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Sample records for inotropic effect binding

  1. Relationship between the stereoselective negative inotropic effects of verapamil enantiomers and their binding to putative calcium channels in human heart.

    PubMed Central

    Ferry, D. R.; Glossmann, H.; Kaumann, A. J.

    1985-01-01

    Ventricular preparations from patients with mitral disease and hypertrophic obstructive cardiomyopathy (HOCM) were set up to contract isometrically. Ventricular membrane particles were also prepared and putative calcium channels were labelled with [3H]-nimodipine. Positive staircase was induced by varying the rate of stimulation of isolated strips from 6 min-1 to 120 min-1 in the presence of 6-60 microM (-)-adrenaline or (-)-noradrenaline. (-)-Verapamil 3-5 microM or (+)-verapamil 20-30 microM reversed the force-frequency relationship (i.e. caused negative staircase) in preparations from patients with mitral disease or HOCM. In subendocardial strips of ventricular septum from 5 patients with HOCM paced at 60 min-1, both (-)-verapamil and (+)-verapamil caused cardiodepression. Half-maximal cardiodepression was observed with 0.4 microM (-)-verapamil and with 3 microM (+)-verapamil. [3H]-nimodipine bound to ventricular membrane particles in a saturable, reversible fashion to a high affinity site with an equilibrium dissociation constant of 0.23 nM. The density of these sites was 95 fmol mg-1 of membrane protein. Binding of the tritiated 1,4-dihydropyridine was stereoselectively inhibited by 1,4-dihydropyridine enantiomers and nifedipine. (-)-Verapamil and (+)-verapamil inhibited high affinity [3H]-nimodipine binding in a negative heterotropic allosteric manner with (-)-verapamil being 5 times more potent than (+)-verapamil on an IC50 basis. At a given [3H]-nimodipine concentration, (+)-verapamil inhibited a greater fraction of specific [3H]-nimodipine binding. The allosteric mode of (+)-verapamil inhibition of [3H]-nimodipine binding was confirmed by kinetic studies. (-)-Verapamil shifted (+)-verapamil-binding inhibition curves to the right in an apparently competitive fashion. The inversion of staircase caused by both verapamil enantiomers suggests that they cause a use-dependent channel blockade. The similar potency ratios for binding and for cardiodepression are

  2. The inotropic effects of UK 14,275, a phosphodiesterase inhibitor, in man.

    PubMed Central

    Jackson, P G; Jackson, G; Kitson, D; Jewitt, D E

    1978-01-01

    1. UK 14,275 (Pfizer) an inotropic agent with cardiac phosphodiesterase inhibitory activity, was administered to ten healthy male volunteers. 2. The inotropic activity was assessed by non-invasive measurement of systolic time intervals (STI). 3. The compound had significant inotropic activity in the doses administered, as judged by the shortening of pre-ejection period (PEP), without any significant chronotropic activity. 4. The inotropic effect was abolished when measurements were repeated following beta-adrenoceptor blockade with oral propranolol. 5. The inotropic activity was compared to that of intravenous isoprenaline. PMID:619936

  3. Positive inotropic effect of porcine left ventricular extract on canine ventricular muscle.

    PubMed Central

    Navaratnam, S.; Chau, T.; Agbanyo, M.; Bose, D.; Khatter, J. C.

    1990-01-01

    1. We previously isolated an extract from porcine left ventricle that possessed digitalis-like properties such as inhibition of cardiac and kidney Na+, K(+)-ATPase, displacement of [3H]-ouabain from its binding sites and cross reactivity with digoxin antibodies. The extract also had a positive inotropic effect on the guinea-pig heart. 2. In the present study the positive inotropic response of the extract was characterized in canine right ventricular trabeculae. Maximum inotropic response (501 +/- 20%) was produced by 300 microliters and the half maximal increase occurred with 125 microliters of the extract. 3. Ouabagenin produced aftercontractions in rapidly paced trabeculae. Equipotent and even greater amounts of the extract did not produce aftercontractions. 4. The extract increased the amplitude of the delayed component (P2) of biphasic contractions produced by replacing about 92-96% of the external Ca with Sr. A smaller increase in the size of the early component (P1) was also seen. 5. The extract decreased post-rest potentiation after rest for 30s and 2 min. After 8 min of rest, post-rest potentiation was converted to post-rest depression. 6. The extract (20 microliters) produced a decrease in the amplitude of the post-rest rapid cooling contracture (RCC) at all rest intervals. The steady state RCC, although greater than that in the control muscle, was increased to a lesser extent than the size of the steady state electrically driven contractions. 7. It is suggested that the extract from porcine left ventricle produces a positive inotropic response by increasing the trans-sarcolemmal influx of Ca.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2257439

  4. Effect of inotropic stimulation on mitochondrial calcium in cardiac muscle.

    PubMed

    Moravec, C S; Bond, M

    1992-03-15

    Ca(2+)-dependent activation of citric acid cycle enzymes has been demonstrated in isolated cardiac mitochondria. These observations led to the hypothesis that Ca2+ is the signal coupling myofibrillar energy use to mitochondrial energy production in vivo. To test this hypothesis we have measured mitochondrial Ca2+ content during increased energy demand, using electron probe microanalysis. Mitochondrial Ca2+ was measured in hamster papillary muscles rapidly frozen at the peak rate of tension rise under control conditions and after stimulation with the beta-adrenergic agonist isoