth elements (REE) to humic acid (HA) was studied by combining ultrafiltration and Inductively Coupled Plasma Mass Spectrometry techniques. REE-HA complexation experiments were performed at pH 3 for REE/C molar ratios ranging from ca 4 × 10 -4 to 2.7 × 10 -2. Results show that the relative amount of REE bound to HA strongly increases with decreasing REE/C. A middle-REE (MREE) downward concavity is shown by patterns at high metal loading, whereas patterns at low metal loading display a regular increase from La to Lu. Humic Ion Model VI modelling are close to the experimental data variations, provided that (i) the ΔLK 2 parameter (i.e. the Model VI parameter taken into account the presence of strong but low density binding sites) is allowed to increase regularly from La to Lu (from 1.1 to 2.1) and (ii) the published log KMA values (i.e. the REE-HA binding constants specific to Model VI) are slightly modified, in particular with respect to heavy REE. Modelling approach provided evidence that logKdREE patterns with varying REE/C likely arises because REE binding to HA occurs through two types of binding sites in different density: (i) a few strong sites that preferentially complex the heavy REE and thus control the logKdREE atterns at low REE/C; (ii) a larger amount of weaker binding sites that preferentially complex the middle-REE and thus control the logKdREE pattern at high REE/C. Hence, metal loading exerts a major effect on HA-mediated REE binding, which could explain the diversity of published conditional constants for REE binding with HA. A literature survey suggests that the few strong sites activated at low REE/C could be multidentate carboxylic sites, or perhaps N-, or P-functional groups. Finally, an examination of the literature field data proposed that the described loading effect could account for much of the variation in REE patterns observed in natural organic-rich waters (DOC > 5 mg L -1 and 4

  5. Effects of gene carrier polyethyleneimines on the structure and binding capability of bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Guo, Zhiyong; Kong, Zhijie; Wei, Yanshan; Li, Hua; Wang, Yajing; Huang, Aimin; Ma, Lin

    2017-02-01

    Polyethyleneimine (PEI), one of the most effective non-viral gene carriers, is also cytotoxic, however the molecular basis is poorly understood. Little is known about the effects of PEI on the structure and functions of the biomacromolecules. In this work, fluorescence, UV-vis absorption, circular dichroism (CD) spectroscopy and zeta-potential measurement were conducted to reveal the interaction between PEIs (average molecular weight 25, 10 and 1.8 kDa) and bovine serum albumin (BSA), and to evaluate the effects on the conformation of BSA as long as its binding capability to the model compounds, 8-anilino-1-naphthalenesulfonic acid (ANS) and quercetin. PEIs were found to complex with BSA and induced a conformational change of the protein by a major reduction of α-helix at PEI concentration < 0.2 mg·mL- 1 and an increase at higher PEI concentration. The binding efficacy of ANS and quercetin to BSA was greatly reduced by the competitive binding by PEI and influenced by the conformational change of BSA, which was found to display a similar trend to the change of the α-helix content of the protein. The polymer size played an important role in PEI-BSA interaction. PEI of higher molecular weight was more favorable to interact with BSA and more efficient to perturb the conformation and binding capability of the protein.

  6. Effects of gene carrier polyethyleneimines on the structure and binding capability of bovine serum albumin.

    PubMed

    Guo, Zhiyong; Kong, Zhijie; Wei, Yanshan; Li, Hua; Wang, Yajing; Huang, Aimin; Ma, Lin

    2017-02-15

    Polyethyleneimine (PEI), one of the most effective non-viral gene carriers, is also cytotoxic, however the molecular basis is poorly understood. Little is known about the effects of PEI on the structure and functions of the biomacromolecules. In this work, fluorescence, UV-vis absorption, circular dichroism (CD) spectroscopy and zeta-potential measurement were conducted to reveal the interaction between PEIs (average molecular weight 25, 10 and 1.8kDa) and bovine serum albumin (BSA), and to evaluate the effects on the conformation of BSA as long as its binding capability to the model compounds, 8-anilino-1-naphthalenesulfonic acid (ANS) and quercetin. PEIs were found to complex with BSA and induced a conformational change of the protein by a major reduction of α-helix at PEI concentration <0.2mg·mL(-1) and an increase at higher PEI concentration. The binding efficacy of ANS and quercetin to BSA was greatly reduced by the competitive binding by PEI and influenced by the conformational change of BSA, which was found to display a similar trend to the change of the α-helix content of the protein. The polymer size played an important role in PEI-BSA interaction. PEI of higher molecular weight was more favorable to interact with BSA and more efficient to perturb the conformation and binding capability of the protein.

  7. Glucocorticoid interactions with ethanol effects on synaptic plasma membranes: influence on [125I]calmodulin binding.

    PubMed

    Sze, P Y

    1996-02-01

    Ca(++)-dependent binding of calmodulin (CaM) to brain synaptic plasma membranes is known to be inhibited by ethanol and stimulated by glucocorticoids. These opposite neurochemical actions between ethanol and the steroids in vitro are consistent with glucocorticoid antagonism of ethanol-induced sedation reported to occur in vivo. The present study was undertaken to characterize the interactions of corticosterone with ethanol effects on [125I]CaM binding in synaptic plasma membranes. From the shift of concentration-response curves when corticosterone and ethanol were present in combination, the interaction between steroid stimulation and ethanol inhibition occurred in an additive relationship over the range of their effective concentrations. From Scatchard analyses, ethanol-induced decrease in membrane affinity for [125I]CaM was antagonized by steroid-induced increase in the membrane affinity, indicating that the convergent event in their interaction was the alteration of membrane affinity for CaM. Glucocorticoid antagonism of ethanol inhibition of [125I]CaM binding exhibited a high degree of steroid specificity; steroids with glucocorticoid activity including cortisol, dexamethasone and triamcinolone were effective, whereas gonadal steroids and excitatory neuroactive steroid metabolites were ineffective. The demonstration that glucocorticoids antagonized the inhibition of CaM binding by ethanol provides support for the hypothesis that these steroids are among the endogenous factors that modulate neuronal sensitivity to ethanol.

  8. Effect of antibodies against distinctive rat liver estrogen-binding protein on hormone-binding activity of this protein and steroid hormone receptors

    SciTech Connect

    Smirnov, A.N.; Shchelkunova, T.A.; Smirnova, O.V.; Rozen, V.B.

    1986-12-10

    The effect of rabbit polyclonal antibodies (AB) against a distinctive estrogen-binding protein (DEBP) of rat liver, isolated using an immunosorbent, on the interaction of (/sup 3/H)estradiol with the DEBP and estrogen receptors of the uterus and other tissues, as well as of (/sup 3/H)dihydrotestosterone with prostate androgen receptors, (/sup 3/H)progesterone with uterine progesterone receptors, and (/sup 3/H)dexamethasone with rat thymus glucocorticoid receptors was investigated. It was found that preincubation of the cytosol of the tissues under investigation with the antibodies decreases the capacity of the DEBP of the estrogen and androgen receptors to bind the corresponding ligand. The hormone-binding activity of progesterone and the glucocorticoid receptors does not change in the presence of AB. The binding activity of DEBP in the presence of AB decreases as a result of a decrease in the concentration of binding sites of the protein, while that of the estrogen and androgen receptors drops as a result of a decrease in affinity for the ligand, due to a drop in the association rate constant. A cross effect of AB on the activity of uterine estrogen receptors of the rabbit, guinea pig, and mouse was found. It was concluded that there is a definite similarity in the structure of DEBP and sex steroid receptors.

  9. Substrate and Transition State Binding in Alkaline Phosphatase Analyzed by Computation of Oxygen Isotope Effects.

    PubMed

    Roston, Daniel; Cui, Qiang

    2016-09-14

    Enzymes are powerful catalysts, and a thorough understanding of the sources of their catalytic power will facilitate many medical and industrial applications. Here we have studied the catalytic mechanism of alkaline phosphatase (AP), which is one of the most catalytically proficient enzymes known. We have used quantum mechanics calculations and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to model a variety of isotope effects relevant to the reaction of AP. We have calculated equilibrium isotope effects (EIEs), binding isotope effects (BIEs), and kinetic isotope effects (KIEs) for a range of phosphate mono- and diester substrates. The results agree well with experimental values, but the model for the reaction's transition state (TS) differs from the original interpretation of those experiments. Our model indicates that isotope effects on binding make important contributions to measured KIEs on V/K, which complicated interpretation of the measured values. Our results provide a detailed interpretation of the measured isotope effects and make predictions that can test the proposed model. The model indicates that the substrate is deformed in the ground state (GS) of the reaction and partially resembles the TS. The highly preorganized active site preferentially binds conformations that resemble the TS and not the GS, which induces the substrate to adapt to the enzyme, rather than the other way around-as with classic "induced fit" models. The preferential stabilization of the TS over the GS is what lowers the barrier to the chemical step.

  10. Action-effect binding is decreased in motor conversion disorder: implications for sense of agency.

    PubMed

    Kranick, Sarah M; Moore, James W; Yusuf, Nadia; Martinez, Valeria T; LaFaver, Kathrin; Edwards, Mark J; Mehta, Arpan R; Collins, Phoebe; Harrison, Neil A; Haggard, Patrick; Hallett, Mark; Voon, Valerie

    2013-07-01

    The abnormal movements seen in motor conversion disorder are affected by distraction and entrainment, similar to voluntary movement. Unlike voluntary movement, however, patients lack a sense of control for the abnormal movements, a failure of "self-agency." The action-effect binding paradigm has been used to quantify the sense of self-agency, because subjective contraction of time between an action and its effect only occurs if the patient feels that they are the agent responsible for the action. We used this paradigm, coupled with emotional stimuli, to investigate the sense of agency with voluntary movements in patients with motor conversion disorder. Twenty patients with motor conversion disorder and 20 age-matched and sex-matched healthy volunteers used a rotating clock to judge the time of their own voluntary key presses (action) and a subsequent auditory tone (effect) after they completed conditioning blocks in which high, medium, and low tones were coupled to images of happy, fearful, and neutral faces. The results replicated those produced previously: it was reported that an effect after a voluntary action occurred earlier, and the preceding action occurred later, compared with trials that used only key presses or tones. Patients had reduced overall binding scores relative to healthy volunteers, suggesting a reduced sense of agency. There was no effect of the emotional stimuli (faces) or other interaction effects. Healthy volunteers with subclinical depressive symptoms had higher overall binding scores. We demonstrate that patients with motor conversion disorder have decreased action-effect binding for normal voluntary movements compared with healthy volunteers, consistent with the greater experience of lack of control.

  11. Action-effect bindings and ideomotor learning in intention- and stimulus-based actions.

    PubMed

    Herwig, Arvid; Waszak, Florian

    2012-01-01

    According to ideomotor theory, action-effect associations are crucial for voluntary action control. Recently, a number of studies started to investigate the conditions that mediate the acquisition and application of action-effect associations by comparing actions carried out in response to exogenous stimuli (stimulus-based) with actions selected endogenously (intention-based). There is evidence that the acquisition and/or application of action-effect associations is boosted when acting in an intention-based action mode. For instance, bidirectional action-effect associations were diagnosed in a forced choice test phase if participants previously experienced action-effect couplings in an intention-based but not in a stimulus-based action mode. The present study aims at investigating effects of the action mode on action-effect associations in more detail. In a series of experiments, we compared the strength and durability of short-term action-effect associations (binding) immediately following intention- as well as stimulus-based actions. Moreover, long-term action-effect associations (learning) were assessed in a subsequent test phase. Our results show short-term action-effect associations of equal strength and durability for both action modes. However, replicating previous results, long-term associations were observed only following intention-based actions. These findings indicate that the effect of the action mode on long-term associations cannot merely be a result of accumulated short-term action-effect bindings. Instead, only those episodic bindings are selectively perpetuated and retrieved that integrate action-relevant aspects of the processing event, i.e., in case of intention-based actions, the link between action and ensuing effect.

  12. The neural correlates of age effects on verbal-spatial binding in working memory.

    PubMed

    Meier, Timothy B; Nair, Veena A; Meyerand, Mary E; Birn, Rasmus M; Prabhakaran, Vivek

    2014-06-01

    In this study, we investigated the neural correlates of age-related differences in the binding of verbal and spatial information utilizing event-related working memory tasks. Twenty-one right handed younger adults and twenty-one right handed older adults performed two versions of a dual task of verbal and spatial working memory. In the unbound dual task version letters and locations were presented simultaneously in separate locations, while in the bound dual task version each letter was paired with a specific location. In order to identify binding-specific differences, mixed-effects ANOVAs were run with the interaction of age and task as the effect of interest. Although older adults performed worse in the bound task than younger adults, there was no significant interaction between task and age on working memory performance. However, interactions of age and task were observed in brain activity analyses. Older adults did not display the greater unbound than bound task activity that younger adults did at the encoding phase in bilateral inferior parietal lobule, right putamen, and globus pallidus as well as at the maintenance phase in the cerebellum. We conclude that the binding of letters and locations in working memory is not as efficient in older adults as it is in younger adults, possibly due to the decline of cognitive control processes that are specific to working memory binding.

  13. Effect of linker structure on surface density of aptamer monolayers and their corresponding protein binding efficiency.

    PubMed

    Balamurugan, Subramanian; Obubuafo, Anne; McCarley, Robin L; Soper, Steven A; Spivak, David A

    2008-12-15

    A systematic study is reported on the effect of linker size and its chemical composition toward ligand binding to a surface-immobilized aptamer, measured using surface plasmon resonance. The results, using thrombin as the model system, showed that as the number of thymidine (T) units in the linker increases from 0 to 20 in four separate increments (T(0), T(5), T(10), T(20)), the surface density of the aptamer decreased linearly from approximately 25 to 12 pmol x cm(-2). The decrease in aptamer surface density occurred due to the increased size of the linker molecules. In addition, thrombin binding capacity was shown to increase as the linker length increased from 0 to 5 thymidine nucleotides and then decreased as the number of thymidine residues increased to 20 due to a balance between two different effects. The initial increase was due to increased access of thrombin to the aptamer as the aptamer was moved away from the surface. For linkers greater in length than T(5), the overall decrease in binding capacity was primarily due to a decrease in the surface density. Incorporation of a hexa(ethylene glycol) moiety into the linker did not affect the surface density but increased the amount of thrombin bound. In addition, the attachment of the linker at the 3'- versus the 5'-end of the aptamer resulted in increased aptamer surface density. However, monolayers formed with equal surface densities showed similar amounts of thrombin binding irrespective of the point of attachment.

  14. Changes of insulin effect on lipogenesis and insulin binding receptors during hypokinesia

    NASA Astrophysics Data System (ADS)

    Macho, L.; Fickova, M.; Zorad, S.

    The effect of hypokinesia on insulin action and insulin binding to specific receptors in fat cells was studied. Male Wistar rats were exposed to hypokinesia in special adjustable plastic cages for 1, 7, 21 and 60 days, and the stimulatory effect of insulin (10 and 100 mU) on the incorporation of radiocarbon labelled glucose into lipids of fat tissue and the binding of insulin to receptors of isolated adipocytes was estimated. The stimulation of lipogenesis by insulin was slightly diminished after hypokinesia for 1 day, however, an important increase of insulin action was found in rats exposed to hypokinesia for 60 days. The decrease of insulin binding capacity of the number of binding sites per cell and of the insulin receptor density was found after 1 day of hypokinesia. In rats exposed to hypokinesia for 60 days, in agreement with the higher stimulatory affect of insulin, an increase of insulin receptor density was observed. These results showed that hypokinesia has an important influence on stimulatory action of insulin and on insulin receptors in adipocytes.

  15. Effects of bromocriptine on (/sup 3/H)estradiol binding in cytosol of anterior pituitary

    SciTech Connect

    De Nicola, A.F.; Weisenberg, L.S.; Arakelian, M.C.; Libertun, C.

    1981-07-01

    The hypothalamus may control hormone receptors in the anterior pituitary either by a direct trophic effect or indirectly by regulation of serum pituitary hormone levels. Rats whose medial basal hypothalamus had been destroyed in order to suppress neural control of the gland showed a reduction in (/sup 3/H)estradiol binding in the anterior pituitary and high serum PRL levels; both changes were reversed by treatment of the lesioned rats with daily injections of bromocriptine, a dopamine agonist. In nonlesioned animals, the same treatment did not modify significantly those parameters. In another hyperprolactinemic model (rats with anterior pituitaries transplanted under the kidney capsule), (/sup 3/H)estradiol binding by the in situ pituitaries of the host rats was similar to that in the nongrafted controls. These results suggest that changes due to median eminence lesion are reversible and that bromocriptine is able to act as a substitutive therapy which restores binding of estradiol in glands whose receptors have been decreased by the effect of the lesion. High PRL levels due to pituitary transplant do not account for the observed changes in the pituitary estradiol binding.

  16. Binding and Inhibitory Effect of the Dyes Amaranth and Tartrazine on Amyloid Fibrillation in Lysozyme.

    PubMed

    Basu, Anirban; Suresh Kumar, Gopinatha

    2017-02-16

    Interaction of two food colorant dyes, amaranth and tartrazine, with lysozyme was studied employing multiple biophysical techniques. The dyes exhibited hypochromic changes in the presence of lysozyme. The intrinsic fluorescence of lysozyme was quenched by both dyes; amaranth was a more efficient quencher than tartrazine. The equilibrium constant of amaranth was higher than that of tartarzine. From FRET analysis, the binding distances for amaranth and tartrazine were calculated to be 4.51 and 3.93 nm, respectively. The binding was found to be dominated by non-polyelectrolytic forces. Both dyes induced alterations in the microenvironment surrounding the tryptophan and tyrosine residues of the protein, with the alterations being comparatively higher for the tryptophans than the tyrosines. The interaction caused significant loss in the helicity of lysozyme, the change being higher with amaranth. The binding of both dyes was exothermic. The binding of amaranth was enthalpy driven, while that of tartrazine was predominantly entropy driven. Amaranth delayed lysozyme fibrillation at 25 μM, while tartrazine had no effect even at 100 μM. Nevertheless, both dyes had a significant inhibitory effect on fibrillogenesis. The present study explores the potential antiamyloidogenic property of these azo dyes used as food colorants.

  17. Sensing (un)binding events via surface plasmons: effects of resonator geometry

    NASA Astrophysics Data System (ADS)

    Antosiewicz, Tomasz J.; Claudio, Virginia; Käll, Mikael

    2016-04-01

    The resonance conditions of localized surface plasmon resonances (LSPRs) can be perturbed in any number ways making plasmon nanoresonators viable tools in detection of e.g. phase changes, pH, gasses, and single molecules. Precise measurement via LSPR of molecular concentrations hinge on the ability to confidently count the number of molecules attached to a metal resonator and ideally to track binding and unbinding events in real-time. These two requirements make it necessary to rigorously quantify relations between the number of bound molecules and response of plasmonic sensors. This endeavor is hindered on the one hand by a spatially varying response of a given plasmonic nanosensor. On the other hand movement of molecules is determined by stochastic effects (Brownian motion) as well as deterministic flow, if present, in microfluidic channels. The combination of molecular dynamics and the electromagnetic response of the LSPR yield an uncertainty which is little understood and whose effect is often disregarded in quantitative sensing experiments. Using a combination of electromagnetic finite-difference time-domain (FDTD) calculations of the plasmon resonance peak shift of various metal nanosensors (disk, cone, rod, dimer) and stochastic diffusion-reaction simulations of biomolecular interactions on a sensor surface we clarify the interplay between position dependent binding probability and inhomogeneous sensitivity distribution. We show, how the statistical characteristics of the total signal upon molecular binding are determined. The proposed methodology is, in general, applicable to any sensor and any transduction mechanism, although the specifics of implementation will vary depending on circumstances. In this work we focus on elucidating how the interplay between electromagnetic and stochastic effects impacts the feasibility of employing particular shapes of plasmonic sensors for real-time monitoring of individual binding reactions or sensing low concentrations

  18. Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump.

    PubMed

    Marcoux, Julien; Wang, Sheila C; Politis, Argyris; Reading, Eamonn; Ma, Jerome; Biggin, Philip C; Zhou, Min; Tao, Houchao; Zhang, Qinghai; Chang, Geoffrey; Morgner, Nina; Robinson, Carol V

    2013-06-11

    Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent KD values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.

  19. A comprehensive study of clinical biomarkers, use of inotropic medications and fluid resuscitation in newborns with persistent pulmonary hypertension.

    PubMed

    Mydam, Janardhan; Zidan, Marwan; Chouthai, Nitin Shashikant

    2015-01-01

    Persistent pulmonary hypertension of the newborn (PPHN) is associated with high morbidity and mortality. This study evaluated clinical outcomes in PPHN in relation to echocardiographic (EC) markers, score of neonatal acute physiology, perinatal extension, version II (SNAPPE II) scores, inotropic agent use, and the amount of fluid received as boluses. In this retrospective chart analysis of 98 neonates with PPHN born at >34 weeks' gestation, we compared two cohorts of newborns: those who received inhaled nitric oxide and mechanical ventilation only, and who survived to discharge (Group 1); and those who required extracorporeal membrane oxygenation (ECMO) or who died (Group 2). Of 21 EC parameters assessed, seven were significantly different between Group 1 and Group 2. Eleven (24.4%) newborns in Group 2 had decreased left ventricular (LV) function, compared with three (5.1%) in Group 1 (p = 0.011). Median SNAPPE II scores were significantly higher in Group 2 than in Group 1 (p < 0.001). Newborns in Group 2 also received a significantly higher amount of fluid as boluses during the first 7 days of hospitalization compared with Group 1 (p = 0.018). Following logistic regression analysis, only the difference in total SNAPPE II score retained statistical significance (p < 0.001); however, the total amount of fluid administered as boluses trended higher (p = 0.087) for newborns in Group 2. Our findings show that SNAPPE II scores may help guide counseling for parents of newborns with PPHN regarding the likelihood of death or the need for ECMO. Limiting fluid boluses may improve outcomes in newborns with high SNAPPE II scores and decreased LV function.

  20. Novel oral phosphate binder with nanocrystalline maghemite-phosphate binding capacity and pH effect.

    PubMed

    Nguyen, T M-H; Müller, R H; Taupitz, M; Schnorr, J; Hamm, B; Wagner, S

    2015-03-30

    Hyperphosphatemia is one of the main risk factors contributing to morbidity and mortality in patients with end stage renal disease. The demand for a new phosphate binder is continuously increasing since the number of patients suffering under hyperphosphatemia is growing. However, side effects and high pill burden of currently available phosphate binders are the main reasons for low compliance and uncontrolled serum phosphate levels. Therefore, the aim of this study was to develop a novel phosphate binder with a high phosphate binding capacity over the entire gastrointestinal (GI) pH range. This novel phosphate binder C-PAM-10 is based on d-mannose coated nanocrystalline maghemite and belongs to the new class of phosphate binders, called the "iron based agents". It was possible to obtain a phosphate binding product that showed very high phosphate binding capacities with the characteristic of being pH independent at relevant pH ranges. The simulation of a GI passage ranging from pH 1.2 to pH 7.5 showed a 2.5 times higher phosphate binding capacity compared to the commonly used phosphate binder sevelamer carbonate. The simulation of a pH sensitive coating that releases the iron based phosphate binder at pH values ≥4.5 still showed a very high phosphate binding capacity combined with very low iron release which might decrease iron related side effects in vivo. Therefore, C-PAM-10 and its variations may be very promising candidates as a superior phosphate binder.

  1. Polydispersity of Bacillus thuringiensis Cry1 toxins in solution and its effect on receptor binding kinetics.

    PubMed

    Masson, Luke; Mazza, Alberto; Sangadala, Sreedhara; Adang, Michael J; Brousseau, Roland

    2002-02-11

    Dynamic light scattering and surface plasmon resonance techniques were used to investigate the influence of ionic strength, buffer composition and pH on the multimerization of trypsin-activated Cry1Ac and Cry1C toxins over time and the subsequent effects of the different multimers on receptor binding models. In carbonate buffer at pH 10.5, Cry1Ac and Cry1C assumed a monomeric state. After 24 h, a complete conversion of monomeric toxin to a dimeric or trimeric form was observed only for Cry1Ac under low ionic strength condition. Cry1C and Cry1Ac in high ionic strength buffer remained monomeric. Substitution of CAPS pH 11 for carbonate buffer suppressed this Cry1Ac oligomerization effect. Once Cry1Ac toxin was in an aggregated form, increases in ionic strength failed to revert the aggregated toxin back to a monomeric form. Monomeric Cry1Ac bound to a purified 115 kDa aminopeptidase N receptor from Manduca sexta in a 2:1 molar ratio thus confirming the existence of two binding sites on this receptor. Binding rates of dimeric or higher aggregated Cry1Ac toxin forms were different from those generated using the monomeric form and could not be fitted to existing binding models. In summary, our results confirm that the M. sexta 115 kDa aminopeptidase N receptor possesses two Cry1Ac binding sites. They further suggest that although high pH and low salt conditions promote Cry1Ac aggregation, this observation cannot be applied universally to other members of the Cry family.

  2. Effects of conformational ordering on protein/polyelectrolyte electrostatic complexation: ionic binding and chain stiffening

    NASA Astrophysics Data System (ADS)

    Cao, Yiping; Fang, Yapeng; Nishinari, Katsuyoshi; Phillips, Glyn O.

    2016-03-01

    Coupling of electrostatic complexation with conformational transition is rather general in protein/polyelectrolyte interaction and has important implications in many biological processes and practical applications. This work studied the electrostatic complexation between κ-carrageenan (κ-car) and type B gelatin, and analyzed the effects of the conformational ordering of κ-car induced upon cooling in the presence of potassium chloride (KCl) or tetramethylammonium iodide (Me4NI). Experimental results showed that the effects of conformational ordering on protein/polyelectrolyte electrostatic complexation can be decomposed into ionic binding and chain stiffening. At the initial stage of conformational ordering, electrostatic complexation can be either suppressed or enhanced due to the ionic bindings of K+ and I‑ ions, which significantly alter the charge density of κ-car or occupy the binding sites of gelatin. Beyond a certain stage of conformational ordering, i.e., helix content θ > 0.30, the effect of chain stiffening, accompanied with a rapid increase in helix length ζ, becomes dominant and tends to dissociate the electrostatic complexation. The effect of chain stiffening can be theoretically interpreted in terms of double helix association.

  3. Investigation of the binding of Salvianolic acid B to human serum albumin and the effect of metal ions on the binding

    NASA Astrophysics Data System (ADS)

    Chen, Tingting; Cao, Hui; Zhu, Shajun; Lu, Yapeng; Shang, Yanfang; Wang, Miao; Tang, Yanfeng; Zhu, Li

    2011-10-01

    The studies on the interaction between HSA and drugs have been an interesting research field in life science, chemistry and clinical medicine. There are also many metal ions present in blood plasma, thus the research about the effect of metal ions on the interaction between drugs and plasma proteins is crucial. In this study, the interaction of Salvianolic acid B (Sal B) with human serum albumin (HSA) was investigated by the steady-state, synchronous fluorescence and circular dichroism (CD) spectroscopies. The results showed that Sal B had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching mechanism. Binding parameters calculated showed that Sal B was bound to HSA with the binding affinities of 10 5 L mol -1. The thermodynamic parameters studies revealed that the binding was characterized by positive enthalpy and positive entropy changes, and hydrophobic interactions were the predominant intermolecular forces to stabilize the complex. The specific binding distance r (2.93 nm) between donor (HSA) and acceptor (Sal B) was obtained according to Förster non-radiative resonance energy transfer theory. The synchronous fluorescence experiment revealed that Sal B cannot lead to the microenvironmental changes around the Tyr and Trp residues of HSA, and the binding site of Sal B on HSA is located in hydrophobic cavity of subdomain IIA. The CD spectroscopy indicated the secondary structure of HSA is not changed in the presence of Sal B. Furthermore, The effect of metal ions (e.g. Zn 2+, Cu 2+, Co 2+, Ni 2+, Fe 3+) on the binding constant of Sal B-HSA complex was also discussed.

  4. The Effect of Binding Groups on the Seebeck Coefficient of Phenyl Derivative Molecular Junctions

    NASA Astrophysics Data System (ADS)

    Chang, William; Mai, Chengkang; Kotiuga, Michele; Urban, Jeffrey; Neaton, Jeffrey; Bazan, Gui; Segalman, Rachel

    2013-03-01

    Thermoelectrics currently suffer from low efficiencies due to inverse coupling of the Seebeck coefficient and electrical conductivity, limiting the power factor. Decoupling of these two physical properties has previously been demonstrated in molecular junctions. Using an STM break junction measurement technique, we demonstrate the effect that the direct binding group Au-C has on the Seebeck coefficient. Phenyl derivative molecules with an Au-C direct binding group show a significantly lower Seebeck coefficient than molecules with an Au-S binding group. This lower Seebeck coefficient is explained by theoretical calculations as a broadening in the transmission function due to the direct bonding group. This demonstrates the importance of the metal-molecule interface and binding group selection in tuning the transmission function, and the resultant conductance and Seebeck coefficient. This result will lend further insight in rational design for molecules with higher power factors. We would like to acknowledge support from Office of Naval Research - ONR/AFOSR BAA 10-026

  5. Effect of diet on insulin binding and glucose transport in rat sarcolemmal vesicles

    SciTech Connect

    Grimditch, G.K.; Barnard, R.J.; Sternlicht, E.; Whitson, R.H.; Kaplan, S.A.

    1987-03-01

    The purpose of this study was to compare the effects of a high-fat, high-sucrose diet (HFS) and a low-fat, high-complex carbohydrate diet (LFC) on glucose tolerance, insulin binding, and glucose transport in rat skeletal muscle. During the intravenous glucose tolerance test, peak glucose values at 5 min were significantly higher in the HFS group; 0-, 20-, and 60-min values were similar. Insulin values were significantly higher in the HFS group at all time points (except 60 min), indicating whole-body insulin resistance. Skeletal muscle was responsible, in part, for this insulin resistance, because specific D-glucose transport in isolated sarcolemmal (SL) vesicles under basal conditions was similar between LFC and HFS rats, despite the higher plasma insulin levels. Scatchard analyses of insulin binding curves to sarcolemmal vesicles revealed that the K/sub a/ of the high-affinity binding sites was significantly reduced by the HFS diet; no other binding changes were noted. Specific D-glucose transport in SL vesicles after maximum insulin stimulation (1 U/kg) was significantly depressed in the HFS group, indicating that HFS feeding also caused a postbinding defect. These results indicate that the insulin resistance in skeletal muscle associated with a HFS diet is due to both a decrease in the K/sub a/ of the high-affinity insulin receptors and a postbinding defect.

  6. Effects of binding factors on structural elements in F-actin.

    PubMed

    Scoville, Damon; Stamm, John D; Altenbach, Christian; Shvetsov, Alexander; Kokabi, Kaveh; Rubenstein, Peter A; Hubbell, Wayne L; Reisler, Emil

    2009-01-20

    Understanding the dynamics of the actin filament is essential to a detailed description of their interactions and role in the cell. Previous studies have linked the dynamic properties of actin filaments (F-actin) to three structural elements contributing to a hydrophobic pocket, namely, the hydrophobic loop, the DNase I binding loop, and the C-terminus. Here, we examine how these structural elements are influenced by factors that stabilize or destabilize F-actin, using site-directed spin-labeled (SDSL) electron paramagnetic resonance (EPR), fluorescence, and cross-linking techniques. Specifically, we employ cofilin, an actin destabilizing protein that binds and severs filaments, and phalloidin, a fungal toxin that binds and stabilizes F-actin. We find that cofilin shifts both the DNase I binding loop and the hydrophobic loop away from the C-terminus in F-actin, as demonstrated by weakened spin-spin interactions, and alters the environment of spin probes on residues of these two loops. In contrast, although phalloidin strongly stabilizes F-actin, it causes little or no local change in the environment of the loop residues. This indicates that the stabilizing effect of phalloidin is achieved mainly through constraining structural fluctuations in F-actin and suggests that factors and interactions that control these fluctuations have an important role in the cytoskeleton dynamics.

  7. The effect of hyperthyroidism on opiate receptor binding and pain sensitivity

    SciTech Connect

    Edmondson, E.A. ); Bonnet, K.A.; Friedhoff, A.J. )

    1990-01-01

    This study was conducted to determine the effect of thyroid hormone on opiate receptor ligand-binding and pain sensitivity. Specific opiate receptor-binding was performed on brain homogenates of Swiss-Webster mice. There was a significant increase in {sup 3}H-naloxone-binding in thyroxine-fed subjects (hyperthyroid). Scatchard analysis revealed that the number of opiate receptors was increased in hyperthyroid mice (Bmax = 0.238 nM for hyperthyroid samples vs. 0.174 nM for controls). Binding affinity was unaffected (Kd = 1.54 nM for hyperthyroid and 1.58 nM for control samples). When mice were subjected to hotplate stimulation, the hyperthyroid mice were noted to be more sensitive as judged by pain aversion response latencies which were half that of control animals. After morphine administration, the hyperthyroid animals demonstrated a shorter duration of analgesia. These findings demonstrate that thyroxine increases opiate receptor number and native pain sensitivity but decreases the duration of analgesia from morphine.

  8. Effect of van der Waals interactions on the structural and binding properties of GaSe

    SciTech Connect

    Sarkisov, Sergey Y.; Kosobutsky, Alexey V.; Shandakov, Sergey D.

    2015-12-15

    The influence of van der Waals interactions on the lattice parameters, band structure, elastic moduli and binding energy of layered GaSe compound has been studied using projector-augmented wave method within density functional theory. We employed the conventional local/semilocal exchange-correlation functionals and recently developed van der Waals functionals which are able to describe dispersion forces. It is found that application of van der Waals density functionals allows to substantially increase the accuracy of calculations of the lattice constants a and c and interlayer distance in GaSe at ambient conditions and under hydrostatic pressure. The pressure dependences of the a-parameter, Ga–Ga, Ga–Se bond lengths and Ga–Ga–Se bond angle are characterized by a relatively low curvature, while c(p) has a distinct downward bowing due to nonlinear shrinking of the interlayer spacing. From the calculated binding energy curves we deduce the interlayer binding energy of GaSe, which is found to be in the range 0.172–0.197 eV/layer (14.2–16.2 meV/Å{sup 2}). - Highlights: • Effects of van der Waals interactions are analyzed using advanced density functionals. • Calculations with vdW-corrected functionals closely agree with experiment. • Interlayer binding energy of GaSe is estimated to be 14.2–16.2 meV/Å{sup 2}.

  9. Effect of glucose on the insulin production and insulin binding of Tetrahymena.

    PubMed

    Lajkó, Eszter; Pállinger, Eva; Csaba, G

    2012-12-01

    As the unicellular ciliate, Tetrahymena has insulin receptors and produces insulin itself, which can regulate its glucose metabolism and other cell functions, in the present experiments the feed-back, the effect of glucose on the insulin binding and insulin production was studied. The cells were kept partly in tryptone-yeast medium, partly in Losina salt solution. The duration of treatment (in 0.1, 1.0, 10.0 mg/ml glucose) in the binding study was 10 min, in the hormone production study 30 min. FITC-insulin binding was significantly decreased only by 0.1 mg/ml glucose treatment in medium and by 10 mg/ml glucose in salt. The insulin production was significantly lower only in cells treated with 10 mg/ml glucose in medium. The insulin binding in salt was always higher and the insulin production always lower, than in medium. Earlier results demonstrated that the hormonal system (presence of hormones, receptors and signal pathways) of higher ranked animals can be deduced to a unicellular level, however, the feed-back mechanism is not really present here, only the traces can be observed in these protozoa.

  10. Binding effect of proline-rich-proteins (PRPs) on in vitro antimicrobial activity of the flavonoids

    PubMed Central

    Ansari, Jawaad Ahmed; Naz, Shahina; Tarar, Omer Mukhtar; Siddiqi, Rahmanullah; Haider, Muhammad Samee; Jamil, Khalid

    2015-01-01

    The interaction of the cyanidin, pelargonidin, catechin, myrecetin and kaempferol with casein and gelatin, as proline rich proteins (PRPs), was studied. The binding constants calculated for both flavonoid-casein and flavonoid-gelatin were fairly large (10 5 –10 7 M −1 ) indicating strong interaction. Due to higher proline content in gelatin, the binding constants of flavonoid-gelatin (2.5 × 10 5 –6.2 × 10 7 M −1 ) were found to be higher than flavonoid-casein (1.2 × 10 5 –5.0 × 10 7 M −1 ). All the flavonoids showed significant antibacterial activity against the tested strains. Significant loss in activity was observed due to the complexation with PRPs confirming that binding effectively reduced the concentration of the free flavonoids to be available for antibacterial activity. The decline in activity was corresponding to the values of the binding constants. Though the activities of free catechin and myrecetin were higher compared to pelargonidin, cyanidin and kaempferol yet the decline in activity of catechin and myrecetin due to complexation with casein and gelatin was more pronounced. PMID:26221106

  11. Effect of polymorphisms on ligand binding by mouse major urinary proteins

    PubMed Central

    Darwish Marie, Amr; Veggerby, Christina; Robertson, Duncan H.L.; Gaskell, Simon J.; Hubbard, Simon J.; Martinsen, Line; Hurst, Jane L.; Beynon, Robert J.

    2001-01-01

    Mouse urine contains an abundance of major urinary proteins, lipocalins, whose roles include slow release of semiochemicals. These proteins are highly polymorphic, with small sequence differences between individual members. In this study, we purified to homogeneity four of these proteins from two strains of inbred mice and characterized them by mass spectrometry. This analysis has led to the discovery of another variant in this group of proteins. Three of the polymorphic variants that map to the surface have no effect on the binding of a fluorescent probe in the binding cavity, but the fourth, characterized by a Phe to Val substitution in the cavity, shows a substantially lower affinity and fluorescence yield for the probe. These results are interpreted in light of the known crystal structure of the protein and molecular modeling calculations, which rationalize the experimental findings. This work raises the possibility that the calyx-binding site can show specificity for different ligands, the implications of which on pheromone binding and chemical communication are discussed. PMID:11266626

  12. The effect of prolactin and relaxin on insulin binding by adipocytes from pregnant women.

    PubMed

    Jarrett JC2nd; Ballejo, G; Saleem, T H; Tsibris, J C; Spellacy, W N

    1984-06-01

    The effects of prolactin and relaxin on insulin binding by isolated human adipocytes from women at term gestation were studied in vitro. It was found that prolactin decreases, and relaxin increases, insulin binding to the adipocytes. Both changes appear to be due to alterations in the affinity of the insulin receptors. These effects seem to be mediated through specific prolactin and relaxin receptors of the adipocyte and require the presence of an intact cellular cytoskeleton. This suggests that one hormone, for example, prolactin, can interact with its own specific receptor and thereby after the affinity of a heterologous receptor for its hormone (insulin). Heterologous hormone-receptor complex interactions ("cross-talk") may be widespread and could represent a fundamental mechanism in the functioning of the endocrine system.

  13. Electrostatic effect of H1-histone protein binding on nucleosome repeat length

    NASA Astrophysics Data System (ADS)

    Cherstvy, Andrey G.; Teif, Vladimir B.

    2014-08-01

    Within a simple biophysical model we describe the effect of electrostatic binding of H1 histone proteins on the nucleosome repeat length in chromatin. The length of wrapped DNA optimizes its binding energy to the histone core and the elastic energy penalty of DNA wrapping. The magnitude of the effect predicted from our model is in agreement with the systematic experimental data on the linear variation of nucleosome repeat lengths with H1/nucleosome ratio (Woodcock C L et al 2006 Chromos. Res. 14 17-25). We compare our model to the data for different cell types and organisms, with a widely varying ratio of bound H1 histones per nucleosome. We underline the importance of this non-specific histone-DNA charge-balance mechanism in regulating the positioning of nucleosomes and the degree of compaction of chromatin fibers in eukaryotic cells.

  14. Effect of mutations in putative hormone binding sites on V2 vasopressin receptor function.

    PubMed

    Sebti, Y; Rabbani, M; Sadeghi, H Mir Mohammad; Sardari, S; Ghahremani, M H; Innamorati, G

    2015-01-01

    The vasopressin V2 receptor belongs to the large family of the G-protein coupled receptors and is responsible for the antidiuretic effect of the neurohypophyseal hormone arginine vasopressin (AVP). Based on bioinformatic studies it seems that Ala300 and Asp297 of the V2 vasopressin receptor (V2R) are involved in receptor binding. Ala300Glu mutation resulted in lower energy while Asp297Tyr mutation resulted in higher energy in AVP-V2R docked complex rather than the wild type. Therefore we hypothesized that the Ala300Glu mutation results in stronger and Asp297Tyr mutation leads to weaker ligand-receptor binding. Site directed mutagenesis of Asp297Tyr and Ala300Glu was performed using nested polymerase chain reaction. After restriction enzyme digestion, the inserts were ligated into the pcDNA3 vector and Escherichia coli XL1-Blue competent cells were transformed using commercial kit and electroporation methods. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using ESCORT™ IV transfection reagent, the adenylyl cyclase activity assay was performed for functional studies. The cell surface expression of V2R was analyzed by indirect ELISA method. Based on the obtained results, the Ala300Glu mutation of V2R led to reduced levels of cAMP production without a marked effect on the receptor expression and the receptor binding. Effect of Asp297Tyr mutation on cell surface expression of V2R was the same as the wild type receptor. Pretreatment with 1 nM vasopressin showed an increased level of Asp297Tyr mutant receptor internalization as compared to the wild type receptor, while the effect of 100 nM vasopressin was similar in the mutant and wild type receptors. These data suggest that alterations in Asp297 but not Ala300 would affect the hormone receptor binding.

  15. Quantum electrodynamics analysis of optical binding in counterpropagating beams and effect of particle size.

    PubMed

    Rodriguez, Justo

    2008-10-01

    A general expression for optical binding energy between particles of any size, in counterpropagating beams with and without interference, is derived using quantum electrodynamics. The effect of particle size on the optically induced interparticle energy surface, which has been the subject of recent research, is explored. Significant changes in this surface when particle size approaches the wavelength of the optical field are revealed. Finally, optically induced particle arrays that may be fabricated with these potentials are briefly discussed.

  16. Effect of Sodium and Chloride Binding on a Lecithin Bilayer. A Molecular Dynamics Study

    PubMed Central

    Reif, Maria M.; Kallies, Christopher; Knecht, Volker

    2017-01-01

    The effect of ion binding on the structural, mechanical, dynamic and electrostatic properties of a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer in a 0.5 M aqueous NaCl solution is investigated using classical atomistic molecular dynamics simulation with different force-field descriptions for ion-ion and ion-lipid interactions. Most importantly, the repulsive Lennard–Jones parameters for the latter were modified, such that approximately similar binding of cations and anions to the lipid membrane is achieved. This was done to qualitatively improve the apparent ion-lipid binding constants obtained from simulations with the original force field (Berger lipids and GROMOS87 ions in combination with the SPC water model) in comparison to experimental data. Furthermore, various parameters characterizing membrane structure, elasticity, order and dynamics are analyzed. It is found that ion binding as observed in simulations involving the modified in comparison to the original force-field description leads to: (i) a smaller salt-induced change in the area per lipid, which is in closer agreement with the experiment; (ii) a decrease in the area compressibility and bilayer thickness to values comparable to a bilayer in pure water; (iii) lipid deuterium order parameters and lipid diffusion coefficients on nanosecond timescales that are very similar to the values for a membrane in pure water. In general, salt effects on the structural properties of a POPC bilayer in an aqueous sodium-chloride solution appear to be reproduced reasonably well by the new force-field description. An analysis of membrane-membrane disjoining pressure suggests that the smaller salt-induced change in area per lipid induced by the new force-field description is not due to the alteration of membrane-associated net charge, but must rather be understood as a consequence of ion-specific effects on the arrangement of lipid molecules. PMID:28125062

  17. Hyper-Binding across Time: Age Differences in the Effect of Temporal Proximity on Paired-Associate Learning

    ERIC Educational Resources Information Center

    Campbell, Karen L.; Trelle, Alexandra; Hasher, Lynn

    2014-01-01

    Older adults show hyper- (or excessive) binding effects for simultaneously and sequentially presented distraction. Here, we addressed the potential role of hyper-binding in paired-associate learning. Older and younger adults learned a list of word pairs and then received an associative recognition task in which rearranged pairs were formed from…

  18. Assessment of the in vitro binding of JHW 007, a dopamine transport inhibitor that blocks the effects of cocaine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Benztropine (BZT) and its analogues, like cocaine, bind to the dopamine transporter and block dopamine uptake. However, while BZT analogues bind the DAT with high affinity, they generally do not have cocaine-like behavioral effects. JHW 007 is a BZT analogue that displaces [3H]WIN 35,428 from the D...

  19. Effect of Osmolytes on the Binding of EGR1 Transcription Factor to DNA

    PubMed Central

    Mikles, David C.; Bhat, Vikas; Schuchardt, Brett J.; McDonald, Caleb B.; Farooq, Amjad

    2014-01-01

    Osmolytes play a key role in maintaining protein stability and mediating macromolecular interactions within the intracellular environment of the cell. Herein, we show that osmolytes such as glycerol, sucrose and PEG400 mitigate the binding of EGR1 transcription factor to DNA in a differential manner. Thus, while physiological concentrations of glycerol only moderately reduce the binding affinity, addition of sucrose and PEG400 is concomitant with a loss in the binding affinity by an order of magnitude. This salient observation suggests that EGR1 is most likely subject to conformational equilibrium and that the osmolytes exert their effect via favorable interactions with the unliganded conformation. Consistent with this notion, our analysis reveals that while EGR1 displays rather high structural stability in complex with DNA, the unliganded conformation becomes significantly destabilized in solution. In particular, while liganded EGR1 adopts a well-defined arc-like architecture, the unliganded protein samples a comparatively large conformational space between two distinct states that periodically interconvert between an elongated rod-like shape and an arc-like conformation on a sub-microsecond time scale. Consequently, the ability of osmolytes to favorably interact with the unliganded conformation so as to stabilize it could account for the negative effect of osmotic stress on EGR1-DNA interaction observed here. Taken together, our study sheds new light on the role of osmolytes in modulating a key protein-DNA interaction. PMID:25269753

  20. Renormalization of myoglobin–ligand binding energetics by quantum many-body effects

    PubMed Central

    Weber, Cédric; Cole, Daniel J.; O’Regan, David D.; Payne, Mike C.

    2014-01-01

    We carry out a first-principles atomistic study of the electronic mechanisms of ligand binding and discrimination in the myoglobin protein. Electronic correlation effects are taken into account using one of the most advanced methods currently available, namely a linear-scaling density functional theory (DFT) approach wherein the treatment of localized iron 3d electrons is further refined using dynamical mean-field theory. This combination of methods explicitly accounts for dynamical and multireference quantum physics, such as valence and spin fluctuations, of the 3d electrons, while treating a significant proportion of the protein (more than 1,000 atoms) with DFT. The computed electronic structure of the myoglobin complexes and the nature of the Fe–O2 bonding are validated against experimental spectroscopic observables. We elucidate and solve a long-standing problem related to the quantum-mechanical description of the respiration process, namely that DFT calculations predict a strong imbalance between O2 and CO binding, favoring the latter to an unphysically large extent. We show that the explicit inclusion of the many-body effects induced by the Hund’s coupling mechanism results in the correct prediction of similar binding energies for oxy- and carbonmonoxymyoglobin. PMID:24717844

  1. Effects of AMPARs trafficking and glutamate-receptors binding probability on stochastic variability of EPSC.

    PubMed

    Ventriglia, Francesco; Di Maio, Vito

    2013-06-01

    Mathematical models of the excitatory synapse are providing valuable information about the synaptic response. The effects of several synaptic components on EPSC variability have been tested by computer simulation. Our model, based on Brownian diffusion of glutamate in the synaptic cleft, is basically the same we have used in previous papers but parameters have been upgraded according to the new experimental findings. The presence of filaments into the synaptic cleft and the number and the ratio of AMPA and NMDA receptors have been the main parameters upgraded. A different way of computing the binding probability of glutamate molecules to receptors by means of geometrical considerations has been also used. The obtained results were more precise and they suggested that the new elements can play a significant role in the stochastic variability of the synaptic response. Nevertheless, new problems arise concerning the value of the lower limit of the binding probability.

  2. Levodopa effects on [ 11C]raclopride binding in the resting human brain

    PubMed Central

    Black, Kevin J.; Piccirillo, Marilyn L.; Koller, Jonathan M.; Hseih, Tiffany; Wang, Lei; Mintun, Mark A.

    2015-01-01

    Rationale: Synaptic dopamine (DA) release induced by amphetamine or other experimental manipulations can displace [ 11C]raclopride (RAC*) from dopamine D2-like receptors. We hypothesized that exogenous levodopa might increase dopamine release at striatal synapses under some conditions but not others, allowing a more naturalistic assessment of presynaptic dopaminergic function. Presynaptic dopaminergic abnormalities have been reported in Tourette syndrome (TS). Objective: Test whether levodopa induces measurable synaptic DA release in healthy people at rest, and gather pilot data in TS. Methods: This double-blind crossover study used RAC* and positron emission tomography (PET) to measure synaptic dopamine release 4 times in each of 10 carbidopa-pretreated, neuroleptic-naïve adults: before and during an infusion of levodopa on one day and placebo on another (in random order). Five subjects had TS and 5 were matched controls. RAC* binding potential (BP ND) was quantified in predefined anatomical volumes of interest (VOIs). A separate analysis compared BP ND voxel by voxel over the entire brain. Results: DA release declined between the first and second scan of each day (p=0.012), including on the placebo day. Levodopa did not significantly reduce striatal RAC* binding and striatal binding did not differ significantly between TS and control groups. However, levodopa’s effect on DA release differed significantly in a right midbrain region (p=0.002, corrected), where levodopa displaced RAC* by 59% in control subjects but increased BP ND by 74% in TS subjects. Discussion: Decreased DA release on the second scan of the day is consistent with the few previous studies with a similar design, and may indicate habituation to study procedures. We hypothesize that mesostriatal DA neurons fire relatively little while subjects rest, possibly explaining the non-significant effect of levodopa on striatal RAC* binding. The modest sample size argues for caution in interpreting the

  3. Probing the binding sites and the effect of berbamine on the structure of bovine serum albumin

    NASA Astrophysics Data System (ADS)

    Cheng, Xiao-Xia; Lui, Yi; Zhou, Bo; Xiao, Xiao-He; Liu, Yi

    2009-06-01

    Berbamine, a naturally occurring isoquinoline alkaloid extracted from Berberis sp., is the active constituent of some Chinese herbal medicines and exhibits a variety of pharmacological activities. The effects of berbamine on the structure of bovine serum albumin (BSA) were investigated by circular dichroism, fluorescence and absorption spectroscopy under physiological conditions. Berbamine caused a static quenching of the intrinsic fluorescence of BSA, and the quenching data were analyzed by application of the Stern-Volmer equation. There was a single primary berbamine-binding site on BSA with a binding constant of 2.577 × 10 4 L mol -1 at 298 K. The thermodynamic parameters, enthalpy change (Δ H0) and entropy change (Δ S0) for the reaction were -76.5 kJ mol -1 and -173.4 J mol -1 K -1 according to the van't Hoff equation. The results showed that the hydrogen bond and van der Waals interaction were the predominant forces in the binding process. Competitive experiments revealed a displacement of warfarin by berbamine, indicating that the binding site was located at Drug sites I. The distance r between the donor (BSA) and the acceptor (berbamine) was obtained according to the Förster non-radiation energy transfer theory. The results of three-dimensional fluorescence spectra, UV-vis absorption difference spectra and circular dichroism of BSA in the presence of berbamine showed that the conformation of BSA was changed. The results provide a quantitative understanding of the effect of berbamine on the structure of bovine serum albumin, providing a useful guideline for further drug design.

  4. Tryptic digestion of the human erythrocyte glucose transporter: effects on ligand binding and tryptophan fluorescence.

    PubMed

    May, J M; Qu, Z C; Beechem, J M

    1993-09-21

    The conformation of the human erythrocyte glucose transport protein has been shown to determine its susceptibility to enzymatic cleavage on a large cytoplasmic loop. We took the converse approach and investigated the effects of tryptic digestion on the conformational structure of this protein. Exhaustive tryptic digestion of protein-depleted erythrocyte ghosts decreased the affinity of the residual transporter for cytochalasin B by 3-fold but did not affect the total number of binding sites. Tryptic digestion also increased the affinity of the residual transporter for D-glucose and inward-binding sugar phenyl beta-D-glucopyranoside but decreased that for the outward-binding 4,6-O-ethylidene glucose. These results suggest that tryptic cleavage stabilized the remaining transporter in an inward-facing conformation, but one with decreased affinity for cytochalasin B. The steady-state fluorescence emission scan of the purified reconstituted glucose transport protein was unaffected by tryptic digestion. Addition of increasing concentrations of potassium iodide resulted in linear Stern-Volmer plots, which were also unaffected by prior tryptic digestion. The tryptophan oxidant N-bromosuccinimide was investigated to provide a more sensitive measure of tryptophan environment. This agent irreversibly inhibited 3-O-methylglucose transport in intact erythrocytes and cytochalasin B binding in protein-depleted ghosts, with a half-maximal effect observed for each activity at about 0.3-0.4 nM. Treatment of purified glucose transport protein with N-bromosuccinimide resulted in a time-dependent quench of tryptophan fluorescence, which was resolved into two components by nonlinear regression using global analysis. Tryptic digestion retarded the rate of oxidation of the more slowly reacting class of tryptophans. (ABSTRACT TRUNCATED AT 250 WORDS)

  5. Effect of pH on antigen binding by clonotypic antibodies with different isoelectric points

    SciTech Connect

    Endo, Y.; Miyai, K.; Hata, N.; Iijima, Y.

    1987-02-01

    Polyclonal rabbit antibodies to thyroxine, human myoglobin, human growth hormone, human thyrotropin, human alpha-fetoprotein, and human thyroglobulin were fractionated into clonotypic antibodies with different isoelectric points by agarose isoelectric focusing or chromatofocusing. The effect of pH on the binding of these antigens by their respective clonotypic antibodies was assessed by radioimmunoassay. The profiles of the pH effect differed both for different antigens and for different pI's of the antibodies used. The pH optima in the radioimmunoassays for protein antigens were found to be expressed as a function of pI and molecular weight of both antigen and antibody molecules.

  6. Binding of coumarins to site I of human serum albumin. Effect of the fatty acids.

    PubMed

    Zatón, A M; Ferrer, J M; Ruiz de Gordoa, J C; Marquínez, M A

    1995-07-14

    It is known that binding site I on human serum albumin (HSA) consists of a zone of two overlapping regions: the specific binding region represented by warfarin binding and the specific binding region represented by azapropazone and phenylbutazone binding. In this paper binding parameters to defatted HSA and to HSA with fatty acids (molar ratio of fatty acid/HSA = 4) were compared. High-affinity binding sites for warfarin, 4-chromanol, 4-hydroxycoumarin, coumarin, 3-acetylcoumarin and phenylbutazone (759,549 M-1 > Ka > 67,024 M-1) constitute binding site I on HSA. In this binding area defatted HSA can bind two molecules of warfarin, but the presence of fatty acids diminish the binding capacity of warfarin to HSA (2 > n > 1).

  7. Participation of central imidazoline binding sites in antinociceptive effect of ethanol and nicotine in rats.

    PubMed

    Aglawe, Manish Manohar; Taksande, Brijesh Gulabrao; Kuldhariya, Sharvari Shambabu; Chopde, Chandrabhan Tukaram; Umekar, Milind Janrao; Kotagale, Nandkishor Ramdas

    2014-06-01

    Despite synergistic morbidity and mortality, concomitant consumption of alcohol and tobacco is increasing, and their antinociceptive effect has been linked with co-abuse. Present study was designed to investigate the role of imidazoline binding sites in the antinociceptive effect of nicotine, ethanol, and their combination. Separate group of male Sprague-Dawley rats (200-250 g) were treated with different doses of alcohol (0.50-2 g/kg, i.p.) or nicotine (0.25-1 mg/kg, i.p.), and their combination evaluated in tail flick test. Influence of endogenous imidazoline binding site ligands, agonist, and antagonists were determined by their prior treatment with effective or subeffective doses of either ethanol or nicotine. Ethanol, nicotine, or their subeffective dose combination exhibited significant antinociceptive effects in dose-dependent manner. Antinociceptive effect of ethanol and nicotine was significantly augmented by intracerebroventricular (i.c.v.) administration of endogenous imidazoline receptor ligands, harmane (25 μg/rat, i.c.v.) and agmatine (10 μg/rat, i.c.v.), as well as imidazoline I1 /α2 adrenergic receptor agonist, clonidine (2 μg/rat, i.c.v.), I1 agonist moxonidine (25 μg/rat, i.c.v.), and imidazoline I2 agonist, 2-BFI (10 μg/rat, i.c.v.). Conversely, antinociception elicited by ethanol or nicotine or their subeffective dose combination was antagonized by pretreatment with imidazoline I1 antagonist, efaroxan (10 μg/rat, i.c.v.), and I2 antagonist, idazoxan (4 μg/rat, i.c.v.), at their per se ineffective doses. These findings project imidazoline binding ligands as important therapeutic molecules for central antinociceptive activity as well as may reduce the co-abuse potential of alcohol and nicotine.

  8. Joint interaction of ethidium bromide and methylene blue with DNA. The effect of ionic strength on binding thermodynamic parameters.

    PubMed

    Vardevanyan, Poghos O; Antonyan, Ara P; Parsadanyan, Marine A; Torosyan, Margarita A; Karapetian, Armen T

    2016-07-01

    Large amount of data of experimental and theoretical studies have shown that ethidium bromide (EtBr) and methylene blue (MB) may bind to nucleic acids via three modes: intercalation between two adjacent base pairs, insertion into the plane between neighboring bases in the same strand (semi-intercalation), and outside binding with negatively charged backbone phosphate groups. The aim of the given research is to examine the behavior of these two ligands at both separate and joint DNA binding. The obtained experimental data show that the effect of simultaneous binding of EtBr and MB on double-stranded DNA has a non-additive effect of separate binding. The analyses of the melting thermodynamic parameters of DNA complexes with two bound ligands suggest competitive mechanism of interaction.

  9. Effect of 5-FU substitution and mutation on Sm protein binding to human U4 snRNA.

    PubMed

    Cui, Wei; Gmeiner, William H

    2002-01-01

    The effects of native and non-native nucleotide substitution on the binding of Sm proteins to human U4 snRNA were investigated to determine if the Sm site was a likely target for the RNA-mediated effects of the anticancer drug 5-FU, and other nucleoside analogues. The Sm binding site of human U4 snRNA was prepared by in vitro transcription, and Sm protein binding was assessed using gel mobility shift assays. The U4:Sm RNA:protein complex was identified by immunoprecipitation with the Sm-specific Y12 antibody. The effects of 5-FU substitution were assessed by including FUTP in the in vitro transcription reactions. The effects of native nucleotide substitution were assessed by mutagenesis. Deletion mutants were used to assess the relative importance of the two stem-loops that flank the Sm binding site for protein binding. Point mutation (U-->G) to the 5'-Urd in the Sm site reduced Sm protein binding while similar point mutation to the 3'-Urd had a lesser effect. Mutation (U-->G) of all Urd in the Sm site completely inhibited Sm protein binding. The central stem-loop contributed significantly to Sm protein complex formation but the 3' stem-loop had little effect. Substitution of Urd by 5-fluorourdine (FUrd) did not inhibit Sm protein binding, but reduced the stability of the resulting complex. The results indicate that 5-FU, or other Uracil analogues, are unlikely to exert RNA-mediated effects through inhibition of Sm protein binding.

  10. Effective tight-binding models for excitons in branched conjugated molecules

    NASA Astrophysics Data System (ADS)

    Li, Hao; Malinin, Sergey V.; Tretiak, Sergei; Chernyak, Vladimir Y.

    2013-08-01

    Effective tight-binding models have been introduced to describe vertical electronic excitations in branched conjugated molecules. The excited-state electronic structure is characterized by quantum particles (excitons) that reside on an irregular lattice (graph) that reflects the molecular structure. The methodology allows for the exciton spectra and energy-dependent exciton scattering matrices to be described in terms of a small number of lattice parameters which can be obtained from quantum-chemical computations using the exciton scattering approach as a tool. We illustrate the tight-binding model approach using the time-dependent Hartree-Fock computations in phenylacetylene oligomers. The on-site energies and hopping constants have been identified from the exciton dispersion and scattering matrices. In particular, resonant, as well as bound states, are reproduced for a symmetric quadruple branching center. The capability of the tight-binding model approach to describe the exciton-phonon coupling and energetic disorder in large branched conjugated molecules is briefly discussed.

  11. Iron Oxide Surface Chemistry: Effect of Chemical Structure on Binding in Benzoic Acid and Catechol Derivatives.

    PubMed

    Korpany, Katalin V; Majewski, Dorothy D; Chiu, Cindy T; Cross, Shoronia N; Blum, Amy Szuchmacher

    2017-03-13

    The excellent performance of functionalized iron oxide nanoparticles (IONPs) in nanomaterial and biomedical applications often relies on achieving the attachment of ligands to the iron oxide surface both in sufficient number and with proper orientation. Toward this end, we determine relationships between the ligand chemical structure and surface binding on magnetic IONPs for a series of related benzoic acid and catechol derivatives. Ligand exchange was used to introduce the model ligands, and the resultant nanoparticles were characterized using Fourier transform infrared-attenuated internal reflectance spectroscopy, transmission electron microscopy, and nanoparticle solubility behavior. An in-depth analysis of ligand electronic effects and reaction conditions reveals that the nature of ligand binding does not solely depend on the presence of functional groups known to bind to IONPs. The structure of the resultant ligand-surface complex was primarily influenced by the relative positioning of hydroxyl and carboxylic acid groups within the ligand and whether or not HCl(aq) was added to the ligand-exchange reaction. Overall, this study will help guide future ligand-design and ligand-exchange strategies toward realizing truly custom-built IONPs.

  12. The effect on structural and solvent water molecules of substrate binding to ferric horseradish peroxidase.

    PubMed

    Simpson, Niall; Adamczyk, Katrin; Hithell, Gordon; Shaw, Daniel J; Greetham, Gregory M; Towrie, Michael; Parker, Anthony W; Hunt, Neil T

    2015-01-01

    Ultrafast, multi-dimensional infrared spectroscopy, in the form of 2D-IR and pump-probe measurements, has been employed to investigate the effect of substrate binding on the structural dynamics of the horseradish peroxidase (HRP) enzyme. Using nitric oxide bound to the ferric haem of HRP as a sensitive probe of local dynamics, we report measurements of the frequency fluctuations (spectral diffusion) and vibrational lifetime of the NO stretching mode with benzohydroxamic acid (BHA) located in the substrate-binding position at the periphery of the haem pocket, in both D2O and H2O solvents. The results reveal that, with BHA bound to the enzyme, the local structural dynamics are insensitive to H/D exchange. These results are in stark contrast to those found in studies of the substrate-free enzyme, which demonstrated that the local chemical and dynamic environment of the haem ligand is influenced by water molecules. In light of the large changes in solvent accessibility caused by substrate binding, we discuss the potential for varying roles for the solvent in the haem pocket of HRP at different stages along the reaction coordinate of the enzymatic mechanism.

  13. Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

    PubMed Central

    Howes, Stuart C.; Alushin, Gregory M.; Shida, Toshinobu; Nachury, Maxence V.; Nogales, Eva

    2014-01-01

    Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments. PMID:24227885

  14. Effective DNA binding and cleaving tendencies of malonic acid coupled transition metal complexes

    NASA Astrophysics Data System (ADS)

    Pravin, Narayanaperumal; Utthra, Ponnukalai Ponya; Kumaravel, Ganesan; Raman, Natarajan

    2016-11-01

    Eight transition metal complexes were designed to achieve maximum biological efficacy. They were characterized by elemental analysis and various other spectroscopic techniques. The monomeric complexes were found to espouse octahedral geometry and non-electrolytic nature. The DNA interaction propensity of the complexes with calf thymus DNA (CT-DNA), studied at physiological pH by spectrophotometric, spectrofluorometric, cyclic voltammetry, and viscometric techniques revealed intercalation as the possible binding mode. Fascinatingly, the complexes were found to exhibit greater binding strength than that of the free ligands. A strong hypochromism and a slight red shift were exhibited by complex 5 among the other complexes. The intrinsic binding constant values of all the complexes compared to cisplatin reveal that they are excellent metallonucleases than that of cisplatin. The complexes were also shown to reveal displacement of the ethidium bromide, a strong intercalator using fluorescence titrations. Gel electrophoresis was used to divulge the competence of the complexes in cleaving the supercoiled pBR322 plasmid DNA. From the results, it is concluded that the complexes, especially 5, are excellent chemical nucleases in the presence of H2O2. Furthermore, the in vitro antimicrobial screening of the complexes exposes that these complexes are excellent antimicrobial agents. Overall the effect of coligands is evident from the results of all the investigations.

  15. Effect of ouabain binding on the fluorescent properties of the Na+/K+-ATPase.

    PubMed

    Grimaldi, S; Pascale, E; Pozzi, D; D'Onofrio, M; Giganti, M G; Verna, R

    1988-09-15

    The influence of occupancy by ouabain of its specific binding site on the stability and conformation of the Na+/K+-ATPase has been investigated. When native Na+/K+-ATPase is exposed to guanidinium chloride or diluted acid, tryptophanyl fluorescence falls to 50% of the initial value. If ouabain is bound, higher concentrations of GdmCl or acidity are needed to reach the same decrease in fluorescence. The rotational diffusion coefficient (relaxation time), shows higher values for the Na+/K+-ATPase (ouabain) complex compared to the enzyme alone, suggesting an increase in molecular asymmetry. This observation is confirmed by the Stern-Volmer analysis that shows an increase in the accessibility of the fluorophores in the Na+/K+-ATPase (ouabain) (KSV = 15.6 M-1) with respect to the native enzyme (KSV = 12.5 M-1). Iodine perturbation of the enzyme labelled with FITC, demonstrates a decrease in the accessibility of the fluorescein probe in the Na+/K+-ATPase(ouabain) (KSV = 4 M-1) compared to the Na+/K+-ATPase (KSV = 7 M-1) indicating that after ouabain binding this site of the enzyme is less exposed to the solvent. These data, in agreement with other reports, suggest an allosteric effect of ouabain binding on the Na+/K+-ATPase conformation.

  16. The same pocket in menin binds both MLL and JUND but has opposite effects on transcription

    SciTech Connect

    Huang, Jing; Gurung, Buddha; Wan, Bingbing; Matkar, Smita; Veniaminova, Natalia A.; Wan, Ke; Merchant, Juanita L.; Hua, Xianxin; Lei, Ming

    2013-04-08

    Menin is a tumour suppressor protein whose loss or inactivation causes multiple endocrine neoplasia 1 (MEN1), a hereditary autosomal dominant tumour syndrome that is characterized by tumorigenesis in multiple endocrine organs. Menin interacts with many proteins and is involved in a variety of cellular processes. Menin binds the JUN family transcription factor JUND and inhibits its transcriptional activity. Several MEN1 missense mutations disrupt the menin-JUND interaction, suggesting a correlation between the tumour-suppressor function of menin and its suppression of JUND-activated transcription. Menin also interacts with mixed lineage leukaemia protein 1 (MLL1), a histone H3 lysine 4 methyltransferase, and functions as an oncogenic cofactor to upregulate gene transcription and promote MLL1-fusion-protein-induced leukaemogenesis. A recent report on the tethering of MLL1 to chromatin binding factor lens epithelium-derived growth factor (LEDGF) by menin indicates that menin is a molecular adaptor coordinating the functions of multiple proteins. Despite its importance, how menin interacts with many distinct partners and regulates their functions remains poorly understood. Here we present the crystal structures of human menin in its free form and in complexes with MLL1 or with JUND, or with an MLL1-LEDGF heterodimer. These structures show that menin contains a deep pocket that binds short peptides of MLL1 or JUND in the same manner, but that it can have opposite effects on transcription. The menin-JUND interaction blocks JUN N-terminal kinase (JNK)-mediated JUND phosphorylation and suppresses JUND-induced transcription. In contrast, menin promotes gene transcription by binding the transcription activator MLL1 through the peptide pocket while still interacting with the chromatin-anchoring protein LEDGF at a distinct surface formed by both menin and MLL1.

  17. Effects of hyperthermia on binding, internalization, and degradation of epidermal growth factor. [/sup 125/I

    SciTech Connect

    Magun, B.E.; Fennie, C.W.

    1981-04-01

    /sup 125/I-epidermal growth factor was used as a molecular probe to study the effects of hyperthermia and local anesthetics on cultured Rat-1 cells. Heating cells at 45/sup 0/C for times up to 1 h caused a continuous decrease in EGF binding. Scatchard analysis showed that the decreased binding resulted from a decrease in the affinity of the EGF receptors rather than from a decrease in receptor number. Exposure to 42/sup 0/C had no effect on degradation. We compared the effects of heat to those caused by the local anesthetics procaine the lidocaine, which have been shown to prevent EGF degradation. Because procaine and lidocaine have been shown by others to potentiate the killing effects of hyperthermia on tumors and in cultured cells, we suggest that hyperthermia and the local anesthetics may act at the same cellular site. By inhibiting the action of lysosomes, hyperthermia and local anesthetics may permit potentially toxic materials to enter the cell by endocytosis, where they would accumulate and induce lethal damage.

  18. Many-body dispersion effects in the binding of adsorbates on metal surfaces

    SciTech Connect

    Maurer, Reinhard J.; Ruiz, Victor G.; Tkatchenko, Alexandre

    2015-09-14

    A correct description of electronic exchange and correlation effects for molecules in contact with extended (metal) surfaces is a challenging task for first-principles modeling. In this work, we demonstrate the importance of collective van der Waals dispersion effects beyond the pairwise approximation for organic–inorganic systems on the example of atoms, molecules, and nanostructures adsorbed on metals. We use the recently developed many-body dispersion (MBD) approach in the context of density-functional theory [Tkatchenko et al., Phys. Rev. Lett. 108, 236402 (2012) and Ambrosetti et al., J. Chem. Phys. 140, 18A508 (2014)] and assess its ability to correctly describe the binding of adsorbates on metal surfaces. We briefly review the MBD method and highlight its similarities to quantum-chemical approaches to electron correlation in a quasiparticle picture. In particular, we study the binding properties of xenon, 3,4,9,10-perylene-tetracarboxylic acid, and a graphene sheet adsorbed on the Ag(111) surface. Accounting for MBD effects, we are able to describe changes in the anisotropic polarizability tensor, improve the description of adsorbate vibrations, and correctly capture the adsorbate–surface interaction screening. Comparison to other methods and experiment reveals that inclusion of MBD effects improves adsorption energies and geometries, by reducing the overbinding typically found in pairwise additive dispersion-correction approaches.

  19. Substituent effects on axle binding in amide pseudorotaxanes: comparison of NMR titration and ITC data with DFT calculations.

    PubMed

    Kaufmann, Lena; Dzyuba, Egor V; Malberg, Friedrich; Löw, Nora L; Groschke, Matthias; Brusilowskij, Boris; Huuskonen, Juhani; Rissanen, Kari; Kirchner, Barbara; Schalley, Christoph A

    2012-08-14

    The binding behaviour of differently substituted diamide axle molecules to Hunter/Vögtle tetralactam macrocycles was studied with a combination of NMR titration, isothermal titration calorimetry (ITC) experiments and calculations employing density functional theory (DFT), along with dispersion-corrected exchange-correlation functionals. Guests with alkyl or alkenyl chains attached to the diamide carbonyl groups have a significantly higher binding affinity to the macrocycle than guests with benzoyl amides and their substituted analogues. While the binding of the benzoyl and alkenyl substituted axles is enthalpically driven, the alkyl-substituted guest binds mainly because of a positive binding entropy. The electronic effects of para-substituents at the benzoyl moieties have an influence on the binding affinities. Electron donating substituents increase, while electron-withdrawing substituents decrease the binding energies. The binding affinities obtained from both NMR titration and ITC experiments correlate well with each other. The substituent effects observed in the experimental data are reflected in adiabatic interaction energies calculated with density functional methods. The calculated structures also agree well with pseudorotaxane crystal structures.

  20. Elevated glucocorticoid receptor binding in cultured human lymphoblasts following hydroxyurea treatment: lack of effect on steroid responsiveness

    SciTech Connect

    Littlefield, B.A.; Hoagland, H.C.; Greipp, P.R.

    1986-08-01

    While studying the effects of chemotherapy on glucocorticoid receptor (GR) binding levels in hematological malignancies, we observed a sizable increase in nuclear GR binding of (/sup 3/H)dexamethasone in peripheral leukocytes from a chronic basophilic leukemia patient following treatment with hydroxyurea plus prednisone, but not after prednisone alone. This apparent clinical effect of hydroxyurea led to an examination of hydroxyurea effects on GR binding and sensitivity in the glucocorticoid-sensitive human lymphoblast cell line GM4672A. GR binding levels in GM4672A cells were measured following a 3-day exposure to 50 microM hydroxyurea, a concentration chosen to have a minimal but measurable effect on cellular growth rates with little or no effect on cellular viability. Under these conditions, nuclear (/sup 3/H)dexamethasone receptor binding measured by Scatchard analysis using a whole-cell assay was elevated 2.4-fold over control values (P less than 0.05), while cytosolic residual receptor binding (measured at 37/sup 0/C) remained unchanged. Thus, the total cellular content of measurable GR was increased, and this increase was totally accounted for by GR capable of nuclear binding. Hydroxyurea treatment of GM4672A cells had no effect on the affinity of nuclear or cytosolic GR for (/sup 3/H)dexamethasone. The increase in measurable nuclear-bound receptors occurred in a time-dependent manner over a period of 3 days and was fully reversible within 3 days following removal of hydroxyurea. The increase in receptor binding could not be explained by the slight alterations in cell cycle kinetics which occur at this low level of hydroxyurea. Despite increased receptor binding, cellular glucocorticoid responsiveness was unaltered as assessed by dexamethasone inhibition of cell growth and dexamethasone inhibition of a urokinase-like plasminogen activator.

  1. Crystal structures of the ligand-binding region of uPARAP: effect of calcium ion binding.

    PubMed

    Yuan, Cai; Jürgensen, Henrik J; Engelholm, Lars H; Li, Rui; Liu, Min; Jiang, Longguang; Luo, Zhipu; Behrendt, Niels; Huang, Mingdong

    2016-08-01

    The proteins of the mannose receptor (MR) family share a common domain organization and have a broad range of biological functions. Urokinase plasminogen activator receptor-associated protein (uPARAP) (or Endo180) is a member of this family and plays an important role in extracellular matrix remodelling through interaction with its ligands, including collagens and urokinase plasminogen activator receptor (uPAR). We report the crystal structures of the first four domains of uPARAP (also named the ligand-binding region, LBR) at pH 7.4 in Ca(2+)-bound and Ca(2+)-free forms. The first domain (cysteine-rich or CysR domain) folds into a new and unique conformation different from the β-trefoil fold of typical CysR domains. The so-called long loop regions (LLRs) of the C-type lectin-like domain (CTLD) 1 and 2 (the third and fourth domain) mediate the direct contacts between these domains. These LLRs undergo a Ca(2+)-dependent conformational change, and this is likely to be the key structural determinant affecting the overall conformation of uPARAP. Our results provide a molecular mechanism to support the structural flexibility of uPARAP, and shed light on the structural flexibility of other members of the MR family.

  2. Chloride diffusivity in hardened cement paste from microscale analyses and accounting for binding effects.

    PubMed

    Carrara, P; De Lorenzis, L; Bentz, D P

    2016-08-01

    The diffusion of chloride ions in hardened cement paste (HCP) under steady-state conditions and accounting for the highly heterogeneous nature of the material is investigated. The HCP microstructures are obtained through segmentation of X-ray images of real samples as well as from simulations using the cement hydration model CEMHYD3D. Moreover, the physical and chemical interactions between chloride ions and HCP phases (binding), along with their effects on the diffusive process, are explicitly taken into account. The homogenized diffusivity of the HCP is then derived through a least square homogenization technique. Comparisons between numerical results and experimental data from the literature are presented.

  3. Effects of water molecules on binding kinetics of peptide receptor on a piezoelectric microcantilever

    NASA Astrophysics Data System (ADS)

    Hui Kim, Sang; Kyoung Yoo, Yong; Chae, Myung-Sic; Yoon Kang, Ji; Song Kim, Tae; Seon Hwang, Kyo; Hoon Lee, Jeong

    2012-12-01

    The use of highly selective reversible peptide receptors is essential for cantilever-based electronic nose systems. Here, we present the effects of water molecules on the binding kinetics of 2,4-dinitrotoluene (DNT) molecules with DNT selective peptide receptors linked with a tri(ethylene glycol)-based (TEG) self-assembled monolayer (SAM) in a gas phase in a piezoelectric microcantilever sensor. We observed 1.5-times faster reaction kinetics in wet conditions compared with dry conditions. In a dissociation step, distinctive differences in the recovery time were observed in wet conditions, which could be attributed to water retention efficiency of TEG-linkers for the conformation of biomolecules.

  4. Quantitative Assessment of the Effects of Oxidants on Antigen-Antibody Binding In Vitro

    PubMed Central

    Han, Shuang; Wang, Guanyu; Xu, Naijin; Liu, Hui

    2016-01-01

    Objective. We quantitatively assessed the influence of oxidants on antigen-antibody-binding activity. Methods. We used several immunological detection methods, including precipitation reactions, agglutination reactions, and enzyme immunoassays, to determine antibody activity. The oxidation-reduction potential was measured in order to determine total serum antioxidant capacity. Results. Certain concentrations of oxidants resulted in significant inhibition of antibody activity but had little influence on total serum antioxidant capacity. Conclusions. Oxidants had a significant influence on interactions between antigen and antibody, but minimal effect on the peptide of the antibody molecule. PMID:27313823

  5. Chloride diffusivity in hardened cement paste from microscale analyses and accounting for binding effects

    NASA Astrophysics Data System (ADS)

    Carrara, P.; De Lorenzis, L.; Bentz, D. P.

    2016-08-01

    The diffusion of chloride ions in hardened cement paste (HCP) under steady-state conditions and accounting for the highly heterogeneous nature of the material is investigated. The three-dimensional HCP microstructures are obtained through segmentation of x-ray images of real samples as well as from simulations using the cement hydration model CEMHYD3D. Moreover, the physical and chemical interactions between chloride ions and HCP phases (binding), along with their effects on the diffusive process, are explicitly taken into account. The homogenized diffusivity of the HCP is then derived through a least square homogenization technique. Comparisons between numerical results and experimental data from the literature are presented.

  6. Some dipeptides reverse the inhibitory effect of GABA on /sup 35/S-TBPS binding

    SciTech Connect

    Squires, R.F.; Saederup, E.

    1987-05-01

    All known GABA-A receptor blocker reverse the inhibitory effect of GABA on /sup 35/S-t-butylphosphorothionate (TBPS) binding to rat brain membranes in vitro. This system has already been used to identify several novel GABA antagonists. The authors now report that 12 out of 52 dipeptides tested (all containing L-amino acids), at 1 mM, significantly reverse the inhibitory effect of 1 ..mu..M GABA, which inhibits specific /sup 35/S-TBPS binding about 60%. Most of the active dipeptides contain an aromatic and a basic amino acid. Tryptophan usually conferred greater activity than phe or tyr, while arg usually conferred greater activity than lys or his. Several larger peptides containing the HFRW sequence found in ACTH were also GABA antagonists; ACTH(1-24), ACTH(1-18), ACTH(1-13), ACTH(4-10) and ..gamma..-MSH while ACTH(11-24) was inactive. The excitatory effects of these later peptides may be in part due to blockade of GABA-A receptors.

  7. The Effects of Chronic Alcohol Self-Administration on Serotonin-1A Receptor Binding in Nonhuman Primates

    PubMed Central

    Hillmer, Ansel T.; Wooten, Dustin W.; Tudorascu, Dana L.; Barnhart, Todd E.; Ahlers, Elizabeth O.; Resch, Leslie M.; Larson, Julie A.; Converse, Alexander K.; Moore, Colleen F.; Schneider, Mary L.; Christian, Bradley T.

    2014-01-01

    Background Previous studies have found interrelationships between the serotonin system and alcohol self-administration. The goal of this work was to directly observe in vivo effects of chronic ethanol self-administration on serotonin 5-HT1A receptor binding with [18F]mefway PET neuroimaging in rhesus monkeys. Subjects were first imaged alcohol-naïve and again during chronic ethanol self-administration to quantify changes in 5-HT1A receptor binding. Methods Fourteen rhesus monkey subjects (10.7-12.8 years) underwent baseline [18F]mefway PET scans prior to alcohol exposure. Subjects then drank gradually increasing ethanol doses over four months as an induction period, immediately followed by at least nine months ad libidum ethanol access. A post [18F]mefway PET scan was acquired during the final three months of ad libidum ethanol self-administration. 5-HT1A receptor binding was assayed with binding potential (BPND) using the cerebellum as a reference region. Changes in 5-HT1A binding during chronic ethanol self-administration were examined. Relationships of binding metrics with daily ethanol self-administration were also assessed. Results Widespread increases in 5-HT1A binding were observed during chronic ethanol self-administration, independent of the amount of ethanol consumed. A positive correlation between 5-HT1A binding in the raphe nuclei and average daily ethanol self-administration was also observed, indicating that baseline 5-HT1A binding in this region predicted drinking levels. Conclusions The increase in 5-HT1A binding levels during chronic ethanol self-administration demonstrates an important modulation of the serotonin system due to chronic alcohol exposure. Furthermore, the correlation between 5-HT1A binding in the raphe nuclei and daily ethanol self-administration indicates a relationship between the serotonin system and alcohol self-administration. PMID:25220896

  8. [Effect of calcium binding on the quenching time of self-fluorescence of Ca-binding proteins].

    PubMed

    Permiakov, E A; Burshteĭn, E A; Emel'ianenko, V I; Aleksandrov, Iu M; Glagolev, K V

    1983-01-01

    Lifetimes of phenylalanine, tyrosine and tryptophan self-fluorescence of three Ca2+-binding proteins (parvalbumins pI 4.47 and 3.95 and bovine alpha-lactalbumin) in the Ca2+-saturated state and without Ca2+ were measured on a device functioning in a channel of synchrotron radiation of the Lebedev Physical Institute electron accelerator C-60 with a single photon counting system. The decay curve of phenylalanine fluorescence of Ca2+-saturated parvalbumin pI 4.47 is two-exponential, which results from the presence of two subsystems of phenylalanine residues in this protein. Radiation of these subsystems is almost independent of one another. Detachment of Ca2+ from protein disturbs these subsystems. In case of tyrosine fluorescence of carp parvalbumin pI 3.95 a change in the quantum yield value of the stationary fluorescence induced by elimination of Ca2+ proceeds without a change of fluorescence lifetime. This seems to be related to the existence of static quenching of fluorescence in this case at the expense of complex formation between the chromophore and some adjacent quenching groups. Detachment of Ca2+ from alpha-lactalbumin induces conformational changes in its structure. The latter result in a transition of a number of tryptophane residues from its interior to the surface of the globule which is reflected in an increase of fluorescence quantum yield duration. It is concluded that in Ca2+-saturated alpha-lactalbumin some tryptophane residues are located near the quenching groups (dynamic quenching), most likely the disulfide bridges.

  9. Binding motifs in bacterial gene promoters modulate transcriptional effect of global regulators

    SciTech Connect

    Leuze, Michael Rex; Karpinets, Tatiana V; Syed, Mustafa H; Beliaev, Alexander S; Uberbacher, Edward C

    2012-01-01

    Bacterial gene regulation involves transcription factors (TFs) that influence the expression of many genes. Global regulators, including CRP (cAMP Receptor Protein), ArcA, and FNR, can modulate the transcriptional activity of multiple operons. The similarity of a regulatory element s sequence to a TF s consensus binding site (BS) and the position of the regulatory element in an operon promoter are considered the most important determinants of this TF s regulatory influence. In this study we explore the hypothesis that the number of TFBS half-sites (where a half-site is one half of the palindromic BS consensus sequence, which we shall refer to as a binding motif or a BM) of a global regulator in an operon s promoter plays an important role in the operon s transcriptional regulation. We examine empirical data from transcriptional profiling of the CRP regulon in Shewanella oneidenses MR 1 and Escherichia coli, and of the ArcA regulon in S. oneidenses MR 1. We compare the power of CRP BM counts and of full, symmetrical CRP TFBS characteristics, namely similarity to consensus and location, to predict CRP-induced transcriptional activity. We find that CRP BM counts have a nonlinear effect on CRP-dependent transcriptional activity and predict this activity better than full-length TFBS quality or location. Regression analysis indicates that IHF (Integration Host Factor) and ArcA have synergistic effects on CRP-induced gene transcription, positive and negative, respectively. Based on these results, we propose that the fine-tuning of bacterial transcriptional activity by CRP may involves not only the bending of the operon promoter, facilitated by CRP in cooperation with the histone-like protein IHF, but also the cumulative binding affinity of multiple weak BMs.

  10. Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding.

    PubMed

    Smith, Kathryn A; Conboy, John C

    2011-06-01

    The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.

  11. Dihydrocytochalasin B. Biological effects and binding to 3T3 cells

    PubMed Central

    Atlas, S. J.; Lin, S.

    1978-01-01

    Dihydrocytochalasin B (H2CB) does not inhibit sugar uptake in BALB/c 3T3 cells. Excess H2CB does not affect inhibition of sugar uptake by cytochalasin B (CB), indicating that it does not compete with CB for binding to high-affinity sites. As in the case of CB, H2CB inhibits cytokinesis and changes the morphology of the cells. These results demonstrate that the effects of CB on sugar transport and on cell motility and morphology involve separate and independent sites. Comparison of the effects of H2CB, CB, and cytochalasin D (CD) indicates that treatment of cells with any one of the compounds results in the same series of morphological changes; the cells undergo zeiosis and elongation at 2-4 microM CB and become arborized and rounded up at 10-50 microM CB. H2CB is slightly less potent than CB, whereas CD is five to eight times more potent than CB in causing a given state of morphological change. These results indicate that the cytochalasin-induced changes in cell morphology are mediated by a specific site(s) which can distinguish the subtle differences in the structures of the three compounds. Competitive binding studies indicate that excess H2CB displaces essentially all of the high-affinity bound [3H]CB, but, at less than 5 x 10(-5) M H2CB is not so efficient as unlabeled CB in the displacement reaction. In contrast, excess CD displaces up to 40% of the bound [3H]CB. These results suggest that three different classes of high-affinity CB binding sites exist in 3T3 cells: sites related to sugar transport, sites related to cell motility and morphology, and sites with undetermined function. PMID:10605443

  12. Quantitative Genetics of CTCF Binding Reveal Local Sequence Effects and Different Modes of X-Chromosome Association

    PubMed Central

    Lee, Bum-Kyu; Battenhouse, Anna; Louzada, Sandra; Yang, Fengtang; Dunham, Ian; Crawford, Gregory E.; Lieb, Jason D.; Durbin, Richard; Iyer, Vishwanath R.; Birney, Ewan

    2014-01-01

    Associating genetic variation with quantitative measures of gene regulation offers a way to bridge the gap between genotype and complex phenotypes. In order to identify quantitative trait loci (QTLs) that influence the binding of a transcription factor in humans, we measured binding of the multifunctional transcription and chromatin factor CTCF in 51 HapMap cell lines. We identified thousands of QTLs in which genotype differences were associated with differences in CTCF binding strength, hundreds of them confirmed by directly observable allele-specific binding bias. The majority of QTLs were either within 1 kb of the CTCF binding motif, or in linkage disequilibrium with a variant within 1 kb of the motif. On the X chromosome we observed three classes of binding sites: a minority class bound only to the active copy of the X chromosome, the majority class bound to both the active and inactive X, and a small set of female-specific CTCF sites associated with two non-coding RNA genes. In sum, our data reveal extensive genetic effects on CTCF binding, both direct and indirect, and identify a diversity of patterns of CTCF binding on the X chromosome. PMID:25411781

  13. Effect of direct albumin binding to sphingosylphosphorylcholine in Jurkat T cells.

    PubMed

    Han, Mijin; Kim, Yu-Lee; Sacket, Santosh J; Kim, Kyeok; Kim, Hyo-Lim; Jo, Ji-Yeong; Ha, Nam-Chul; Im, Dong-Soon

    2007-11-01

    We investigated the effects of serum on lysophospholipid-induced cytotoxicity in Jurkat T cells. We found that sphingosylphosphorylcholine (SPC, also known as lysosphingomyelin) induced cytotoxicity and that albumin in serum could protect cells by binding directly to SPC. Furthermore, we also found that SPC induced ROS generation, increased [Ca(2+)](i), and decreased MMP. However, those effects were only observed at concentrations higher than 10 microM and were only induced in albumin-free media. Therefore, SPC may be trapped by albumin in plasma and unable to exert its effects under normal conditions, although at high concentrations, SPC could induce several responses such as ROS generation, increased [Ca(2+)](i), and decreased MMP in Jurkat T cells.

  14. Effect of geometry on the screened acceptor binding energy in a quantum wire

    SciTech Connect

    Shanthi, R. Vijaya Nithiananthi, P.

    2014-04-24

    The effect of various Geometries G(x, y) of the GaAs/Al{sub x}Ga{sub 1−x}As Quantum wire like G{sub 1}: (L, L) {sub 2}: (L, L/2) {sub 3}: (L/2, L/4) on the binding energy of an on-center acceptor impurity has been investigated through effective mass approximation using variational technique. The observations were made including the effect of spatial dependent dielectric screening for different concentration of Al, at T=300K. The influence of spatial dielectric screening on different geometries of the wire has been compared and hence the behavior of the acceptor impurity in GaAs/Al{sub x}Ga{sub 1−x}As Quantum wire has been discussed.

  15. The effect of various naturally occurring metal-binding compounds on the electrochemical behavior of aluminum

    SciTech Connect

    Hansen, D.C.; McCafferty, E.

    1996-01-01

    Naturally occurring biological molecules are of considerable interest as possible corrosion inhibitors because of increased attention on the development of environmentally compatible, nonpolluting corrosion inhibitors. A hydroxamate yeast siderophore (rhodotorulic acid), a catecholate bacterial siderophore (parabactin), an adhesive protein from the blue mussel Mytilus edulis, and two metal-binding compounds isolated from the tomato and sunflower roots, namely, chlorogenic and caffeic acid, respectively, were adsorbed from solution onto pure aluminum (99.9995%) and their effect on the critical pitting potential and polarization resistance in deaerated 0.1 M NaCl was measured. These measurements were made using anodic polarization and ac impedance spectroscopy. The catechol-containing siderophore has an inhibitive effect on the critical pitting potential of aluminum in 0.1 M NaCl and increases the polarization resistance of the metal over time. The adhesive protein from the blue mussel is also effective in inhibiting the pitting of aluminum.

  16. Peptide Chain Termination: Effect of Protein S on Ribosomal Binding of Release Factors

    PubMed Central

    Goldstein, J. L.; Caskey, C. T.

    1970-01-01

    The protein factor S, previously shown to stimulate polypeptide chain termination in bacterial extracts, has two effects upon the complex formed between ribosomes, release factor, and terminator (trinucleotide) codon: (1) in the absence of GTP or GDP, S stimulates formation of an [R·UAA·ribosome] intermediate, and (2) in the presence of GTP or GDP, S participates in dissociation of this intermediate. Factor S can stimulate fMet release from [fMet-tRNAf·AUG·ribosome] intermediates in either the presence or absence of GTP or GDP. A model is proposed which relates the in vitro effects of S ± GTP (or GDP) on fMet release to the effects of S ± GTP (or GDP) on the binding and dissociation of R factor from ribosomes. PMID:5289007

  17. Global effects of the energetics of coenzyme binding: NADPH controls the protein interaction properties of human cytochrome P450 reductase.

    PubMed

    Grunau, Alex; Paine, Mark J; Ladbury, John E; Gutierrez, Aldo

    2006-02-07

    The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry. Binding of 2',5'-ADP, NADP(+), and H(4)NADP, an isosteric NADPH analogue, is described in terms of the dissociation binding constant (K(d)), the enthalpy (DeltaH(B)) and entropy (TDeltaS(B)) of binding, and the heat capacity change (DeltaC(p)). This systematic approach allowed the effect of coenzyme redox state on binding to CPR to be determined. The recognition and stability of the coenzyme-CPR complex are largely determined by interaction with the adenosine moiety (K(d2)(')(,5)(')(-ADP) = 76 nM), regardless of the redox state of the nicotinamide moiety. Similar heat capacity change (DeltaC(p)) values for 2',5'-ADP (-210 cal mol(-)(1) K(-)(1)), NADP(+) (-230 cal mol(-)(1) K(-)(1)), and H(4)NADP (-220 cal mol(-)(1) K(-)(1)) indicate no significant contribution from the nicotinamide moiety to the binding interaction surface. The coenzyme binding stoichiometry to CPR is 1:1. This result validates a recently proposed one-site kinetic model [Daff, S. (2004) Biochemistry 43, 3929-3932] as opposed to a two-site model previously suggested by us [Gutierrez, A., Lian, L.-Y., Wolf, C. R., Scrutton, N. S., and Roberts, C. G. K. (2001) Biochemistry 40, 1964-1975]. Calorimetric studies in which binding of 2',5'-ADP to CPR (TDeltaS(B) = -13400 +/- 200 cal mol(-)(1), 35 degrees C) was compared with binding of the same ligand to the isolated FAD-binding domain (TDeltaS(B) = -11200 +/- 300 cal mol(-)(1), 35 degrees C) indicate that the number of accessible conformational substates of the protein increases upon 2',5'-ADP binding in the presence of the FMN-binding domain. This pattern was consistently observed along the temperature range that was studied (5-35 degrees C). This contribution of coenzyme binding energy to domain dynamics in CPR agrees with conclusions from previous temperature-jump studies [Gutierrez

  18. Effects of microgravity on the binding of acetylsalicylic acid by Rhizobium leguminosarum bv. trifolii

    NASA Astrophysics Data System (ADS)

    Urban, James E.; Gerren, Richard; Zoelle, Jeffery

    1995-07-01

    Bacteroids can be induced in vitro by treating growing Rhizobium leguminosarum bv. trifolii with succinic acid or succinic acid structural analogs like acetylsalicylic acid. Quantitating bacteroid induction by measuring acetylsalicylic binding under normal (1 g) conditions showed two forms of binding to occur. In one form of binding cells immediately bound comparatively high levels of acetylsalicylic acid, but the binding was quickly reversed. The second form of binding increased with time by first-order kinetics, and reached saturation in 40 s. Similar experiments performed in the microgravity environment aboard the NASA 930 aircraft showed only one form of binding and total acetylsalicylic acid bound was 32% higher than at 1 g.

  19. Amnesic effects of the anticholinergic drugs, trihexyphenidyl and biperiden: differences in binding properties to the brain muscarinic receptor.

    PubMed

    Kimura, Y; Ohue, M; Kitaura, T; Kihira, K

    1999-07-10

    An amnesic effect of anticholinergic drugs was previously described from several behavioral studies. We examined this effect induced by trihexyphenidyl and biperiden, clinically used in the parkinsonism and schizophrenic patients, by using passive avoidance tasks. Both of these drugs (0.1-10 mg/kg, s.c.) showed dose-dependent amnesic effects in the acquisition and retrieval phases. However, the effect induced by trihexyphenidyl was transient, whereas that of biperiden was long-lasting. To clarify the reason for the different duration of the amnesic activity, binding to the muscarinic receptor was examined. In the Scatchard analysis, trihexyphenidyl competed with [(3)H]quinuclidinyl benzilate ([(3)H]QNB) on the muscarinic receptor (showed increased K(d) and unchanged B(max) value), while biperiden decreased [(3)H]QNB binding (B(max) value) significantly. Furthermore, in an exchange assay for receptor inactivation, trihexyphenidyl binding to muscarinic receptors was exchanged by [(3)H]QNB completely, but biperiden decreased the exchangeable binding of [(3)H]QNB in a dose dependent manner (0.1-100 nM). These results suggested that the binding of trihexyphenidyl and biperiden to muscarinic receptor might be completely reversible and partially irreversible, respectively, whereas the K(i) values of these two drugs were similar. In conclusion, this difference in binding property may explain the difference in the time-course of the amnesic effect induced by trihexyphenidyl and biperiden.

  20. Effect of fullerenol surface chemistry on nanoparticle binding-induced protein misfolding

    NASA Astrophysics Data System (ADS)

    Radic, Slaven; Nedumpully-Govindan, Praveen; Chen, Ran; Salonen, Emppu; Brown, Jared M.; Ke, Pu Chun; Ding, Feng

    2014-06-01

    Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and dynamics of ubiquitin. We found that all derivatives bound to the model protein. Specifically, the more hydrophilic nanoparticles with a higher number of hydroxyl groups bound to the surface of the protein via hydrogen bonds, which stabilized the protein without inducing large conformational changes in the protein structure. In contrast, fullerene derivatives with a smaller number of hydroxyl groups buried their hydrophobic surface inside the protein, thereby causing protein denaturation. Overall, our results revealed a distinct role of surface chemistry on nanoparticle-protein binding and binding-induced protein misfolding.Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation -- a common strategy for solubilizing and functionalizing fullerene -- on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and

  1. Macromolecular crowding effects on protein-protein binding affinity and specificity

    NASA Astrophysics Data System (ADS)

    Kim, Young C.; Best, Robert B.; Mittal, Jeetain

    2010-11-01

    Macromolecular crowding in cells is recognized to have a significant impact on biological function, yet quantitative models for its effects are relatively undeveloped. The influence of crowding on protein-protein interactions is of particular interest, since these mediate many processes in the cell, including the self-assembly of larger complexes, recognition, and signaling. We use a residue-level coarse-grained model to investigate the effects of macromolecular crowding on the assembly of protein-protein complexes. Interactions between the proteins are treated using a fully transferable energy function, and interactions of protein residues with the spherical crowders are repulsive. We show that the binding free energy for two protein complexes, ubiquitin/UIM1 and cytochrome c/cytochrome c peroxidase, decreases modestly as the concentration of crowding agents increases. To obtain a quantitative description of the stabilizing effect, we map the aspherical individual proteins and protein complexes onto spheres whose radii are calculated from the crowder-excluded protein volumes. With this correspondence, we find that the change in the binding free energy due to crowding can be quantitatively described by the scaled particle theory model without any fitting parameters. The effects of a mixture of different-size crowders—as would be found in a real cell—are predicted by the same model with an additivity ansatz. We also obtain the remarkable result that crowding increases the fraction of specific complexes at the expense of nonspecific transient encounter complexes in a crowded environment. This result, due to the greater excluded volume of the nonspecific complexes, demonstrates that macromolecular crowding can have subtle functional effects beyond the relative stability of bound and unbound complexes.

  2. The propeptides of VEGF-D determine heparin binding, receptor heterodimerization, and effects on tumor biology.

    PubMed

    Harris, Nicole C; Davydova, Natalia; Roufail, Sally; Paquet-Fifield, Sophie; Paavonen, Karri; Karnezis, Tara; Zhang, You-Fang; Sato, Teruhiko; Rothacker, Julie; Nice, Edouard C; Stacker, Steven A; Achen, Marc G

    2013-03-22

    VEGF-D is an angiogenic and lymphangiogenic glycoprotein that can be proteolytically processed generating various forms differing in subunit composition due to the presence or absence of N- and C-terminal propeptides. These propeptides flank the central VEGF homology domain, that contains the binding sites for VEGF receptors (VEGFRs), but their biological functions were unclear. Characterization of propeptide function will be important to clarify which forms of VEGF-D are biologically active and therefore clinically relevant. Here we use VEGF-D mutants deficient in either propeptide, and in the capacity to process the remaining propeptide, to monitor the functions of these domains. We report for the first time that VEGF-D binds heparin, and that the C-terminal propeptide significantly enhances this interaction (removal of this propeptide from full-length VEGF-D completely prevents heparin binding). We also show that removal of either the N- or C-terminal propeptide is required for VEGF-D to drive formation of VEGFR-2/VEGFR-3 heterodimers which have recently been shown to positively regulate angiogenic sprouting. The mature form of VEGF-D, lacking both propeptides, can also promote formation of these receptor heterodimers. In a mouse tumor model, removal of only the C-terminal propeptide from full-length VEGF-D was sufficient to enhance angiogenesis and tumor growth. In contrast, removal of both propeptides is required for high rates of lymph node metastasis. The findings reported here show that the propeptides profoundly influence molecular interactions of VEGF-D with VEGF receptors, co-receptors, and heparin, and its effects on tumor biology.

  3. The Effects of the Amount of Information on Episodic Memory Binding

    PubMed Central

    Torres-Trejo, Frine; Cansino, Selene

    2016-01-01

    The effects of increasing the number of items to be remembered on associative recognition and cued recall were examined. Thirty participants were asked during encoding to determine whether two- and three-item stimuli contained natural objects, artificial objects, or both. In an associative recognition task, the participants indicated whether the stimuli were identical to those presented during encoding, were rearranged by exchanging one of the two-item stimuli for one of the three-item stimuli, or represented a new stimulus. The correctly identified rearranged item pairs and triads were included in a subsequent cued-recall task in which participants verbally reported the missing item. As the number of items increased, the discrimination of rearranged stimuli diminished, but that of identical trials remained the same. Furthermore, the ability to retrieve the missing item was unaffected. It was concluded that the effect of the amount of information on binding depends on how the information must be retrieved. PMID:27512526

  4. The effect of pyridyl substituents on the thermodynamics of porphyrin binding to G-quadruplex DNA.

    PubMed

    Rowland, Gerald B; Barnett, Kerry; Dupont, Jesse I; Akurathi, Gopalakrishna; Le, Vu H; Lewis, Edwin A

    2013-12-01

    Most of the G-quadruplex interactive molecules reported to date contain extended aromatic flat ring systems and are believed to bind principally by π-π stacking on the end G-tetrads of the quadruplex structure. One such molecule, TMPyP4, (5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin), exhibits high affinity and some selectivity for G-quadruplex DNA over duplex DNA. Although not a realistic drug candidate, TMPyP4 is used in many nucleic acid research laboratories as a model ligand for the study of small molecule G-quadruplex interactions. Here we report on the synthesis and G-quadruplex interactions of four new cationic porphyrin ligands having only 1, 2, or 3 (N-methyl-4-pyridyl) substituents. The four new ligands are: P(5) (5-(N-methyl-4-pyridyl)porphyrin), P(5,10) (5,10-di(N-methyl-4-pyridyl)porphyrin), P(5,15) (5,15-di(N-methyl-4-pyridyl)porphyrin), and P(5,10,15) (5,10,15-tri(N-methyl-4-pyridyl)porphyrin). Even though these compounds have been previously synthesized, we report alternative synthetic routes that are more efficient and that result in higher yields. We have used ITC, CD, and ESI-MS to explore the effects of the number of N-methyl-4-pyridyl substituents and the substituent position on the porphyrin on the G-quadruplex binding energetics. The relative affinities for binding these ligands to the WT Bcl-2 promoter sequence G-quadruplex are: K(TMPyP4)≈K(P)(5,15)>KP(5,10,15)>KP(5,10), KP(5). The saturation stoichiometry is 2:1 for both P(5,15) and P(5,10,15), while neither P(5) nor P(5,10) exhibit significant complex formation with the WT Bcl-2 promoter sequence G-quadruplex. Additionally, binding of P(5,15) appears to interact by an 'intercalation mode' while P(5,10,15) appears to interact by an 'end-stacking mode'.

  5. Investigation of binding features: effects on the interaction between CYP2A6 and inhibitors.

    PubMed

    Ai, Chunzhi; Li, Yan; Wang, Yonghua; Li, Wei; Dong, Peipei; Ge, Guangbo; Yang, Ling

    2010-07-15

    A computational investigation has been carried out on CYP2A6 and its naphthalene inhibitors to explore the crucial molecular features contributing to binding specificity. The molecular bioactive orientations were obtained by docking (FlexX) these compounds into the active site of the enzyme. And the density functional theory method was further used to optimize the molecular structures with the subsequent analysis of molecular lipophilic potential (MLP) and molecular electrostatic potential (MEP). The minimal MLPs, minimal MEPs, and the band gap energies (the energy difference between the highest occupied molecular orbital and lowest unoccupied molecular orbital) showed high correlations with the inhibition activities (pIC(50)s), illustrating their significant roles in driving the inhibitor to adopt an appropriate bioactive conformation oriented in the active site of CYP2A6 enzyme. The differences in MLPs, MEPs, and the orbital energies have been identified as key features in determining the binding specificity of this series of compounds to CYP2A6 and the consequent inhibitory effects. In addition, the combinational use of the docking, MLP and MEP analysis is also demonstrated as a good attempt to gain an insight into the interaction between CYP2A6 and its inhibitors.

  6. Effect of lysine modification on the stability and cellular binding of human amyloidogenic light chains.

    PubMed

    Davern, S; Murphy, C L; O'Neill, H; Wall, J S; Weiss, D T; Solomon, A

    2011-01-01

    AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescence microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichroism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

  7. Tight binding model of conformational disorder effects on the optical absorption spectrum of polythiophenes.

    PubMed

    Bombile, Joel H; Janik, Michael J; Milner, Scott T

    2016-05-14

    Semiconducting polymers are soft materials with many conformational degrees of freedom. The limited understanding of how conformational disorder affects their optoelectronic properties is a key source of difficulties that limits their widespread usage in electronic devices. We develop a coarse-grained approach based on the tight binding approximation to model the electronic degrees of freedom of polythiophene chains, taking into account conformational degrees of freedom. Particularly important is dihedral disorder, which disrupts extended electronic states. Our tight binding model is parameterized using density functional theory (DFT) calculations of the one-dimensional band structures for chains with imposed periodic variations in dihedral angles. The model predicts valence and conduction bands for these chain conformations that compare well to DFT results. As an initial application of our model, we compute the optical absorption spectrum of poly(3-hexylthiophene) chains in solution. We observe a broadening of the absorption edge resulting from dihedral disorder, just shy of the experimental broadening. We conclude that the effects of molecular disorder on the optoelectronic properties of conjugated polymer single chains can be mostly accounted for by torsional disorder alone.

  8. Effect of Lysine Modification on the Stability and Cellular Binding of Human Amyloidogenic Light Chains

    SciTech Connect

    O'Neill, Hugh Michael; Davern, Sandra M.; Murphy, Charles L.; Wall, Jonathan; Deborah, Weiss T.; Solomon, Alan

    2011-01-01

    AL amyloidosis is characterized by the pathologic deposition as fibrils of monoclonal light chains (i.e., Bence Jones proteins [BJPs]) in particular organs and tissues. This phenomenon has been attributed to the presence in amyloidogenic proteins of particular amino acids that cause these molecules to become unstable, as well as post-translational modifications and, in regard to the latter, we have investigated the effect of biotinylation of lysyl residues on cell binding. We utilized an experimental system designed to test if BJPs obtained from patients with AL amyloidosis or, as a control, multiple myeloma (MM), bound human fibroblasts and renal epithelial cells. As documented by fluorescent microscopy and ELISA, the amyloidogenic BJPs, as compared with MM components, bound preferentially and this reactivity increased significantly after chemical modification of their lysyl residues with sulfo-NHS-biotin. Further, based on tryptophan fluorescence and circular dichorism data, it was apparent that their conformation was altered, which we hypothesize exposed a binding site not accessible on the native protein. The results of our studies indicate that post-translational structural modifications of pathologic light chains can enhance their capacity for cellular interaction and thus may contribute to the pathogenesis of AL amyloidosis and multiple myeloma.

  9. Effects of stress and. beta. -funal trexamine pretreatment on morphine analgesia and opioid binding in rats

    SciTech Connect

    Adams, J.U.; Andrews, J.S.; Hiller, J.M.; Simon, E.J.; Holtzman, S.G.

    1987-12-28

    This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opiods which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 yr or unstressed were injected ICV with either saline or 2.5 ..mu..g of ..beta..-funaltrexamine (..beta..-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia or were sacrificed and opioid binding in brain was determined. (/sup 3/H)D-Ala/sup 2/NMePhe/sup 4/-Gly/sup 5/(ol)enkephalin (DAGO) served as a specific ligand for mu-opioid receptors, and (/sup 3/H)-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. ..beta..-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with ..beta..-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received ..beta..-FNA while unstressed. ..beta..-FNA caused small and similar decreases in (/sup 3/H)-DAGO binding in brain of both stressed and unstressed animals. 35 references, 2 figures, 2 tables.

  10. Engineering an effective Mn-binding MRI reporter protein by subcellular targeting

    PubMed Central

    Bartelle, Benjamin B.; Mana, Miyeko D.; Suero-Abreu, Giselle A.; Rodriguez, Joe J.; Turnbull, Daniel H.

    2014-01-01

    Purpose Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely utilized for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as an genetic reporter for MEMRI. Methods The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry and MR imaging and relaxometry. Results Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co-expression with the divalent metal transporter DMT1, a previously described Mn-based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone. Conclusion This second-generation reporter system both expands the capabilities of genetically-encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast. PMID:25522343

  11. Multiple ligand simultaneous docking (MLSD): A novel approach to study the effect of inhibitors on substrate binding to PPO.

    PubMed

    Raghavendra, S; Aditya Rao, S J; Kumar, Vadlapudi; Ramesh, C K

    2015-12-01

    Multiple ligand simultaneous docking, a computational approach is used to study the concurrent interactions between substrate and the macromolecule binding together in the presence of an inhibitor. The present investigation deals with the study of the effect of different inhibitors on binding of substrate to the protein Polyphenoloxidase (PPO). The protein was isolated from Mucuna pruriens and confirmed as tyrosinases involved in L-DOPA production. The activity was measured using different inhibitors at different concentrations taking catechol as substrate. A high-throughput binding study was conducted to compare the binding orientations of individual ligands and multiple ligands employing Autodock 4.2. The results of single substrate docking showed a better binding of urea with the binding energy of -3.48 kJ mol(-1) and inter molecular energy of -3.48 kJ mol(-1) while the results of MLSD revealed that ascorbic acid combined with the substrate showed better inhibition with a decreased binding energy of -2.37 kJ mol(-1).

  12. OmpA Binding Mediates the Effect of Antimicrobial Peptide LL-37 on Acinetobacter baumannii

    PubMed Central

    Lin, Ming-Feng; Tsai, Pei-Wen; Chen, Jeng-Yi; Lin, Yun-You; Lan, Chung-Yu

    2015-01-01

    Multidrug-resistant Acinetobacter baumannii has recently emerged as an important pathogen in nosocomial infection; thus, effective antimicrobial regimens are urgently needed. Human antimicrobial peptides (AMPs) exhibit multiple functions and antimicrobial activities against bacteria and fungi and are proposed to be potential adjuvant therapeutic agents. This study examined the effect of the human cathelicidin-derived AMP LL-37 on A. baumannii and revealed the underlying mode of action. We found that LL-37 killed A. baumannii efficiently and reduced cell motility and adhesion. The bacteria-killing effect of LL-37 on A. baumannii was more efficient compared to other AMPs, including human ß–defensin 3 (hBD3) and histatin 5 (Hst5). Both flow cytometric analysis and immunofluorescence staining showed that LL-37 bound to A. baumannii cells. Moreover, far-western analysis demonstrated that LL-37 could bind to the A. baumannii OmpA (AbOmpA) protein. An ELISA assay indicated that biotin-labelled LL-37 (BA-LL37) bound to the AbOmpA74-84 peptide in a dose-dependent manner. Using BA-LL37 as a probe, the ~38 kDa OmpA signal was detected in the wild type but the ompA deletion strain did not show the protein, thereby validating the interaction. Finally, we found that the ompA deletion mutant was more sensitive to LL-37 and decreased cell adhesion by 32% compared to the wild type. However, ompA deletion mutant showed a greatly reduced adhesion defect after LL-37 treatment compared to the wild strain. Taken together, this study provides evidence that LL-37 affects A. baumannii through OmpA binding. PMID:26484669

  13. Morphine-6-glucuronide: analgesic effects and receptor binding profile in rats

    SciTech Connect

    Abbott, F.V.; Palmour, R.M.

    1988-01-01

    The antinociceptive effects of morphine-6-glucuronide (M6G) were examined in two animal models of pain, the tail immersion test (reflex withdrawal to noxious heat) and the formalin test (behavioral response to minor tissue injury). In the tail immersion test, M6G produced and increase in withdrawal latency that rose rapidly between 0.01 and 0.025 ug ICV or 1 and 2 mg/kg SC. A further increase occurred at doses greater than 0.2 ug ICV or 4 mg/kg SC and was associated with marked catelepsy and cyanosis. Naloxone, 0.1 mg/kg SC, shifted the lower component of the dose-effect relation by a factor of 24. In the formalin test, 0.01 ug M6G ICV produced hyperalgesia, while between 0.05 and 0.2 ug ICV, antinociception increased rapidly without toxicity. The dose effect relations for hyperalgesia and antinociception were shifted to the right by factors of 20- and 3-fold, respectively. By comparison, ICV morphine was 60 (formalin test) to 145-200 (tail immersion test) times less potent than M6G. At sub-nanomolar concentrations, M6G enhanced the binding of (/sup 3/H)-etorphine, (/sup 3/H)-dihydromorphine and (/sup 3/H)-naloxone to rat brain membrane receptors by 20-40%. At higher concentrations, M6G displaced each ligand from binding sites, with K/sub i/ values of about 30 nM, as compared to morphine K/sub i/ values of about 3 nM.

  14. The forward rate of binding of surface-tethered reactants: effect of relative motion between two surfaces.

    PubMed Central

    Chang, K C; Hammer, D A

    1999-01-01

    The reaction of molecules confined to two dimensions is of interest in cell adhesion, specifically for the reaction between cell surface receptors and substrate-bound ligand. We have developed a model to describe the overall rate of reaction of species that are bound to surfaces under relative motion, such that the Peclet number is order one or greater. The encounter rate between reactive species is calculated from solution of the two-dimensional convection-diffusion equation. The probability that each encounter will lead to binding depends on the intrinsic rate of reaction and the encounter duration. The encounter duration is obtained from the theory of first passage times. We find that the binding rate increases with relative velocity between the two surfaces, then reaches a plateau. This plateau indicates that the increase in the encounter rate is counterbalanced by the decrease in the encounter duration as the relative velocity increases. The binding rate is fully described by two dimensionless parameters, the Peclet number and the Damköhler number. We use this model to explain data from the cell adhesion literature by incorporating these rate laws into "adhesive dynamics" simulations to model the binding of a cell to a surface under flow. Leukocytes are known to display a "shear threshold effect" when binding selectin-coated surfaces under shear flow, defined as an increase in bind rate with shear; this effect, as calculated here, is due to an increase in collisions between receptor and ligand with increasing shear. The model can be used to explain other published data on the effect of wall shear rate on the binding of cells to surfaces, specifically the mild decrease in binding within a fixed area with increasing shear rate. PMID:10049312

  15. Effects of binding materials on microaggregate size distribution in bauxite residues.

    PubMed

    Zhu, Feng; Huang, Nan; Xue, Shengguo; Hartley, William; Li, Yiwei; Zou, Qi

    2016-12-01

    It is recognized that for successful establishment of a vegetation cover on bauxite residue disposal areas, soil formation and a greater understanding of the processes of soil development are crucial. The stability of microaggregates is a very important physical property that prevents erosion in bauxite residues. Samples were collected from a disposal area in Central China to determine not only the mechanism of aggregation but also clay dispersion. Colloidal stability was assessed by determining organic matter, carbonate, electrolyte, clay mineral, and iron-aluminum oxide forms, as these would contribute to their stability. Organic matter improved microaggregate stability by combining with clay particles and polyvalent cations to form macroaggregates. Polyvalent cations such as calcium had a positive effect on particle flocculation, while organic molecules were more effective at stabilizing microaggregates. Removal of salinity dispersed silt-size aggregates into clay-size aggregates and reduced microaggregate stability. Calcium improved particle aggregation, while sodium had the reverse effect. Quartz powder was added to the residues but did not show any cementing effect, while free and amorphous iron-aluminum oxides were effective binding agents for microaggregate formation. We propose that the presence of organic matter and polyvalent cations, together with incorporation of organic carbon and calcium minerals, may enhance the stability of this material and prove beneficial toward improving its physical condition.

  16. Ion exchange chromatography of monoclonal antibodies: effect of resin ligand density on dynamic binding capacity.

    PubMed

    Hardin, Ann Marie; Harinarayan, Chithkala; Malmquist, Gunnar; Axén, Andreas; van Reis, Robert

    2009-05-15

    Dynamic binding capacity (DBC) of a monoclonal antibody on agarose based strong cation exchange resins is determined as a function of resin ligand density, apparent pore size of the base matrix, and protein charge. The maximum DBC is found to be unaffected by resin ligand density, apparent pore size, or protein charge within the tested range. The critical conductivity (conductivity at maximum DBC) is seen to vary with ligand density. It is hypothesized that the maximum DBC is determined by the effective size of the proteins and the proximity to which they can approach one another. Once a certain minimum resin ligand density is supplied, additional ligand is not beneficial in terms of resin capacity. Additional ligand can provide flexibility in designing ion exchange resins for a particular application as the critical conductivity could be matched to the feedstock conductivity and it may also affect the selectivity.

  17. 3D calculation of Tucson-Melbourne 3NF effect in triton binding energy

    SciTech Connect

    Hadizadeh, M. R.; Tomio, L.; Bayegan, S.

    2010-08-04

    As an application of the new realistic three-dimensional (3D) formalism reported recently for three-nucleon (3N) bound states, an attempt is made to study the effect of three-nucleon forces (3NFs) in triton binding energy in a non partial wave (PW) approach. The spin-isospin dependent 3N Faddeev integral equations with the inclusion of 3NFs, which are formulated as function of vector Jacobi momenta, specifically the magnitudes of the momenta and the angle between them, are solved with Bonn-B and Tucson-Melbourne NN and 3N forces in operator forms which can be incorporated in our 3D formalism. The comparison with numerical results in both, novel 3D and standard PW schemes, shows that non PW calculations avoid the very involved angular momentum algebra occurring for the permutations and transformations and it is more efficient and less cumbersome for considering the 3NF.

  18. Distinct effects of Broncho-Vaxom (OM-85 BV) on gp130 binding cytokines

    PubMed Central

    Roth, M; Block, L

    2000-01-01

    BACKGROUND—Broncho-Vaxom (OM-85 BV) is known to support respiratory tract resistance to bacterial infections. In vivo and in vitro studies in animals and humans have shown that the action of the drug is based on the modulation of the host immune response, and it has been found to upregulate interferon γ (IFN-γ) and interleukin (IL)-2, IL-6, and IL-8. These immunomodulatory effects of the compound may explain its stimulation on T helper cells and natural killer cells. Following earlier findings that OM-85 BV induces the synthesis of IL-6, a study was undertaken to investigate its possible effect on other gp130 binding cytokines including IL-11, IL-12, leukaemia inhibitory factor (LIF), oncostatin M (OSM), and ciliary neutrophil factor (CNTF). Its modulation of the corresponding receptors of the above mentioned cytokines and of the signal transducer gp130 in human pulmonary fibroblasts and peripheral blood lymphocytes was also studied.
METHODS—Transcription of cytokines was assessed by Northern blot analysis. Secretion of cytokines was analysed using commercially available enzyme linked immunosorbent assay kits. Cytokine receptors and gp130 proteins were determined by Western blot analysis.
RESULTS—OM-85 BV increased the expression of IL-11 in human lung fibroblasts, but not in lymphocytes, in a dose and time dependent manner by maximal fivefold within 20 hours. The compound inhibited serum induced IL-12 expression in peripheral blood lymphocytes but did not induce OSM, LIF, or CNTF at any concentration. In lung fibroblasts the expression of the IL-6 receptor was enhanced fourfold at a concentration of 10 µg/ml OM-85 BV while that of the IL-11 receptor was not altered. In peripheral blood lymphocytes LIF receptor α expression was downregulated in the presence of 10 µg/ml OM-85 BV. At a concentration of 10 µg/ml OM-85 BV enhanced gp130 gene transcription fivefold and increased gp130 protein accumulation in cell membranes by 2.5times

  19. The effective opening of nicotinic acetylcholine receptors with single agonist binding sites

    PubMed Central

    Williams, Dustin K.; Stokes, Clare; Horenstein, Nicole A.

    2011-01-01

    We have identified a means by which agonist-evoked responses of nicotinic receptors can be conditionally eliminated. Modification of α7L119C mutants by the sulfhydryl reagent 2-aminoethyl methanethiosulfonate (MTSEA) reduces responses to acetylcholine (ACh) by more than 97%, whereas corresponding mutations in muscle-type receptors produce effects that depend on the specific subunits mutated and ACh concentration. We coexpressed α7L119C subunits with pseudo wild-type α7C116S subunits, as well as ACh-insensitive α7Y188F subunits with wild-type α7 subunits in Xenopus laevis oocytes using varying ratios of cRNA. When mutant α7 cRNA was coinjected at a 5:1 ratio with wild-type cRNA, net charge responses to 300 µM ACh were retained by α7L119C-containing mutants after MTSEA modification and by the ACh-insensitive Y188F-containing mutants, even though the expected number of ACh-sensitive wild-type binding sites would on average be fewer than two per receptor. Responses of muscle-type receptors with one MTSEA-sensitive subunit were reduced at low ACh concentrations, but much less of an effect was observed when ACh concentrations were high (1 mM), indicating that saturation of a single binding site with agonist can evoke strong activation of nicotinic ACh receptors. Single-channel patch clamp analysis revealed that the burst durations of fetal wild-type and α1β1γδL121C receptors were equivalent until the α1β1γδL121C mutants were exposed to MTSEA, after which the majority (81%) of bursts were brief (≤2 ms). The longest duration events of the receptors modified at only one binding site were similar to the long bursts of native receptors traditionally associated with the activation of receptors with two sites containing bound agonists. PMID:21444659

  20. Effects of a variety of food extracts and juices on the specific binding ability of norovirus GII.4 P particles.

    PubMed

    Li, Dan; Baert, Leen; Xia, Ming; Zhong, Weiming; Jiang, Xi; Uyttendaele, Mieke

    2012-07-01

    The effects of 13 food extracts and juices, including shellfish, fruits, and vegetables, on the binding ability of human norovirus (NoV) were examined, using P particles of human NoV GII.4 as a research surrogate. The enhancements (positive values) or reductions (negative values) of NoV P particle detection (changes in optical density at 450 nm) in the presence of different food extracts and juices as compared with P particles diluted in phosphate-buffered saline were tested by saliva-binding, enzyme-linked immunosorbent assay in triplicate. In the presence of different food extracts and juices at different concentrations, an increase or decrease of the receptor-binding ability of the NoV P particles was observed. Due to a higher specific binding and thus a higher accumulation of the viral particles, oysters may be contaminated with human NoV more often than other shellfish species (mussel, hard clams, and razor clams). Cranberry and pomegranate juices were shown to reduce the specific binding ability of human NoV P particles. No such binding inhibition effects were observed for the other tested extracts of fresh produce (strawberry, blackberry, blueberry, cherry tomato, spinach, romaine lettuce) or, notably, for raspberry, which has been associated with human NoV outbreaks.

  1. Human guanylate binding proteins potentiate the anti-chlamydia effects of interferon-gamma.

    PubMed

    Tietzel, Illya; El-Haibi, Christelle; Carabeo, Rey A

    2009-08-04

    Chlamydiae are obligate intracellular pathogens that are sensitive to pro-inflammatory cytokine interferon-gamma. IFN-gamma-inducible murine p47 GTPases have been demonstrated to function in resistance to chlamydia infection in vivo and in vitro. Because the human genome does not encode IFN-gamma-inducible homologues of these proteins, the significance of the p47 GTPase findings to chlamydia pathogenesis in humans is unclear. Here we report a pair of IFN-gamma-inducible proteins, the human guanylate binding proteins (hGBPs) 1 and 2 that potentiate the anti-chlamydial properties of IFN-gamma. hGBP1 and 2 localize to the inclusion membrane, and their anti-chlamydial functions required the GTPase domain. Alone, hGBP1 or 2 have mild, but statistically significant and reproducible negative effects on the growth of Chlamydia trachomatis, whilst having potent anti-chlamydial activity in conjunction with treatment with a sub-inhibitory concentration of IFN-gamma. Thus, hGBPs appear to potentiate the anti-chlamydial effects of IFN-gamma. Indeed, depletion of hGBP1 and 2 in cells treated with IFN-gamma led to an increase in inclusion size, indicative of better growth. Interestingly, chlamydia species/strains harboring the full-length version of the putative cytotoxin gene, which has been suggested to confer resistance to IFN-gamma was not affected by hGBP overexpression. These findings identify the guanylate binding proteins as potentiators of IFN-gamma inhibition of C. trachomatis growth, and may be the targets of the chlamydial cytotoxin.

  2. Effects of active site cleft residues on oligosaccharide binding, hydrolysis, and glycosynthase activities of rice BGlu1 and its mutants.

    PubMed

    Pengthaisong, Salila; Ketudat Cairns, James R

    2014-12-01

    Rice BGlu1 (Os3BGlu7) is a glycoside hydrolase family 1 β-glucosidase that hydrolyzes cellooligosaccharides with increasing efficiency as the degree of polymerization (DP) increases from 2 to 6, indicating six subsites for glucosyl residue binding. Five subsites have been identified in X-ray crystal structures of cellooligosaccharide complexes with its E176Q acid-base and E386G nucleophile mutants. X-ray crystal structures indicate that cellotetraose binds in a similar mode in BGlu1 E176Q and E386G, but in a different mode in the BGlu1 E386G/Y341A variant, in which glucosyl residue 4 (Glc4) interacts with Q187 instead of the eliminated phenolic group of Y341. Here, we found that the Q187A mutation has little effect on BGlu1 cellooligosaccharide hydrolysis activity or oligosaccharide binding in BGlu1 E176Q, and only slight effects on BGlu1 E386G glycosynthase activity. X-ray crystal structures showed that cellotetraose binds in a different position in BGlu1 E176Q/Y341A, in which it interacts directly with R178 and W337, and the Q187A mutation had little effect on cellotetraose binding. Mutations of R178 and W337 to A had significant and nonadditive effects on oligosaccharide hydrolysis by BGlu1, pNPGlc cleavage and cellooligosaccharide inhibition of BGlu1 E176Q and BGlu1 E386G glycosynthase activity. Hydrolysis activity was partially rescued by Y341 for longer substrates, suggesting stacking of Glc4 on Y341 stabilizes binding of cellooligosaccharides in the optimal position for hydrolysis. This analysis indicates that complex interactions between active site cleft residues modulate substrate binding and hydrolysis.

  3. Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4.

    PubMed

    Nookala, Anantha R; Li, Junhao; Ande, Anusha; Wang, Lei; Vaidya, Naveen K; Li, Weihua; Kumar, Santosh; Kumar, Anil

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir

  4. Overexpression of Drosophila juvenile hormone esterase binding protein results in anti-JH effects and reduced pheromone abundance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The titer of juvenile hormone (JH), which has wide ranging physiological effects in insects, is regulated in part by JH esterase (JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent anti-JH effects. We hypothesize that DmP29 ...

  5. Cationic Gold Clusters Ligated with Differently Substituted Phosphines: Effect of Substitution on Ligand Reactivity and Binding

    SciTech Connect

    Johnson, Grant E.; Olivares, Astrid M.; Hill, David E.; Laskin, Julia

    2015-01-01

    We present a systematic study of the effect of the number of methyl (Me) and cyclohexyl (Cy) functional groups in monodentate phosphine ligands on the solution-phase synthesis of ligated sub-nanometer gold clusters and their gas-phase fragmentation pathways. Small mixed ligand cationic gold clusters were synthesized using ligand exchange reactions between pre-formed triphenylphosphine ligated (PPh3) gold clusters and monodentate Me- and Cy-substituted ligands in solution and characterized using electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation (CID) experiments. Under the same experimental conditions, larger gold-PPh3 clusters undergo efficient exchange of unsubstituted PPh3 ligands for singly Me- and Cy-substituted PPh2Me and PPh2Cy ligands. The efficiency of ligand exchange decreases with an increasing number of Me or Cy groups in the substituted phosphine ligands. CID experiments performed for a series of ligand-exchanged gold clusters indicate that loss of a neutral Me-substituted ligand is preferred over loss of a neutral PPh¬3 ligand while the opposite trend is observed for Cy-substituted ligands. The branching ratio of the competing ligand loss channels is strongly correlated with the electron donating ability of the phosphorous lone pair as determined by the relative proton affinity of the ligand. The results indicate that the relative ligand binding energies increase in the order PMe3 < PPhMe2 < PPh2Me < PPh3< PPh2Cy < PPhCy2< PCy3. Furthermore, the difference in relative ligand binding energies increases with the number of substituted PPh3-mMem or PPh3-mCym ligands (L) exchanged onto each cluster. This study provides the first experimental determination of the relative binding energies of ligated gold clusters containing differently substituted monophosphine ligands, which are important to controlling their synthesis and reactivity in solution. The results also indicate that ligand substitution is an important

  6. Determination of binding constant of transcription factor myc-max/max-max and E-box DNA: the effect of inhibitors on the binding.

    PubMed

    Park, Seyeon; Chung, Sunah; Kim, Kyung-Mee; Jung, Kyung-Chae; Park, Chihoon; Hahm, Eun-Ryeong; Yang, Chul-Hak

    2004-02-24

    The truncated myc and max proteins, only containing basic regions and helix-loop-helix/zipper (b/HLH/Zip) regions were over-expressed in E. coli and used for the determination of the binding constant and of the inhibitory mechanism on myc-max (or max-max)-DNA complex formation. The association kinetic constants (k(1) and k(-1)) of truncated max-max or myc-max dimer and DNA were determined as k(1)=(1.7+/-0.6)x10(5) M(-1) s(-1), k(-1)=(3.4+/-1.2)x10(-2) s(-1) for max-max and DNA or k(1)=(2.1+/-0.7)x10(5) M(-1) s(-1), k(-1)=(3.2+/-1.4)x10(-2) s(-1) for myc-max and DNA. The equilibrium binding constant (K(1)) was determined using these kinetic parameters [K(XXD)=(7.8+/-2.6)x10(6) M(-1) for max-max and DNA or K(XYD)=(6.9+/-2.2)x10(6) M(-1) for myc-max and DNA]. The binding constants of myc-max or max-max dimer formation were K(XX)=(2.6+/-0.9)x10(5) M(-1) or K(XY)=(1.3+/-0.4)x10(4) M(-1), respectively. When truncated proteins were used, the max-max dimer formation was easier than the myc-max dimer formation, contrary to the physiologically determined case. This leads us to deduce that domains other than b/HLH/Zip are very important for the transcriptional regulatory activity in physiological conditions. The truncated myc and max proteins, which were expressed in E. coli and contained only b/HLH/Zip regions were also used for the screening of inhibitors of myc-max-DNA complex formation. A synthesized curcuminoid, 1,7-bis(4-methyl-3-nitrophenyl)-1,6-heptadiene-3,5-dione (curcuminoid 004), showed the most potent inhibition out of the synthesized curcuminoids, in competition with DNA. The dissociation constant of max-max dimer and the inhibitor was 9 microM, when investigated using in vitro expressed b/HLH/Zip dimer proteins. The curcuminoid 004 showed an inhibitory effect on the binding of myc-max protein to the E-box element in SNU16 cells, and suppressed the expression of myc target genes including ornithine decarboxylase (ODC), cdc25a and c-myc in myc over

  7. Effective tight-binding model for MX2 under electric and magnetic fields

    DOE PAGES

    Shanavas, Kavungal Veedu; Satpathy, S.

    2015-06-15

    We present a systematic method for developing a five band Hamiltonian for the metal d orbitals that can be used to study the effect of electric and magnetic fields on multilayer MX2 (M=Mo,W and X=S,Se) systems. On a hexagonal lattice of d orbitals, the broken inversion symmetry of the monolayers is incorporated via fictitious s orbitals at the chalcogenide sites. A tight-binding Hamiltonian is constructed and then downfolded to get effective d orbital overlap parameters using quasidegenerate perturbation theory. The steps to incorporate the effects of multiple layers, external electric and magnetic fields are also detailed. We find that anmore » electric field produces a linear-k Rashba splitting around the Γ point, while a magnetic field removes the valley pseudospin degeneracy at the ±K points. Lastly, our model provides a simple tool to understand the recent experiments on electric and magnetic control of valley pseudospin in monolayer dichalcogendies.« less

  8. In vitro effect of temperature on the conformational structure and collagen binding of SdrF, a Staphylococcus epidermidis adhesin.

    PubMed

    Di Poto, Antonella; Papi, Massimiliano; Trivedi, Sheetal; Maiorana, Alessandro; Gavazzo, Paola; Vassalli, Massimo; Lowy, Franklin D; De Spirito, Marco; Montanaro, Lucio; Imbriani, Marcello; Arciola, Carla Renata; Visai, Livia

    2015-07-01

    Staphylococcus epidermidis is the leading etiologic agent of device-related infections. S. epidermidis is able to bind, by means of the adhesins of its cell wall, the host matrix proteins filming the artificial surfaces. Thence, bacteria cling to biomaterials and infection develops. The effect of temperature on integrity, structure, and biological activity of the collagen-binding adhesin (SdrF) of S. epidermidis has been here investigated. By cloning in E. coli XL1-Blue, a recombinant of the SdrF binding domain B (rSdrFB), carrying an N-terminal polyhistidine, was obtained. Purification was by HiTrap(TM) Chelating HP columns. Assessment of purity, molecular weight, and integrity was by SDS-PAGE. The rSdrFB-collagen binding was investigated by ELISA. A full three-dimensional reconstruction of rSdrFB was achieved by small-angle X-ray scattering (SAXS). At 25 °C, rSdrFB bound to type I collagen in a dose-dependent, saturable manner, with a Kd of 2.48 × 10(-7) M. When temperature increased from 25 to 37 °C, a strong conformational change occurred, together with the abolition of the rSdrFB-collagen binding. The rSdrFB integrity was not affected by temperature variation. SdrFB-collagen binding is switched on/off depending on the temperature. Implications with the infection pathogenesis are enlightened.

  9. Highly effective recognition of carbohydrates by phenanthroline-based receptors: alpha- versus beta-anomer binding preference.

    PubMed

    Mazik, Monika; Hartmann, Andrè; Jones, Peter G

    2009-09-14

    (1)H NMR spectroscopic titrations in competitive and non-competitive media, as well as binding studies in two-phase systems, such as phase transfer of sugars from aqueous into organic solvents and dissolution of solid carbohydrates in apolar media revealed both highly effective recognition of neutral carbohydrates and interesting binding preferences of an acyclic phenanthroline-based receptor 1. Compared to the previously described acyclic receptors, compound 1 displays significantly higher binding affinities, the rare capability to extract sugars from water into non-polar organic solutions and alpha- versus beta-anomer binding preference in the recognition of glycosides, which differs from those observed for other receptor systems. X-ray crystallographic investigations revealed the presence of water molecules in the binding pocket of 1 that are engaged in the formation of hydrogen-bonding motifs similar to those suggested by molecular modelling for the sugar OH groups in the receptor-sugar complexes. The molecular modelling calculations, synthesis, crystal structure and binding properties of 1 are described and compared with those of the previously described receptors.

  10. The effect of polyamines on the binding of anti-DNA antibodies for patients with SLE and normal human subjects

    PubMed Central

    Wang, Xiao; Stearns, Nancy A.; Li, Xingfu; Pisetsky, David S.

    2014-01-01

    Antibodies to DNA (anti-DNA) are the serological hallmark of systemic lupus erythematosus (SLE). To elucidate specificity further, the effect of polyamines on the binding of anti-DNA antibodies from patients with lupus was tested by ELISA to calf thymus (CT) DNA; we also assessed the binding of plasmas of patients and normal human subjects (NHS) to Micrococcus luteus (MC) DNA. As these studies showed, spermine can dose-dependently inhibit SLE anti-DNA binding to CT DNA and can promote dissociation of preformed immune complexes. With MC DNA as antigen, spermine failed to inhibit the NHS anti-DNA binding. Studies using plasmas adsorbed to a CT DNA cellulose affinity indicated that SLE plasmas are mixtures of anti-DNA that differ in inhibition by spermine and binding to conserved and non-conserved determinants. Together, these studies demonstrate that spermine can influence the binding of anti-DNA autoantibodies and may contribute to the antigenicity of DNA. PMID:24732074

  11. Studies of the effect of maltose on the direct binding of porcine pancreatic α-amylase to maize starch.

    PubMed

    Warren, Frederick J; Butterworth, Peter J; Ellis, Peter R

    2012-09-01

    For a two phase system comprising an enzyme in solution acting on an insoluble substrate such as starch, adsorption of the enzyme is a key initial step in the reaction but few studies of agents affecting direct binding have been performed. The effect of maltose on the interaction of maize starch with porcine pancreatic α-amylase was studied by using a method in which the direct binding of starch to amylase is measured under conditions of negligible catalytic activity. The dissociation constant for starch binding increased with maltose concentration and analysis of the binding showed that the kinetic action of maltose was entirely competitive. This result accords with results described in the literature in which maltose was shown to be a competitive inhibitor of amylase action. If the maltose concentration is sufficiently high, a second molecule may bind at the active site but the affinity of the second binding step is approximately 6.5-fold weaker. Because of the relatively low affinity for maltose, it seems unlikely that inhibition by maltose of the initial stage of starch-amylase interaction normally plays any role in regulating intestinal digestion of starch.

  12. Differential effect of detergents on (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195 binding to peripheral-type benzodiazepine-binding sites

    SciTech Connect

    Awad, M.; Gavish, M.

    1988-01-01

    The present study demonstrates a differential effect of various detergent treatments on (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195 binding to peripheral benzodiazepine binding sites (PBS). Triton X-100 caused a decrease of about 70% in (/sup 3/H)Ro 5-4864 binding to membranes from various peripheral tissues of rat, but had only a negligible effect on (/sup 3/H)PK 11195 binding. A similar effect of Triton X-100 was observed on guinea pig and rabbit kidney membranes. The decrease in (/sup 3/H)Ro 5-4864 binding after treatment with Triton X-100 was apparently due to a decrease in the density of PBS, since the affinity remained unaltered. The detergents 3-((3-cholamidopropyl)-dimethylammonio)-1-propane sulfonate (CHAPS), Tween 20, deoxycholic acid, or digitonin (0.0125%) caused only a minor change in (/sup 3/H)Ro 5-4864 and (/sup 3/H)PK 11195 binding to rat kidney membranes; but when concentrations were substantially increased (0.1%), all detergents caused a decrease of at least 50% in (/sup 3/H)Ro 5-4864 binding, while (/sup 3/H)PK 11195 binding to rat kidney membranes remained unaffected by the first three detergents, with only a minor decrease (15%) after treatment with digitonin.

  13. pH-dependent effects of sodium tungstate on the steroid-binding properties of the hen oviduct progesterone receptor.

    PubMed

    Murakami, N; Quattrociocchi, T M; Szocik, J F; Moudgil, V K

    1982-11-24

    Effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor were examined and were found to be pH-dependent. When freshly prepared hen oviduct cytosol containing progesterone receptor was heated at 37 degrees C for 20 min, its ability to bind [3H]progesterone decreased to 20% level of unheated samples. At pH 7, presence of 2-3 mM tungstate during the above incubation period reduced this loss of binding. At higher tungstate concentrations (greater than 5 mM), this stabilizing effect was gradually abolished. Similar results were obtained with preparations that contained [3H]progesterone-receptor complexes; 70-80% of which remained after a 20 min incubation at 37 degrees C in the presence of 2-3 mM tungstate at pH 7. At pH 8, presence of tungstate (1-10 mM) during the 37 degrees C incubation stabilized both the steroid-bound and the unoccupied progesterone receptor in a concentration-dependent manner. The extent of steroid binding by the receptor at 4 degrees C remained unchanged in the presence of up to 10 mM tungstate at both pH 7 and pH 8 assay conditions while presence of 20 mM tungstate lowered this binding capacity. These results indicate that tungstate effects may be mediated via its interaction with the progesterone receptor.

  14. Effect of cysteamine on cytosolic somatostatin binding sites in rabbit duodenal mucosa

    SciTech Connect

    Gonzalez-Guijarro, L.; Lopez-Ruiz, M.P.; Bodegas, G.; Prieto, J.C.; Arilla, E.

    1987-04-01

    Administration of cysteamine in rabbits elicited a rapid depletion of both duodenal mucosa and plasma somatostatin. A significant reduction was observed within 5 min, returning toward control values by 150 min. The depletion of somatostatin was associated with an increase in the binding capacity and a decrease in the affinity of both high- and low-affinity binding sites present in cytosol of duodenal mucosa. Incubation of cytosolic fraction from control rabbits with 1 mM cysteamine did not modify somatostatin binding. Furthermore, addition of cysteamine at the time of binding assay did not affect the integrity of /sup 125/I-Tyr11-somatostatin. It is concluded that in vivo administration of cysteamine to rabbits depletes both duodenal mucosa and plasma somatostatin and leads to up-regulation of duodenal somatostatin binding sites.

  15. Highly effective binding of viologens by p-sulfonatocalixarenes for the treatment of viologen poisoning.

    PubMed

    Wang, Kui; Guo, Dong-Sheng; Zhang, Hong-Qing; Li, Dong; Zheng, Xi-Long; Liu, Yu

    2009-10-22

    Viologens are showing an increasing number of scientific and technical applications in addition to their use as herbicides. However, their high toxicity poses considerable risks to human health, society, and the environment. In this context, we propose a new therapeutic protocol for the treatment of viologen poisoning. The mechanism of this new protocol is based on host-guest chemistry and involves the effective inhibition of viologen toxicity by the complexation of p-sulfonatocalix[n]arenes. NMR, ITC, and X-ray crystallography studies indicated that p-sulfonatocalix[n]arenes could form highly stable complexes with viologens. Electrochemical results showed that the highly effective binding could induce the reduction potentials of viologens to shift to more negative values. Further studies in mice showed that the ingestion of p-sulfonatocalix[n]arenes significantly decreased the mortality rate of viologen-poisoned mice with lung and liver protection. As a result, p-sulfonatocalix[n]arenes may have potential application in the clinical treatment of viologen poisoning.

  16. Memory Effects of Benzodiazepines: Memory Stages and Types Versus Binding-Site Subtypes

    PubMed Central

    Savić, Miroslav M.; Obradović, Dragan I.; Ugrešić, Nenad D.; Bokonjić, Dubravko R.

    2005-01-01

    Benzodiazepines are well established as inhibitory modulators of memory processing. This effect is especially prominent when applied before the acquisition phase of a memory task. This minireview concentrates on the putative subtype selectivity of the acquisition-impairing action of benzodiazepines. Namely, recent genetic studies and standard behavioral tests employing subtype-selective ligands pointed to the predominant involvement of two subtypes of benzodiazepine binding sites in memory modulation. Explicit memory learning seems to be affected through the GABAA receptors containing the α1 and α1 subunits, whereas the effects on procedural memory can be mainly mediated by the α1 subunit. The pervading involvement of the α1 subunit in memory modulation is not at all unexpected because this subunit is the major subtype, present in 60% of all GABAA receptors. On the other hand, the role of α5 subunits, mainly expressed in the hippocampus, in modulating distinct forms of memory gives promise of selective pharmacological coping with certain memory deficit states. PMID:16444900

  17. The cognitive representation of intending not to act: Evidence for specific non-action-effect binding.

    PubMed

    Kühn, Simone; Brass, Marcel

    2010-10-01

    The question how we represent voluntary action on a cognitive level has recently become of increasing interest to researchers studying motor control. However, so far it has been neglected how we represent the voluntary omission of an action. In our attempt to investigate the representation of voluntary non-actions we demonstrated binding effects between voluntary non-actions and subsequent action effects (Kühn, Elsner, Prinz, & Brass, 2009). That study, however, only distinguished between acting or not acting, and did not address the question of whether non-actions are coded as general omissions or whether they can be encoded specifically as the actual negation of the action in question ("not-right" or "not-left"). Our current study provides first evidence for the specificity of representations of intentional non-actions. Additionally, we compare two ways in which the specific non-actions might be represented: an ironic representation account implying that negations are prone to be omitted and a reformulated representation account assuming that the negated action is suppressed and/or the alternative action is facilitated. Our results suggest that the representation of non-actions contains a facilitation of the alternative action rather than a suppression of the action in question.

  18. Cooperative Effects of Rigor and Cycling Cross-Bridges on Calcium Binding to Troponin C

    PubMed Central

    Cantino, Marie E.; Quintanilla, Abraham

    2007-01-01

    The effects of rigor and cycling cross-bridges on distributions of calcium (Ca) bound within sarcomeres of rabbit psoas muscle fibers were compared using electron probe x-ray microanalysis. Calcium in the overlap region of rigor fibers, after correction for that bound to thick filaments, was significantly higher than in the I-band at all pCa levels tested between 6.9 and 4.8, but the difference was greatest at pCa 6.9. With addition of MgATP, differences were significant at high levels of activation (pCa 5.6 and 4.9); near and below the threshold for activation, Ca was the same in I-band and overlap regions. Comparison of Ca and mass profiles at the A-I junction showed elevation of Ca extending 55–110 nm (up to three regulatory units) into the I-band. Extraction of TnC-reduced I-band and overlap Ca in rigor fibers at pCa 5.6 to the same levels found in unextracted fibers at pCa 8.9, suggesting that variations reported here reflect changes in Ca bound to troponin C (TnC). Taken together, these observations provide evidence for near-neighbor cooperative effects of both rigor and cycling cross-bridges on Ca2+ binding to TnC. PMID:17056730

  19. Effect of the Protein Corona on Antibody-Antigen Binding in Nanoparticle Sandwich Immunoassays.

    PubMed

    de Puig, Helena; Bosch, Irene; Carré-Camps, Marc; Hamad-Schifferli, Kimberly

    2017-01-18

    We investigated the effect of the protein corona on the function of nanoparticle (NP) antibody (Ab) conjugates in dipstick sandwich immunoassays. Ab specific for Zika virus nonstructural protein 1 (NS1) were conjugated to gold NPs, and another anti-NS1 Ab was immobilized onto the nitrocellulose membrane. Sandwich immunoassay formation was influenced by whether the strip was run in corona forming conditions, i.e., in human serum. Strips run in buffer or pure solutions of bovine serum albumin exhibited false positives, but those run in human serum did not. Serum pretreatment of the nitrocellulose also eliminated false positives. Corona formation around the NP-Ab in serum was faster than the immunoassay time scale. Langmuir binding analysis determined how the immobilized Ab affinity for the NP-Ab/NS1 was impacted by corona formation conditions, quantified as an effective dissociation constant, KD(eff). Results show that corona formation mediates the specificity and sensitivity of the antibody-antigen interaction of Zika biomarkers in immunoassays, and plays a critical but beneficial role.

  20. Effect of polarization on HIV-1protease and fluoro-substituted inhibitors binding energies by large scale molecular dynamics simulations

    PubMed Central

    Duan, Li L.; Zhu, T.; Li, Yu C.; Zhang, Qing G.; Zhang, John Z. H.

    2017-01-01

    Molecular dynamics simulations in explicit water are carried out to study the binding of six inhibitors to HIV-1 protease (PR) for up to 700 ns using the standard AMBER force field and polarized protein-specific charge (PPC). PPC is derived from quantum mechanical calculation for protein in solution and therefore it includes electronic polarization effect. Our results show that in all six systems, the bridging water W301 drifts away from the binding pocket in AMBER simulation. However, it is very stable in all six complexes systems using PPC. Especially, intra-protease, protease-inhibitor hydrogen bonds are dynamic stabilized in MD simulation. The computed binding free energies of six complexes have a significantly linear correlation with those experiment values and the correlation coefficient is found to be 0.91 in PPC simulation. However, the result from AMBER simulation shows a weaker correlation with the correlation coefficient of −0.51 due to the lack of polarization effect. Detailed binding interactions of W301, inhibitors with PR are further analyzed and discussed. The present study provides important information to quantitative understanding the interaction mechanism of PR-inhibitor and PR-W301 and these data also emphasizes the importance of both the electronic polarization and the bridging water molecule in predicting precisely binding affinities. PMID:28155907

  1. Examining the Effects of Sodium Ions on the Binding of Antagonists to Dopamine D2 and D3 Receptors

    PubMed Central

    Wood, Martyn D.; Strange, Philip G.

    2016-01-01

    Many G protein-coupled receptors have been shown to be sensitive to the presence of sodium ions (Na+). Using radioligand competition binding assays, we have examined and compared the effects of sodium ions on the binding affinities of a number of structurally diverse ligands at human dopamine D2 and dopamine D3 receptor subtypes, which are important therapeutic targets for the treatment of psychotic disorders. At both receptors, the binding affinities of the antagonists/inverse agonists SB-277011-A, L,741,626, GR 103691 and U 99194 were higher in the presence of sodium ions compared to those measured in the presence of the organic cation, N-methyl-D-glucamine, used to control for ionic strength. Conversely, the affinities of spiperone and (+)-butaclamol were unaffected by the presence of sodium ions. Interestingly, the binding of the antagonist/inverse agonist clozapine was affected by changes in ionic strength of the buffer used rather than the presence of specific cations. Similar sensitivities to sodium ions were seen at both receptors, suggesting parallel effects of sodium ion interactions on receptor conformation. However, no clear correlation between ligand characteristics, such as subtype selectivity, and sodium ion sensitivity were observed. Therefore, the properties which determine this sensitivity remain unclear. However these findings do highlight the importance of careful consideration of assay buffer composition for in vitro assays and when comparing data from different studies, and may indicate a further level of control for ligand binding in vivo. PMID:27379794

  2. Reflex cardiovascular effects of intracoronary acetylstrophanthidin in the conscious dog.

    PubMed

    Barron, K W; Bishop, V S

    1985-06-01

    The present experiments were designed to examine the reflex cardiovascular effects of intracoronary administration of acetylstrophanthidin in the conscious dog. Administration of 4 micrograms/kg of this agent into the left circumflex coronary artery increased left ventricular dP/dtmax but had no effect on mean arterial pressure, heart rate, renal resistance, or iliac resistance. The positive inotropic effects of acetylstrophanthidin were less under control conditions (+599 mmHg/s) than during bilateral cervical vagal cold block (+850 mmHg/s, P less than 0.05); however, interruption of vagal efferent influences (atropine) alone did not alter the contractile effects of acetylstrophanthidin. Interruption of sympathetic efferent influences on the heart with either the nicotinic ganglionic receptor antagonist, hexamethonium, or the beta 1-adrenergic receptor antagonist, metoprolol, also augmented the inotropic effects of acetylstrophanthidin to a degree similar to that observed with vagal cold block. In contrast to the effects observed with acetylstrophanthidin, the inotropic effects of intracoronary administration of calcium gluconate were not altered by vagal cold block or any other conditions examined in this study. We conclude that interruption of vagal afferents results in an augmentation of the positive inotropic actions of acetylstrophanthidin and that this augmented inotropic effect can be accounted for by interruption of cardiac vagal afferent-mediated restraint on sympathetic outflow to the heart.

  3. Effect of abdominal binding on respiratory mechanics during exercise in athletes with cervical spinal cord injury

    PubMed Central

    West, Christopher R.; Goosey-Tolfrey, Victoria L.; Campbell, Ian G.

    2014-01-01

    We asked whether elastic binding of the abdomen influences respiratory mechanics during wheelchair propulsion in athletes with cervical spinal cord injury (SCI). Eight Paralympic wheelchair rugby players with motor-complete SCI (C5-C7) performed submaximal and maximal incremental exercise tests on a treadmill, both with and without abdominal binding. Measurements included pulmonary function, pressure-derived indices of respiratory mechanics, operating lung volumes, tidal flow-volume data, gas exchange, blood lactate, and symptoms. Residual volume and functional residual capacity were reduced with binding (77 ± 18 and 81 ± 11% of unbound, P < 0.05), vital capacity was increased (114 ± 9%, P < 0.05), whereas total lung capacity was relatively well preserved (99 ± 5%). During exercise, binding introduced a passive increase in transdiaphragmatic pressure, due primarily to an increase in gastric pressure. Active pressures during inspiration were similar across conditions. A sudden, sustained rise in operating lung volumes was evident in the unbound condition, and these volumes were shifted downward with binding. Expiratory flow limitation did not occur in any subject and there was substantial reserve to increase flow and volume in both conditions. V̇o2 was elevated with binding during the final stages of exercise (8–12%, P < 0.05), whereas blood lactate concentration was reduced (16–19%, P < 0.05). V̇o2/heart rate slopes were less steep with binding (62 ± 35 vs. 47 ± 24 ml/beat, P < 0.05). Ventilation, symptoms, and work rates were similar across conditions. The results suggest that abdominal binding shifts tidal breathing to lower lung volumes without influencing flow limitation, symptoms, or exercise tolerance. Changes in respiratory mechanics with binding may benefit O2 transport capacity by an improvement in central circulatory function. PMID:24855136

  4. Effect of abdominal binding on respiratory mechanics during exercise in athletes with cervical spinal cord injury.

    PubMed

    West, Christopher R; Goosey-Tolfrey, Victoria L; Campbell, Ian G; Romer, Lee M

    2014-07-01

    We asked whether elastic binding of the abdomen influences respiratory mechanics during wheelchair propulsion in athletes with cervical spinal cord injury (SCI). Eight Paralympic wheelchair rugby players with motor-complete SCI (C5-C7) performed submaximal and maximal incremental exercise tests on a treadmill, both with and without abdominal binding. Measurements included pulmonary function, pressure-derived indices of respiratory mechanics, operating lung volumes, tidal flow-volume data, gas exchange, blood lactate, and symptoms. Residual volume and functional residual capacity were reduced with binding (77 ± 18 and 81 ± 11% of unbound, P < 0.05), vital capacity was increased (114 ± 9%, P < 0.05), whereas total lung capacity was relatively well preserved (99 ± 5%). During exercise, binding introduced a passive increase in transdiaphragmatic pressure, due primarily to an increase in gastric pressure. Active pressures during inspiration were similar across conditions. A sudden, sustained rise in operating lung volumes was evident in the unbound condition, and these volumes were shifted downward with binding. Expiratory flow limitation did not occur in any subject and there was substantial reserve to increase flow and volume in both conditions. V̇o2 was elevated with binding during the final stages of exercise (8-12%, P < 0.05), whereas blood lactate concentration was reduced (16-19%, P < 0.05). V̇o2/heart rate slopes were less steep with binding (62 ± 35 vs. 47 ± 24 ml/beat, P < 0.05). Ventilation, symptoms, and work rates were similar across conditions. The results suggest that abdominal binding shifts tidal breathing to lower lung volumes without influencing flow limitation, symptoms, or exercise tolerance. Changes in respiratory mechanics with binding may benefit O2 transport capacity by an improvement in central circulatory function.

  5. Effects of ligand binding on the mechanical stability of protein GB1 studied by steered molecular dynamics simulation.

    PubMed

    Su, Ji-Guo; Zhao, Shu-Xin; Wang, Xiao-Feng; Li, Chun-Hua; Li, Jing-Yuan

    2016-08-01

    Regulation of the mechanical properties of proteins plays an important role in many biological processes, and sheds light on the design of biomaterials comprised of protein. At present, strategies to regulate protein mechanical stability focus mainly on direct modulation of the force-bearing region of the protein. Interestingly, the mechanical stability of GB1 can be significantly enhanced by the binding of Fc fragments of human IgG antibody, where the binding site is distant from the force-bearing region of the protein. The mechanism of this long-range allosteric control of protein mechanics is still elusive. In this work, the impact of ligand binding on the mechanical stability of GB1 was investigated using steered molecular dynamics simulation, and a mechanism underlying the enhanced protein mechanical stability is proposed. We found that the external force causes deformation of both force-bearing region and ligand binding site. In other words, there is a long-range coupling between these two regions. The binding of ligand restricts the distortion of the binding site and reduces the deformation of the force-bearing region through a long-range allosteric communication, which thus improves the overall mechanical stability of the protein. The simulation results are very consistent with previous experimental observations. Our studies thus provide atomic-level insights into the mechanical unfolding process of GB1, and explain the impact of ligand binding on the mechanical properties of the protein through long-range allosteric regulation, which should facilitate effective modulation of protein mechanical properties.

  6. A scale of metal ion binding strengths correlating with ionic charge, Pauling electronegativity, toxicity, and other physiological effects.

    PubMed

    Kinraide, Thomas B; Yermiyahu, Uri

    2007-09-01

    Equilibrium constants for binding to plant plasma membranes have been reported for several metal ions, based upon adsorption studies and zeta-potential measurements. LogK values for the ions are these: Al(3+), 4.30; La(3+), 3.34; Cu(2+), 2.60; Ca(2+) and Mg(2+), 1.48; Na(+) and K(+), 0 M(-1). These values correlate well with logK values for ion binding to many organic and inorganic ligands. LogK values for metal ion binding to 12 ligands were normalized and averaged to produce a scale for the binding of 49 ions. The scale correlates well with the values presented above (R(2)=0.998) and with ion binding to cell walls and other biomass. The scale is closely related to the charge (Z) and Pauling electronegativity (PE) of 48 ions (all but Hg(2+)); R(2)=0.969 for the equation (Scale values)=-1.68+Z(1.22+0.444PE). Minimum rhizotoxicity of metal ions appears to be determined by binding strengths: log a(PM,M)=1.60-2.41exp[0.238(Scale values)] determines the value of ion activities at the plasma membrane surface (a(PM,M)) that will ensure inhibition of root elongation. Additional toxicity appears to be related to softness, accounting for the great toxicity of Ag(+), for example. These binding-strength values correlate with additional physiological effects and are suitable for the computation of cell-surface electrical potentials.

  7. Silencing IFN-γ binding/signaling in astrocytes vs. microglia leads to opposite effects on CNS autoimmunity

    PubMed Central

    Ding, Xiaoli; Yan, Yaping; Li, Xing; Li, Ke; Ciric, Bogoljub; Yang, Jingxian; Zhang, Yuan; Wu, Shuai; Xu, Hui; Chen, Wanjun; Lovett-Racke, Amy E.; Zhang, Guang-Xian; Rostami, Abdolmohamad

    2015-01-01

    IFN-γ, the hallmark cytokine of Th1 cells, plays an important role in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Thus far, the role of IFN-γ in EAE has been largely studied through its effects on immune cells, while much less is known about its effects on central nervous system (CNS) cells, especially in vivo. In this study we dissected the in vivo effects and mechanisms of IFN-γ binding/signaling in astrocytes and microglia and found that IFN-γ signaling in these cell types has opposite effects in EAE pathogenesis. Silencing IFN-γ binding/signaling in astrocytes ameliorated EAE, while in microglia, and likely in some infiltrating macrophages, it increased disease severity. Silencing IFN-γ signaling in astrocytes resulted in diminished expression of chemokines and fewer inflammatory cells infiltrating into the CNS, while blocking IFN-γ binding/signaling in microglia, probably infiltrating macrophages as well, increased disease severity through augmented activation and proliferation of microglia. Further, blocking IFN-γ binding/signaling in astrocytes ameliorated both Th1- and Th17-mediated adoptive EAE, indicating an important role for IFN-γ signaling in astrocytes in autoimmune CNS inflammation. Thus, our study defines novel mechanisms of action of IFN-γ in EAE pathogenesis, and also highlights an opportunity for development of MS therapies directed at CNS cells. PMID:25795755

  8. Development of molecular docking-based binding energy to predict the joint effect of BPA and its analogs.

    PubMed

    Zhang, Hong-Chang; Hu, Xia-Lin; Yin, Da-Qiang; Lin, Zhi-Fen

    2011-04-01

    A general proposal for predicting the joint effect of endocrine disrupting chemicals by examining binding energy models was developed in this study. 2,2-bis(4-hydroxyphenyl)propane (BPA) and 11 of its analogs were chosen, and the estrogenic activity of each compound was measured by determining its EC50 value using a recombinant gene yeast assay. Binding energies (BEs) were calculated using Surflex-Docking software. The analysis of the relationship between EC50 values and BEs showed that there is a linear correlation between the BEs and EC50 values. Furthermore, the analysis of the given binary and quaternary mixtures of BPA and three of its analogs showed that the joint effects of the mixtures were affected by the proportions of the chemicals in each mixture and their relative binding energy. The correlation between the joint effects of mixtures and the binding energy of the individual compounds has been described using one formula, which can be used to predict the joint effects of other mixtures.

  9. Interaction between curcumin and human serum albumin in the presence of excipients and the effect of binding on curcumin photostability.

    PubMed

    Vukićević, Milica; Tønnesen, Hanne Hjorth

    2016-01-01

    Curcumin (Cur) is known to bind to human serum albumin (HSA) which may lead to a reduced phototoxic effect of the compound in the presence of serum or saliva. The influence of excipients on the Cur-HSA binding was studied by HSA florescence quenching and Cur absorption and emission spectroscopy in the presence and absence of the selected excipients. Photostabilty of Cur in the presence of HSA was evaluated, as well as the effect of excipients on HSA bound Cur photodegradation. Cyclodextrins (CDs) (2-hydroxypropyl-β-cyclodextrin and 2-hydroxypropyl-γ-cyclodextrin) and polymers (polyethylene glycol 400, PEG 400 and Pluronic F-127, PF-127) were selected for the study. CDs and PF-127 seem to decrease Cur binding to HSA, probably through competitive binding. Cur was still bound to HSA in polyethylene glycol (PEG) solutions at the highest investigated concentration (5% w/v). However, high PEG concentration appears to have effect on the protein conformation, as shown by the fluorescence quenching study. Low Cur photostability in the presence of HSA could be improved by the addition of hydroxylpropyl-γ-cyclodextrin (HPγCD) to the samples, whereas PEG and PF-127 showed no effect.

  10. The effect of neurotoxin on rabies virus binding to mouse neuroblastoma cells.

    PubMed

    Briggs, D J; Phillips, R M

    1991-08-01

    Mouse neuroblastoma cells were exposed to alpha bungarotoxin, a neurotoxin known to inhibit rabies virus binding to the nicotinic acetylcholine receptor located at the neuromuscular junction in muscle tissue. The total amount of 3H-CVS virus that bound to neurotoxin treated cells was separated into specific and non-specific binding using a cold competition assay. Comparison of untreated and neurotoxin treated cells demonstrated that the majority of cell-associated virus in untreated cells was of a specific nature whereas the majority of the cell-associated virus in neurotoxin treated cells was due to non-specific binding.

  11. An autoradiographic map of (3H)diprenorphine binding in rat brain: effects of social interaction

    SciTech Connect

    Panksepp, J.; Bishop, P.

    1981-10-01

    (3H)Diprenorphine binding was analyzed autoradiographically in the brains of 33 day old rat pups. A photographic atlas of diprenorphine binding in the coronal plane is provided to highlight the dispersion of opioid receptor systems through the brain. To determine whether brain opioid release may be induced by social interactions, half the animals were sacrificed following a 30 min period of social interaction while the other half were sacrificed following 30 min of social isolation. Opioid binding was higher in isolate-tested animals than socially-tested ones, suggesting that social interaction may promote endogenous brain opioid release.

  12. Branching Reaction in Melanogenesis: The Effect of Intramolecular Cyclization on Thiol Binding

    NASA Astrophysics Data System (ADS)

    Kishida, Ryo; Kasai, Hideaki; Aspera, Susan Meñez; Arevalo, Ryan Lacdao; Nakanishi, Hiroshi

    2017-01-01

    With the aid of density functional theory-based first principles calculations, we investigated energetics and electronic structure changes in reactions involving dopaquinone to give insights into the branching behaviors in melanogenesis. The reactions we investigated are the intramolecular cyclization and thiol binding, which are competing with each other. It was found that, in order to accomplish thiol binding, charge transfer of around one electron from thiol to dopaquinone occurs. Furthermore, intramolecular cyclization of dopaquinone increases the lowest unnoccupied molecular orbital level substantially. This result clearly shows prevention of the binding of thiol by intramolecular cyclization.

  13. Effects of Major Spore-Specific DNA Binding Proteins on Bacillus subtilis Sporulation and Spore Properties

    PubMed Central

    Setlow, Barbara; McGinnis, Kelly A.; Ragkousi, Katerina; Setlow, Peter

    2000-01-01

    Sporulation of a Bacillus subtilis strain (termed α− β−) lacking the majority of the α/β-type small, acid-soluble spore proteins (SASP) that are synthesized in the developing forespore and saturate spore DNA exhibited a number of differences from that of the wild-type strain, including delayed forespore accumulation of dipicolinic acid, overexpression of forespore-specific genes, and delayed expression of at least one mother cell-specific gene turned on late in sporulation, although genes turned on earlier in the mother cell were expressed normally in α− β− strains. The sporulation defects in α− β− strains were corrected by synthesis of chromosome-saturating levels of either of two wild-type, α/β-type SASP but not by a mutant SASP that binds DNA poorly. Spores from α− β− strains also exhibited less glutaraldehyde resistance and slower outgrowth than did wild-type spores, but at least some of these defects in α− β− spores were abolished by the synthesis of normal levels of α/β-type SASP. These results indicate that α/β-type SASP may well have global effects on gene expression during sporulation and spore outgrowth. PMID:11092849

  14. Rational optimization of conformational effects induced by hydrocarbon staples in peptides and their binding interfaces.

    PubMed

    Lama, Dilraj; Quah, Soo T; Verma, Chandra S; Lakshminarayanan, Rajamani; Beuerman, Roger W; Lane, David P; Brown, Christopher J

    2013-12-13

    eIF4E is frequently over-expressed in different cancers and causes increased translation of oncogenic proteins via deregulated cap-dependent translation. Inhibitors of the eIF4E:eIF4G interactions represent an approach that would normalize cap-dependent translation. Stapled peptides represent an emerging class of therapeutics that can target protein: protein interactions. We present here molecular dynamics simulations for a set of rationally designed stapled peptides in solution and in complex with eIF4E, supported with biophysical and crystallographic data. Clustering of the simulated structures revealed the favoured conformational states of the stapled peptides in their bound or free forms in solution. Identifying these populations has allowed us to design peptides with improved affinities by introducing mutations into the peptide sequence to alter their conformational distributions. These studies emphasise the effects that engineered mutations have on the conformations of free and bound peptides, and illustrate that both states must be considered in efforts to attain high affinity binding.

  15. Rational Optimization of Conformational Effects Induced By Hydrocarbon Staples in Peptides and their Binding Interfaces

    NASA Astrophysics Data System (ADS)

    Lama, Dilraj; Quah, Soo T.; Verma, Chandra S.; Lakshminarayanan, Rajamani; Beuerman, Roger W.; Lane, David P.; Brown, Christopher J.

    2013-12-01

    eIF4E is frequently over-expressed in different cancers and causes increased translation of oncogenic proteins via deregulated cap-dependent translation. Inhibitors of the eIF4E:eIF4G interactions represent an approach that would normalize cap-dependent translation. Stapled peptides represent an emerging class of therapeutics that can target protein: protein interactions. We present here molecular dynamics simulations for a set of rationally designed stapled peptides in solution and in complex with eIF4E, supported with biophysical and crystallographic data. Clustering of the simulated structures revealed the favoured conformational states of the stapled peptides in their bound or free forms in solution. Identifying these populations has allowed us to design peptides with improved affinities by introducing mutations into the peptide sequence to alter their conformational distributions. These studies emphasise the effects that engineered mutations have on the conformations of free and bound peptides, and illustrate that both states must be considered in efforts to attain high affinity binding.

  16. Translating slow-binding inhibition kinetics into cellular and in vivo effects

    PubMed Central

    Walkup, Grant K.; You, Zhiping; Ross, Philip L.; Allen, Eleanor K. H.; Daryaee, Fereidoon; Hale, Michael R.; O’Donnell, John; Ehmann, David E.; Schuck, Virna J. A.; Buurman, Ed T.; Choy, Allison L.; Hajec, Laurel; Murphy-Benenato, Kerry; Marone, Valerie; Patey, Sara A.; Grosser, Lena A.; Johnstone, Michele; Walker, Stephen G.; Tonge, Peter J.; Fisher, Stewart L.

    2015-01-01

    Many drug candidates fail in clinical trials due to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug:target complex has a longer half-life than off-target:drug complexes. However, while slow-binding inhibition kinetics are a key feature of many marketed drugs1,2, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection. PMID:25894085

  17. Tyr-301 Phosphorylation Inhibits Pyruvate Dehydrogenase by Blocking Substrate Binding and Promotes the Warburg Effect*

    PubMed Central

    Fan, Jun; Kang, Hee-Bum; Shan, Changliang; Elf, Shannon; Lin, Ruiting; Xie, Jianxin; Gu, Ting-Lei; Aguiar, Mike; Lonning, Scott; Chung, Tae-Wook; Arellano, Martha; Khoury, Hanna J.; Shin, Dong M.; Khuri, Fadlo R.; Boggon, Titus J.; Kang, Sumin; Chen, Jing

    2014-01-01

    The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect. PMID:25104357

  18. Tyr-301 phosphorylation inhibits pyruvate dehydrogenase by blocking substrate binding and promotes the Warburg effect.

    PubMed

    Fan, Jun; Kang, Hee-Bum; Shan, Changliang; Elf, Shannon; Lin, Ruiting; Xie, Jianxin; Gu, Ting-Lei; Aguiar, Mike; Lonning, Scott; Chung, Tae-Wook; Arellano, Martha; Khoury, Hanna J; Shin, Dong M; Khuri, Fadlo R; Boggon, Titus J; Kang, Sumin; Chen, Jing

    2014-09-19

    The mitochondrial pyruvate dehydrogenase complex (PDC) plays a crucial role in regulation of glucose homoeostasis in mammalian cells. PDC flux depends on catalytic activity of the most important enzyme component pyruvate dehydrogenase (PDH). PDH kinase inactivates PDC by phosphorylating PDH at specific serine residues, including Ser-293, whereas dephosphorylation of PDH by PDH phosphatase restores PDC activity. The current understanding suggests that Ser-293 phosphorylation of PDH impedes active site accessibility to its substrate pyruvate. Here, we report that phosphorylation of a tyrosine residue Tyr-301 also inhibits PDH α 1 (PDHA1) by blocking pyruvate binding through a novel mechanism in addition to Ser-293 phosphorylation. In addition, we found that multiple oncogenic tyrosine kinases directly phosphorylate PDHA1 at Tyr-301, and Tyr-301 phosphorylation of PDHA1 is common in EGF-stimulated cells as well as diverse human cancer cells and primary leukemia cells from human patients. Moreover, expression of a phosphorylation-deficient PDHA1 Y301F mutant in cancer cells resulted in increased oxidative phosphorylation, decreased cell proliferation under hypoxia, and reduced tumor growth in mice. Together, our findings suggest that phosphorylation at distinct serine and tyrosine residues inhibits PDHA1 through distinct mechanisms to impact active site accessibility, which act in concert to regulate PDC activity and promote the Warburg effect.

  19. Heme binding site in apomyoglobin may be effectively targeted with small molecules to control aggregation.

    PubMed

    Azami-Movahed, Mehrnaz; Shariatizi, Sajad; Sabbaghian, Marjan; Ghasemi, Atiyeh; Ebrahim-Habibi, Azadeh; Nemat-Gorgani, Mohsen

    2013-02-01

    A number of ligands with affinities for the heme binding site of apomyoglobin were tested to control amorphous and fibrillar aggregation in the protein. Several techniques, including fluorescence, dynamic light scattering, transmission electron microscopy, dot blot analysis combined with viability studies were employed for structural characterization and cytotoxicity assessment of the intermediate and final protein structures formed during the aggregation process. Of the small molecules investigated, chrysin and Nile red with high structural similarities to heme were chosen for further studies. Only fibril formation was found to be prevented by Nile red, while chrysin, with a greater structural flexibility, was able to prevent both types of aggregate formation. The two ligands were found to influence aggregation at different stages of intermediate structure formation, an ability determined by their degrees of similarities with heme. Based on structural characterization and toxicity studies, it is concluded that ligands similar in structure to heme may be effective in influencing various stages of aggregate formation and toxicity potencies of the protein structures. Since metalloproteins constitute more than thirty percent of all known proteins, it is concluded that the present strategy may be of general significance.

  20. Effect of Methamphetamine Exposure on Expression of Calcium Binding Proteins in Rat Frontal Cortex and Hippocampus.

    PubMed

    Veerasakul, Siriluk; Thanoi, Samur; Reynolds, Gavin P; Nudmamud-Thanoi, Sutisa

    2016-10-01

    Methamphetamine (METH) is a psychostimulant drug with potent effects on the central nervous system that can cause psychotic symptoms similar to those of schizophrenia. Specific alterations in GABAergic neuronal markers have been reported in schizophrenia and animal models of psychotic illness. The aim of this study was to determine whether there are changes in subpopulations of GABAergic neurons, defined by the presence of calcium binding proteins (CBPs), in animal models of METH abuse. Rats received acute (Binge) doses of 4 × 6 mg/kg, a chronic escalating dose regime (0.1-4 mg/kg over 14 days) or a combination of the two and were compared with a vehicle-administered control group. Brains were taken and sections of frontal cortex (Cg1) and hippocampus (dentate gyrus and CA1-3 regions) underwent immunostaining for three CBPs [parvalbumin (PV), calbindin (CB), and calretinin (CR)]. Significant decreases in PV-immunoreactive (IR) neurons in each METH group and all regions were observed. Smaller METH-induced deficits in CB-IR cells were observed, reaching significance primarily following chronic METH regimes, while CR-IR was significantly reduced only in frontal cortex following chronic administration. These results suggest that METH regimes in rats can induce selective deficits in GABAergic neuronal subtypes similar to those seen in schizophrenia and may underlie the psychosis and/or cognitive impairment that can occur in METH abuse and dependence.

  1. Genetic effects of sterol regulatory element binding proteins and fatty acid-binding protein4 on the fatty acid composition of Korean cattle (Hanwoo)

    PubMed Central

    Oh, Dong-Yep; Lee, Jea-Young; Jang, Ji-Eun; Lee, Seung-Uk

    2017-01-01

    Objective This study identifies single-nucleotide polymorphisms (SNP) or gene combinations that affect the flavor and quality of Korean cattle (Hanwoo) by using the SNP Harvester method. Methods Four economic traits (oleic acid [C18:1], saturated fatty acids), monounsaturated fatty acids, and marbling score) were adjusted for environmental factors in order to focus solely on genetic effects. The SNP Harvester method was used to investigate gene combinations (two-way gene interactions) associated with these economic traits. Further, a multifactor dimensionality reduction method was used to identify superior genotypes in gene combinations. Results Table 3 to 4 show the analysis results for differences between superior genotypes and others for selected major gene combinations using the multifactor dimensionality reduction method. Environmental factors were adjusted for in order to evaluate only the genetic effect. Table 5 shows the adjustment effect by comparing the accuracy before and after correction in two-way gene interactions. Conclusion The g.3977-325 T>C and (g.2988 A>G, g.3977-325 T>C) combinations of fatty acid-binding protein4 were the superior gene, and the superior genotype combinations across all economic traits were the CC genotype at g.3977-325 T>C and the AACC, GACC, GGCC genotypes of (g.2988 A>G, g.3977-325 T>C). PMID:27492349

  2. Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase

    NASA Astrophysics Data System (ADS)

    Singh, Warispreet; Karabencheva-Christova, Tatyana G.; Black, Gary W.; Ainsley, Jon; Dover, Lynn; Christov, Christo Z.

    2016-01-01

    Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.

  3. The effect of Pot1 binding on the repair of thymine analogs in a telomeric DNA sequence.

    PubMed

    Theruvathu, Jacob A; Darwanto, Agus; Hsu, Chia Wei; Sowers, Lawrence C

    2014-08-01

    Telomeric DNA can form duplex regions or single-stranded loops that bind multiple proteins, preventing it from being processed as a DNA repair intermediate. The bases within these regions are susceptible to damage; however, mechanisms for the repair of telomere damage are as yet poorly understood. We have examined the effect of three thymine (T) analogs including uracil (U), 5-fluorouracil (5FU) and 5-hydroxymethyluracil (5hmU) on DNA-protein interactions and DNA repair within the GGTTAC telomeric sequence. The replacement of T with U or 5FU interferes with Pot1 (Pot1pN protein of Schizosaccharomyces pombe) binding. Surprisingly, 5hmU substitution only modestly diminishes Pot1 binding suggesting that hydrophobicity of the T-methyl group likely plays a minor role in protein binding. In the GGTTAC sequence, all three analogs can be cleaved by DNA glycosylases; however, glycosylase activity is blocked if Pot1 binds. An abasic site at the G or T positions is cleaved by the endonuclease APE1 when in a duplex but not when single-stranded. Abasic site formation thermally destabilizes the duplex that could push a damaged DNA segment into a single-stranded loop. The inability to enzymatically cleave abasic sites in single-stranded telomere regions would block completion of the base excision repair cycle potentially causing telomere attrition.

  4. Binding motifs in bacterial gene promoters modulate transcriptional effects of global regulators CRP and ArcA

    SciTech Connect

    Leuze, Mike; Karpinets, Tatiana V.; Syed, Mustafa H.; Beliaev, Alex S.; Uberbacher, Edward

    2012-05-30

    Bacterial gene regulation involves transcription factors (TF) that bind to DNA recognition sequences in operon promoters. These recognition sequences, many of which are palindromic, are known as regulatory elements or transcription factor binding sites (TFBS). Some TFs are global regulators that can modulate the expression of hundreds of genes. In this study we examine global regulator half-sites, where a half-site, which we shall call a binding motif (BM), is one half of a palindromic TFBS. We explore the hypothesis that the number of BMs plays an important role in transcriptional regulation, examining empirical data from transcriptional profiling of the CRP and ArcA regulons. We compare the power of BM counts and of full TFBS characteristics to predict induced transcriptional activity. We find that CRP BM counts have a nonlinear effect on CRP-dependent transcriptional activity and predict this activity better than full TFBS quality or location.

  5. An astrocytic binding site for neuronal Thy-1 and its effect on neurite outgrowth.

    PubMed Central

    Dreyer, E B; Leifer, D; Heng, J E; McConnell, J E; Gorla, M; Levin, L A; Barnstable, C J; Lipton, S A

    1995-01-01

    Thy-1, a member of the immunoglobulin superfamily, is one of the most abundant glycoproteins on mammalian neurons. Nevertheless, its role in the peripheral or central nervous system is poorly understood. Certain monoclonal antibodies to Thy-1 promote neurite outgrowth by rodent central nervous system neurons in vitro, suggesting that Thy-1 functions, in part, by modulating neurite outgrowth. We describe a binding site for Thy-1 on astrocytes. This Thy-1-binding protein has been characterized by immunofluroesence with specific anti-idiotype monoclonal antibodies and by three competitive binding assays using (i) anti-idiotype antibodies, (ii) purified Thy-1, and (iii) Thy-1-transfected cells. The Thy-1-binding protein may participate in axonal or dendritic development in the nervous system. Images Fig. 1 Fig. 3 PMID:7479964

  6. Biological effects of individually synthesized TNF-binding domain of variola virus CrmB protein.

    PubMed

    Tsyrendorzhiev, D D; Orlovskaya, I A; Sennikov, S V; Tregubchak, T V; Gileva, I P; Tsyrendorzhieva, M D; Shchelkunov, S N

    2014-06-01

    The biological characteristics of a 17-kDa protein synthesized in bacterial cells, a TNF-binding domain (VARV-TNF-BP) of a 47-kDa variola virus CrmB protein (VARV-CrmB) consisting of TNF-binding and chemokine-binding domains, were studied. Removal of the C-terminal chemokine-binding domain from VARV-CrmB protein was inessential for the efficiency of its inhibition of TNF cytotoxicity towards L929 mouse fibroblast culture and for TNF-induced oxidative metabolic activity of mouse blood leukocytes. The results of this study could form the basis for further studies of VARV-TNF-BP mechanisms of activity for prospective use in practical medicine.

  7. Measurement in vivo of dopamine receptor density I: Effect of endogenous dopamine on spiroperidol binding

    SciTech Connect

    De Jesus, O.T.; Van Moffaert, G.J.C.; Friedman, A.M.; Dinerstein, R.J.

    1984-01-01

    Non-invasive localization of brain dopamine (DA) receptors has been achieved by us and others using gamma emitting derivatives of the DA antagonist spiroperidol (SP). To accurately characterize this localization, the authors have previously described an equilibrium binding model involving SP and DA for a single DA receptor. It is the purpose of this study to establish experimentally the significance of endogenous DA on the ability of SP to bind a group of DA receptors. Several mice were administered different doses of SP. To one group of mice L-dopa was given with peripheral decarboxylase inhibitor, RO-4-4602, in order to elevate brain DA levels while a separate group served as control. /sup 3/H-SP binding and DA levels were measured in each brain sample. The results reflect a significant competition between DA and SP for caudate DA binding sites.

  8. Effect of chronic exposure to methylxanthines on diazepam cerebral binding in female rats and their offsprings.

    PubMed

    Daval, J L; Vert, P

    1986-06-01

    Caffeine, theophylline or saline were injected daily into female rats during the gestation and lactation periods. Crude synaptosomal membranes were isolated from the brains of offsprings at various stages of development and their ability to specifically bind [3H]diazepam was tested. An other approach consisted of injecting [3H]diazepam into offsprings and cerebral specifically bound diazepam was measured. It was shown that methylxanthines were able to inhibit [3H]diazepam binding by reducing total number of binding sites in the brain of 5- and 15-day-old rats born from treated mothers, with a total recovery of control values at 25 days of age. Moreover, in vivo percentage of cerebral bound diazepam dramatically fell when rats were exposed to methylxanthines in utero and through the mother's milk. Since caffeine and theophylline displace diazepam binding not necessarily in a competitive manner, it is suggested that they could interfere with diazepam as adenosine antagonists.

  9. ARSENITE BINDING TO SYNTHETIC PEPTIDES: THE EFFECT OF INCREASING LENGTH BETWEEN TWO CYSTEINES

    EPA Science Inventory

    Binding of trivalent arsenicals to peptides and proteins can alter peptide/protein structure and enzyme function and thereby contribute to arsenic toxicity and carcinogenicity. We utilized radioactive 73As- labeled arsenite and vacuum filtration methodology to determine the bindi...

  10. Effect of membrane protein concentration on binding of /sup 3/H-imipramine in human platelets

    SciTech Connect

    Barkai, A.I.; Kowalik, S.; Baron, M.

    1985-02-01

    Binding of /sup 3/H-imipramine to platelet membranes has been implicated as a marker for depression. Comparing /sup 3/H-IMI binding between depressed patients and normal subjects we observed an increase in the dissociation constant Kd with increasing membrane protein. This phenomenon was studied more rigorously in five normal subjects. Platelet membranes were prepared and adjusted to four concentrations of protein ranging from 100 to 800 micrograms/ml. The /sup 3/H-IMI binding parameters of maximum binding sites number (Bmax) and Kd were obtained by Scatchard analysis at each membrane concentration. A positive linear relationship was found between K/sub d/ values and the concentration of membrane protein in the assay, but no change was observed in Bmax. The variability in Kd values reported in the literature may be accounted for in part by the different concentrations of membrane protein used in various studies.

  11. The effect of structural alterations of three mammalian serum albumins on their binding properties

    NASA Astrophysics Data System (ADS)

    Równicka-Zubik, J.; Sułkowski, L.; Maciążek-Jurczyk, M.; Sułkowska, A.

    2013-07-01

    The binding of piroxicam (PIR) to human (HSA), bovine (BSA) and sheep (SSA) serum albumin in native and destabilized/denaturated state was studied by the fluorescence quenching technique. Quenching of the intrinsic fluorescence of three analyzed serum albumins was observed due to selective exciting of tryptophanyl and tyrosil residues at 295 nm and 280 nm. Based on fluorescence emission spectra the quenching (KQ) and binding constants (Ka) were determined. The results showed that PIR is bound mainly in IIA subdomain of HSA and is additionally able to interact with tyrosil groups located in subdomains IB, IIB or IIIA. PIR interacts only with tryptophanyl residues of BSA and SSA [Trp-214, Trp-237 (IIA) and Trp-135, Trp-158 (IB)]. The presence of denaturating factors modified the mechanism of fluorescence quenching of SSA by PIR. Linear Scatchard plots suggest that HSA, BSA and SSA bind PIR in one class of binding sites.

  12. Multiple binding sites involved in the effect of choline esters on decarbamoylation of monomethylcarbamoyl- or dimethylcarbamoly-acetylcholinesterase.

    PubMed Central

    Sok, D E; Kim, Y B; Choi, S J; Jung, C H; Cha, S H

    1994-01-01

    Multiple binding sites for inhibitory choline esters in spontaneous decarbamoylation of dimethylcarbamoyl-acetylcholinesterase (AChE) were suggested from a wide range of IC50 values, in contrast with a limited range of AC50 values (concentration giving 50% of maximal activation) at a peripheral activatory site. Association of choline esters containing a long acyl chain (C7-C12) with the hydrophobic zone in the active site could be deduced from a linear relationship between the size of the acyl group and the inhibitory potency in either spontaneous decarbamoylation or acetylthiocholine hydrolysis. Direct support for laurylcholine binding to the active site might come from the competitive inhibition (Ki 33 microM) of choline-catalysed decarbamoylation by laurylcholine. Moreover, its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE, where there is a greater steric hindrance at the active centre. In further support, the inhibition of pentanoylthiocholine-induced decarbamoylation by laurylcholine was suggested to be due to laurylcholine binding to a central site rather than a peripheral site, similar to the inhibition of spontaneous decarbamoylation by laurylcholine. Supportive data for acetylcholine binding to the active site are provided by the results that acetylcholine is a competitive inhibitor (Ki 7.6 mM) of choline-catalysed decarbamoylation, and its inhibitory action was greater for monomethylcarbamoyl-AChE than for dimethylcarbamoyl-AChE. Meanwhile, choline esters with an acyl group of an intermediate size (C4-C6), more subject to steric exclusion at the active centre, and less associable with the hydrophobic zone, appear to bind preferentially to a peripheral activity site. Thus the multiple effects of choline esters may be governed by hydrophobicity and/or a steric effect exerted by the acyl moiety at the binding sites. PMID:8053896

  13. Effects of linalool on [(3)H]MK801 and [(3)H] muscimol binding in mouse cortical membranes.

    PubMed

    Brum, L F; Elisabetsky, E; Souza, D

    2001-08-01

    Linalool is a monoterpene compound reported to be a major component of essential oils of several aromatic species. Several linalool-producing species are used in traditional medical systems for sedative purposes, including the interruption and prevention of seizures. Previous studies in mice revealed that linalool modulates glutamatergic (competitive antagonism of L-[(3)H]glutamate binding, delayed intraperitoneal NMDA-induced convulsions and blockade of intracerebroventricular Quin-induced convulsions) and GABAergic transmission (protection against pentylenetetrazol and picrotoxin-induced convulsions). To further clarify the anticonvulsive mechanisms of linalool, we studied the effects of linalool on binding of [(3)H]MK801 (NMDA antagonist) and [(3)H]muscimol (GABA(A) agonist) to mouse cortical membranes. Linalool showed a dose dependent non-competitive inhibition of [(3)H]MK801 binding (IC(50) = 2.97 mM) but no effect on [(3)H]muscimol binding. The data suggest that the anticonvulsant mode of action of linalool includes a direct interaction with the NMDA receptor complex. The data do not, however, support a direct interaction of linalool with GABA(A) receptors, although changes in GABA-mediated neuronal inhibition or effects on GABA release and uptake cannot be ruled out.

  14. Spectroscopic characterization of effective components anthraquinones in Chinese medicinal herbs binding with serum albumins

    NASA Astrophysics Data System (ADS)

    Bi, Shuyun; Song, Daqian; Kan, Yuhe; Xu, Dong; Tian, Yuan; Zhou, Xin; Zhang, Hanqi

    2005-11-01

    The interactions of serum albumins such as human serum albumin (HSA) and bovine serum albumin (BSA) with emodin, rhein, aloe-emodin and aloin were assessed employing fluorescence quenching and absorption spectroscopic techniques. The results obtained revealed that there are relatively strong binding affinity for the four anthraquinones with HSA and BSA and the binding constants for the interactions of anthraquinones with HSA or BSA at 20 °C were obtained. Anthraquinone-albumin interactions were studied at different temperatures and in the presence of some metal ions. And the competition binding of anthraquinones with serum albumins was also discussed. The Stern-Volmer curves suggested that the quenching occurring in the reactions was the static quenching process. The binding distances and transfer efficiencies for each binding reactions were calculated according to the Föster theory of non-radiation energy transfer. Using thermodynamic equations, the main action forces of these reactions were also obtained. The reasons of the different binding affinities for different anthraquinone-albumin reactions were probed from the point of view of molecular structures.

  15. Specific binding of lactoferrin to brush-border membrane: Ontogeny and effect of glycan chain

    SciTech Connect

    Davidson, L.A.; Loennerdal, B. )

    1988-04-01

    Bioavailability of iron from human milk is exceptionally high. It has been suggested that lactoferrin, the major iron-binding protein in human milk, may participate in this high iron bioavailability from milk. The authors examined the interaction of lactoferrin with the intestinal brush-border membrane using the rhesus monkeys as a model. Brush-border membrane vesicles were prepared from monkeys of various ages. Binding studies with {sup 59}Fe-labeled human and monkey lactoferrin were performed to examine interaction of lactoferrin with the brush-border membrane. Specific saturable binding of lactoferrin was found at all ages studied. The dissociation constant for lactoferrin-receptor binding was 9 {times} 10{sup {minus}6} M. In contrast, no binding of serum transferrin or bovine lactoferrin occurred. Removal of fucose from the lactoferrin glycans resulted in a significant decrease in binding. It was concluded that lactoferrin in milk may function in the process of iron absorption through interaction with a small intestinal receptor and that fucosylated glycans on the carbohydrate chain of lactoferrin are necessary for receptor recognition.

  16. Comparative effects of cryosolvents on tubulin association, thermal stability, and binding of microtubule-associated proteins.

    PubMed

    Pajot-Augy, E

    1993-06-01

    Organic cryosolvents essential for cryopreservation of living cells have a colligative effect on water properties, but also affect cellular structures such as the membrane, actin, or tubulin cytoskeleton. The effects of cryosolvents on actin and its binding proteins are starting to be well investigated. In parallel, tubulin assembly characteristics were investigated comparatively, with 0-30% 1,2-propanediol, dimethyl sulfoxide, or glycerol, and with or without microtubule-associated proteins, at 37 or 4 degrees C. Tubulin association was monitored by spectrometry and sedimentation, providing the concentration in free protein, cold-depolymerizable microtubules, and cold-resistant associations. At 37 degrees C, 1,2-propanediol and dimethyl sulfoxide induce a similar association level and cold stability of the assemblies. Glycerol yields a lower level of tubulin association. Cold stability of the assemblies requires the presence of solvent, the amount of which is modulated by microtubule-associated proteins (MAPs): 15% 1,2-propanediol or dimethyl sulfoxide, decreasing down to 10% with MAPs, or 10% glycerol with MAPs only. At 4 degrees C, some cold-stable association is promoted by 1,2-propanediol or dimethyl sulfoxide above 10-15%, in the presence or absence of MAPs, but not with glycerol. In addition, protein content of the various fractions obtained with MAPs and 30% solvent was examined by densitometry of electrophoresis gels. Cold-labile associations obtained at 37 degrees C with 1,2-propanediol or dimethyl sulfoxide are lacking in tubulin and enriched in tau proteins relative to control or glycerol. Associations formed at 37 degrees C and stable to subsequent cold treatment, or at 4 degrees C, regardless of the solvent, present a large tubulin content, as well as few tau proteins and high-molecular-weight MAPs.

  17. Intra- and Interdomain Effects Due to Mutation of Calcium-binding Sites in Calmodulin*

    PubMed Central

    Xiong, Liang-Wen; Kleerekoper, Quinn K.; Wang, Xu; Putkey, John A.

    2010-01-01

    The IQ-motif protein PEP-19, binds to the C-domain of calmodulin (CaM) with significantly different kon and koff rates in the presence and absence of Ca2+, which could play a role in defining the levels of free CaM during Ca2+ transients. The initial goal of the current study was to determine whether Ca2+ binding to sites III or IV in the C-domain of CaM was responsible for affecting the kinetics of binding PEP-19. EF-hand Ca2+-binding sites were selectively inactivated by the common strategy of changing Asp to Ala at the X-coordination position. Although Ca2+ binding to both sites III and IV appeared necessary for native-like interactions with PEP-19, the data also indicated that the mutations caused undesirable structural alterations as evidenced by significant changes in amide chemical shifts for apoCaM. Mutations in the C-domain also affected chemical shifts in the unmodified N-domain, and altered the Ca2+ binding properties of the N-domain. Conversion of Asp93 to Ala caused the greatest structural perturbations, possibly due to the loss of stabilizing hydrogen bonds between the side chain of Asp93 and backbone amides in apo loop III. Thus, although these mutations inhibit binding of Ca2+, the mutated CaM may not be able to support potentially important native-like activity of the apoprotein. This should be taken into account when designing CaM mutants for expression in cell culture. PMID:20048169

  18. [3H]-verapamil binding to rat cardiac sarcolemmal membrane fragments; an effect of ischaemia.

    PubMed Central

    Dillon, J. S.; Nayler, W. G.

    1987-01-01

    The [3H]-verapamil binding activity of rat cardiac sarcolemmal fragments was studied, using membranes harvested from non-perfused, aerobically-perfused and ischaemic hearts. Glass-fibre filters were found to contain specific, high affinity--(KD 38 +/- 3.1 nM) [3H]-verapamil binding sites--making them unsuitable for use in [3H]-verapamil binding studies. Incubation of membranes from non-perfused hearts in a medium containing 150 mM NaCl, 1 mM CaCl2 and 50 mM Tris revealed two populations of [3H]-verapamil binding sites. When centrifugation instead of filtration was used to separate bound and free [3H]-verapamil, high affinity sites with a KD of 0.57 +/- 0.19 microM and a Bmax of 38 +/- 5.2 pmol mg-1 protein, and low affinity sites with a KD of 78 +/- 27.5 microM and a Bmax of 2.9 +/- 1.3 nmol mg-1 protein were detected. However, only low affinity binding sites could be detected in membranes which had been incubated in a cation-free medium containing 50 mM Tris. [3H]-verapamil binding to the low and high affinity sites was saturable, reversible, stereospecific and displaceable by D600 greater than diltiazem greater than Ca2+ but not by nifedipine, nitrendipine, nisoldipine or prazosin. The two populations of binding sites survived aerobic perfusion and 60 min ischaemia at 37 degrees C. Ischaemia reduced the Bmax and KD but selectivity was maintained. PMID:3028561

  19. Bile acid-binding activity of young persimmon (Diospyros kaki) fruit and its hypolipidemic effect in mice.

    PubMed

    Matsumoto, Kenji; Yokoyama, Shin-ichiro; Gato, Nobuki

    2010-02-01

    The hypolipidemic effects and bile acid-binding properties of young persimmon (Diospyros kaki) fruit were examined. In an animal experiment, male C57BL/6.Cr mice (n = 5) were fed an AIN-76-modified high fat diet supplemented with 2% or 5% (w/w) dried young persimmon fruit (YP) for 10 weeks. The intake of YP significantly enhanced fecal bile acid excretion and lowered the concentration of hepatic lipids and plasma cholesterol. Analysis of gene expression in liver tissue showed that 2% or 5% YP up-regulated the expression of the sterol regulatory element-binding protein-2 gene. In the 5% group, there were increased expressions of the genes for cholesterol 7alpha-hydroxylase and the low-density lipoprotein receptor. Next, the bile acid-binding ability of YP was analysed in vitro using cholic acid (CA). In 100-2000 microM CA solutions, 1% (w/v) YP adsorbed approximately 60% of CA, while dried mature persimmon fruit adsorbed approximately 20% of CA. The positive control, cholestyramine, adsorbed approximately 80% of CA in the 100-2000 microM CA solutions. A crude tannin extract from YP, which contained 54.7% condensed tannins, adsorbed approximately 78% of CA in the 2000 microM CA solutions. These results suggest that the ability of YP to bind bile acid contributes to its hypolipidemic effect in mice.

  20. Effect of DNA Binding on Geminate CO Recombination Kinetics in CO-sensing Transcription Factor CooA*

    PubMed Central

    Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan; Poulos, Thomas L.; Champion, Paul M.

    2012-01-01

    Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in the CooA proteins relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases, and the distribution of the conformations available in the heme domain becomes restricted. The kinetic studies also offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role. PMID:22544803

  1. Binding Enthalpy Calculations for a Neutral Host-Guest Pair Yield Widely Divergent Salt Effects across Water Models

    PubMed Central

    Gao, Kaifu; Yin, Jian; Henriksen, Niel M.; Fenley, Andrew T.; Gilson, Michael K.

    2015-01-01

    Dissolved salts are a part of the physiological milieu and can significantly influence the kinetics and thermodynamics of varied biomolecular processes, such as binding and catalysis, so it is important for molecular simulations to reliably describe their effects. The present study uses a simple, non-ionized host-guest model system to study the sensitivity of computed binding enthalpies to the choice of water and salt models. Molecular dynamics simulations of a cucurbit[7]uril host with a neutral guest molecule show striking differences in the salt dependency of the binding enthalpy across four water models, TIP3P, SPC/E, TIP4P-Ew and OPC, with additional sensitivity to the choice of parameters for sodium and chloride. In particular, although all of the models predict that binding will be less exothermic with increasing NaCl concentration, the strength of this effect varies by 7 kcal/mol across models. The differences appear to result primarily from differences in the number of sodium ions displaced from the host on binding the guest, rather than from differences in the enthalpy associated with this displacement; and it is the electrostatic energy that contributes most to the changes in enthalpy with increasing salt concentration. That a high sensitivity of salt effects to the choice of water model is observed for the present host-guest system, despite its being non-ionized, raises issues regarding the selection and adjustment of water models for use with biological macromolecules, especially as these typically possess multiple ionized groups which can interact relatively strongly with ions in solution. PMID:26574247

  2. CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing.

    PubMed

    Maita, Hiroshi; Kitaura, Hirotake; Ariga, Hiroyoshi; Iguchi-Ariga, Sanae M M

    2005-02-15

    We have reported that PAP-1, a product of a causative gene for autosomal retinitis pigmentosa, plays a role in splicing. In this study, CIR, a protein originally identified as a CBF1-interacting protein and reported to act as a transcriptional corepressor, was identified as a PAP-1 binding protein and its function as a splicing factor was investigated. In addition to a basic lysine and acidic serine-rich (BA) domain and a zinc knuckle-like motif, CIR has an arginine/serine dipeptide repeat (RS) domain in its C terminal region. The RS domain has been reported to be present in the superfamily of SR proteins, which are involved in splicing reactions. We generated CIR mutants with deletions of each BA and RS domain and studied their subcellular localizations and interactions with PAP-1 and other SR proteins, including SC35, SF2/ASF, and U2AF35. CIR was found to interact with U2AF35 through the BA domain, with SC35 and SF2/ASF through the RS domain, and with PAP-1 outside the BA domain in vivo and in vitro. CIR was found to be colocalized with SC35 and PAP-1 in nuclear speckles. Then the effect of CIR on splicing was investigated using the E1a minigene as a reporter in HeLa cells. Ectopic expression of CIR with the E1a minigene changed the ratio of spliced isoforms of E1a that were produced by alternative selection of 5'-splice sites. These results indicate that CIR is a member of the family of SR-related proteins and that CIR plays a role in splicing regulation.

  3. CIR, a corepressor of CBF1, binds to PAP-1 and effects alternative splicing

    SciTech Connect

    Maita, Hiroshi; Kitaura, Hirotake; Ariga, Hiroyoshi . E-mail: hiro@pharm.hokudai.ac.jp; Iguchi-Ariga, Sanae M.M.

    2005-02-15

    We have reported that PAP-1, a product of a causative gene for autosomal retinitis pigmentosa, plays a role in splicing. In this study, CIR, a protein originally identified as a CBF1-interacting protein and reported to act as a transcriptional corepressor, was identified as a PAP-1 binding protein and its function as a splicing factor was investigated. In addition to a basic lysine and acidic serine-rich (BA) domain and a zinc knuckle-like motif, CIR has an arginine/serine dipeptide repeat (RS) domain in its C terminal region. The RS domain has been reported to be present in the superfamily of SR proteins, which are involved in splicing reactions. We generated CIR mutants with deletions of each BA and RS domain and studied their subcellular localizations and interactions with PAP-1 and other SR proteins, including SC35, SF2/ASF, and U2AF{sup 35}. CIR was found to interact with U2AF{sup 35} through the BA domain, with SC35 and SF2/ASF through the RS domain, and with PAP-1 outside the BA domain in vivo and in vitro. CIR was found to be colocalized with SC35 and PAP-1 in nuclear speckles. Then the effect of CIR on splicing was investigated using the E1a minigene as a reporter in HeLa cells. Ectopic expression of CIR with the E1a minigene changed the ratio of spliced isoforms of E1a that were produced by alternative selection of 5'-splice sites. These results indicate that CIR is a member of the family of SR-related proteins and that CIR plays a role in splicing regulation.

  4. Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.

    PubMed

    Geronimo, Inacrist; Denning, Catherine A; Rogers, W Eric; Othman, Thaer; Huxford, Tom; Heidary, David K; Glazer, Edith C; Payne, Christina M

    2016-06-28

    Cytochrome P450BM3 is a heme-containing enzyme from Bacillus megaterium that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450BM3 mutants often exhibit lower stability, thereby hindering their industrial application. This study demonstrated that the heme domain R47L/F87V/L188Q/E267V/F81I pentuple mutant (PM) is destabilized because of the disruption of hydrophobic contacts and salt bridge interactions. This was directly observed from crystal structures of PM in the presence and absence of ligands (palmitic acid and metyrapone). The instability of the tertiary structure and heme environment of substrate-free PM was confirmed by pulse proteolysis and circular dichroism, respectively. Binding of the inhibitor, metyrapone, significantly stabilized PM, but the presence of the native substrate, palmitic acid, had no effect. On the basis of high-temperature molecular dynamics simulations, the lid domain, β-sheet 1, and Cys ligand loop (a β-bulge segment connected to the heme) are the most labile regions and, thus, potential sites for stabilizing mutations. Possible approaches to stabilization include improvement of hydrophobic packing interactions in the lid domain and introduction of new salt bridges into β-sheet 1 and the heme region. An understanding of the molecular factors behind the loss of stability of P450BM3 variants therefore expedites site-directed mutagenesis studies aimed at developing thermostability.

  5. Synergistic effects of multiple treatments, and both DNA and RNA direct bindings on, green tea catechins.

    PubMed

    Kuzuhara, Takashi; Tanabe, Akitoshi; Sei, Yoshihisa; Yamaguchi, Kentaro; Suganuma, Masami; Fujiki, Hirota

    2007-08-01

    This article reviews two main topics: (1) the synergistic effects of multiple treatments with green tea catechin and (2) the direct binding of (-)-epigallocatechin gallate (EGCG) to both DNA and RNA molecules. Japanese drink green tea throughout the day, so we studied whether multiple treatments of cells with EGCG would enhance the expression of apoptosis-related genes, such as growth arrest and DNA damage-inducible gene (GADD153) and cyclin-dependent kinase inhibitor gene (p21(waf1)): The results suggest that the synergistic enhancement of both GADD153 and p21(waf1) gene expressions by multiple treatments plays a significant role in human cancer prevention with green tea beverage. Our previous observation-that nucleic acids extracted from catechin-treated cells are colored-allowed us to speculate that catechins directly interact with nucleic acids. Surface plasmon resonance assay (Biacore) indicated that four catechins, EGCG, (-)-epicatechin gallate (ECG), (+)-gallocatechin gallate (GCG), and (+)-catechin gallate (CG), bound to DNA oligomers. Cold spray ionization mass spectrometry (CSI-MS) analysis showed that one to three EGCG molecules bound to single-stranded 18 mers of DNA and RNA. Moreover, one or two molecules of EGCG bound to double-stranded AG:CT oligomers of various nucleotide lengths. Double-stranded DNA (dsDNA) oligomers were detected only as EGCG-bound forms at high temperature, whereas at low temperature both the free and bound forms were detected, suggesting that EGCG protects double-stranded DNA oligomers from double-stranded melting into single-stranded DNA. We assume that catechins accumulate in both double-stranded DNA and RNA molecules through multiple administrations of green tea beverage in in vivo, and that the accumulated green tea catechins play a significant role for human cancer prevention.

  6. A Review of the Receptor-Binding Properties of p-Synephrine as Related to Its Pharmacological Effects

    PubMed Central

    Stohs, Sidney J.; Preuss, Harry G.; Shara, Mohd

    2011-01-01

    Bitter orange (Citrus aurantium) extract and its primary protoalkaloid p-synephrine are used widely in weight loss/weight management and sports performance products. Because of structural similarities, the pharmacological effects of p-synephrine are widely assumed to be similar to those of ephedrine, m-synephrine (phenylephrine), and endogenous amine neurotransmitters as norepinephrine and epinephrine. However, small structural changes result in the receptor binding characteristics of these amines that are markedly different, providing a plausible explanation for the paucity of adverse effects associated with the wide-spread consumption of p-synephrine in the form of dietary supplements as well as in various Citrus foods and juices. This paper summarizes the adrenoreceptor binding characteristics of p-synephrine relative to m-synephrine, norepinephrine, and other amines as related to the observed pharmacological effects. PMID:21904645

  7. Molecular biology of beta-estradiol-estrogen receptor complex binding to estrogen response element and the effect on cell proliferation.

    PubMed

    Heger, Zbynek; Zitka, Ondrej; Krizkova, Sona; Beklova, Miroslava; Kizek, Rene; Adam, Vojtech

    2013-01-01

    Group of estrogen pollutants, where the highest estrogen activity is reported at estradiol, is characterized by the fact that even at very low concentrations have potential to cause xenoestrogenic effects. During exposure of excessive amounts of estradiols may be produced undesirable effects resulting in the feminization of males of water organisms. The presence of estradiols in drinking water implies also a risk for the human population in the form of cancers of endocrine systems, abnormalities in reproduction or dysfunctions of neuronal and immune system. Currently, the research is focused mainly to uncover the relationship between the estrogen receptors binding affinity with an estrogen response element and estradiol. In this review we summarized facts about molecular biological principles of β estradiol-estrogen receptor complex binding with estrogen response element and its successive effect on cancer genes expression.

  8. Effects of pH and ionic strength on the thermodynamics of human serum albumin-photosensitizer binding

    PubMed Central

    Jones, Cecil L.; Dickson, TiReJe; Hayes, Ronald; Thomas, Lana

    2013-01-01

    Fluorescence spectroscopy was used to measure the effects of pH and ionic strength on thermodynamic parameters governing the interaction of human serum albumin with zinc phthalocyanine tetrasulfonic acid. Fluorescence emission of zinc phthalocyanine increases at 686 nm with increasing concentrations of the protein. The non-linear correlation between protein concentration and emission of the photosensitizer was fitted using Chipman’s analysis to calculate the binding affinities. The standard enthalpy and entropy changes were estimated from van’t Hoff analysis of data that were acquired from temperature ramping studies. Results show that reaction is primarily driven by solution dynamics and that the change in enthalpy for the system becomes increasingly unfavorable with increasing pH and ionic strength. The effect of ionic strength on the entropy change for binding is shown to be significantly greater than the effects of pH. The interplay between entropy and enthalpy changes is demonstrated. PMID:24058218

  9. Effect of thymol on calcium handling in mammalian ventricular myocardium.

    PubMed

    Szentandrássy, Norbert; Szigeti, Gyula; Szegedi, Csaba; Sárközi, Sándor; Magyar, János; Bányász, Tamás; Csernoch, László; Kovács, László; Nánási, Péter P; Jóna, István

    2004-01-02

    Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC(50) = 297 +/- 12 microM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V(max) = 0.47 +/- 0.04 nmol s(-1), EC(50) = 258 +/- 21 microM, Hill coefficient = 3.0 +/- 0.54), and decreased the activity of the calcium pump (EC(50) = 253 +/- 4.7 microM, Hill coefficient = 1.62 +/- 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, this effect was significant from 50 microM, whereas the thymol-induced calcium release only from 100 microM. In single ryanodine receptors incorporated into artificial lipid bilayer thymol induced long lasting openings, having mean open times increased with 3 orders of magnitude, however, the specific conductance of the channel remained unaltered. This effect of thymol was not voltage-dependent and failed to prevent the binding of ryanodine. In conclusion, the negative inotropic action of thymol can be explained by reduction in calcium content of the sarcoplasmic reticulum due to the combination of the thymol-induced calcium release and inhibition of the calcium pump. The calcium-sensitizer effect, observed at lower thymol concentrations, indicates that thymol is likely to interact with the contractile machinery also.

  10. The consequences of fruit and vegetable fibre fermentation on their binding capacity for MeIQx and the effects of soluble fibre sources on the binding affinity of wheat bran preparations.

    PubMed

    Ryden, P; Robertson, J A

    1995-08-01

    Fruits and vegetables provide dietary fibre some of which is partly soluble in the upper gut; in the colon it is highly fermentable. Using alcohol-insoluble residues prepared from a range of fruits and vegetables the effects of fermentation on the changes in composition and binding capacity have been assessed for the hydrophobic mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). Fermentation was extensive and resulted in destruction of most of the pectic polysaccharides. Of the unfermented vegetable fibre only cabbage had a measurable hydrophobic binding capacity. The binding capacities of unfermented apple, carrot and sugar beet were negligible. After fermentation, binding capacities, (per mg of fermented residue), increased. Although fermented cabbage was found to have the highest capacity of the fruit and vegetable fibres this remained less than the least effective of unfermented wheat bran samples which had a relatively high affinity for MeIQx. Mucin inhibited the binding of MeIQx to wheat bran fibre but apple fibre did not. The results show that the contribution of fruit and vegetable fibre to a hydrophobic binding matrix in the colon is insignificant and the suggested harmful effect of fruit and vegetable fibre, maintaining hydrophobic mutagens in solution, can be prevented by the presence of wheat bran fibre.

  11. Effects of organic solvents and substrate binding on trypsin in acetonitrile and hexane media.

    PubMed

    Meng, Yanyan; Yuan, Yuan; Zhu, Yanyan; Guo, Yanzhi; Li, Menglong; Wang, Zhimeng; Pu, Xuemei; Jiang, Lin

    2013-09-01

    In this work, we used molecular dynamic (MD) simulation to study trypsin with and without a six-amino-acid peptide bound in three different solvents (water, acetonitrile and hexane) in order to provide molecular information for well understanding the structure and function of enzymes in non-aqueous media. The results show that the enzyme is more compact and less native-like in hexane than in the other two polar solvents. The substrate could stabilize the native protein structure in the two polar media, but not in the non-polar hexane. There are no significant differences in the conformation of the S1 pocket upon the substrate binding in water and acetonitrile media while a reverse behavior is observed in hexane media, implying a possible induced fit binding mechanism in the non-polar media. The substrate binding enhances the stability of catalytic H-bond network since it could expel the solvent molecules from the active site. The enzyme and the substrate appear to be more appropriate to the reactive conformation in the organic solvents compared with aqueous solution. There is much greater substrate binding strength in hexane media than the water and acetonitrile ones since the polar solvent significantly weakens electrostatic interactions, which are observed to be the main driving force to the binding. In addition, some residues of the S1 pocket could remain favorable contribution to the binding despite the solvent change, but with differences in the contribution extent, the number and the type of residues between the three media.

  12. Effects of DNA-binding drugs on T4 DNA ligase.

    PubMed Central

    Montecucco, A; Pedrali-Noy, G; Spadari, S; Lestingi, M; Ciarrocchi, G

    1990-01-01

    A number of DNA intercalating and externally binding drugs have been found to inhibit nick sealing, cohesive and blunt end ligation, AMP-dependent DNA topoisomerization and EDTA-induced DNA nicking mediated by bacteriophage T4 DNA ligase. The inhibition seems to arise from drug-substrate interaction so that formation of active DNA-Mg2(+)-AMP-enzyme complex is impaired while assembled and active complexes are not disturbed by drug binding to the substrate. Images Fig. 2. Fig. 4. Fig. 5. PMID:2156493

  13. Binding enthalpy calculations for a neutral host-guest pair yield widely divergent salt effects across water models.

    PubMed

    Gao, Kaifu; Yin, Jian; Henriksen, Niel M; Fenley, Andrew T; Gilson, Michael K

    2015-10-13

    Dissolved salts are a part of the physiological milieu and can significantly influence the kinetics and thermodynamics of various biomolecular processes, such as binding and catalysis; thus, it is important for molecular simulations to reliably describe their effects. The present study uses a simple, nonionized host-guest model system to study the sensitivity of computed binding enthalpies to the choice of water and salt models. Molecular dynamics simulations of a cucurbit[7]uril host with a neutral guest molecule show striking differences in the salt dependency of the binding enthalpy across four water models, TIP3P, SPC/E, TIP4P-Ew, and OPC, with additional sensitivity to the choice of parameters for sodium and chloride. In particular, although all of the models predict that binding will be less exothermic with increasing NaCl concentration, the strength of this effect varies by 7 kcal/mol across models. The differences appear to result primarily from differences in the number of sodium ions displaced from the host upon binding the guest rather than from differences in the enthalpy associated with this displacement, and it is the electrostatic energy that contributes most to the changes in enthalpy with increasing salt concentration. That a high sensitivity of salt affecting the choice of water model, as observed for the present host-guest system despite it being nonionized, raises issues regarding the selection and adjustment of water models for use with biological macromolecules, especially as these typically possess multiple ionized groups that can interact relatively strongly with ions in solution.

  14. Effects of norepinephrine transporter gene variants on NET binding in ADHD and healthy controls investigated by PET.

    PubMed

    Sigurdardottir, Helen L; Kranz, Georg S; Rami-Mark, Christina; James, Gregory M; Vanicek, Thomas; Gryglewski, Gregor; Kautzky, Alexander; Hienert, Marius; Traub-Weidinger, Tatjana; Mitterhauser, Markus; Wadsak, Wolfgang; Hacker, Marcus; Rujescu, Dan; Kasper, Siegfried; Lanzenberger, Rupert

    2016-03-01

    Attention deficit hyperactivity disorder (ADHD) is a heterogeneous disorder with a strong genetic component. The norepinephrine transporter (NET) is a key target for ADHD treatment and the NET gene has been of high interest as a possible modulator of ADHD pathophysiology. Therefore, we conducted an imaging genetics study to examine possible effects of single nucleotide polymorphisms (SNPs) within the NET gene on NET nondisplaceable binding potential (BPND ) in patients with ADHD and healthy controls (HCs). Twenty adult patients with ADHD and 20 HCs underwent (S,S)-[18F]FMeNER-D2 positron emission tomography (PET) and were genotyped on a MassARRAY MALDI-TOF platform using the Sequenom iPLEX assay. Linear mixed models analyses revealed a genotype-dependent difference in NET BPND between groups in the thalamus and cerebellum. In the thalamus, a functional promoter SNP (-3081 A/T) and a 5'-untranslated region (5'UTR) SNP (-182 T/C), showed higher binding in ADHD patients compared to HCs depending on the major allele. Furthermore, we detected an effect of genotype in HCs, with major allele carriers having lower binding. In contrast, for two 3'UTR SNPs (*269 T/C, *417 A/T), ADHD subjects had lower binding in the cerebellum compared to HCs depending on the major allele. Additionally, symptoms of hyperactivity and impulsivity correlated with NET BPND in the cerebellum depending on genotype. Symptoms correlated positively with cerebellar NET BPND for the major allele, while symptoms correlated negatively to NET BPND in minor allele carriers. Our findings support the role of genetic influence of the NE system on NET binding to be pertubated in ADHD.

  15. Effects of norepinephrine transporter gene variants on NET binding in ADHD and healthy controls investigated by PET

    PubMed Central

    Sigurdardottir, Helen L.; Kranz, Georg S.; Rami‐Mark, Christina; James, Gregory M.; Vanicek, Thomas; Gryglewski, Gregor; Kautzky, Alexander; Hienert, Marius; Traub‐Weidinger, Tatjana; Mitterhauser, Markus; Wadsak, Wolfgang; Hacker, Marcus; Rujescu, Dan; Kasper, Siegfried

    2016-01-01

    Abstract Attention deficit hyperactivity disorder (ADHD) is a heterogeneous disorder with a strong genetic component. The norepinephrine transporter (NET) is a key target for ADHD treatment and the NET gene has been of high interest as a possible modulator of ADHD pathophysiology. Therefore, we conducted an imaging genetics study to examine possible effects of single nucleotide polymorphisms (SNPs) within the NET gene on NET nondisplaceable binding potential (BPND) in patients with ADHD and healthy controls (HCs). Twenty adult patients with ADHD and 20 HCs underwent (S,S)‐[18F]FMeNER‐D2 positron emission tomography (PET) and were genotyped on a MassARRAY MALDI‐TOF platform using the Sequenom iPLEX assay. Linear mixed models analyses revealed a genotype‐dependent difference in NET BPND between groups in the thalamus and cerebellum. In the thalamus, a functional promoter SNP (−3081 A/T) and a 5′‐untranslated region (5′UTR) SNP (−182 T/C), showed higher binding in ADHD patients compared to HCs depending on the major allele. Furthermore, we detected an effect of genotype in HCs, with major allele carriers having lower binding. In contrast, for two 3′UTR SNPs (*269 T/C, *417 A/T), ADHD subjects had lower binding in the cerebellum compared to HCs depending on the major allele. Additionally, symptoms of hyperactivity and impulsivity correlated with NET BPND in the cerebellum depending on genotype. Symptoms correlated positively with cerebellar NET BPND for the major allele, while symptoms correlated negatively to NET BPND in minor allele carriers. Our findings support the role of genetic influence of the NE system on NET binding to be pertubated in ADHD. Hum Brain Mapp 37:884–895, 2016. © 2015 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc. PMID:26678348

  16. Effect of Magnetic Field and Shell Thickness on Binding Energies of a ZnSe/ZnS Core Shell Quantum Dot

    NASA Astrophysics Data System (ADS)

    Bhat, Bashir Mohi ud din; Parvaiz, Muhammad Shunaid; Sen, Pratima

    2017-02-01

    We investigated the effect of external magnetic field and shell thickness on the binding energies of a ZnSe/ZnS core shell quantum dot. The binding energies were calculated using the variational method within the effective mass approximation and confinement potential. The binding energy of the 2 s and 2 p + states was found to increase with magnetic field. However, the 2 p 0 state was found to be independent of the magnetic field at a shell thickness of 0.5 nm. Degeneracy of the lifted 2 p states was found to occur. The results also showed that the electron binding energy increases at the outset with the increasing shell thickness, and at larger shell thicknesses, the binding energy saturates. The binding energy was found to be decreasing with increasing core diameter and becomes appreciably smaller at core radius of 0.42 nm. The observed results were compared with the previously reported results.

  17. Small chemicals with inhibitory effects on PtdIns(3,4,5)P3 binding of Btk PH domain.

    PubMed

    Yoon, Youngdae

    2014-05-15

    Phosphatidylinositol-3,4-5-triphosphates (PtdIns(3,4,5)P3) formed by phosphoinositide-3-kinase (PI3K) had been known as a signaling molecule that plays important roles in diverse cellular processes such as cell signaling, metabolism, cell differentiation, and apoptosis. PtdIns(3,4,5)P3 regulates diverse cellular processes by recruiting effector proteins to the specific cellular locations for correct functions. In this study, we reported the inhibitory effect of small chemicals on the interaction between PtdIns(3,4,5)P3-Btk PH domain. Small chemicals were synthesized based on structural similarity of PtdInsP head-groups, and tested the inhibitory effects in vitro via surface plasmon resonance (SPR). As a result, the chemical 8 showed highest inhibitory effect with 17μM of IC50 value. To elucidate diverse inhibitory effects of different small chemicals we employed in silico docking experiment using molecular modeling and simulation. The result of docking experiments showed chemical 8 has more hydrogen bonding with the residues in PtdIns(3,4,5)P3 binding site of Btk PH domain than others. Overall, our studies demonstrate the efficient approach to develop lipid binding inhibitors, and further we can use these chemicals to regulate effector proteins. In addition, our study would provide new insight that lipid binding domain may be the attractive therapeutic targets to treat severe human diseases.

  18. Ligand Binding and Activation of PPARγ by Firemaster® 550: Effects on Adipogenesis and Osteogenesis in Vitro

    PubMed Central

    Pillai, Hari K.; Fang, Mingliang; Beglov, Dmitri; Kozakov, Dima; Vajda, Sandor; Stapleton, Heather M.; Webster, Thomas F.

    2014-01-01

    Background: The use of alternative flame retardants has increased since the phase out of pentabromodiphenyl ethers (pentaBDEs). One alternative, Firemaster® 550 (FM550), induces obesity in rats. Triphenyl phosphate (TPP), a component of FM550, has a structure similar to that of organotins, which are obesogenic in rodents. Objectives: We tested the hypothesis that components of FM550 are biologically active peroxisome proliferator-activated receptor γ (PPARγ) ligands and estimated indoor exposure to TPP. Methods: FM550 and its components were assessed for ligand binding to and activation of human PPARγ. Solvent mapping was used to model TPP in the PPARγ binding site. Adipocyte and osteoblast differentiation were assessed in bone marrow multipotent mesenchymal stromal cell models. We estimated exposure of children to TPP using a screening-level indoor exposure model and house dust concentrations determined previously. Results: FM550 bound human PPARγ, and binding appeared to be driven primarily by TPP. Solvent mapping revealed that TPP interacted with binding hot spots within the PPARγ ligand binding domain. FM550 and its organophosphate components increased human PPARγ1 transcriptional activity in a Cos7 reporter assay and induced lipid accumulation and perilipin protein expression in BMS2 cells. FM550 and TPP diverted osteogenic differentiation toward adipogenesis in primary mouse bone marrow cultures. Our estimates suggest that dust ingestion is the major route of exposure of children to TPP. Conclusions: Our findings suggest that FM550 components bind and activate PPARγ. In addition, in vitro exposure initiated adipocyte differentiation and antagonized osteogenesis. TPP likely is a major contributor to these biological actions. Given that TPP is ubiquitous in house dust, further studies are warranted to investigate the health effects of FM550. Citation: Pillai HK, Fang M, Beglov D, Kozakov D, Vajda S, Stapleton HM, Webster TF, Schlezinger JJ. 2014

  19. Effect of atrazine and fenitrothion at no-observed-effect-levels (NOEL) on amphibian and mammalian corticosterone-binding-globulin (CBG).

    PubMed

    Hernández, Sandra E; Sernia, Conrad; Bradley, Adrian J

    2014-11-04

    This study determines the effect of atrazine and fenitrothion no-observed-effect-levels (NOEL) on the binding of corticosterone (B) to corticosterone-binding-globulin (CBG) in an amphibian and a mammal. Plasma from five cane toads and five Wistar rats was exposed to atrazine and fenitrothion at the NOEL approved for Australian fresh water residues and by the World Health Organization (WHO). The concentration required to displace 50% (IC50) of B binding to CBG was determined by a competitive microdialysis protein assay. Competition studies showed that both atrazine and fenitrothion at NOEL are able to compete with B for CBG binding sites in toad and rat plasma. The IC50 levels for atrazine in toads and rats were 0.004 nmol/l and 0.09 nmol/l respectively. In the case of fenitrothion the IC50 level found in toads was 0.007 nmol/l, and 0.025 nmol/l in rats. Plasma dilution curves showed parallelism with the curve of B, demonstrating that these agro-chemicals are competitively inhibiting binding to CBG. The displacement of B by atrazine and fenitrothion would affect the total:free ratio of B and consequently disrupt the normal stress response. This is the first time that the potential disruptive effect of atrazine and fenitrothion on B-CBG interaction at the NOELs has been demonstrated in amphibian and mammalian models.

  20. Snyder-Robinson Syndrome: Rescuing the Disease-Causing Effect of G56S mutant by Small Molecule Binding

    NASA Astrophysics Data System (ADS)

    Zhang, Zhe; Martiny, Virginie; Lagorce, David; Alexov, Emil; Miteva, Maria; Clemson University Team; Université Paris Diderot Team

    2013-03-01

    Snyder-Robinson Syndrome (SRS) is an X-linked mental retardation disorder, which is caused by defects in a particular gene coding for the spermine synthase (SMS) protein. Among the missense mutations known to be disease-causing is the G56S, which is positioned at the interface of the SMS homo-dimer. Previous computational and experimental investigations have shown that G56S mutation destabilizes the homo-dimer and thus greatly reduces the SMS enzymatic activity. In this study, we explore the possibility of mitigating the effect of G56S mutation by binding small molecules to suitable pockets around the mutation site. It is done by combined efforts of molecular dynamics simulations and in silico screening. The binding of selected molecules was calculated to fully compensate the effect of the mutation and rescue the wild type dimer affinity. This work was supported by NIH, NLM grant. No. 1R03LM009748

  1. Effects of cardiac Myosin binding protein-C on actin motility are explained with a drag-activation-competition model.

    PubMed

    Walcott, Sam; Docken, Steffen; Harris, Samantha P

    2015-01-06

    Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin.

  2. Effects of Cardiac Myosin Binding Protein-C on Actin Motility Are Explained with a Drag-Activation-Competition Model

    PubMed Central

    Walcott, Sam; Docken, Steffen; Harris, Samantha P.

    2015-01-01

    Although mutations in cardiac myosin binding protein-C (cMyBP-C) cause heart disease, its role in muscle contraction is not well understood. A mechanism remains elusive partly because the protein can have multiple effects, such as dual biphasic activation and inhibition observed in actin motility assays. Here we develop a mathematical model for the interaction of cMyBP-C with the contractile proteins actin and myosin and the regulatory protein tropomyosin. We use this model to show that a drag-activation-competition mechanism accurately describes actin motility measurements, while models lacking either drag or competition do not. These results suggest that complex effects can arise simply from cMyBP-C binding to actin. PMID:25564844

  3. The Effect of Macromolecular Crowding, Ionic Strength and Calcium Binding on Calmodulin Dynamics

    PubMed Central

    Wang, Qian; Liang, Kao-Chen; Czader, Arkadiusz; Waxham, M. Neal; Cheung, Margaret S.

    2011-01-01

    The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM, we combined methods of computer simulation and experiments based on circular dichroism (CD) to investigate the structural characteristics of CaM that influence its target recognition in crowded cell-like conditions. We developed a unique multiscale solution of charges computed from quantum chemistry, together with protein reconstruction, coarse-grained molecular simulations, and statistical physics, to represent the charge distribution in the transition from apoCaM to holoCaM upon calcium binding. Computationally, we found that increased levels of macromolecular crowding, in addition to calcium binding and ionic strength typical of that found inside cells, can impact the conformation, helicity and the EF hand orientation of CaM. Because EF hand orientation impacts the affinity of calcium binding and the specificity of CaM's target selection, our results may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells. PMID:21829336

  4. The Cognitive Representation of Intending Not to Act: Evidence for Specific Non-Action-Effect Binding

    ERIC Educational Resources Information Center

    Kuhn, Simone; Brass, Marcel

    2010-01-01

    The question how we represent voluntary action on a cognitive level has recently become of increasing interest to researchers studying motor control. However, so far it has been neglected how we represent the voluntary omission of an action. In our attempt to investigate the representation of voluntary non-actions we demonstrated binding effects…

  5. Effects of heat treatment on oil-binding ability of rice flour.

    PubMed

    Tabara, Aya; Nakagawa, Mariko; Ushijima, Yuki; Matsunaga, Kotaro; Seguchi, Masaharu

    2015-01-01

    Heat-treated (120 °C for 120 min) rice flour showed high affinity to oil (oil-binding ability). This oil-binding ability could be observed by shaking the heat-treated rice flour (2.0 g), oil (4.0 mL), and water (20 mL) vigorously in a test tube, and the oil bound to the rice flour sank into the water. To examine the time-dependent levels of the oil-binding ability, rice flour was heat-treated at 120 °C for 10, 20, 40, 60, and 120 min, and the precipitated volume of oil/rice flour complex increased with an increase of the heating time. The oil-binding ability of the rice flour was not affected by the treatments with diethyl ether or boiled chloroform/methanol (2:1) solutions, which suggested no relationship to the oil in the rice flour, but was lost upon alkali (0.2% NaOH solution) or pepsin treatment, which suggested its relationship to the rice proteins.

  6. Milk matrix effects on antibody binding analyzed by elisa and biolayer interferometry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biolayer interferometry (BLI) was employed to study the impact of the milk matrix on the binding of ricin to asialofetuin (ASF) and to antibodies. This optical sensing platform utilized ligands immobilized covalently or via biotin-streptavidin linkage, and the results were compared to those obtained...

  7. Effect of trastuzumab interchain disulfide bond cleavage on Fcγ receptor binding and antibody-dependent tumour cell phagocytosis.

    PubMed

    Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Ootsubo, Michiko; Izawa, Ken-ichi; Kohroki, Junya; Masuho, Yasuhiko

    2016-01-01

    The Fc domain of human IgG1 binds to Fcγ receptors (FcγRs) to induce effector functions such as phagocytosis. There are four interchain disulfide bonds between the H and L chains. In this study, the disulfide bonds within the IgG1 trastuzumab (TRA), which is specific for HER2, were cleaved by mild S-sulfonation or by mild reduction followed by S-alkylation with three different reagents. The cleavage did not change the binding activities of TRA to HER2-bearing SK-BR-3 cells. The binding activities of TRA to FcγRIIA and FcγRIIB were greatly enhanced by modification with mild reduction and S-alkylation with ICH2CONH2 or N-(4-aminophenyl) maleimide, while the binding activities of TRA to FcγRI and FcγRIIIA were decreased by any of the four modifications. However, the interchain disulfide bond cleavage by the different modifications did not change the antibody-dependent cell-mediated phagocytosis (ADCP) of SK-BR-3 cells by activated THP-1 cells. The order of FcγR expression levels on the THP-1 cells was FcγRII > FcγRI > FcγRIII and ADCP was inhibited by blocking antibodies against FcγRI and FcγRII. These results imply that the effect of the interchain disulfide bond cleavage on FcγRs binding and ADCP is dependent on modifications of the cysteine residues and the FcγR isotypes.

  8. Structural studies of the effect that dimethyl sulfoxide (DMSO) has on cisplatin and carboplatin binding to histidine in a protein.

    PubMed

    Tanley, Simon W M; Schreurs, Antoine M M; Kroon-Batenburg, Loes M J; Meredith, Joanne; Prendergast, Richard; Walsh, Danielle; Bryant, Patrick; Levy, Colin; Helliwell, John R

    2012-05-01

    The anticancer complexes cisplatin and carboplatin target the DNA major groove, forming intrastrand and interstrand cross-links between guanine bases through their N7 atoms, causing distortion of the DNA helix and apoptotic cell death. A major side effect of these drugs is toxicity, which is caused via binding to many proteins in the body. A range of crystallographic studies have been carried out involving the cocrystallization of hen egg-white lysozyme (HEWL) as a test protein with cisplatin and carboplatin in aqueous and dimethyl sulfoxide (DMSO) conditions. Different cryoprotectants, glycerol and Paratone, were used for each of the cisplatin and carboplatin cocrystallization cases, while silicone oil was used for studies involving N-acetylglucosamine (NAG). Both cisplatin and carboplatin do not bind to HEWL in aqueous media on the timescales of the conditions used here, but upon addition of DMSO two molecules of cisplatin or carboplatin bind either side of His15, which is the only His residue in lysozyme and is assumed to be an imidazolyl anion or a chemical resonance moiety, i.e. both imidazole N atoms are chemically reactive. To identify the platinum-peak positions in the 'with DMSO conditions', anomalous scattering maps were calculated as a cross-check with the F(o) - F(c) OMIT maps. Platinum-occupancy σ values were established using three different software programs in each case. The use of EVAL15 to process all of the diffraction data sets provided a consistent platform for a large ensemble of data sets for the various protein and platinum-compound model refinements with REFMAC5 and then SHELXTL. Overall, this extensive set of crystallization and cryoprotectant conditions allowed a systematic evaluation of cisplatin and carboplatin binding to lysozyme as a test protein via detailed X-ray crystal structure characterizations. DMSO is used as a super-solvent for drug delivery as it is deemed to cause no effect upon drug binding. However, these results show

  9. Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

    PubMed Central

    Wen, Yi; Dick, Robert A.

    2016-01-01

    ABSTRACT The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes. IMPORTANCE Retroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order

  10. Modulating Uranium Binding Affinity in Engineered Calmodulin EF-Hand Peptides: Effect of Phosphorylation

    PubMed Central

    Pardoux, Romain; Sauge-Merle, Sandrine; Lemaire, David; Delangle, Pascale; Guilloreau, Luc; Adriano, Jean-Marc; Berthomieu, Catherine

    2012-01-01

    To improve our understanding of uranium toxicity, the determinants of uranyl affinity in proteins must be better characterized. In this work, we analyzed the contribution of a phosphoryl group on uranium binding affinity in a protein binding site, using the site 1 EF-hand motif of calmodulin. The recombinant domain 1 of calmodulin from A. thaliana was engineered to impair metal binding at site 2 and was used as a structured template. Threonine at position 9 of the loop was phosphorylated in vitro, using the recombinant catalytic subunit of protein kinase CK2. Hence, the T9TKE12 sequence was substituted by the CK2 recognition sequence TAAE. A tyrosine was introduced at position 7, so that uranyl and calcium binding affinities could be determined by following tyrosine fluorescence. Phosphorylation was characterized by ESI-MS spectrometry, and the phosphorylated peptide was purified to homogeneity using ion-exchange chromatography. The binding constants for uranyl were determined by competition experiments with iminodiacetate. At pH 6, phosphorylation increased the affinity for uranyl by a factor of ∼5, from Kd = 25±6 nM to Kd = 5±1 nM. The phosphorylated peptide exhibited a much larger affinity at pH 7, with a dissociation constant in the subnanomolar range (Kd = 0.25±0.06 nM). FTIR analyses showed that the phosphothreonine side chain is partly protonated at pH 6, while it is fully deprotonated at pH 7. Moreover, formation of the uranyl-peptide complex at pH 7 resulted in significant frequency shifts of the νas(P-O) and νs(P-O) IR modes of phosphothreonine, supporting its direct interaction with uranyl. Accordingly, a bathochromic shift in νas(UO2)2+ vibration (from 923 cm−1 to 908 cm−1) was observed upon uranyl coordination to the phosphorylated peptide. Together, our data demonstrate that the phosphoryl group plays a determining role in uranyl binding affinity to proteins at physiological pH. PMID:22870263

  11. Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I

    PubMed Central

    1993-01-01

    Insulin-like growth factor binding proteins (IGFBPs) have been shown to serve as carrier proteins for the insulin-like growth factors (IGFs) and to modulate their biologic effects. Since extracellular matrix (ECM) has been shown to be a reservoir for IGF-I and IGF-II, we examined the ECM of cultured human fetal fibroblasts and found that IGFBP-5 was incorporated intact into ECM, while mostly inert proteolytic fragments were found in the medium. In contrast, two other forms of IGFBP that are secreted by these cells were either present in ECM in minimal amounts (IGFBP-3) or not detected (IGFBP-4). Likewise, when purified IGFBPs were incubated with ECM, IGFBP-5 bound preferentially. IGFBP-5 was found to bind to types III and IV collagen, laminin, and fibronectin. Increasing salt concentrations inhibited the binding of IGFBP-5 to ECM and accelerated the release of IGFBP-5 from ECM, suggesting an ionic basis for this interaction. ECM-associated IGFBP-5 had a sevenfold decrease in affinity for IGF-I compared to IGFBP-5 in solution. Furthermore, when IGFBP-5 was present in cell culture substrata, it potentiated the growth stimulatory effects of IGF- I on fibroblasts. When IGFBP-5 was present only in the medium, it was degraded to a 22-kD fragment and had no effect on IGF-I-stimulated growth. We conclude that IGFBP-5 is present in fibroblast ECM, where it is protected from degradation and can potentiate the biologic actions of IGF-I. These findings provide a molecular explanation for the association of the IGF's with the extracellular matrix, and suggest that the binding of the IGF's to matrix, via IGFBP-5, may be important in mediating the cellular growth response to these growth factors. PMID:7683690

  12. Importance of Many-Body Effects in the Kernel of Hemoglobin for Ligand Binding

    NASA Astrophysics Data System (ADS)

    Weber, Cédric; O'Regan, David D.; Hine, Nicholas D. M.; Littlewood, Peter B.; Kotliar, Gabriel; Payne, Mike C.

    2013-03-01

    We propose a mechanism for binding of diatomic ligands to heme based on a dynamical orbital selection process. This scenario may be described as bonding determined by local valence fluctuations. We support this model using linear-scaling first-principles calculations, in combination with dynamical mean-field theory, applied to heme, the kernel of the hemoglobin metalloprotein central to human respiration. We find that variations in Hund’s exchange coupling induce a reduction of the iron 3d density, with a concomitant increase of valence fluctuations. We discuss the comparison between our computed optical absorption spectra and experimental data, our picture accounting for the observation of optical transitions in the infrared regime, and how the Hund’s coupling reduces, by a factor of 5, the strong imbalance in the binding energies of heme with CO and O2 ligands.

  13. Effects of rare earth elements and REE-binding proteins on physiological responses in plants.

    PubMed

    Liu, Dongwu; Wang, Xue; Chen, Zhiwei

    2012-02-01

    Rare earth elements (REEs), which include 17 elements in the periodic table, share chemical properties related to a similar external electronic configuration. REEs enriched fertilizers have been used in China since the 1980s. REEs could enter the cell and cell organelles, influence plant growth, and mainly be bound with the biological macromolecules. REE-binding proteins have been found in some plants. In addition, the chlorophyll activities and photosynthetic rate can be regulated by REEs. REEs could promote the protective function of cell membrane and enhance the plant resistance capability to stress produced by environmental factors, and affect the plant physiological mechanism by regulating the Ca²⁺ level in the plant cells. The focus of present review is to describe how REEs and REE-binding proteins participate in the physiological responses in plants.

  14. Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction.

    PubMed Central

    Risse, B.; Stempfer, G.; Rudolph, R.; Möllering, H.; Jaenicke, R.

    1992-01-01

    Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size-exclusion high-performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 micrograms/mL, whereas the apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of < 20 micrograms/mL. In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect. Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of Ka = 2 x 10(7) M-1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 microM FAD

  15. Metal centre effects on HNO binding in porphyrins and the electronic origin: metal's electronic configuration, position in the periodic table, and oxidation state.

    PubMed

    Yang, Liu; Fang, Weihai; Zhang, Yong

    2012-04-21

    HNO binds to many different metals in organometallic and bioinorganic chemistry. To help understand experimentally observed metal centre effects, a quantum chemical investigation was performed, revealing clear general binding trends with respect to metal centre characteristics and the electronic origin for the first time.

  16. The moral ties that bind . . . Even to out-groups: the interactive effect of moral identity and the binding moral foundations.

    PubMed

    Smith, Isaac H; Aquino, Karl; Koleva, Spassena; Graham, Jesse

    2014-08-01

    Throughout history, principles such as obedience, loyalty, and purity have been instrumental in binding people together and helping them thrive as groups, tribes, and nations. However, these same principles have also led to in-group favoritism, war, and even genocide. Does adhering to the binding moral foundations that underlie such principles unavoidably lead to the derogation of out-group members? We demonstrated that for people with a strong moral identity, the answer is "no," because they are more likely than those with a weak moral identity to extend moral concern to people belonging to a perceived out-group. Across three studies, strongly endorsing the binding moral foundations indeed predicted support for the torture of out-group members (Studies 1a and 1b) and withholding of necessary help from out-group members (Study 2), but this relationship was attenuated among participants who also had a strong moral identity.

  17. Effective binding of a phosphatidylserine-targeting antibody to Ebola virus infected cells and purified virions.

    PubMed

    Dowall, S D; Graham, V A; Corbin-Lickfett, K; Empig, C; Schlunegger, K; Bruce, C B; Easterbrook, L; Hewson, R

    2015-01-01

    Ebola virus is responsible for causing severe hemorrhagic fevers, with case fatality rates of up to 90%. Currently, no antiviral or vaccine is licensed against Ebola virus. A phosphatidylserine-targeting antibody (PGN401, bavituximab) has previously been shown to have broad-spectrum antiviral activity. Here, we demonstrate that PGN401 specifically binds to Ebola virus and recognizes infected cells. Our study provides the first evidence of phosphatidylserine-targeting antibody reactivity against Ebola virus.

  18. Effects of continuous administration of paroxetine on ligand binding site and expression of serotonin transporter protein in mouse brain.

    PubMed

    Hirano, Kazufumi; Seki, Takahiro; Sakai, Norio; Kato, Yasuhiro; Hashimoto, Hisakuni; Uchida, Shinya; Yamada, Shizuo

    2005-08-16

    Selective serotonin reuptake inhibitors (SSRIs), such as paroxetine, are utilized in the treatment of depression and anxiety disorders. Although SSRIs potently interfere with the activity of brain serotonin transporter (SERT) after acute treatment, clinical improvement of psychiatric diseases is observed only after the repeated administration for several weeks (2-6 weeks). The present study was undertaken to investigate the effects of continuous administration of paroxetine on specific [3H]paroxetine binding sites and expression of SERT protein in mouse brain. Mice continuously and subcutaneously received paroxetine at doses of 2.67 or 13.3 mumol/kg/day for 21 days by using osmotic minipumps, and the steady-state plasma drug levels were within the range of reported concentrations in the clinical therapy. Continuous administration of paroxetine at theses doses produced significant (25-46%) reduction of [3H]paroxetine binding in each brain region (cerebral cortex, striatum, hippocampus, thalamus, midbrain) of mice. In Western blot analysis, expression levels of SERT protein in the thalamus and midbrain of mice were significantly (51% and 61%, respectively) decreased on day 21 after the implantation of minipumps at the higher dose. In conclusion, this study has firstly shown that continuous administration of paroxetine induces significant reduction of not only ligand binding sites of SERT but the protein expression level in mouse brain. Such down-regulation of SERT may partly underlie the therapeutic effect of long-term treatment with SSRIs in human.

  19. Effective reduction of non-specific binding by bovine serum albumin modified quantum dot surface for cell targeted imaging

    NASA Astrophysics Data System (ADS)

    Wang, Qian; Zhang, Min; Wang, Ling; Gu, Yueqing

    2014-09-01

    Exploiting the bright and colorful photophysical properties of semiconductor quantum dots (QDs) is an onward trend in biotechnology and QDs have been widely used as fluorescent probes in cell-targeted imaging. However, nonspecific binding to cellular membranes has been a major challenge. In this study, high quality cadmium telluride (CdTe) quantum dots with different particle sizes were prepared via hydrothermal method. The surface of the QDs was modified with the chemically reduced bovine serum albumin (BSA) for effective reduction of nonspecific binding in cell targeting. Here,the as-prepared QDs exhibits tunable photoluminescence (PL) emission between 525nm and 620 nm.BSA-coated QDs which also provide reaction sites for conjugation of targeting ligands is mainly achieved by multiple mercapto groups in BSA macromolecules as multidentate ligands and partially by ligand exchange interaction between BSA and QDs. The BSA-coated QDs, with an overwhelming majority of hydrodynamic diameter size of 15nm ,are found to exhibit strong fluorescent intensities. The cellular uptake and localization of QDs was studied using laser confocal scanning microscopy. The results indicated that the nonspecific cellular binding is effectively reduced by BSA-coated QDs, compared with the N-acetyl-L-cysteine (NAC) -coated CdTe QDs.

  20. Effect of Cordycepin-Enriched WIB801C from Cordyceps militaris Suppressing Fibrinogen Binding to Glycoprotein IIb/IIIa

    PubMed Central

    Lee, Dong-Ha; Kim, Hyun-Hong; Lim, Deok Hwi; Kim, Jong-Lae; Park, Hwa-Jin

    2015-01-01

    In this study, we investigated the effects of cordycepin-enriched (CE)-WIB801C, a n-butanol extract of Cordyceps militaris-hypha on collagen-stimulated platelet aggregation. CE-WIB801C dose dependently inhibited collagen-induced platelet aggregation, and had a synergistic effect together with cordycepin (W-cordycepin) from CE-WIB801C on the inhibition of collagen-induced platelet aggregation. CE-WIB801C and cordycepin stimulated the phosphorylation of VASP (Ser157) and the dephosphorylation of PI3K and Akt, and inhibited the binding of fibrinogen to glycoprotein IIb/IIIa (αIIb/β3) and the release of ATP and serotonin in collagen-induced platelet aggregation. A-kinase inhibitor Rp-8-Br-cAMPS reduced CE-WIB801C-, and cordycepin-increased VASP (Ser157) phosphorylation, and increased CE-WIB801C-, and cordycepin-inhibited the fibrinogen binding to αIIb/β3. Therefore, we demonstrate that CE-WIB801C-, and cordycepin-inhibited fibrinogen binding to αIIb/β3 are due to stimulation of cAMP-dependent phosphorylation of VASP (Ser157), and inhibition of PI3K/Akt phosphorylation. These results strongly indicate that CE-WIB801C and cordycepin may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease. PMID:25593645

  1. Action and perception in social contexts: intentional binding for social action effects

    PubMed Central

    Pfister, Roland; Obhi, Sukhvinder S.; Rieger, Martina; Wenke, Dorit

    2014-01-01

    The subjective experience of controlling events in the environment alters the perception of these events. For instance, the interval between one's own actions and their consequences is subjectively compressed—a phenomenon known as intentional binding. In two experiments, we studied intentional binding in a social setting in which actions of one agent prompted a second agent to perform another action. Participants worked in pairs and were assigned to a “leader” and a “follower” role, respectively. The leader's key presses triggered (after a variable interval) a tone and this tone served as go signal for the follower to perform a keypress as well. Leaders and followers estimated the interval between the leader's keypress and the following tone, or the interval between the tone and the follower's keypress. The leader showed reliable intentional binding for both intervals relative to the follower's estimates. These results indicate that human agents experience a pre-reflective sense of agency for genuinely social consequences of their actions. PMID:25228869

  2. [Effect of conditions of monoclonal antibody adsorption on antigen-binding activity].

    PubMed

    Tarakanova, Iu N; Dmitriev, D A; Massino, Iu S; Smirnova, M B; Segal, O L; Fartushnaia, O V; Iakovleva, D A; Koliaskina, G I; Lavrov, V F; Dmitriev, A D

    2012-01-01

    The dependence of the antigen-binding activity of immobilized antibodies on pH of a saturating buffer has been investigated. We analyzed 28 monoclonal antibodies (MCAs) produced by various hybridomas to three virus antigens, i.e., the nuclear p23 protein of hepatitis C virus (C core protein p23), p24 protein of HIV 1, and the surface antigen of hepatitis B virus (HBsAg). Antibodies were adsorbed on the surfaces of immune plates in acidic (pH 2.8), neutral (pH 7.5), and alkaline (pH 9.5) buffers. The binding of labeled antigens, i.e., biotinylated or conjugated with horseradish peroxidase, with immobilized antigens was tested. It was shown that 10 out of 28 analyzed MCAs (36%) considerably better preserved their antigen-binding activity if their passive adsorption was carried out on the surface of polystyrene plates in an acidic buffer (pH 2.8). This approach allowed constructing a highly sensitive sandwich method for HBsAg assay with a minimal reliably determined antigen concentration of 0.013-0.017 ng/ml. The described approach may be recommended for the optimization of sandwich methods and solid-phase competitive methods.

  3. Effect of acoustic field parameters on arc acoustic binding during ultrasonic wave-assisted arc welding.

    PubMed

    Xie, Weifeng; Fan, Chenglei; Yang, Chunli; Lin, Sanbao

    2016-03-01

    As a newly developed arc welding method, power ultrasound has been successfully introduced into arc and weld pool during ultrasonic wave-assisted arc welding process. The advanced process for molten metals can be realized by utilizing additional ultrasonic field. Under the action of the acoustic wave, the plasma arc as weld heat source is regulated and its characteristics make an obvious change. Compared with the conventional arc, the ultrasonic wave-assisted arc plasma is bound significantly and becomes brighter. To reveal the dependence of the acoustic binding force on acoustic field parameters, a two-dimensional acoustic field model for ultrasonic wave-assisted arc welding device is established. The influences of the radiator height, the central pore radius, the radiator radius, and curvature radius or depth of concave radiator surface are discussed using the boundary element method. Then the authors analyze the resonant mode by this relationship curve between acoustic radiation power and radiator height. Furthermore, the best acoustic binding ability is obtained by optimizing the geometric parameters of acoustic radiator. In addition, three concave radiator surfaces including spherical cap surface, paraboloid of revolution, and rotating single curved surface are investigated systematically. Finally, both the calculation and experiment suggest that, to obtain the best acoustic binding ability, the ultrasonic wave-assisted arc welding setup should be operated under the first resonant mode using a radiator with a spherical cap surface, a small central pore, a large section radius and an appropriate curvature radius.

  4. Effects of healthy ageing on precision and binding of object location in visual short term memory.

    PubMed

    Pertzov, Yoni; Heider, Maike; Liang, Yuying; Husain, Masud

    2015-03-01

    Visual short term memory (STM) declines as people get older, but the nature of this deterioration is not well understood. We tested 139 healthy subjects (19-83 years) who were first required to identify a previously seen object and then report its location using a touchscreen. Results demonstrated an age-related decline in both object identification and localization. Deterioration in localization performance was apparent even when only 1 item had to be remembered, worsening disproportionately with increasing memory load. Thus, age-dependent memory degradation cannot be explained simply by a decrease in the number of items that can be held in visual STM but rather by the precision with which they are recalled. More important, there was no evidence for a significant decrease in object-location binding with increasing age. Thus, although precision for object identity and location declines with age, the ability to associate object identity to its location seems to remain unimpaired. As it has been reported that binding deficits in STM might be the first cognitive signs of early Alzheimer's disease (AD), the finding that object-location binding processes are relatively intact with normal aging supports the possible suitability of using misbinding as an index measures for probing early diagnosis of AD.

  5. Ligand binding stepwise disrupts water network in thrombin: enthalpic and entropic changes reveal classical hydrophobic effect.

    PubMed

    Biela, Adam; Sielaff, Frank; Terwesten, Felix; Heine, Andreas; Steinmetzer, Torsten; Klebe, Gerhard

    2012-07-12

    Well-ordered water molecules are displaced from thrombin's hydrophobic S3/4-pocket by P3-varied ligands (Gly, d-Ala, d-Val, d-Leu to d-Cha with increased hydrophobicity and steric requirement). Two series with 2-(aminomethyl)-5-chlorobenzylamide and 4-amidinobenzylamide at P1 were examined by ITC and crystallography. Although experiencing different interactions in S1, they display almost equal potency. For both scaffolds the terminal benzylsulfonyl substituent differs in binding, whereas the increasingly bulky P3-groups address S3/4 pocket similarly. Small substituents leave the solvation pattern unperturbed as found in the uncomplexed enzyme while increasingly larger ones stepwise displace the waters. Medium-sized groups show patterns with partially occupied waters. The overall 40-fold affinity enhancement correlates with water displacement and growing number of van der Waals contacts and is mainly attributed to favorable entropy. Both Gly derivatives deviate from the series and adopt different binding modes. Nonetheless, their thermodynamic signatures are virtually identical with the homologous d-Ala derivatives. Accordingly, unchanged thermodynamic profiles are no reliable indicator for conserved binding modes.

  6. The effect of the cellulose-binding domain from Clostridium cellulovorans on the supramolecular structure of cellulose fibers.

    PubMed

    Ciolacu, Diana; Kovac, Janez; Kokol, Vanja

    2010-03-30

    The cellulose-binding domain (CBD) is the second important and the most wide-spread element of cellulase structure involved in cellulose transformation with a great structural diversity and a range of adsorption behavior toward different types of cellulosic materials. The effect of the CBD from Clostridium cellulovorans on the supramolecular structure of three different sources of cellulose (cotton cellulose, spruce dissolving pulp, and cellulose linters) was studied. Fourier-transform infrared spectroscopy (FTIR) was used to record amides I and II absorption bands of cotton cellulose treated with CBD. Structural changes as weakening and splitting of the hydrogen bonds within the cellulose chains after CBD adsorption were observed. The decrease of relative crystallinity index of the treated celluloses was confirmed by FTIR spectroscopy and X-ray diffraction (XRD). X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to confirm the binding of the CBD on the cellulose surface and the changing of the cellulose morphology.

  7. The effect of quantity and timing of brine addition on water binding and textural characteristics of cooked beef rolls.

    PubMed

    Pietrasik, Z; Shand, P J

    2003-10-01

    The combined influence of quantity and timing of water/sodium chloride/phosphate addition on quality characteristics of beef rolls processed with 25 or 50% brine level was investigated. Properties of beef rolls were determined by measuring hydration and textural characteristics. The higher level of brine addition (50%) had detrimental effects on product water binding and textural characteristics. Late addition of brine/water during tumbling (i.e. during the last hour) resulted in rolls which were less hard, chewy and elastic, and had poorer water holding properties. Addition of brine in two parts favourably affected hydration properties and thermal stability, yielding lower cooking loss and purge and higher WHC, irrespective of level of brine addition. It also increased hardness and chewiness and improved springiness, cohesiveness and bind of cooked beef rolls.

  8. Polycystin-2 (TRPP2) Regulation by Ca2+ Is Effected and Diversified by Actin-Binding Proteins

    PubMed Central

    Cantero, María del Rocío; Cantiello, Horacio F.

    2015-01-01

    Calcium regulation of Ca2+-permeable ion channels is an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the transient receptor potential superfamily, is a nonselective cation channel with Ca2+ permeability. The molecular mechanisms associated with PC2 regulation by Ca2+ remain ill-defined. We recently demonstrated that PC2 from human syncytiotrophoblast (PC2hst) but not the in vitro translated protein (PC2iv), functionally responds to changes in intracellular (cis) Ca2+. In this study we determined the regulatory effect(s) of Ca2+-sensitive and -insensitive actin-binding proteins (ABPs) on PC2iv channel function in a lipid bilayer system. The actin-bundling protein α-actinin increased PC2iv channel function in the presence of cis Ca2+, although instead was inhibitory in its absence. Conversely, filamin that shares actin-binding domains with α-actinin had a strong inhibitory effect on PC2iv channel function in the presence, but no effect in the absence of cis Ca2+. Gelsolin stimulated PC2iv channel function in the presence, but not the absence of cis Ca2+. In contrast, profilin that shares actin-binding domains with gelsolin, significantly increased PC2iv channel function both in the presence and absence of Ca2+. The distinct effect(s) of the ABPs on PC2iv channel function demonstrate that Ca2+ regulation of PC2 is actually mediated by direct interaction(s) with structural elements of the actin cytoskeleton. These data indicate that specific ABP-PC2 complexes would confer distinct Ca2+-sensitive properties to the channel providing functional diversity to the cytoskeletal control of transient receptor potential channel regulation. PMID:25954877

  9. Kinetics of CO binding to the haem domain of murine inducible nitric oxide synthase: differential effects of haem domain ligands.

    PubMed Central

    Stevenson, T H; Gutierrez, A F; Alderton, W K; Lian , L; Scrutton, N S

    2001-01-01

    The binding of CO to the murine inducible nitric oxide synthase (iNOS) oxygenase domain has been studied by laser flash photolysis. The effect of the (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)) cofactor L-arginine and several Type I L-arginine analogues/ligands on the rates of CO rebinding has been evaluated. The presence of BH(4) in the iNOS active site has little effect on the rebinding of protein-caged haem-CO pairs (geminate recombination), but decreases the bimolecular association rates 2-fold. Addition of L-arginine to the BH(4)-bound complex completely abolishes geminate recombination and results in a further 80-fold decrease in the overall rate of bimolecular association. Three of the Type I ligands, S-ethylisothiourea, L-canavanine and 2,5-lutidine, displaced the CO from the haem iron upon addition to the iNOS oxygenase domain. The Type I ligands significantly decreased the rate of bimolecular binding of CO to the haem iron after photolysis. Most of these ligands also completely abolished geminate recombination. These results are consistent with a relatively open distal pocket that allows CO to bind unhindered in the active site of murine iNOS in the absence of L-arginine or BH(4). In the presence of BH(4) and L-arginine, however, the enzyme adopts a more closed structure that can greatly reduce ligand access to the haem iron. These observations are discussed in terms of the known structure of iNOS haem domain and solution studies of ligand binding in iNOS and neuronal NOS isoenzymes. PMID:11485568

  10. Effects of serum, its protein and lipid extracts, and commercial serum proteins and lipid on the isolated frog heart.

    PubMed

    Singh, J; Hutton, T; Hussain, M; Waring, J J

    1991-01-01

    This study investigates the inotropic effects of serum, its protein and lipid extracts, and commercial serum proteins and lipid on the isolated, spontaneously-beating heart and superfused, hypodynamic ventricle of the frog. Serum taken from either man, horse, calf, frog, or rabbit evoked marked positive inotropic responses which were unaffected by cholinergic, serotonergic, and adrenergic receptor antagonists. Dialysed serum (dialisand) and void volume fractions from Sephadex G200-120 columns corresponding to large molecular weight constituents evoked marked positive inotropic responses. When serum was separated into fractions containing either proteins or lipids/lipoproteins by high-density ultracentrifugation or activated charcoal, both extracts evoked marked positive inotropic responses. Commercial serum globulins and serum containing a high proportion of immunoglobulins elicited large increases in contractile force, whereas serum albumin evoked a negative inotropic effect. Serum which was either boiled and/or treated with chymotrypsin to denature proteins also caused a marked increase in isometric twitch tension in the frog heart. Similar inotropic response was obtained with fractions of boiled serum eluted on columns of Sephadex G200-120. These fractions corresponded to molecular weight in the region of 60-70 kDa. However, the inotropic effect of boiled serum was abolished following pretreatment with lipase. Superfusion of frog hearts with commercial cardiolipin resulted in marked dose-dependent increases in contractile force. The results demonstrate the presence of at least two large molecular weight cardioactive principles in serum. These substances are comparable in size to constituents of serum proteins (e.g., globulins and immunoglobulins) and serum lipids/lipoproteins (e.g., cardiolipin) and may serve as physiological regulators of cardiac function.

  11. Potential effects of calcium binding protein S100A12 on severity evaluation and curative effect of severe acute pancreatitis.

    PubMed

    Feng, Zhang; Yinchu, Zhan; Yinsheng, Shi; Fengqing, Wu; Xiaoyang, Zhou; Jin, Li; Xiaofei, Gao

    2015-02-01

    Severe acute pancreatitis is a life threatening disease with a high rate of mortality, but its treatments are still controversial. The purpose of this study is to investigate the potential effects of calcium binding protein S100A12 on severity evaluation and curative effect of severe acute pancreatitis induced by caerulein and lipopolysaccharide in mice. Intraperitoneal injection of 50 μg/kg caerulein for seven times (every interval time was an hour) and intraperitoneal injection of 10 mg/kg lipopolysaccharide for once to establish acute pancreatitis mice models. One hundred sixty specific pathogen-free imprinting control region (ICR) female mice were randomly divided into the control group (group A, normal saline), the mild group (group B, caerulein), the severe group (group C, caerulein + lipopolysaccharide), and the intervention group (group D, S100A12 recombinant antibodies + caerulein + lipopolysaccharide); each group had 40 mice. We sampled the blood at 8, 12, and 24 h after the beginning of building animal models. In each period of time, we respectively detected the serum S100A12, amylase (AMY), C-reactive protein (CRP), interleukin (IL-1β, IL-6), and tumor necrosis factor (TNF-α) levels. In addition, we observed and scored the pancreas and lungs histopathology of the mice. In each same period of time compared with group C, serum AMY, CRP, IL-1β, IL-6, TNF-α levels of group D were significantly decreased (p < 0.05). In each same period of time compared with group B and group C, serum S100A12 concentration of group D was significantly decreased (p < 0.05), and the pancreas and lungs histopathology were also much improved. These observations demonstrate that S100A12 recombinant antibodies were able to significantly reduce the severity of acute pancreatitis induced by caerulein and lipopolysaccharide in mice. Serum S100A12 may serve as a useful marker for disease severity and curative effect in mice with severe acute pancreatitis.

  12. Effects of dopamine on the in vivo binding of dopamine D2 receptor radioligands in rat striatum.

    PubMed

    Moresco, R M; Loc'h, C; Ottaviani, M; Guibert, B; Leviel, V; Maziere, M; Fazio, F; Maziere, B

    1999-01-01

    The effects of moderate changes in extracellular dopamine concentrations on the in vivo binding of specific dopaminergic D2 radioligands with different affinities and kinetics were investigated in rats. Either [125I]NCQ298 (Kd = 19 pM), or [25I]iodolisuride (Kd = 0.27 nM) or [3H]raclopride (Kd = 1.5 nM) were administered intravenously (IV) to animals 1 h after the intraperitoneal (IP) injection of either alpha-methyl-p-tyrosine (AMPT) (250 mg/kg) or nomifensine (15 mg/kg), or saline. The kinetics of radioactivity concentration in the striatum, cerebellum, and plasma were measured for up to 4 h after [125I]NCQ298 or [125I]iodolisuride injection and up to 1.5 h after [3H]raclopride injection. For each tracer, the striatum-to-cerebellum radioactivity concentration ratios (S/C) and the binding potential (BP), calculated as the association to dissociation binding rate constant ratios (k3/k4), were assessed and related to the changes in extracellular dopamine concentration induced by drug treatments. Results show that S/C and BP of [3H]raclopride were significantly diminished by pretreatment with nomifensine, a drug that increases extracellular dopamine concentration. Nomifensine pretreatment induced no changes in the in vivo binding indexes of the high affinity [125I]NCQ298 and a slight but not significant decrease of the binding indexes of 125I]iodolisuride. Treatment with AMPT, which induced a 40% reduction in dopamine concentration, did not change [125I]NCQ298 binding indexes but slightly increased those of [3H]raclopride and [125I]iodolisuride. In conclusion, the change of dopamine concentration induces modification of radiotracer kinetics. Thus, the combined use of tracers with high and low affinities could allow us to obtain information both on receptor density and neurotransmitter release in vivo. However, as indicated by the [3H]raclopride study with AMPT, small changes in the concentration of intrasynaptic dopamine cannot be easily detected.

  13. Effect of pH on the Structure and DNA Binding of the FOXP2 Forkhead Domain.

    PubMed

    Blane, Ashleigh; Fanucchi, Sylvia

    2015-06-30

    Forkhead box P2 (FOXP2) is a transcription factor expressed in cardiovascular, intestinal, and neural tissues during embryonic development and is implicated in language development. FOXP2 like other FOX proteins contains a DNA binding domain known as the forkhead domain (FHD). The FHD interacts with DNA by inserting helix 3 into the major groove. One of these DNA-protein interactions is a direct hydrogen bond that is formed with His554. FOXP2 is localized in the nuclear compartment that has a pH of 7.5. Histidine contains an imidazole side chain in which the amino group typically has a pKa of ~6.5. It seems possible that pH fluctuations around 6.5 may result in changes in the protonation state of His554 and thus the ability of the FOXP2 FHD to bind DNA. To investigate the effect of pH on the FHD, both the structure and the binding affinity were studied in the pH range of 5-9. This was done in the presence and absence of DNA. The structure was assessed using size exclusion chromatography, far-UV circular dichroism, and intrinsic and extrinsic fluorescence. The results indicated that while pH did not affect the secondary structure in the presence or absence of DNA, the tertiary structure was pH sensitive and the protein was less compact at low pH. Furthermore, the presence of DNA caused the protein to become more compact at low pH and also had the potential to increase the dimerization propensity. Fluorescence anisotropy was used to investigate the effect of pH on the FOXP2 FHD DNA binding affinity. It was found that pH had a direct effect on binding affinity. This was attributed to the altered hydrogen bonding patterns upon protonation or deprotonation of His554. These results could implicate pH as a means of regulating transcription by the FOXP2 FHD, which may also have repercussions for the behavior of this protein in cancer cells.

  14. Effect of topical microbicides on infectious human immunodeficiency virus type 1 binding to epithelial cells.

    PubMed

    Roth, Susan; Monsour, Michael; Dowland, Amanda; Guenthner, Patricia C; Hancock, Kelly; Ou, Chin-Yi; Dezzutti, Charlene S

    2007-06-01

    Topical microbicides (cellulose acetate 1,2 benzene dicarboxylate [CAP], PRO 2000, SPL7013, and UC781) are being investigated to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1). These products were shown to prevent the transfer of infectious HIV-1 from urogenital and colorectal epithelial cell lines to peripheral blood mononuclear cells. However, it was unclear if the topical microbicides rendered the virus noninfectious and/or reduced the binding to the epithelial cells. To test this, epithelial cells were cultured with HIV-1 in the presence or absence of topical microbicides or their placebos. The cells were washed, RNA lysates were made, and real-time PCR was performed for HIV-1. PRO 2000 and SPL7013 significantly (P < 0.0001) reduced the amount of bound HIV-1 to the colorectal epithelial cell line across clades A, B, C, and CRF01-AE. While none of the products reduced the binding of HIV-1 clades A and C to the urogenital cell line, CAP, PRO 2000, and SPL7013 significantly (P binding of clades B and CRF01-AE. In general, PRO 2000 and SPL7013 placebos significantly (P < 0.0001) reduced the amount of bound HIV-1 but were less than the active products. UC781, its placebo, and hydroxyethyl cellulose (placebo for CAP) minimally affected the amount of bound HIV-1. These results suggest that rendering HIV-1 noninfectious may not correlate to the amount of HIV-1 bound to epithelial cells and possible shedding into mucosal secretions. Therefore, functional virological assays in addition to measuring viral RNA should be included when clinically evaluating topical microbicide use by infected persons.

  15. Effect of Topical Microbicides on Infectious Human Immunodeficiency Virus Type 1 Binding to Epithelial Cells▿

    PubMed Central

    Roth, Susan; Monsour, Michael; Dowland, Amanda; Guenthner, Patricia C.; Hancock, Kelly; Ou, Chin-Yi; Dezzutti, Charlene S.

    2007-01-01

    Topical microbicides (cellulose acetate 1,2 benzene dicarboxylate [CAP], PRO 2000, SPL7013, and UC781) are being investigated to reduce the sexual transmission of human immunodeficiency virus type 1 (HIV-1). These products were shown to prevent the transfer of infectious HIV-1 from urogenital and colorectal epithelial cell lines to peripheral blood mononuclear cells. However, it was unclear if the topical microbicides rendered the virus noninfectious and/or reduced the binding to the epithelial cells. To test this, epithelial cells were cultured with HIV-1 in the presence or absence of topical microbicides or their placebos. The cells were washed, RNA lysates were made, and real-time PCR was performed for HIV-1. PRO 2000 and SPL7013 significantly (P < 0.0001) reduced the amount of bound HIV-1 to the colorectal epithelial cell line across clades A, B, C, and CRF01-AE. While none of the products reduced the binding of HIV-1 clades A and C to the urogenital cell line, CAP, PRO 2000, and SPL7013 significantly (P ≤ 0.002) reduced the binding of clades B and CRF01-AE. In general, PRO 2000 and SPL7013 placebos significantly (P < 0.0001) reduced the amount of bound HIV-1 but were less than the active products. UC781, its placebo, and hydroxyethyl cellulose (placebo for CAP) minimally affected the amount of bound HIV-1. These results suggest that rendering HIV-1 noninfectious may not correlate to the amount of HIV-1 bound to epithelial cells and possible shedding into mucosal secretions. Therefore, functional virological assays in addition to measuring viral RNA should be included when clinically evaluating topical microbicide use by infected persons. PMID:17404008

  16. Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor

    PubMed Central

    Stylianou, DC; Auf der Maur, A; Kodack, DP; Henke, RT; Hohn, S; Toretsky, JA; Riegel, AT; Wellstein, A

    2013-01-01

    The tyrosine kinase receptor anaplastic lymphoma kinase (ALK) and its ligand, the growth factor pleiotrophin (PTN), are highly expressed during the development of the nervous system and have been implicated in the malignant progression of different tumor types. Here, we describe human single-chain variable fragment (scFv) antibodies that target the ligand-binding domain (LBD) in ALK and show the effect in vitro and in vivo. The ALK LBD was used as a bait in a yeast two-hybdrid system to select human scFv from a library with randomized complementarity-determining region 3 domains. Surface plasmon resonance showed high-affinity binding of the selected scFv. The anti-ALK scFv competed for binding of PTN to ALK in intact cells and inhibited PTN-dependent signal transduction through endogenous ALK. Invasion of an intact endothelial cell monolayer by U87MG human glioblastoma cells was inhibited by the anti-ALK scFv. In addition, the growth of established tumor xenografts in mice was reversed after the induction of the conditional expression of the anti-ALK scFv. In archival malignant brain tumors expression levels of ALK and PTN were found elevated and appear correlated with poor patient survival. This suggests a rate-limiting function of the PTN/ALK interaction that may be exploited therapeutically. PMID:19633684

  17. Effect of Reactor Turbulence on the Binding-Protein-Mediated Aspartate Transport System in Thin Wastewater Biofilms

    PubMed Central

    Eighmy, T. Taylor; Bishop, P. L.

    1985-01-01

    This research documents an effect of reactor turbulence on the ability of gram-negative wastewater biofilm bacteria to actively transport l-aspartate via a binding-protein-mediated transport system. Biofilms which were not preadapted to turbulence and which possessed two separate and distinct aspartate transport systems (systems 1 and 2) were subjected to a turbulent flow condition in a hydrodynamically defined closed-loop reactor system. A shear stress treatment of 3.1 N · m−2 for 10 min at a turbulent Reynolds number (Re = 11,297) inactivated the low-affinity, high-capacity binding-protein-mediated transport system (system 2) and resolved the high-affinity, low-capacity membrane-bound proton symport system (system 1). The Kt and Vmax values for the resolved system were statistically similar to Kt and Vmax values for system 1 when system 2 was inactivated either by osmotic shock or arsenate, two treatments which are known to inactivate binding-protein-mediated transport systems. We hypothesize that shear stress disrupts system 2 by deforming the outer membranes of the firmly adhered gram-negative bacteria. PMID:16346830

  18. Effects of perfusion pressure and insulin on (/sup 3/H) cytochalasin B (CB) binding to control and diabetic rat hearts

    SciTech Connect

    Pleta, M.; Chan, T.

    1987-05-01

    Using (/sup 3/H) CB, they attempted to quantitate the changes in the amount of glucose transporters in the plasma membrane (PM) and intracellular membranes (HSP) prepared from rat hearts perfused with insulin, under low and high pressure. Membranes isolated from non-perfused hearts showed a PM/HSP ratio of (0.593). Hearts perfused with low pressure showed a lower ratio of (0.474). Perfusion with insulin increased the ratio to (1.8), almost a 3-4 fold increase from low perfusion pressure. These data correlate with insulin effects in glucose transport and CB binding in the fat cells. High pressure perfusion increased the PM/HSP ratio by 1-2 fold. (/sup 3/H) 2-DG transport indicates a comparable increase in glucose uptake with high pressure, but with insulin only a 1.5 fold increase was observed. Initial data obtained from streptozotocin (STZ) injected diabetic rats indicate low CB binding in the PM fraction. Only insulin, but not high perfusion pressure increased PM/HSP ratio in the STZ-diabetic hearts. Their data imply that while both caused apparent translocation of glucose transporters, influences on cardiac glucose metabolism by work load are different. Furthermore, STZ induced diabetes affected only the high perfusion pressure-induced and not the insulin-stimulated change in CB binding.

  19. Effects of oxidation on copper-binding properties of Aβ1-16 peptide: a pulse radiolysis study.

    PubMed

    Ramteke, S N; Ginotra, Y P; Walke, G R; Joshi, B N; Kumbhar, A S; Rapole, S; Kulkarni, P P

    2013-12-01

    The reaction of hydroxyl radicals ((•)OH) with Aβ1-16 peptide was carried out using pulse radiolysis to understand the effect of oxidation of peptide on its copper-binding properties. This reaction produced oxidized, dimeric and trimeric Aβ1-16 peptide species. The formation of these products was established with the help of fluorescence spectroscopy and mass spectrometry. The mass spectral data indicate that the major site of oxidation is at His6, while the site for dimerization is at Tyr10. Diethyl pyrocarbonate-treated Aβ1-16 peptide did not produce any trimeric species upon oxidation with (•)OH. The quantitative chemical modification studies indicated that one of the three histidine residues is covalently modified during pulse radiolysis. The copper-binding studies of the oxidized peptide revealed that it has similar copper-binding properties as the unoxidized peptide. Further, the cytotoxicity studies point out that both oxidized and unoxidized Aβ1-16 peptide are equally efficient in producing free radicals in presence of copper and ascorbate that resulted in comparable cell death.

  20. Effects of nucleotide binding to LmrA: A combined MAS-NMR and solution NMR study.

    PubMed

    Hellmich, Ute A; Mönkemeyer, Leonie; Velamakanni, Saroj; van Veen, Hendrik W; Glaubitz, Clemens

    2015-12-01

    ABC transporters are fascinating examples of fine-tuned molecular machines that use the energy from ATP hydrolysis to translocate a multitude of substrates across biological membranes. While structural details have emerged on many members of this large protein superfamily, a number of functional details are still under debate. High resolution structures yield valuable insights into protein function, but it is the combination of structural, functional and dynamic insights that facilitates a complete understanding of the workings of their complex molecular mechanisms. NMR is a technique well-suited to investigate proteins in atomic resolution while taking their dynamic properties into account. It thus nicely complements other structural techniques, such as X-ray crystallography, that have contributed high-resolution data to the architectural understanding of ABC transporters. Here, we describe the heterologous expression of LmrA, an ABC exporter from Lactococcus lactis, in Escherichia coli. This allows for more flexible isotope labeling for nuclear magnetic resonance (NMR) studies and the easy study of LmrA's multidrug resistance phenotype. We use a combination of solid-state magic angle spinning (MAS) on the reconstituted transporter and solution NMR on its isolated nucleotide binding domain to investigate consequences of nucleotide binding to LmrA. We find that nucleotide binding affects the protein globally, but that NMR is also able to pinpoint local dynamic effects to specific residues, such as the Walker A motif's conserved lysine residue.

  1. Direct detection of thrombin binding to 8-bromodeoxyguanosine-modified aptamer: effects of modification on affinity and kinetics.

    PubMed

    Goji, Shou; Matsui, Jun

    2011-01-01

    The affinity of an 8-bromodeoxyguanosine- (8-BrdG-) substituted thrombin-binding aptamer (TBA-Br), which has the 1st and 10th guanosine residues replaced with 8-BrdG, was estimated using reflectometric interference spectroscopy (RIfS). When comparing TBA-Br with unmodified TBA (TBA-H), it was demonstrated that the modification effectively improved the affinity of TBA; dissociation constants (K(D)) of TBA-H and TBA-Br were 45.4 nM and 1.99 nM, respectively. These values, which were obtained by direct observation of thrombin binding using RIfS, have the same order of magnitude as those obtained in our previous study utilizing conformational changes in TBA to detect thrombin binding, thus confirming the validity of the obtained K(D) values. RIfS measurements also revealed that the 8-BrdG modification resulted in a lower dissociation rate constant (k(d)), which suggests that the enhancement of affinity can be attributed to the stabilization of the G-quadruplex structure on introduction of 8-BrdG.

  2. Dose dependent effects of platelet derived chondroitinsulfate A on the binding of CCL5 to endothelial cells

    PubMed Central

    Weingart, Christian; Nelson, Peter J; Krämer, Bernhard K; Mack, Matthias

    2008-01-01

    Background Chemokines immobilized on endothelial cells play a central role in the induced firm adhesion and transendothelial migration of leukocytes. Activation of platelets at sites of vascular injury is considered to support leukocyte adhesion and extravasation. However, activated platelets also secrete soluble glycosaminoglycans that can interfere with immobilization of chemokines. We therefore analyzed the impact of platelet derived glycosaminoglycans on the immobilization of the chemokine CCL5 (RANTES) on human microvascular endothelial cells and their influence on CCL5-CCR5 interactions. Results We confirm that undiluted serum in contrast to plasma decreases binding of CCL5 to endothelial cells. However, when lower concentrations of serum were used, CCL5-presentation on endothelial cells was markedly enhanced. This enhancement was neutralized if serum was digested with chondroinitase ABC. Using different chondroitinsulfate-subtypes we demonstrate that chondroitinsulfate A mediates the enhanced presentation of CCL5 on endothelial cells, whereas chondroitinsulfate B/C even at low concentrations block CCL5 binding. CCR5 downregulation on CCR5-transfected CHO cells or human monocytes is increased by preincubation of CCL5 with serum or chondroitinsulfate A. Conclusion We show that chondroitinsulfate A released from platelets increases the binding of chemokines to endothelial cells and supports receptor internalization in a dose dependent manner. These data help to understand the proinflammatory effects of activated platelets. PMID:19068144

  3. Inhibitory effect of pyridoxal 5'-phosphate on the DNA binding site of ATP-dependent deoxyribonuclease from Bacillus laterosporus.

    PubMed

    Fujiyoshi, T; Nakayama, J; Anai, M

    1981-04-01

    Bacillus laterosporus ATP-dependent deoxyribonuclease has been found to be inhibited by pyridoxal 5'-phosphate. The inhibition is specific for pyridoxal 5'-phosphate and pyridoxal which are required in relatively high concentrations. Pyridoxamine 5'-phosphate, pyridoxamine, and pyridoxine are ineffective. The inhibition is reversed by dilution or dialysis but can be changed to an irreversible inactivation by reduction of the enzyme . pyridoxal 5'-phosphate complex with sodium borohydride. The compound is a competitive inhibitor with respect to DNA but not ATP. Moreover, the presence of DNA substrate protects the enzyme against this inactivation but the presence of ATP shows no effect. The reduced enzyme . pyridoxal 5'-phosphate complex displays a new absorption maximum at 325 nm and a fluorescence emission at 390-400 nm when excited at 325 nm which are characteristic for epsilon-N-(phosphopyridoxyl)lysine. Thus, B. laterosporus DNase appears to have an essential lysine residue at the DNA binding site of the enzyme, and the enzyme possess two different active sites, a DNA binding site and an ATP binding site.

  4. Mechanism and rates of exchange of L7/L12 between ribosomes and the effects of binding EF-G.

    PubMed

    Deroo, Stéphanie; Hyung, Suk-Joon; Marcoux, Julien; Gordiyenko, Yuliya; Koripella, Ravi Kiran; Sanyal, Suparna; Robinson, Carol V

    2012-06-15

    The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.

  5. Effects of the endogenous cannabinoid anandamide on voltage-dependent sodium and calcium channels in rat ventricular myocytes

    PubMed Central

    Al Kury, Lina T; Voitychuk, Oleg I; Yang, Keun-Hang Susan; Thayyullathil, Faisal T; Doroshenko, Petro; Ramez, Ali M; Shuba, Yaroslav M; Galadari, Sehamuddin; Howarth, Frank Christopher; Oz, Murat

    2014-01-01

    BACKGROUND AND PURPOSE The endocannabinoid anandamide (N-arachidonoyl ethanolamide; AEA) exerts negative inotropic and antiarrhythmic effects in ventricular myocytes. EXPERIMENTAL APPROACH Whole-cell patch-clamp technique and radioligand-binding methods were used to analyse the effects of anandamide in rat ventricular myocytes. KEY RESULTS In the presence of 1–10 μM AEA, suppression of both Na+ and L-type Ca2+ channels was observed. Inhibition of Na+ channels was voltage and Pertussis toxin (PTX) – independent. Radioligand-binding studies indicated that specific binding of [3H] batrachotoxin (BTX) to ventricular muscle membranes was also inhibited significantly by 10 μM metAEA, a non-metabolized AEA analogue, with a marked decrease in Bmax values but no change in Kd. Further studies on L-type Ca2+ channels indicated that AEA potently inhibited these channels (IC50 0.1 μM) in a voltage- and PTX-independent manner. AEA inhibited maximal amplitudes without affecting the kinetics of Ba2+ currents. MetAEA also inhibited Na+ and L-type Ca2+ currents. Radioligand studies indicated that specific binding of [3H]isradipine, was inhibited significantly by metAEA. (10 μM), changing Bmax but not Kd. CONCLUSION AND IMPLICATIONS Results indicate that AEA inhibited the function of voltage-dependent Na+ and L-type Ca2+ channels in rat ventricular myocytes, independent of CB1 and CB2 receptor activation. PMID:24758718

  6. The non-specific membrane binding properties of delta9-tetrahydrocannabinol and the effects of various solubilizers.

    PubMed

    Roth, S H; Williams, P J

    1979-04-01

    The binding of [3H]delta9-tetrahydrocannabinol to crude and purified synaptosomal membrane suspended in either Krebs solution or 10 mM sodium phosphate buffer was examined. The membrane/buffer partition coefficient was found to be 12,500, and was constant over a free concentration range of 10(-8) to 10(-6)M. Binding was similar in both suspending media, and to both crude and purified synaptosomal membrane. The solubilizing agent Cremophor E.L. (8 microgram ml-1) decreased the partition coefficient by one-half, and by greater than 99% at 0.4 mg ml-1. Similar effects were observed with Tween 80, while ethanol caused a maximum decrease of 60%. Membrane concentrations of THC were calculated at various effective concentrations reported in the literature, and were within the range predicted by the Meyer-Overton rule of anaesthesia. An apparent non-specific interaction with neuronal membranes and effective membrane concentrations of the order 2 x 10(-4) to 1 x 10(-2) mol kg-1 suggests THC may exert some of its effects by a mechanism analogous to the general anaesthetics, and thus may be classified as a partial anaesthetic.

  7. Influence of chemical and environmental stresses on metal-binding proteins: Species-dependent effects

    SciTech Connect

    Baer, K.N.

    1988-01-01

    The development of tolerance to cadmium toxicity was investigated in mammals. In adult mice pretreated with 20 mg Cd/kg, no mortality was observed following administration of a 100 mg/kg cadmium challenge dose. In animals receiving prior exposure to cold stress a mortality of 40% was observed, while in animals receiving no pretreatment an 80% mortality was observed following cadmium challenge. Analysis of the metal-binding proteins using G-75 gel-filtration chromatography revealed that MT-like protein was responsible, in part, for the observed tolerance to cadmium toxicity. For example, following 20 mg Cd/kg and cold pretreatment, the MT-like reserve capacity was 56 and 42 nmoles cadmium, respectively, compared to a control value of 12 nmoles cadmium. The influence of pretreatments on the subcellular distribution of cadmium was also examined. The influence of chemical and environmental stresses on metal-binding proteins in teleosts was investigated. Following cadmium exposure, cadmium increased in the MT fraction in both the gill and liver. However, following exposure to environmental stresses such as cold and hypoxia, significant decreases in zinc and copper were observed in the gill MT fraction, as compared to control. In the liver, no significant alterations were observed in the MT fraction, as compared to control.

  8. Alpha-bungarotoxin binding to hippocampal interneurons: immunocytochemical characterization and effects on growth factor expression.

    PubMed

    Freedman, R; Wetmore, C; Strömberg, I; Leonard, S; Olson, L

    1993-05-01

    The nicotinic cholinergic antagonist alpha-bungarotoxin (alpha-BT) binds throughout the rat hippocampal formation. The binding is displaceable by d-tubocurarine. The most heavily labeled cells are GABA-containing interneurons in the dentate and in Ammon's horn. These neurons have several different morphologies and contain several neuropeptides. alpha-BT-labeled interneurons in the dentate are small cells between the granular and molecular layers that often contain neuropeptide Y. alpha-BT-labeled interneurons in CA1 are medium-sized interneurons, occasionally found in stratum pyramidale, but more often found in stratum radiatum and stratum lacunosum moleculare. These neurons often contain cholecystokinin. The largest alpha-BT-labeled interneurons are found in CA3, in both stratum radiatum and stratum lucidum. These neurons are multipolar and frequently are autofluorescent. They often contain somatostatin or cholecystokinin. These large interneurons have been found to receive medial septal innervation and may also have projections that provide inhibitory feedback directly to the medial septal nucleus. The cholinergic innervation of the hippocampus from the medial septal nucleus is under the trophic regulation of NGF and brain-derived neurotrophic factor, even in adult life. Expression of mRNA for both these factors is increased in CA3 and the dentate after intraventricular administration of alpha-BT, but not after administration of the muscarinic antagonist atropine. alpha-BT-sensitive cholinergic receptors on inhibitory interneurons may be critical to medial septal regulation of the hippocampal activity, including the habituation of response to sensory input.

  9. In Vitro Glycoengineering of IgG1 and Its Effect on Fc Receptor Binding and ADCC Activity

    PubMed Central

    Thomann, Marco; Schlothauer, Tilman; Dashivets, Tetyana; Malik, Sebastian; Avenal, Cecile; Bulau, Patrick; Rüger, Petra; Reusch, Dietmar

    2015-01-01

    The importance and effect of Fc glycosylation of monoclonal antibodies with regard to biological activity is widely discussed and has been investigated in numerous studies. Fc glycosylation of monoclonal antibodies from current production systems is subject to batch-to-batch variability. If there are glycosylation changes between different batches, these changes are observed not only for one but multiple glycan species. Therefore, studying the effect of distinct Fc glycan species such as galactosylated and sialylated structures is challenging due to the lack of well-defined differences in glycan patterns of samples used. In this study, the influence of IgG1 Fc galactosylation and sialylation on its effector functions has been investigated using five different samples which were produced from one single drug substance batch by in vitro glycoengineering. This sample set comprises preparations with minimal and maximal galactosylation and different levels of sialylation of fully galactosylated Fc glycans. Among others, Roche developed the glycosyltransferase enzyme sialyltransferase which was used for the in vitro glycoengineering activities at medium scale. A variety of analytical assays, including Surface Plasmon Resonance and recently developed FcγR affinity chromatography, as well as an optimized cell-based ADCC assay were applied to investigate the effect of Fc galactosylation and sialylation on the in vitro FcγRI, IIa, and IIIa receptor binding and ADCC activity of IgG1. The results of our studies do not show an impact, neither positive nor negative, of sialic acid- containing Fc glycans of IgG1 on ADCC activity, FcγRI, and RIIIa receptors, but a slightly improved binding to FcγRIIa. Furthermore, we demonstrate a galactosylation-induced positive impact on the binding activity of the IgG1 to FcγRIIa and FcγRIIIa receptors and ADCC activity. PMID:26266936

  10. Alkali metal cation-hexacyclen complexes: effects of alkali metal cation size on the structure and binding energy.

    PubMed

    Austin, C A; Rodgers, M T

    2014-07-24

    Threshold collision-induced dissociation (CID) of alkali metal cation-hexacyclen (ha18C6) complexes, M(+)(ha18C6), with xenon is studied using guided ion beam tandem mass spectrometry techniques. The alkali metal cations examined here include: Na(+), K(+), Rb(+), and Cs(+). In all cases, M(+) is the only product observed, corresponding to endothermic loss of the intact ha18C6 ligand. The cross-section thresholds are analyzed to extract zero and 298 K M(+)-ha18C6 bond dissociation energies (BDEs) after properly accounting for the effects of multiple M(+)(ha18C6)-Xe collisions, the kinetic and internal energy distributions of the M(+)(ha18C6) and Xe reactants, and the lifetimes for dissociation of the activated M(+)(ha18C6) complexes. Ab initio and density functional theory calculations are used to determine the structures of ha18C6 and the M(+)(ha18C6) complexes, provide molecular constants necessary for the thermodynamic analysis of the energy-resolved CID data, and theoretical estimates for the M(+)-ha18C6 BDEs. Calculations using a polarizable continuum model are also performed to examine solvent effects on the binding. In the absence of solvent, the M(+)-ha18C6 BDEs decrease as the size of the alkali metal cation increases, consistent with the noncovalent nature of the binding in these complexes. However, in the presence of solvent, the ha18C6 ligand exhibits selectivity for K(+) over the other alkali metal cations. The M(+)(ha18C6) structures and BDEs are compared to those previously reported for the analogous M(+)(18-crown-6) and M(+)(cyclen) complexes to examine the effects of the nature of the donor atom (N versus O) and the number donor atoms (six vs four) on the nature and strength of binding.

  11. Multiple calcium channels in synaptosomes: voltage dependence of 1,4-dihydropyridine binding and effects on function

    SciTech Connect

    Dunn, S.M.J.

    1988-07-12

    The voltage dependence of binding of the calcium channel antagonist, (+)-(/sup 3/H)PN200-110, to rat brain synaptosomes and the effects of dihydropyridines on /sup 45/Ca/sup 2 +/ uptake have been investigated. Under nondepolarizing conditions (+)-(/sup 3/H)PN200-110 binds to a single class of sites with a K/sub d/ of 0.07 nM and a binding capacity of 182 fmol/mg of protein. When the synaptosomal membrane potential was dissipated either by osmotic lysis of the synaptosomes or by depolarization induced by raising the external K/sup +/ concentration, there was a decrease in affinity with no change in the number of sites. The effects of calcium channel ligands on /sup 45/Ca/sup 2 +/ uptake by synaptosomes have been measured as a function of external potassium concentration, i.e., membrane potential. Depolarization led to a rapid influx of /sup 45/Ca/sup 2 +/ whose magnitude was voltage-dependent. Verapamil almost completely inhibited calcium uptake at all potassium concentrations studies. In contrast, the effects of dihydropyridines (2 ..mu..M) appear to be voltage-sensitive. At relatively low levels of depolarization nitrendipine and PN200-110 completely inhibited /sup 45/Ca/sup 2 +/ influx, whereas the agonist Bay K8644 slightly potentiated the response. At higher K/sup +/ concentrations an additional dihydropyridine-insensitive component of calcium uptake was observed. These results provide evidence for the presence of dihydropyridine-sensitive calcium channels in synaptosomes which may be activated under conditions of partial depolarization.

  12. The amplification effect of functionalized gold nanoparticles on the binding of anticancer drug dacarbazine to DNA and DNA bases

    NASA Astrophysics Data System (ADS)

    Shen, Qin; Wang, Xuemei; Fu, Degang

    2008-11-01

    The promising application of functionalized gold nanoparticles to amplify the performance of biosensors and relevant biomolecular recognition processes has been explored in this paper. Our observations illustrate the apparent enhancement effect of the gold nanoparticles on the electrochemical response of the anticancer drug dacarbazine (DTIC) binding to DNA and DNA bases, indicating that these functionalized gold nanoparticles could readily facilitate the specific interactions between DTIC and DNA/DNA bases. This raises the potential valuable applications of these biocompatible nanoparticles in the promising biosensors and biomedical engineering.

  13. Binding Procurement

    NASA Technical Reports Server (NTRS)

    Rao, Gopalakrishna M.; Vaidyanathan, Hari

    2007-01-01

    This viewgraph presentation reviews the use of the binding procurement process in purchasing Aerospace Flight Battery Systems. NASA Engineering and Safety Center (NESC) requested NASA Aerospace Flight Battery Systems Working Group to develop a set of guideline requirements document for Binding Procurement Contracts.

  14. Iron binding efficiency of polyphenols: Comparison of effect of ascorbic acid and ethylenediaminetetraacetic acid on catechol and galloyl groups.

    PubMed

    Tamilmani, Poonkodi; Pandey, Mohan Chandra

    2016-04-15

    Dietary polyphenols are markedly studied for their antioxidant activity. They also have a negative impact on nutrition whereby they interfere with iron absorption. Common dietary polyphenols include: catechins, flavonols, flavanols, flavones, anthocyanins, proanthocyanidins and phenolic acids. Ascorbic acid (AA) and Ethylenediaminetetraacetic acid (EDTA) are commonly used to counter act this reaction and increase iron bioavailability. This study was aimed at determining the effect of AA and EDTA on the catechol or galloyl iron binding ability of pure phenolics, coffee and tea. Phenolic concentrations of 40, 80, 610, 240, 320, 400, 520 and 900 μg/ml were tested against six levels of AA and EDTA. These effects were studied in detail using Multivariate Analysis of Variance (MANOVA) with the hypothesis that there would be one or more mean differences between the ratio of enhancer and the different concentrations of samples tested. AA was found to be more efficient than EDTA in a way that lesser quantity is required for completely overcoming negative iron binding effects of polyphenols and similar samples.

  15. Differences in receptor binding affinity of several phytocannabinoids do not explain their effects on neural cell cultures.

    PubMed

    Rosenthaler, Sarah; Pöhn, Birgit; Kolmanz, Caroline; Huu, Chi Nguyen; Krewenka, Christopher; Huber, Alexandra; Kranner, Barbara; Rausch, Wolf-Dieter; Moldzio, Rudolf

    2014-01-01

    Phytocannabinoids are potential candidates for neurodegenerative disease treatment. Nonetheless, the exact mode of action of major phytocannabinoids has to be elucidated, but both, receptor and non-receptor mediated effects are discussed. Focusing on the often presumed structure-affinity-relationship, Ki values of phytocannabinoids cannabidiol (CBD), cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN), THC acid (THCA) and THC to human CB1 and CB2 receptors were detected by using competitive inhibition between radioligand [(3)H]CP-55,940 and the phytocannabinoids. The resulting Ki values to CB1 range from 23.5 nM (THCA) to 14711 nM (CBDV), whereas Ki values to CB2 range from 8.5 nM (THC) to 574.2 nM (CBDV). To study the relationship between binding affinity and effects on neurons, we investigated possible CB1 related cytotoxic properties in murine mesencephalic primary cell cultures and N18TG2 neuroblastoma cell line. Most of the phytocannabinoids did not affect the number of dopaminergic neurons in primary cultures, whereas propidium iodide and resazurin formation assays revealed cytotoxic properties of CBN, CBDV and CBG. However, THC showed positive effects on N18TG2 cell viability at a concentration of 10 μM, whereas CBC and THCA also displayed slightly positive activities. These findings are not linked to the receptor binding affinity therewith pointing to another mechanism than a receptor mediated one. [Corrected

  16. Malaysian cockle (Anadara granosa) allergy: Identification of IgE-binding proteins and effects of different cooking methods.

    PubMed

    Zailatul, H M Y; Rosmilah, M; Faizal, B; Noormalin, A; Shahnaz, M

    2015-06-01

    The purpose of this study was to evaluate the effect of different cooking methods on the allergenicity of cockle and to identify proteins most frequently bound by IgE antibodies using a proteomics approach. Raw, boiled, fried and roasted extracts of the cockle were prepared. The protein profiles of the extracts were obtained by separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis (2-DE). IgE-immunoblotting was then performed with the use of individual sera from patients with cockle allergy and the major IgE-binding proteins were analyzed by mass-spectrometry. SDS-PAGE of raw extract showed 13 protein bands. Smaller numbers of protein bands were detected in the boiled, fried and roasted extracts. The 2-DE gel profile of the raw extract further separated the protein bands to ~50 protein spots with molecular masses between 13 to 180 kDa and isoelectric point (pI) values ranging from 3 to 10. Immunoblotting of raw extract exhibited 11 IgE-binding proteins with two proteins of 36 and 40 kDa as the major IgE-binding proteins, while the boiled extract revealed 3 IgE-binding proteins. Fried and roasted extracts only showed a single IgE-binding protein at 36 kDa. 2-DE immunoblotting of raw extract demonstrated 5 to 20 IgE reactive spots. Mass spectrometry analysis led to identification of 2 important allergens, tropomyosin (36 kDa) and arginine kinase (40 kDa). Heated extracts showed a reduction in the number of IgE-reactive bands compared with raw extract, which suggest that thermal treatment can be used as a tool in attempting to reduce cockle allergenicity. The degree of allergenicity of cockle was demonstrated in the order raw > boiled > fried ≈ roasted. Two important allergens reacting with more than 50% of patients' sera identified using mass spectrometric approaches were tropomyosin and arginine kinase. Thus, allergens found in this study would help in component based diagnosis, management of

  17. Substituent Effects on Xenon Binding Affinity and Solution Behavior of Water-Soluble Cryptophanes

    PubMed Central

    Hill, P. Aru; Wei, Qian; Troxler, Thomas; Dmochowski, Ivan J.

    2009-01-01

    A water-soluble triacetic acid cryptophane-A derivative (TAAC) was synthesized and determined by isothermal titration calorimetry (ITC) and fluorescence quenching assay to have a xenon association constant of 33,000 M−1 at 293 K, which is the largest value measured for any host molecule to date. Fluorescence lifetime measurements of TAAC in the presence of varying amounts of xenon indicated static quenching by the encapsulated xenon and the presence of a second non-xenon-binding conformer in solution. Acid-base titrations and aqueous NMR spectroscopy of TAAC and a previously synthesized tri-(triazole propionic acid) cryptophane-A derivative (TTPC) showed how solvation of the carboxylate anions can affect the aqueous behavior of the large, nonpolar cryptophane. Specifically, whereas only the crown-crown (CC) conformer of TTPC was observed, a crown-saddle (CS) conformer of TAAC was also detected in aqueous solution. PMID:19239271

  18. Localized Control of Ligand Binding in Hemoglobin: Effect of Tertiary Structure on Picosecond Geminate Recombination

    NASA Astrophysics Data System (ADS)

    Friedman, J. M.; Scott, T. W.; Fisanick, G. J.; Simon, S. R.; Findsen, E. W.; Ondrias, M. R.; MacDonald, V. W.

    1985-07-01

    The picosecond geminate rebinding of molecular oxygen was monitored in a variety of different human, reptilian, and fish hemoglobins. The fast (100 to 200 picoseconds) component of the rebinding is highly sensitive to protein structure. Both proximal and distal perturbations of the heme affect this rebinding process. The rebinding yield for the fast process correlates with the frequency of the stretching motion of the iron-proximal histidine mode (vFe-His) observed in the transient Raman spectra of photodissociated ligated hemoglobins. The high-affinity R-state species exhibit the highest values for vFe-His and the highest yields for fast rebinding, whereas low affinity R-state species and T-state species exhibit lower values of vFe-His and correspondingly reduced yields for this geminate process. These findings link protein control of ligand binding with events at the heme.

  19. Cytochrome c binding to Apaf-1: The effects of dATP and ionic strength

    PubMed Central

    Purring-Koch, Cherie; McLendon, George

    2000-01-01

    In the apoptosis pathway in mammals, cytochrome c and dATP are critical cofactors in the activation of caspase 9 by Apaf-1. Until now, the detailed sequence of events in which these cofactors interact has been unclear. Here, we show through fluorescence polarization experiments that cytochrome c can bind to Apaf-1 in the absence of dATP; when dATP is added to the cytochrome c·Apaf-1 complex, further assembly occurs to produce the apoptosome. These findings, along with the discovery that the exposed heme edge of cytochrome c is involved in the cytochrome c·Apaf-1 interaction, are confirmed through enhanced chemiluminescence visualization of native PAGE gels and through acrylamide fluorescence quenching experiments. We also report here that the cytochrome c·Apaf-1 interaction depends highly on ionic strength, indicating that there is a strong electrostatic interaction between the two proteins. PMID:11035811

  20. Fibulin-1 Binds to Fibroblast Growth Factor 8 with High Affinity: EFFECTS ON EMBRYO SURVIVAL.

    PubMed

    Fresco, Victor M; Kern, Christine B; Mohammadi, Moosa; Twal, Waleed O

    2016-09-02

    Fibulin-1 (FBLN1) is a member of a growing family of extracellular matrix glycoproteins that includes eight members and is involved in cellular functions such as adhesion, migration, and differentiation. FBLN1 has also been implicated in embryonic heart and valve development and in the formation of neural crest-derived structures, including aortic arch, thymus, and cranial nerves. Fibroblast growth factor 8 (FGF8) is a member of a large family of growth factors, and its functions include neural crest cell (NCC) maintenance, specifically NCC migration as well as patterning of structures formed from NCC such as outflow tract and cranial nerves. In this report, we sought to investigate whether FBLN1 and FGF8 have cooperative roles in vivo given their influence on the development of the same NCC-derived structures. Surface plasmon resonance binding data showed that FBLN1 binds tightly to FGF8 and prevents its enzymatic degradation by ADAM17. Moreover, overexpression of FBLN1 up-regulates FGF8 gene expression, and down-regulation of FBLN1 by siRNA inhibits FGF8 expression. The generation of a double mutant Fbln1 and Fgf8 mice (Fbln1(-/-) and Fgf8(-/-)) showed that haplo-insufficiency (Fbln1(+/-) and Fgf8(+/-)) resulted in increased embryonic mortality compared with single heterozygote crosses. The mortality of the FGF8/Fbln1 double heterozygote embryos occurred between 14.5 and 16.5 days post-coitus. In conclusion, FBLN1/FGF8 interaction plays a role in survival of vertebrate embryos, and reduced levels of both proteins resulted in added mortality in utero The FBLN1/FGF8 interaction may also be involved in the survival of neural crest cell population during development.

  1. Mechanical Effects of Dynamic Binding between Tau Proteins on Microtubules during Axonal Injury

    PubMed Central

    Ahmadzadeh, Hossein; Smith, Douglas H.; Shenoy, Vivek B.

    2015-01-01

    The viscoelastic nature of axons plays a key role in their selective vulnerability to damage in traumatic brain injury (TBI). Experimental studies have shown that although axons can tolerate 100% strain under slow loading rates, even strain as small as 5% can rupture microtubules (MTs) during the fast loading velocities relevant to TBI. Here, we developed a computational model to examine rate-dependent behavior related to dynamic interactions between MTs and the MT-associated protein tau under varying strains and strain rates. In the model, inverted pairs of tau proteins can dynamically cross-link parallel MTs via the respective MT-binding domain of each tau. The model also incorporates realistic thermodynamic breaking and reformation of the bonds between the connected tau proteins as they respond to mechanical stretch. With simulated stretch of the axon, the model shows that despite the highly dynamic nature of binding and unbinding events, under fast loading rates relevant to TBI, large tensile forces can be transmitted to the MTs that can lead to mechanical rupture of the MT cylinder, in agreement with experimental observations and as inferred in human TBI. In contrast, at slow loading rates, the progressive breaking and reformation of the bonds between the tau proteins facilitate the extension of axons up to ∼100% strain without any microstructural damage. The model also predicts that under fast loading rates, individual MTs detach from MT bundles via sequential breaking of the tau-tau bonds. Finally, the model demonstrates that longer MTs are more susceptible to mechanical rupture, whereas short MTs are more prone to detachment from the MT bundle, leading to disintegration of the axonal MT ultrastructure. Notably, the predictions from the model are in excellent agreement with the findings of the recent in vitro mechanical testing of micropatterned neuronal cultures. PMID:26636944

  2. Studies on binding and mitogenic effect of insulin and insulin-like growth factor I in glomerular mesangial cells

    SciTech Connect

    Conti, F.G.; Striker, L.J.; Lesniak, M.A.; MacKay, K.; Roth, J.; Striker, G.E.

    1988-06-01

    The mesangial cells are actively involved in regulating glomerular hemodynamics. Their overlying endothelium is fenestrated; therefore, these cells are directly exposed to plasma substances, including hormones such as insulin and insulin-like growth factor I (IGF-I). These peptides may contribute to the mesangial sclerosis and cellular hyperplasia that characterize diabetic glomerulopathy. We report herein the characterization of the receptors and the mitogenic effects of IGF-I and insulin on mouse glomerular mesangial cells in culture. The IGF-I receptor was characterized on intact cells. The Kd of the IGF-I receptor was 1.47 X 10(-9) M, and the estimated number of sites was 64,000 receptors/cell. The binding was time, temperature, and pH dependent, and the receptor showed down-regulation after exposure to serum. The expression of the receptor did not change on cells at different densities. The specific binding for insulin was too low to allow characterization of the insulin receptor on intact cells. However, it was possible to identify the insulin receptor in a wheat germ agglutinin-purified preparation of solubilized mesangial cells. This receptor showed the characteristic features of the insulin receptor, including pH dependence of binding and a curvilinear Scatchard plot. The mitogenic effects of insulin and IGF-I on mesangial cells were measured by the incorporation of (3H)thymidine into DNA. IGF-I was more potent than insulin. The half-maximal response to IGF-I stimulation occurred at 1.3 X 10(-10) M, and a similar increase with insulin was observed at concentrations in the range of 10(-7) M, suggesting that this insulin action was mediated through the IGF-I receptor. These data show that the mouse microvascular smooth muscle cells of the glomerulus express a cell surface receptor for IGF-I in vitro and that this peptide is a potent mitogen for these mesangial cells.

  3. A contralateral repeated bout effect attenuates induction of NF-κB DNA binding following eccentric exercise.

    PubMed

    Xin, Ling; Hyldahl, Robert D; Chipkin, Stuart R; Clarkson, Priscilla M

    2014-06-01

    We investigated the existence of contralateral repeated bout effect and tested if the attenuation of nuclear factor-kappa B (NF-κB; an important regulator of muscle inflammation) induction following eccentric exercise is a potential mechanism. Thirty-one healthy men performed two bouts of knee extension eccentric exercise, initially with one leg and then with the opposite leg 4 wk later. Vastus lateralis muscle biopsies of both exercised and control legs were taken 3 h postexercise. Knee extension isometric and isokinetic strength (60°/sec and 180°/sec) were measured at baseline, pre-exercise, immediately postexercise, and 1/day for 5 days postexercise. Serum creatine kinase (CK) activity and muscle soreness were assessed at baseline and 1/day for 5 days postexercise. NF-κB (p65) DNA-binding activity was measured in the muscle biopsies. Isometric strength loss was lower in bout 2 than in bout 1 at 24, 72, and 96 h postexercise (P < 0.05). Isokinetic strength (60°/s and 180°/s) was reduced less in bout 2 than in bout 1 at 72 h postexercise (P < 0.01). There were no significant differences between bouts for postexercise CK activity or muscle soreness. p65 DNA-binding activity was increased following eccentric exercise (compared with the control leg) in bout 1 (122.9% ± 2.6%; P < 0.001) and bout 2 (109.1% ± 3.0%; P < 0.05). Compared with bout 1, the increase in NF-κB DNA-binding activity postexercise was attenuated after bout 2 (P = 0.0008). Repeated eccentric exercise results in a contralateral repeated bout effect, which could be due to the attenuated increase in NF-κB activity postexercise.

  4. Dlx5 Homeodomain: DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome

    DOE PAGES

    Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael; ...

    2016-01-29

    The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked to SHFM-1;more » this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.« less

  5. Targeting the GPIbα Binding Site of Thrombin To Simultaneously Induce Dual Anticoagulant and Antiplatelet Effects

    PubMed Central

    2015-01-01

    Exosite 2 of human thrombin contributes to two opposing pathways, the anticoagulant pathway and the platelet aggregation pathway. We reasoned that an exosite 2 directed allosteric thrombin inhibitor should simultaneously induce anticoagulant and antiplatelet effects. To assess this, we synthesized SbO4L based on the sulfated tyrosine-containing sequence of GPIbα. SbO4L was synthesized in three simple steps in high yield and found to be a highly selective, direct inhibitor of thrombin. Michelis–Menten kinetic studies indicated a noncompetitive mechanism of inhibition. Competitive inhibition studies suggested ideal competition with heparin and glycoprotein Ibα, as predicted. Studies with site-directed mutants of thrombin indicated that SbO4L binds to Arg233, Lys235, and Lys236 of exosite 2. SbO4L prevented thrombin-mediated platelet activation and aggregation as expected on the basis of competition with GPIbα. SbO4L presents a novel paradigm of simultaneous dual anticoagulant and antiplatelet effects achieved through the GPIbα binding site of thrombin. PMID:24635452

  6. Dlx5 Homeodomain: DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome

    SciTech Connect

    Proudfoot, Andrew; Axelrod, Herbert L.; Geralt, Michael; Fletterick, Robert J.; Yumoto, Fumiaki; Deacon, Ashley M.; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt; Serrano, Pedro

    2016-01-29

    The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked to SHFM-1; this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.

  7. Ouabain receptor binding of hydroxyprogesterone derivatives.

    PubMed Central

    Chow, E.; Kim, R. S.; Labella, F. S.; Queen, G.

    1979-01-01

    1 A specific and sensitive radioreceptor assay ahs been devised which is based on high affinity, saturable binding of 9 nM [3H]-ouabain to the total particulate fraction isolated from dog heart. Ouabain and other cardiac glycosides, including the aglycones, were about equipotent in their ability to displace [3H]-ouabain from its receptor, the IC50s ranging from 10 to 30 nM. 2 The only other substances found to compete significantly in the assay were derivatives of hydroxyprogesterone having a 17 alpha-acetate substituent: chlormadinone acetate, megestrol acetate, cyproterone acetate and medroxyprogesterone acetate, with IC50s of 2, 7.4, 9 and 21 microM, respectively. Prednisolone-3,20-bisguanyl-hydrazone, reported to have inotropic activity, gave an IC50 of 6.4 microM. Cyproterone-17 alpha-OH was less active (IC50 90 microM) than cyproterone-17 alpha-acetate. 3 A large number of peptide and protein hormones, steroid hormones and their metabolites, amines, and drugs were inactive. PMID:497535

  8. Molecular simulations of polycation-DNA binding exploring the effect of peptide chemistry and sequence in nuclear localization sequence based polycations.

    PubMed

    Elder, Robert M; Jayaraman, Arthi

    2013-10-10

    Gene therapy relies on the delivery of DNA into cells, and polycations are one class of vectors enabling efficient DNA delivery. Nuclear localization sequences (NLS), cationic oligopeptides that target molecules for nuclear entry, can be incorporated into polycations to improve their gene delivery efficiency. We use simulations to study the effect of peptide chemistry and sequence on the DNA-binding behavior of NLS-grafted polycations by systematically mutating the residues in the grafts, which are based on the SV40 NLS (peptide sequence PKKKRKV). Replacing arginine (R) with lysine (K) reduces binding strength by eliminating arginine-DNA interactions, but placing R in a less hindered location (e.g., farther from the grafting point to the polycation backbone) has surprisingly little effect on polycation-DNA binding strength. Changing the positions of the hydrophobic proline (P) and valine (V) residues relative to the polycation backbone changes hydrophobic aggregation within the polycation and, consequently, changes the conformational entropy loss that occurs upon polycation-DNA binding. Since conformational entropy loss affects the free energy of binding, the positions of P and V in the grafts affect DNA binding affinity. The insight from this work guides synthesis of polycations with tailored DNA binding affinity and, in turn, efficient DNA delivery.

  9. The effect of organochlorines and heavy metals on sex steroid-binding proteins in vitro in the plasma of nesting green turtles, Chelonia mydas.

    PubMed

    Ikonomopoulou, Maria Petrou; Olszowy, Henry; Hodge, Mary; Bradley, Adrian J

    2009-07-01

    In this study on green turtles, Chelonia mydas, from Peninsular Malaysia, the effect of selected environmental toxicants was examined in vitro. Emphasis was placed on purported hormone-mimicking chemicals such as dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene, dieldrin, lead, zinc and copper. Five concentrations were used: high (1 mg/L), medium (10(-1) mg/L), low (10(-2) mg/L), very low (10(-6) mg/L) and control (diluted carrier solvent but no toxicants). The results suggest that environmental pesticides and heavy metals may significantly alter the binding of steroids [i.e. testosterone (T) and oestradiol] to the plasma proteins in vitro. Competition studies showed that only Cu competed for binding sites with testosterone in the plasma collected from nesting C. mydas. Dieldrin and all heavy metals competed with oestradiol for binding sites. Furthermore, testosterone binding affinity was affected at various DDT concentrations and was hypothesised that DDT in vivo may act to inhibit steroid-protein interactions in nesting C. mydas. Although the precise molecular mechanism is yet to be described, DDT could have an effect upon the protein conformation thus affecting T binding (e.g. the T binding site on the steroid hormone binding protein molecule).

  10. Adhesive and Migratory Effects of Phosphophoryn Are Modulated by Flanking Peptides of the Integrin Binding Motif

    PubMed Central

    Suzuki, Shigeki; Kobuke, Seiji; Haruyama, Naoto; Hoshino, Hiroaki; Kulkarni, Ashok B.; Nishimura, Fusanori

    2014-01-01

    Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). Gene duplications in the ancestor dentin matrix protein-1 (DMP-1) genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Many SIBLING members have been shown to evoke various cell responses through the integrin-binding Arg-Gly-Asp (RGD) domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-463SDESDTNSESANESGSRGDA482-OH) was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-478SRGDASYTSDESSDDDNDSDSH499-OH). This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala482. Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin

  11. Effects of ligand binding on the stability of aldo–keto reductases: Implications for stabilizer or destabilizer chaperones

    PubMed Central

    Kabir, Aurangazeb; Honda, Ryo P.; Kamatari, Yuji O.; Endo, Satoshi; Fukuoka, Mayuko

    2016-01-01

    Abstract Ligands such as enzyme inhibitors stabilize the native conformation of a protein upon binding to the native state, but some compounds destabilize the native conformation upon binding to the non‐native state. The former ligands are termed “stabilizer chaperones” and the latter ones “destabilizer chaperones.” Because the stabilization effects are essential for the medical chaperone (MC) hypothesis, here we have formulated a thermodynamic system consisting of a ligand and a protein in its native‐ and non‐native state. Using the differential scanning fluorimetry and the circular dichroism varying the urea concentration and temperature, we found that when the coenzyme NADP+ was absent, inhibitors such as isolithocholic acid stabilized the aldo–keto reductase AKR1A1 upon binding, which showed actually the three‐state folding, but destabilized AKR1B10. In contrast, in the presence of NADP+, they destabilized AKR1A1 and stabilized AKR1B10. To explain these phenomena, we decomposed the free energy of stabilization (ΔΔG) into its enthalpy (ΔΔH) and entropy (ΔΔS) components. Then we found that in a relatively unstable protein showing the three‐state folding, native conformation was stabilized by the negative ΔΔH in association with the negative ΔΔS, suggesting that the stabilizer chaperon decreases the conformational fluctuation of the target protein or increase its hydration. However, in other cases, ΔΔG was essentially determined by the delicate balance between ΔΔH and ΔΔS. The proposed thermodynamic formalism is applicable to the system including multiple ligands with allosteric interactions. These findings would promote the development of screening strategies for MCs to regulate the target conformations. PMID:27595938

  12. Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells.

    PubMed Central

    Delicado, E G; Sen, R P; Miras-Portugal, M T

    1991-01-01

    Secretagogues inhibited adenosine uptake in chromaffin cells without causing apparent changes in the uptake affinity. The inhibition caused by carbachol, nicotine and acetylcholine reached 50%. This inhibition was reproduced by the action of protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA; 100 nM), phorbol 12,13-dibutyrate (PDBu; 100 nM), dicaproin (10 micrograms/ml) and tricaprylin (10 micrograms/ml), with inhibitions of Vmax. of 18, 20, 37 and 47% respectively. No changes in the affinity of uptake were observed with these effectors. Down-regulation of protein kinase C by phorbol esters decreased the inhibitory effects of carbachol on adenosine uptake. Binding studies with nitrobenzylthioinosine (NBTI) showed a similar decrease in the number of transporters when chromaffin cells were treated with the same effectors used for the uptake studies. The high-affinity dissociation constants showed minor changes with respect to the control. The ratio between maximal uptake capacity and the transporter number per cell was not significantly modified by the action of secretagogues or direct effectors of protein kinase C. The number of high-affinity binding sites for NBTI was decreased in cellular homogenates by the direct action of protein kinase C activators, with staurosporine able to reverse this action. Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes. These data suggest the possibility of a molecular modification at the transporter level. PMID:1953658

  13. Investigation of salt properties with electro-acoustic measurements and their effect on dynamic binding capacity in hydrophobic interaction chromatography.

    PubMed

    Müller, Egbert; Faude, Alexander

    2008-01-11

    The pH dependence in hydrophobic interaction chromatography (HIC) is usually discussed exclusively in terms of protein dependence and there are no clear defined trends. Many of the deviations from an ideal solution are caused solely by the high salt concentration, as protein concentration is usually negligible. So pH dependency in hydrophobic interaction chromatography could also be the result of pH dependent changes of ion properties from the salt solution. The possibility that pH dependent ion hydration or ion association in highly concentrated salt solutions may influence the dynamic protein binding capacity onto HIC resins was investigated. In buffer solutions commonly used in HIC e.g. sodium chloride, ammonium sulphate and sodium citrate pH dependent maxima in the electro-acoustic signals were found. These maxima are related to an increase of the ion sizes by hydration or ion association. At low ionic strength the maxima are in the range between 4.5 and 6 and they increased in concentrated electrolyte solutions to values between 6 and 8. The range of these maxima is in the same region as dynamic protein binding capacity maxima often observed in HIC. For a qualitative interpretation of this phenomenon of increased protein stabilization by volume exclusion effect extended scaling theory can be used. This theory predicts a maximum of protein stabilization if the ratio of salt ion diameter to water is 1.8. According to the hypothesis raised here, if the pH dependent ratio of salt ion diameter to water approaches this value the transport of the protein in the pore system is less restricted and an increase in binding capacity can be produced.

  14. Effect of starch and amylase on the expression of amylase-binding protein A in Streptococcus gordonii.

    PubMed

    Nikitkova, A E; Haase, E M; Scannapieco, F A

    2012-08-01

    Streptococcus gordonii is a common oral commensal bacterial species in tooth biofilm (dental plaque) and specifically binds to salivary amylase through the surface exposed amylase-binding protein A (AbpA). When S. gordonii cells are pretreated with amylase, amylase bound to AbpA facilitates growth with starch as a primary nutrition source. The goal of this study was to explore possible regulatory effects of starch, starch metabolites and amylase on the expression of S. gordonii AbpA. An amylase ligand-binding assay was used to assess the expression of AbpA in culture supernatants and on bacterial cells from S. gordonii grown in defined medium supplemented with 1% starch, 0.5 mg ml(-1) amylase, with starch and amylase together, or with various linear malto-oligosaccharides. Transcription of abpA was determined by reverse transcription quantitative polymerase chain reaction. AbpA was not detectable in culture supernatants containing either starch alone or amylase alone. In contrast, the amount of AbpA was notably increased when starch and amylase were both present in the medium. The expression of abpA was significantly increased (P < 0.05) following 40 min of incubation in defined medium supplemented with starch and amylase. Similar results were obtained in the presence of maltose and other short-chain malto-oligosacchrides. These results suggest that the products of starch hydrolysis produced from the action of salivary α-amylase, particularly maltose and maltotriose, up-regulate AbpA expression in S. gordonii.

  15. The Effect of CRH, Dexamethasone and Naltrexone on the Mu, Delta and Kappa Opioid Receptor Agonist Binding in Lamb Hypothalamic-Pituitary-Adrenal Axis.

    PubMed

    Pierzchała-Koziec, Krystyna; Dziedzicka-Wasylewska, Marta; Oeltgen, Peter; Zubel-Łojek, Joanna; Latacz, Anna; Ocłon, Ewa

    2015-01-01

    The aim of the study was to evaluate changes in the opioid receptor binding (mu, delta and kappa) in the hypothalamus, anterior pituitary and adrenal cortex (HPA) of lambs treated in vivo with corticotrophin releasing hormone (CRH), naltrexone, an opioid receptor antagonist (NAL), and dexamethasone, a potent cortisol analog (DEX). Experiment was carried out on 3 months old female lambs of polish mountain strain. Lambs received a single i.v. injection of NaCl (control), CRH (alone or in combination with naltrexone), naltrexone or dexamethasone. One hour later animals were decapitated under anaesthesia, tissues were dissected out and receptor binding assays were performed with radioligands for each type of opioid receptors--3H-DAGO, 3H-DPDPE and 3H-EKC for mu, delta and kappa receptor, respectively. Coexistence of specific binding sites for each type of opioid receptor was demonstrated in all levels of HPA axis of control lambs, however their distribution was uneven. Acute treatment with CRH, DEX and NAL caused downregulation or upregulation of mu, delta, kappa receptor binding in each level of HPA axis. CRH effects on mu, delta and kappa opioid receptor binding varied within the HPA axis and were modulated by naltrexone. Treatment with naltrexone increased in vitro mu, delta and kappa receptor binding in most tested structures except delta receptor binding in adrenal (decrease by 52%) and kappa receptor binding in pituitary (decrease by 41%). Dexamethasone significantly decreased the mu, delta and kappa opioid receptor binding in adrenal cortex but differentially affected opioid receptor binding in hypothalamus and pituitary. It seems probable that endogenous opioid peptides acting through mu, delta and kappa receptors interact with the hormones released from the hypothalamic-pituitary-adrenal axis in physiological and pathophysiological situations.

  16. Linear poly(ethylene imine)-based hydrogels for effective binding and release of DNA.

    PubMed

    Englert, Christoph; Tauhardt, Lutz; Hartlieb, Matthias; Kempe, Kristian; Gottschaldt, Michael; Schubert, Ulrich S

    2014-04-14

    A series of copolymers containing both amine groups of linear poly(ethylene imine) (LPEI) and double bonds of poly(2-(3-butenyl)-2-oxazoline) (PButEnOx) was prepared. To this end, a poly(2-ethyl-2-oxazoline) (PEtOx) precursor was hydrolyzed to the respective LPEI and functionalized in an amidation reaction with butenyl groups resulting in the double bond containing poly(2-(3-butenyl-2-oxazoline)-co-ethylene imine) (P(ButEnOx-co-EI)). Hydrogels were obtained by cross-linking with dithiols under UV-irradiation resulting in networks with different properties in dependence of the content of double bonds. The developed method allows the exact control of the amount of ethylene imine units within the copolymer and, thus, within the resulting hydrogels. The gel structures were characterized by solid state NMR and infrared spectroscopy. In addition the water uptake behavior from the liquid and the gas phase was investigated. It was shown by an ethidium bromide assay (EBA) that the copolymers and the respective hydrogels were able to bind and release DNA. Furthermore, the influence of the ethylene imine content on this interaction was investigated.

  17. Effect of bioceramic functional groups on drug binding and release kinetics

    NASA Astrophysics Data System (ADS)

    Trujillo, Christopher

    Bioceramics have been studied extensively as drug delivery systems (DDS). Those studies have aimed to tailor the drug binding and release kinetics to successfully treat infections and other diseases. This research suggests that the drug binding and release kinetics are predominantly driven by the functional groups available on the surface of a bioceramic. The goal of the present study is to explain the role of silicate and phosphate functional groups in drug binding to and release kinetics from bioceramics. alpha-cristobalite (Cris; SiO2) particles (90-150 microm) were prepared and doped with 0 microg (P-0), 39.1 microg (P-39.1), 78.2 microg (P-78.2), 165.5 microg (P-165.5) or 331 microg (P-331) of P 2O5 per gram Cris, using 85% orthophosphoric (H3PO 4) acid and thermal treatment. The material structure was analyzed using X-ray diffraction (XRD) with Rietveld Refinement and Fourier Transform Infrared (FTIR) spectroscopy with Gaussian fitting. XRD demonstrated an increase from sample P-0 (170.5373 A3) to P-331 (170.6466 A 3) in the unit cell volume as the P2O5 concentration increased in the material confirming phosphate silicate substitution in Cris. Moreover, FTIR showed the characteristic bands of phosphate functional groups of nu4 PO4/O-P-O bending, P-O-P stretching, P-O-P bending, P=O stretching, and P-O-H bending in doped Cris indicating phosphate incorporation in the silicate structure. Furthermore, FTIR showed that the nu4 PO4/O-P-O bending band around 557.6 cm-1 and P=O stretching band around 1343.9 cm-1 increased in area for samples P-39.1 to P-331 from 3.5 to 10.5 and from 10.1 to 22.4, respectively due to phosphate doping. In conjunction with the increase of the nu4 PO4/O-P-O bending band and P=O stretching band, a decrease in area of the O-Si-O bending bands around 488.1 and 629.8 cm-1 was noticed for samples P-39.1 to P-331 from 5 to 2 and from 11.8 to 5.4, respectively. Furthermore, Cris samples (200 mg, n=5 for each sample) were immersed separately in

  18. Comparative examination of effects of binding of different metals on chromophores of dissolved organic matter.

    PubMed

    Yan, Mingquan; Korshin, Gregory V

    2014-03-18

    This study quantified the binding of dissolved organic matter (DOM) from Suwannee River with nine metals, Ca(II), Mg(II), Fe(III), Al(III), Cu(II), Cd(II), Cr(III), Eu(III), and Th(IV), using a differential absorbance approach. The differential spectra of DOM were closely fitted with six Gaussian bands that were present for all of the metals at varying pH values. Their maxima were located at ca. 200, 240, 276, 316, 385, and 547 nm (denoted as A0, A1, A2, A3, A4, and A5, respectively). The relative contributions and signs of the Gaussian bands were metal-specific and correlated to some degree with the covalent-bonding index of the ions and applicable complexation constants of the NICA-Donnan model. The intensity of band A4 was linearly proportional to the concentration of DOM-complexed metal, although these correlations formed two groups with different slopes, reflecting the nature of DOM-metal interactions. The results demonstrate that differential spectra yield results indicative of the nature and extent of metal and DOM interactions.

  19. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B

    PubMed Central

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  20. Effects of lead on the kidney: Roles of high-affinity lead-binding proteins

    SciTech Connect

    Fowler, B.A. ); DuVal, G. )

    1991-02-01

    Lead-induced nephropathy produces both tubular and interstitial manifestations of cell injury, but the pathophysiology of these lesions is not completely understood. Delineation of the molecular factors underlying renal handling of lead is one of central importance in understanding the mechanisms of renal cell injury from this agent. Recent studies from this laboratory have identified several distinct high-affinity lead-binding proteins (PbBP) from rat kidney and brain that appear to play critical roles in the intracellular bioavailability of lead to several essential cellular processes in these target tissues at low dose levels. These studies have also shown that the real PbBP is selectively localized in only certain nephrons and only specific segments of the renal proximal tubule. The striking nephron and cell-type specificity of the localization reaction could result from physoiological differences in nephron functional activity or selective molecular uptake mechanisms/metabolism differences that act to define target cell populations in the kidney. In addition, other preliminary studies have shown that short-term, high-dose lead exposure produces increased excretion of this protein into the urine with concomitant decreases in renal concentrations.

  1. Effects of Biomolecular Flexibility on Alchemical Calculations of Absolute Binding Free Energies.

    PubMed

    Lawrenz, Morgan; Baron, Riccardo; Wang, Yi; McCammon, J Andrew

    2011-06-02

    The independent trajectory thermodynamic integration (IT-TI) approach (Lawrenz et. al J. Chem. Theory. Comput. 2009, 5:1106-1116(1)) for free energy calculations with distributed computing is employed to study two distinct cases of protein-ligand binding: first, the influenza surface protein N1 neuraminidase bound to the inhibitor oseltamivir, and second, the M. tuberculosis enzyme RmlC complexed with the molecule CID 77074. For both systems, finite molecular dynamics (MD) sampling and varied molecular flexibility give rise to IT-TI free energy distributions that are remarkably centered on the target experimental values, with a spread directly related to protein, ligand, and solvent dynamics. Using over 2 μs of total MD simulation, alternative protocols for the practical, general implementation of IT-TI are investigated, including the optimal use of distributed computing, the total number of alchemical intermediates, and the procedure to perturb electrostatics and van der Waals interactions. A protocol that maximizes predictive power and computational efficiency is proposed. IT-TI outperforms traditional TI predictions and allows a straightforward evaluation of the reliability of free energy estimates. Our study has broad implications for the use of distributed computing in free energy calculations of macromolecular systems.

  2. Effect of Composting on Dissolved Organic Matter in Animal Manure and Its Binding with Cu

    PubMed Central

    Zhang, Fengsong; Li, Yanxia; Xiong, Xiong; Yang, Ming; Li, Wei

    2012-01-01

    The agricultural application of raw animal manure introduces large amounts of dissolved organic matter (DOM) into soil and would increase transport of heavy metals such as Cu which are widely present in animal manure. The purpose of this research was to evaluate the evolution of DOM from pig and cattle manures during composting through excitation-emission matrix (EEM) fluorescence spectroscopy and the binding ability of DOM toward copper (Cu) ions with the aid of fluorescence quenching titration. The excitation-emission matrix spectra indicated that tyrosine-like, tryptophan-like, and soluble microbial byproduct-like fluorescence decreased significantly, while humic-like and fulvic-like fluorescence increased and became the main peaks in composted manure DOM. Fluorescence quenching titration showed that the complexing capacities of pig and cattle manure DOM decreased after composting. Correlation analysis confirmed that complexing capacity of DOM positively and significantly correlates with tyrosine-like and soluble microbial byproduct-like materials which mostly degraded after composting. These results would suggest that the ability of manure DOM to complex with Cu is inhibited as a result of reduced protein-like materials after composting. PMID:23125554

  3. Modeling the dynamics of airway constriction: effects of agonist transport and binding.

    PubMed

    Amin, Samir D; Majumdar, Arnab; Frey, Urs; Suki, Béla

    2010-08-01

    Recent advances have revealed that during exogenous airway challenge, airway diameters cannot be adequately predicted by their initial diameters. Furthermore, airway diameters can also vary greatly in time on scales shorter than a breath. To better understand these phenomena, we developed a multiscale model that allowed us to simulate aerosol challenge in the airways during ventilation. The model incorporates agonist-receptor binding kinetics to govern the temporal response of airway smooth muscle contraction on individual airway segments, which, together with airway wall mechanics, determines local airway caliber. Global agonist transport and deposition are coupled with pressure-driven flow, linking local airway constrictions with global flow dynamics. During the course of challenge, airway constriction alters the flow pattern, redistributing the agonist to less constricted regions. This results in a negative feedback that may be a protective property of the normal lung. As a consequence, repetitive challenge can cause spatial constriction patterns to evolve in time, resulting in a loss of predictability of airway diameters. Additionally, the model offers new insights into several phenomena including the intra- and interbreath dynamics of airway constriction throughout the tree structure.

  4. Small yet effective: the ethylene responsive element binding factor-associated amphiphilic repression (EAR) motif.

    PubMed

    Kagale, Sateesh; Rozwadowski, Kevin

    2010-06-01

    The Ethylene-responsive element binding factor-associated Amphiphilic Repression (EAR) motif is a small yet distinct regulatory motif that is conserved in many plant transcriptional regulator (TR) proteins associated with diverse biological functions. We have previously established a list of high-confidence Arabidopsis EAR repressors, the EAR repressome, comprising 219 TRs belonging to 21 different TR families. This class of proteins and the sequence context of the EAR motif exhibited a high degree of conservation across evolutionarily diverse plant species. Our comprehensive genome-wide analysis enabled refining EAR motifs as comprising either LxLxL or DLNxxP. Comparing the representation of these sequence signatures in TRs to that of other repressor motifs we show that the EAR motif is the one most frequently represented, detected in 10 to 25% of the TRs from diverse plant species. The mechanisms involved in regulation of EAR motif function and the cellular fates of EAR repressors are currently not well understood. Our earlier analysis had implicated amino acid residues flanking the EAR motifs in regulation of their functionality. Here, we present additional evidence supporting possible regulation of EAR motif function by phosphorylation of integral or adjacent Ser and/or Thr residues. Additionally, we discuss potential novel roles of EAR motifs in plant-pathogen interaction and processes other than transcriptional repression.

  5. Effect of malondialdehyde treatment on the IgE binding capacity and conformational structure of shrimp tropomyosin.

    PubMed

    Song, Yongna; Li, Zhenxing; Lin, Hong; Du, Shuyuan; Hao, Zina; Lin, Haixin; Zhu, Zhen

    2015-05-15

    The aim of this work was to determine the effect of malondialdehyde (MDA) treatment of shrimp tropomyosin (TM) with respect to IgE binding capacity and conformational structure. Following treatment with MDA, changes in TM molecular weight were characterized by SDS-PAGE and TM cross-linking was observed, then the aggregates were recognized by IgG/IgE in immunoblot analysis. Meanwhile, TM allergenicity slowly decreased following MDA treatment. These data show a correlation between the loss of TM structure and allergenic potential. TM secondary structure became more disordered following treatment with increasing concentrations of MDA. Moreover, the enhancement of surface hydrophobicity was also in accordance with the effect caused by MDA. Additionally, MDA treatment resulted in an increase in carbonyl content and a decrease in free amine groups and available lysine. The results suggest that MDA-induced conformational changes in TM can significantly influence the antigenicity and allergenicity of TM.

  6. Analysis of oxygen binding to Panulirus japonicus hemocyanin. The effect of divalent cations on the allosteric transition.

    PubMed

    Makino, N

    1986-01-02

    The effects of H+ and divalent cations on the O2 equilibrium of hexameric hemocyanin from a spiny lobster, Panulirus japonicus, were examined. The hemocyanin showed the normal Bohr effect. When divalent cations were removed by EDTA treatment, the protein showed a fivefold increase in the O2 affinity and a considerable decrease in the cooperativity. Several cooperativity models were tested for the conformity with the observed O2-binding isotherms by the least-square curve fitting. Among the models examined, the three-state concerted model was found to be most consistent with the results. It was postulated that in the absence of divalent cations deoxyhemocyanin is mainly in the intermediate-affinity state. The arthropod hemocyanins were found to be classifiable into two groups according to their functional responses to the divalent cations. It was suggested that the cations act differently on the allosteric transitions of the two groups of hemocyanins.

  7. The effect of digestive enzymes on the binding and bacteriostatic properties of lactoferrin and vitamin B12 binder in human milk.

    PubMed

    Samson, R R; Mirtle, C; McClelland, D B

    1980-07-01

    Human milk contains unsaturated lactoferrin and vitamin B12 binding protein. It has been suggested that these proteins may exert antibacterial effects in the intestine of the breast fed infant, but the effect of the intestinal environment on the antibacterial effect of these proteins has not been described. In this study human milk was treated with pepsin and trypsin and the influence of digestion on iron and vitamin B12 binding capacity, bacterial uptake of iron and vitamin B12 from milk and bacteriostatic effect was studied. Pepsin digestion had no effect on vitamin B12 binding capacity, or the ability of bacteria to take up vitamin B12, or the growth inhibitory effect on a vitamin B12 dependent strain. In contrast, trypsin digestion did not affect iron binding or bacteriostatic effects attributable to lactoferrin. The. findings support an in vivo bacteriostatic role for lactoferrin in the breast fed neonate's intestine but do not support a similar role for the vitamin B12 binding protein.

  8. Connecting Effective Immune Response, Fluorescent Granzyme B-like Peptide, Specific Peptide Binding Patterns, Patients with Cancer and Viral Infection, in Remission, Clinical Significance, and Liquid Biopsy.

    PubMed

    Lo, Wai Chun Jennifer; Luther, Donald Gene

    2016-11-01

    Functional cytotoxic-T-lymphocytes (CTL) with granzyme B play an important role in an effective immune response to tumor growth and infection progression. Tumor cells and platelets in peripheral whole blood smears of cancer patients have shown the presence of innate binding targets for GP1R, a fluorescent synthetic Granzyme B-like peptide. It is not known if similar GP1R-binding targets and specific binding patterns are detectable in peripheral blood of patients with viral infection. It is also not known if a specific binding pattern may be associated with an effective immune response to indicate a favorable prognosis. We reviewed the GP1R-binding patterns in the peripheral blood smears of 5 patients in remission at the time of sampling (3 with cancer and 2 with flu-like symptoms) and a negative control. We show with fluoroscopic images that there are: 1) fluorescent GP1R-binding targets mostly in the cytoplasmic areas of nucleated cells in patients with breast and lung cancer who have longer survival, 2) intense fluorescent deposits mostly in the nuclear areas of segmented neutrophils in patients recovered from severe to mild flu-like symptoms, 3) discernible fluorescent deposits in the cytoplasmic areas of small lymphocyte-like elements and overall intense fluorescent stain in large cells in the patient with advanced pancreatic cancer who had shorter survival, 4) GP1R-binding targets in numerous platelet-like elements in all 5 patients. The control sample did not show similar binding patterns. The potential association between specific GP1R-binding patterns in peripheral blood samples and prognostic significance, and its use as liquid biopsy are discussed.

  9. Effects of oral contraceptives, or lanosterol, on ADP-induced aggregation and binding of /sup 125/I-fibrinogen to rat platelets

    SciTech Connect

    McGregor, L.; Toor, B.; McGregor, J.L.; Renaud, S.; Clemetson, K.J.

    1984-03-01

    The aggregation to ADP and the binding of /sup 125/I-fibrinogen to platelets from rats treated with oral contraceptives or normal platelets treated in vitro with lanosterol were compared to their respective controls. Both types of platelets showed a significant increase in ADP-induced aggregation and in binding of fibrinogen, indicating that the effect of oral contraceptives could be partly due to increased levels of lanosterol in platelet membrane.

  10. Electroabsorption Spectroscopy Measurements of the Exciton Binding Energy, ElectronHole Reduced Effective Mass, and Band Gap in the Perovskite CHsub3NHsub3PbIsub3

    DTIC Science & Technology

    2016-07-28

    evidence that the primary photoexcitations (as opposed to recombination mechanisms ) can be described in terms of free carrier generation as transitions to a...Electroabsorption Spectroscopy Measurements of the Exciton Binding Energy , Electron−Hole Reduced Effective Mass, and Band Gap in the Perovskite...98195-1700, United States *S Supporting Information ABSTRACT: We use electroabsorption (EA) spectroscopy to measure the exciton binding energy (EB

  11. Effects of lipophilicity and protein binding on the hepatocellular uptake and hepatic disposition of two anthracyclines, doxorubicin and iododoxorubicin.

    PubMed

    Rivory, L P; Avent, K M; Pond, S M

    1996-01-01

    The anthracyclines, in particular doxorubicin (DOX), have been used for the intra-arterial locoregional therapy of liver tumours for over two decades. However, the results obtained with this form of therapy have been disappointing. It is widely recognised that DOX has a slow and limited tissue uptake, and we hypothesised that lipophilic analogues could be more suitable for locoregional administration. Using rat hepatocyte suspensions and the isolated rat liver, we examined the effects of lipophilicity, as determined from the octanol: buffer partition coefficient (Koct:buf), and protein binding of several anthracyclines on hepatocellular uptake. In particular, we compared DOX with 4'-iodo-4'-deoxy-doxorubicin (IDX), which differs only in the substitution of the daunosamine hydroxyl by an iodine molecule. Using a direct spectrofluorimetric method to evaluate cell uptake, we found that the influx rates correlated with the logarithm of Koct:buf and that IDX had the highest rate. However, the addition of bovine serum albumin (BSA) to the medium reduced the hepatocellular uptake of IDX more extensively than that of DOX such that the DOX uptake exceeded that of IDX with 4% BSA. Experiments in the isolated perfused rat liver confirmed these findings. We suggest that a trade-off of cellular uptake for reduced protein binding is desirable in the selection of drugs for intrahepatic administration. This may be accomplished by choosing anthracyclines with intermediate lipophilicity.

  12. Effect of a high glucose diet on insulin binding and insulin action in rat adipocytes. A longitudinal study.

    PubMed

    Oka, Y; Akanuma, Y; Kasuga, M; Kosaka, K

    1980-11-01

    To elucidate the mechanisms whereby changes in dietary composition affect the action of insulin on glucose metabolism, insulin binding and glucose uptake and oxidation have been studied in epididymal fat pad adipocytes from rats fed high glucose diets for 5 and 10 days. After 5 days, insulin binding was increased, the mainly to an increased number of receptors (3.4 X 10(5) vs. 2.4 X 10(5) sites per cell) in spite of increased plasma insulin levels (3.0 +/- 0.2 vs. 2.1 +/- 0.1 microgram/l; P < 0.05). The maximal response of glucose oxidation to insulin was increased (925 +/- 55 vs. 510 +/- 58 n moles/2 X 10(5) cells/2h; P < 0.01) and the dose-response curve of glucose uptake was shifted to the left. After 10 days, receptor number decreased to the control level and the effect of insulin on glucose uptake and oxidation (% basal) were similar to controls. Thus, in the early stage of high glucose feeding, insulin receptor number, and insulin sensitivity of glucose uptake, and insulin responsiveness of glucose oxidation were increased.

  13. A new class of peroxisome proliferator-activated receptor agonists with a novel binding epitope shows antidiabetic effects.

    PubMed

    Ostberg, Tove; Svensson, Stefan; Selén, Göran; Uppenberg, Jonas; Thor, Markus; Sundbom, Maj; Sydow-Bäckman, Mona; Gustavsson, Anna-Lena; Jendeberg, Lena

    2004-09-24

    The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the NR1 subfamily of nuclear receptors. The PPARs play key roles in the control of glucose and lipid homeostasis, and the synthetic isoform-specific PPAR agonists are used clinically to improve insulin sensitivity and to lower serum triglyceride levels. All of the previously reported PPAR agonists form the same characteristic interactions with the receptor, which have been postulated to be important for the induction of agonistic activity. Here we describe a new class of PPARalpha/gamma modulators, the 5-substituted 2-benzoylaminobenzoic acids (2-BABAs). As shown by x-ray crystallography, the representative compounds BVT.13, BVT.762, and BVT.763, utilize a novel binding epitope and lack the agonist-characteristic interactions. Despite this, some compounds within the 2-BABA family are potent agonists in a cell-based reporter gene assay. Furthermore, BVT.13 displays antidiabetic effects in ob/ob mice. We concluded that the 2-BABA binding mode can be used to design isoform-specific PPAR modulators with biological activity in vivo.

  14. Neuroprotective Effect of Ginseng against Alteration of Calcium Binding Proteins Immunoreactivity in the Mice Hippocampus after Radiofrequency Exposure

    PubMed Central

    Maskey, Dhiraj

    2013-01-01

    Calcium binding proteins (CaBPs) such as calbindin D28-k, parvalbumin, and calretinin are able to bind Ca2+ with high affinity. Changes in Ca2+ concentrations via CaBPs can disturb Ca2+ homeostasis. Brain damage can be induced by the prolonged electromagnetic field (EMF) exposure with loss of interacellular Ca2+ balance. The present study investigated the radioprotective effect of ginseng in regard to CaBPs immunoreactivity (IR) in the hippocampus through immunohistochemistry after one-month exposure at 1.6 SAR value by comparing sham control with exposed and ginseng-treated exposed groups separately. Loss of dendritic arborization was noted with the CaBPs in the Cornu Ammonis areas as well as a decrease of staining intensity of the granule cells in the dentate gyrus after exposure while no loss was observed in the ginseng-treated group. A significant difference in the relative mean density was noted between control and exposed groups but was nonsignificant in the ginseng-treated group. Decrease in CaBP IR with changes in the neuronal staining as observed in the exposed group would affect the hippocampal trisynaptic circuit by alteration of the Ca2+ concentration which could be prevented by ginseng. Hence, ginseng could contribute as a radioprotective agent against EMF exposure, contributing to the maintenance of Ca2+ homeostasis by preventing impairment of intracellular Ca2+ levels in the hippocampus. PMID:24069603

  15. Role of cytosolic liver fatty acid binding protein in hepatocellular oxidative stress: effect of dexamethasone and clofibrate treatment.

    PubMed

    Rajaraman, G; Wang, G Q; Yan, J; Jiang, P; Gong, Y; Burczynski, F J

    2007-01-01

    The presence of cysteine and methionine groups together with an ability to bind long-chain fatty acid (LCFA) oxidation products makes liver fatty acid binding protein (L-FABP) an attractive candidate against hepatocellular oxidative stress. In this report, we show that pharmacological treatment directed at modulating L-FABP level affected hepatocellular oxidant status. L-FABP expressing 1548-hepatoma cells, treated with dexamethasone or clofibrate, decreased and increased intracellular L-FABP levels, respectively. Oxidative stress was induced by H2O2 incubation or hypoxia-reoxygenation. The fluorescent marker, dichlorofluorescein (DCF), was employed to measure intracellular reactive oxygen species (ROS). Hepatocellular damage was assessed by lactate dehydrogenase (LDH) level. Dexamethasone treatment resulted in a significant increase in DCF fluorescence with higher LDH release compared to control cells. Clofibrate treatment, however, resulted in a significant decrease in both parameters (p<0.05). Drug treatments did not affect cytosolic activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), or catalase suggesting that the differences between treated and control cells may likely be associated with varying L-FABP levels. We conclude that L-FABP may act as an effective endogenous cytoprotectant against hepatocellular oxidative stress.

  16. Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects.

    PubMed

    Hervé, Cécile; Rogowski, Artur; Blake, Anthony W; Marcus, Susan E; Gilbert, Harry J; Knox, J Paul

    2010-08-24

    Cell wall degrading enzymes have a complex molecular architecture consisting of catalytic modules and noncatalytic carbohydrate-binding modules (CBMs). The function of CBMs in cell wall degrading processes is poorly understood. Here, we have evaluated the potential enzyme-targeting function of CBMs in the context of intact primary and secondary cell wall deconstruction. The capacity of a pectate lyase to degrade pectic homogalacturonan in primary cell walls was potentiated by cellulose-directed CBMs but not by xylan-directed CBMs. Conversely, the arabinofuranosidase-mediated removal of side chains from arabinoxylan in xylan-rich and cellulose-poor wheat grain endosperm cell walls was enhanced by a xylan-binding CBM but less so by a crystalline cellulose-specific module. The capacity of xylanases to degrade xylan in secondary cell walls was potentiated by both xylan- and cellulose-directed CBMs. These studies demonstrate that CBMs can potentiate the action of a cognate catalytic module toward polysaccharides in intact cell walls through the recognition of nonsubstrate polysaccharides. The targeting actions of CBMs therefore have strong proximity effects within cell wall structures, explaining why cellulose-directed CBMs are appended to many noncellulase cell wall hydrolases.

  17. Regulation of protein binding toward a ligand on chromatographic matrixes by masking and forced-releasing effects using thermoresponsive polymer.

    PubMed

    Yoshizako, Kimihiro; Akiyama, Yoshikatsu; Yamanaka, Hidenori; Shinohara, Yasuro; Hasegawa, Yukio; Carredano, Enrique; Kikuchi, Akihiko; Okano, Teruo

    2002-08-15

    A novel concept of affinity regulation based on masking and forced-releasing effects using a thermoresponsive polymer was elucidated. Affinity chromatographic matrixes were prepared using either poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) or poly(glycidyl methacrylate-co-triethyleneglycol dimethacrylate) beads immobilized with ligand molecule, Cibacron Blue F3G-A (CB), together with poly(N-isopropylacrylamide) (PIPAAm), a polymer with a cloud point of 32 degrees C. Two different lengths of spacer molecules were used for the immobilization of CB while maintaining the PIPAAm size constant. Chromatographic analyses using bovine serum albumin as a model protein showed a clear correlation between spacer length and binding capacity at temperatures lower than the lower critical solution temperature (LCST) of PIPAAm. The binding capacity under the LCST was significantly reduced only when the calculated spacer length was shorter than the mean size of the extended PIPAAm. Furthermore, the adsorbed protein could be desorbed (released) from the matrix surface by lowering the temperature to below the LCST while maintaining other factors such as pH and ion strength. Selective recovery of human albumin from human sera was demonstrated using this newly developed thermoresponsive affinity column.

  18. Interactive effects of waterborne metals in binary mixtures on short-term gill-metal binding and ion uptake in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Niyogi, Som; Nadella, Sunita R; Wood, Chris M

    2015-08-01

    Metal binding to fish gills forms the basis of the biotic ligand model (BLM) approach, which has emerged as a useful tool for conducting site-specific water quality assessments for metals. The current BLMs are designed to assess the toxicity of individual metals, and cannot account for the interactive effects of metal mixtures to aquatic organisms including fish. The present study was designed mainly to examine the interactive effects of waterborne metals (Cd, Zn, Cu, Ag, and Ni) in specific binary combinations on short-term (3h) gill-metal binding and essential ion (Ca(2+) and Na(+)) uptake (a physiological index of toxicity) in fish, using juvenile freshwater rainbow trout (Oncorhynchus mykiss) as the model species. We hypothesized that binary mixtures of metals that share a common mode of uptake and toxicity (e.g., Cd and Zn - Ca(2+) antagonists, Cu and Ag - Na(+) antagonists) would reduce the gill binding of each other via competitive interactions and induce less than additive effects on ion transport. In addition, the mixture of metals that have different modes of uptake and toxicity (e.g., Cd and Cu, or Cd and Ni) would not exhibit any interactive effects either on gill-metal binding or ion transport. We found that both Zn and Cu reduced gill-Cd binding and vice versa, however, Ni did not influence gill-Cd binding in fish. Surprisingly, Ag was found to stimulate gill-Cu binding especially at high exposure concentrations, whereas, Cu had no effect on gill-Ag binding. The inhibitory effect of Cd and Zn in mixture on branchial Ca(2+) uptake was significantly greater than that of Cd or Zn alone. Similarly, the inhibitory effect of Cu and Ag in mixture on branchial Na(+) uptake was significantly greater than that of Cu or Ag alone. The inhibitory effects of Cd and Zn mixture on Ca(2+) uptake as well as Cu and Ag mixture on Na(+) uptake were found to follow the principles of simple additivity. In contrast, no significant additive effect on either Ca(2+) or Na

  19. DNA binding, DNA cleavage and cytotoxicity studies of a new water soluble copper(II) complex: The effect of ligand shape on the mode of binding

    NASA Astrophysics Data System (ADS)

    Kashanian, Soheila; Khodaei, Mohammad Mehdi; Roshanfekr, Hamideh; Shahabadi, Nahid; Mansouri, Ghobad

    2012-02-01

    The interaction of native calf thymus DNA (CT-DNA) with [Cu(ph 2phen)(phen-dione)Cl]Cl was studied at physiological pH by spectrophotometric, spectrofluorometric, circular dichroism, and viscometric techniques. Considerable hypochromicity and red shift are observed in the UV absorption band of the Cu complex. Binding constants ( Kb) of DNA with the complex were calculated at different temperatures. Thermodynamic parameters, enthalpy and entropy changes were calculated according to Van't Hoff equation, which indicated that reaction is predominantly enthalpically driven. All these results indicate that Cu(II) complex interacts with CT-DNA via intercalative mode. Also, this new complex induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) and human T lymphocyte carcinoma-Jurkat cell lines.

  20. Prostasomes of canine seminal plasma - zinc-binding ability and effects on motility characteristics and plasma membrane integrity of spermatozoa.

    PubMed

    Mogielnicka-Brzozowska, M; Strzeżek, R; Wasilewska, K; Kordan, W

    2015-06-01

    Prostasomes are small lipid membrane-confined vesicles that are involved in various fertilization-related processes. The aim of this study was to demonstrate canine seminal plasma prostasomes' ability to bind zinc ions, as well as examining their effects on sperm motility characteristics and plasma membrane integrity during cold storage. Ejaculates, collected from five cross-bred dogs (n = 50), were subjected to ultracentrifugation followed by gel filtration (GF) on a Superose 6 column. Prostasomes appeared as a single fraction in the elution profile. Transmission electron microscopy (TEM) analysis of canine prostasomes revealed the presence of membrane vesicles with diameters ranging from 20.3 to 301 nm. The zinc-affinity chromatography on a Chelating Sepharose Fast Flow - Zn(2 +) showed that from 93 to 100% of the prostasome proteins bind zinc ions (P(+) Zn). SDS-PAGE revealed that canine P(+) Zn comprised four protein bands, with low molecular weights (10.2-12 kDa). We have also shown a positive effect of prostasomes (p < 0.05), especially variant B (2% of total seminal plasma protein) on canine sperm motility parameters after 2 h storage at 5°C (TMOT%, 44.75 ± 5.18) and PMOT%, 12.42 ± 1.59) and VAP, VSL, VCL, when compared with Control (TMOT%, 7.30 ± 1.41 and PMOT%, 1.70 ± 0.42). Higher percentage of spermatozoa with intact plasma membrane (SYBR/PI dual staining) and intact acrosome (Giemsa stained), after 2 h storage at 5°C, was showed, in variant A (1.5% of total seminal plasma protein) and B, when compared with Control and variant C (2.5% of total seminal plasma protein). The prostasomes' effect on motility and plasma membrane integrity of canine cold-stored spermatozoa may be related to their ability to bind zinc ions and regulate their availability to the sperm.

  1. From Outcome Prediction to Action Selection: Developmental Change in the Role of Action-Effect Bindings

    ERIC Educational Resources Information Center

    Verschoor, Stephan A.; Spapé, Michiel; Biro, Szilvia; Hommel, Bernhard

    2013-01-01

    Ideomotor theory considers bidirectional action-effect associations to be the fundamental building blocks for intentional action. The present study employed a novel pupillometric and oculomotor paradigm to study developmental changes in the role of action-effects in the acquisition of voluntary action. Our findings suggest that both 7- and…

  2. The effect of SK&F 95654, a novel phosphodiesterase inhibitor, on cardiovascular, respiratory and platelet function.

    PubMed Central

    Murray, K. J.; Eden, R. J.; Dolan, J. S.; Grimsditch, D. C.; Stutchbury, C. A.; Patel, B.; Knowles, A.; Worby, A.; Lynham, J. A.; Coates, W. J.

    1992-01-01

    1. SK&F 95654 inhibited the guanosine 3':5'-cyclic monophosphate (cyclic GMP)-inhibited phosphodiesterase (cGI-PDE) with an IC50 value of 0.7 microM. The IC50 values were greater than 100 microM for the other four phosphodiesterase isoenzymes tested. The R-enantiomer of SK&F 95654 (IC50 = 0.35 microM) was a more potent inhibitor of cGI-PDE than was the S-enantiomer (IC50 = 5.3 microM). 2. In the guinea-pig working heart, SK&F 95654 produced a positive inotropic response without altering heart rate. 3. Oral administration of SK&F 95654 to conscious dogs caused dose-dependent increases in left ventricular dp/dtmax in the range 10-50 micrograms kg-1. These positive inotropic responses were maintained for 3 h without simultaneous changes in heart rate or blood pressure. The peak effects on left ventricular dp/dtmax were similar for orally and intravenously administered compound, indicating good oral bioavailability. 4. SK&F 95654 caused a potent inhibition of U46619-induced aggregation in both a human washed platelet suspension (WPS) (IC50 = 70 nM) and in human platelet-rich plasma (PRP) (IC50 = 60 nM), indicating that the compound shows negligible plasma binding. 5. The R-enantiomer of SK&F 95654 was twenty fold more potent as an inhibitor of platelet aggregation than was the S-enantiomer. The similarity of this ratio to that obtained on the cGI-PDE suggests that SK&F 95654 inhibits platelet aggregation via its effects on cGI-PDE. This was also indicated by studies which showed that SK&F 95654 increased adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels and activated cyclic AMP-dependent protein kinase in human platelets.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1422592

  3. [The effect of tilidine on left-ventricular function of the human heart (author's transl)].

    PubMed

    Strauer, B E

    1975-04-04

    The effect of tilidine (Valoron, 1.5 mg/kg) on left-ventricular function was measured in ten patients undergoing diagnostic cardiac catheterization. In the course of thirty minutes after administration there was a small fall in systemic arterial pressure, decrease in cardiac output, cardiac index and stroke volume, as well as a change in the isovolumetric inotropic state. Haemodynamic and inotropic changes were small in amount, varying between 3.4 and 14.5% of basal values. This study indicates that tilidine, while having a powerful analgesic effect, has only a mild contractility-inhibiting action. Tilidine is thus a useful alternative as an analgesic in cardiac pain.

  4. Effects of DDT and triclosan on tumor-cell binding capacity and cell-surface protein expression of human natural killer cells.

    PubMed

    Hurd-Brown, Tasia; Udoji, Felicia; Martin, Tamara; Whalen, Margaret M

    2013-06-01

    1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) and triclosan (TCS) are organochlorine (OC) compounds that contaminate the environment, are found in human blood and have been shown to decrease the tumor-cell killing (lytic) function of human natural killer (NK) cells. NK cells defend against tumor cells and virally infected cells. They bind to these targets, utilizing a variety of cell surface proteins. The present study examined concentrations of DDT and TCS that decrease lytic function for alteration of NK binding to tumor targets. Levels of either compound that caused loss of binding function were then examined for effects on expression of cell-surface proteins needed for binding. NK cells exposed to 2.5 μM DDT for 24 h (which caused a greater than 55% loss of lytic function) showed a decrease in NK binding function of about 22%, and a decrease in CD16 cell-surface protein of 20%. NK cells exposed to 5 μM TCS for 24 h showed a decrease in ability to bind tumor cells of 37% and a decrease in expression of CD56 of about 34%. This same treatment caused a decrease in lytic function of greater than 87%. These results indicated that only a portion of the loss of NK lytic function seen with exposures to these compounds could be accounted for by loss of binding function. They also showed that loss of binding function is accompanied by a loss of cell-surface proteins important in binding function.

  5. Effects of temperature, pH and sugar binding on the structures of lectins ebulin f and SELfd.

    PubMed

    Carrillo, Celia; Cordoba-Diaz, Damián; Cordoba-Diaz, Manuel; Girbés, Tomás; Jiménez, Pilar

    2017-04-01

    Ebulin f and SELfd are two lectins of Sambucus ebulus L. that show different stability and digestibility properties in gastric fluid due to their structural differences which may explain their different toxicological profiles. The main aim was to determine the effects of pH, temperature and sugar binding on the intrinsic structures of both proteins by fluorescence analyses. Quenching experiments were conducted, under different pH and temperature conditions, with acrylamide (uncharged) and iodide (charged), to study the possible changes of their intrinsic fluorescence. Results revealed that the native structure of SELfd is more folded than that of ebulin f. At pH 2.0, ebulin f displayed a more open structure than at neutral pH. It can be concluded that this is the main reason why ebulin f is accessible to pepsin action and more sensitive to degradation, in contrast to SELfd as we reported previously.

  6. Molecular-Scale Features that Govern the Effects of O-Glycosylation on a Carbohydrate-Binding Module

    DOE PAGES

    Guan, Xiaoyang; Chaffey, Patrick K.; Zeng, Chen; ...

    2015-09-21

    The protein glycosylation is a ubiquitous post-translational modification in all kingdoms of life. Despite its importance in molecular and cellular biology, the molecular-level ramifications of O-glycosylation on biomolecular structure and function remain elusive. Here, we took a small model glycoprotein and changed the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage while monitoring any corresponding changes to physical stability and cellulose binding affinity. The results of this study reveal the collective importance of all the studied features in controlling the most pronounced effects of O-glycosylation in this system. This study suggests the possibility ofmore » designing proteins with multiple improved properties by simultaneously varying the structures of O-glycans and amino acids local to the glycosylation site.« less

  7. Molecular-Scale Features that Govern the Effects of O-Glycosylation on a Carbohydrate-Binding Module

    SciTech Connect

    Guan, Xiaoyang; Chaffey, Patrick K.; Zeng, Chen; Greene, Eric R.; Chen, Liqun; Drake, Matthew R.; Chen, Claire; Groobman, Ari; Resch, Michael G.; Himmel, Michael E.; Beckham, Gregg T.; Tan, Zhongping

    2015-09-21

    The protein glycosylation is a ubiquitous post-translational modification in all kingdoms of life. Despite its importance in molecular and cellular biology, the molecular-level ramifications of O-glycosylation on biomolecular structure and function remain elusive. Here, we took a small model glycoprotein and changed the glycan structure and size, amino acid residues near the glycosylation site, and glycosidic linkage while monitoring any corresponding changes to physical stability and cellulose binding affinity. The results of this study reveal the collective importance of all the studied features in controlling the most pronounced effects of O-glycosylation in this system. This study suggests the possibility of designing proteins with multiple improved properties by simultaneously varying the structures of O-glycans and amino acids local to the glycosylation site.

  8. Vitrification is not superior to rapid freezing of normozoospermic spermatozoa: effects on sperm parameters, DNA fragmentation and hyaluronan binding.

    PubMed

    Agha-Rahimi, Azam; Khalili, Mohammad Ali; Nabi, Ali; Ashourzadeh, Sareh

    2014-03-01

    Human sperm vitrification is a new cryopreservation method. This study compared the effects of rapid freezing and vitrification on various sperm parameters, hyaluronan-binding assay and DNA fragmentation and assessed the impact of cryoprotectant agents (CPA) with vitrification. A total of 30 normo-ejaculates were prepared by swim up and the motile sperm fraction was divided into four: fresh (control), rapid freezing, and two vitrification groups (a, lacking CPA; b, with CPA). For rapid freezing, a cryovial of sperm suspension was held just above the liquid nitrogen surface, and for vitrification, 30μl suspension was dropped directly into liquid nitrogen. Sperm parameters, including motility, viability and morphology, declined after cryopreservation in both groups. DNA fragmentation was not significantly higher in the vitrification (15.7±4.4%) or rapid freezing (16.6±5.6%) groups when compared with controls (11.6±4.5%). The rates of hyaluronan binding were similar between the control and cryopreserved groups. Moreover, addition of CPA for vitrification had a neutral effect on rates of sperm recovery. In conclusion, vitrification has great potential for human sperm cryopreservation and does not require CPA, with its possible toxicity. However, it is not superior to rapid cryopreservation regarding sperm recovery rate in normozoospermia. Human sperm vitrification is a new cryopreservation method that has been introduced recently. This study compared the effects of rapid freezing with vitrification on rates of sperm parameters, hyaluronan-binding assay and DNA fragmentation after thawing/warming and assessed the impact of cryoprotectant agent (CPA) on vitrification. The study was performed on 30 ejaculates prepared using the swim-up technique. Each motile sperm suspension was divided into four: control (fresh); rapid freezing; and two vitrification groups (a, lacking CPA; b, with CPA). For rapid freezing, a cryovial of sperm suspension was held above the surface of

  9. [The effect of mutations in the synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by Azospirillum brasilense].

    PubMed

    Shelud'ko, A V; Kulibiakina, O V; Shirokov, A A; Petrova, L P; Matora, L Iu; Katsy, E I

    2008-01-01

    The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic surfaces.

  10. Binding interactions of dissolved organic matter with iron and copper: Effects of source and pH

    NASA Astrophysics Data System (ADS)

    Du, Y.; Jaffe, R.

    2014-12-01

    Dissolved organic matter(DOM) fluxes from terrestrial to marine environments have increased as a result of anthropogenic activities and climate change, and DOM is known to facilitate the transport of both metals and trace organics. The nature and composition of DOM may influence its binding properties to metals and thus affect their mobility and fate in aquatic environments. Humic substances make up the majority of DOM in many aquatic ecosystems. Metals such as Fe and Cu are not only important as nutrients in aquatic systems, but also in controlling biogeochemical and ecotoxicological processes respectively. Thus, gaining a better understanding on the interaction of humic substances with Fe or Cu is important in the assessment of the bioavailability of such metals in aquatic systems. In this study, the binding processes of two different DOM samples (Suwanee River humic acid, SRHA and Nordic Lake fulvic acid, NLFA)with Fe(III) and Cu(II) were examined. With the assistance of excitation-emission matrix fluorescence (EEM) and parallel factor analysis (PARAFAC), three humic-like components (C1, C2 and C4), and one microbial humic-like component (C3), were identified in the characterization of the samples. The quenching rates of C2 and C4 fluorescence in the presence of Cu were similar, which were slightly faster than that forC1. In contrast, the C3 fluorescence was significantly less affected by the addition of metals. In the presence of Fe, the most significant quenching was also observed for C2, and the behavior of C4 fluorescence was dependent on the pH value. Some differences in the fluorescence quenching were also observed between the two humic acid standards (one more higher-plant/terrestrially derived vs. the other more microbial/aquatic derived). In addition, pH had a significant effect on the metal binding with significantly lower quenching at lower pH values. Some variations where observed for the higher pH conditions particularly for Cu(II). A modified

  11. Electronic Olfactory Sensor Based on A. mellifera Odorant-Binding Protein 14 on a Reduced Graphene Oxide Field-Effect Transistor.

    PubMed

    Larisika, Melanie; Kotlowski, Caroline; Steininger, Christoph; Mastrogiacomo, Rosa; Pelosi, Paolo; Schütz, Stefan; Peteu, Serban F; Kleber, Christoph; Reiner-Rozman, Ciril; Nowak, Christoph; Knoll, Wolfgang

    2015-11-02

    An olfactory biosensor based on a reduced graphene oxide (rGO) field-effect transistor (FET), functionalized by the odorant-binding protein 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the in situ and real-time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand-receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K(d)=4 μM to K(d)=3.3 mM. The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.

  12. Mechanisms of Zn(II) binded to collagen and its effect on the capacity of eco-friendly Zn-Cr combination tanning system.

    PubMed

    Cao, Shan; Liu, Bing; Cheng, Baozhen; Lu, Fuping; Wang, Yanping; Li, Yu

    2017-01-05

    The eco-friendly combination tanning process has been developed to reduce chromium in existing researches, which is based on zinc tanning agents. This can be considered as a less-chrome substitute for current tanning process. To gain deeper understanding of the binding mechanisms of zinc-collagen interaction, which are affected by tanning pH, experiments have been carried out. Analysis in this paper reveals how chemical bonds from the collagen's main function groups combine with zinc. XPS and NIR data was analyzed for further understanding of where the zinc binding sites lie on collagen fibers at different pH. The results indicate that high pH is helpful to amino-binding sites while low pH promotes carboxyl-binding sites on collagen fibers. Furthermore, from the effect of Zinc-chrome combination tanning, we can see that the new method reduces the chromium dosage in tanning process compared to the conventional chrome tanning method.

  13. Effects of atrial and brain natriuretic peptides upon cyclic GMP levels, potassium transport, and receptor binding in rat astrocytes

    SciTech Connect

    Beaumont, K.; Tan, P.K. )

    1990-02-01

    The ability of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) to alter cyclic GMP levels and NaKCl cotransport in rat neocortical astrocytes was determined. At concentrations of 10(-9)-10(-6) M, rat ANP99-126 (rANF), rat ANP102-126 (auriculin B), and rat ANP103-126 (atriopeptin III) stimulated 6- to 100-fold increases in cyclic GMP levels. Porcine BNP (pBNP) and rat BNP (rBNP) were 20%-90% as effective as rANF over most of this concentration range, although 10(-6) M pBNP produced a greater effect than rANF. NaKCl cotransport as measured by bumetanide-sensitive 86Rb+ influx was not altered by exposure of astrocytes to 10(-6)M rANF, pBNP, or rBNP. Both pBNP and rBNP, as well as rat ANP103-123 (atriopeptin I) and des(gl18, ser19, gly20, leu21, gly22) ANF4-23-NH2 (C-ANF4-23) strongly competed for specific 125I-rANF binding sites in astrocyte membranes with affinities ranging from 0.03 to 0.4 nM, suggesting that virtually all binding sites measured at subnanomolar concentrations of 125I-rANF were of the ANP-C (ANF-R2) receptor subtype. These receptors are thought to serve a clearance function and may be linked to a guanylate cyclase activity that is chemically and pharmacologically distinct from that coupled to ANP-A (ANF-R1) receptors. ANP receptors on astrocytes may function in limiting the access of ANP and BNP to neurons involved in body fluid and cardiovascular regulation.

  14. Lorentz approach to static magnetic field effects on bound-ion dynamics and binding kinetics: Thermal noise considerations

    SciTech Connect

    Muehsam, D.J.; Pilla, A.A.

    1996-05-01

    The present study characterizes an ion-binding site, a molecular cleft in a signaling molecule such as calmodulin or troponin C, as a damped linear isotropic oscillator potential for small displacements about the origin. Quantitative assessments of the effects of thermal noise and exogenous static magnetic fields are made through a statistical mechanical treatment of the Lorentz-Langevin equation for an ion bound in a molecular cleft. Thermal noise causes a bound ion to e ejected from the site after a bound life-time dependent upon the thermal noise spectral density. It is shown that the Lorentz-Langevin model requires values of the viscous damping parameter many orders of magnitude below those for bulk water in order to characterize the binding site and to obtain realistic lifetimes for a bound ion. The model predicts that milliTesla-range magnetic fields are required for static field effects on dissociation kinetics. The Lorentz equation also yields a classic coherent solution describing precession of the bound-ion oscillator orientation at the Larmor frequency. The bound-ion dynamics described by this coherent solution are sensitive to microTesla-range static magnetic fields in the presence of thermal noise. Numerical integration of the contribution of thermal noise forces to these dynamics is in good agreement with the results of statistical mechanical analysis, also producing realistic bound lifetimes for only very low viscous damping values. The mechanisms by which modulation of precessional motion might enable a signaling molecule such as calmodulin to detect an exogenous magnetic field are presently unclear.

  15. Opposing effects of estradiol- and testosterone-membrane binding sites on T47D breast cancer cell apoptosis

    SciTech Connect

    Kampa, Marilena; Nifli, Artemissia-Phoebe; Charalampopoulos, Ioannis; Alexaki, Vassilia-Ismini; Theodoropoulos, Panayiotis A.; Stathopoulos, Efstathios N.; Gravanis, Achille; Castanas, Elias . E-mail: castanas@med.uoc.gr

    2005-07-01

    Classical steroid mode of action involves binding to intracellular receptors, the later acting as ligand-activated nuclear transcription factors. Recently, membrane sites for different steroids have been also identified, mediating rapid, non-genomic, steroid actions. Membrane sites for estrogen and androgen have been found in a number of different cell types, bearing or not classical intracellular receptors. In the present study, with the use of radioligand binding, flow cytometry and confocal laser microscopy, we report that T47D human breast cancer cells express specific and saturable membrane receptors for both estrogen (K {sub D} 4.06 {+-} 3.31 nM) and androgen (K {sub D} 7.64 {+-} 3.15 nM). Upon activation with BSA-conjugated, non-permeable ligands (E{sub 2}-BSA and testosterone-BSA), membrane estrogen receptors protect cells from serum-deprivation-induced apoptosis, while androgen receptors induce apoptosis in serum-supplemented T47D cells. In addition, co-incubation of cells with a fixed concentration of one steroid and varying concentrations of the other reversed the abovementioned effect (apoptosis for androgen, and anti-apoptosis for E{sub 2}), suggesting that the fate of the cell depends on the relative concentration of either steroid in the culture medium. We also report the identification of membrane receptors for E{sub 2} and androgen in biopsy slides from breast cancer patients. Both sites are expressed, with the staining for membrane E{sub 2} being strongly present in ER-negative, less differentiated, more aggressive tumors. These findings suggest that aromatase inhibitors may exert their beneficial effects on breast cancer by also propagating the metabolism of local steroids towards androgen, inducing thus cell apoptosis through membrane androgen receptor activation.

  16. Lunar and Martian soil stimulants have different effects on L-[14C]glutamate binding to brain nerve terminals

    NASA Astrophysics Data System (ADS)

    Borisova, Tatiana; Krisanova, Natalia; Nazarova, Anastasiya; Borysov, Arseniy; Chunihin, Olexander

    Nano-sized particles can be deleterious to human physiology because they may be internalized by lung epithelium and overcome the blood-brain barrier. The health effects from exposure to Lunar and Martian dust are almost completely unknown, whereas they can be deleterious to human physiology. The effects of Lunar and Martian Soil Simulants (Orbital Technologies Corporation, Madison, USA) on the conductance of planar lipid membrane, membrane potential, acidification of synaptic vesicles, glutamate uptake, and ambient level of glutamate in isolated rat brain nerve terminals (synaptosomes) were studied using photon correlation spectroscopy, Planar Lipid Bilayer technique, spectrofluorimetry, radiolabeled assay, respectively. Lunar and Martian Soil Simulants did not influence the conductance of planar lipid membrane. It was revealed that nerve terminals were not indifferent to the exposure to inorganic particles of Lunar and Martian Soil Simulants. Using Zetasizer Nanosystem (Malvern Instruments) with helium-neon laser for dynamic light scattering (DLS), the synaptosomal size before and after the addition of Lunar and Martian Soil Simulants was measured and the binding of Lunar and Martian Soil Simulants inorganic particles to nerve terminals was demonstrated. Using potential-sensitive fluorescent dye rhodamine 6G, we showed that Lunar and Martian Soil particles did not influence the potential of the plasma membrane of nerve terminals. Acidification of synaptic vesicles of nerve terminals was not changed in the presence of Lunar and Martian Soil particles that was revealed with pH-sensitive fluorescent dye acridine orange. Martian Soil Simulant particles did not change binding of L-[14C]glutamate to brain nerve terminals, in contrast, Lunar ones changed this parameter and this fact may have harmful consequences to human physiology, in particular, glutamate homeostasis in the mammalian CNS.

  17. Differential Effects of Structural Modifications on the Competition of Chalcones for the PIB Amyloid Imaging Ligand-Binding Site in Alzheimer's Disease Brain and Synthetic Aβ Fibrils.

    PubMed

    Fosso, Marina Y; McCarty, Katie; Head, Elizabeth; Garneau-Tsodikova, Sylvie; LeVine, Harry

    2016-02-17

    Alzheimer's disease (AD) is a complex brain disorder that still remains ill defined. In order to understand the significance of binding of different clinical in vivo imaging ligands to the polymorphic pathological features of AD brain, the molecular characteristics of the ligand interacting with its specific binding site need to be defined. Herein, we observed that tritiated Pittsburgh Compound B ((3)H-PIB) can be displaced from synthetic Aβ(1-40) and Aβ(1-42) fibrils and from the PIB binding complex purified from human AD brain (ADPBC) by molecules containing a chalcone structural scaffold. We eva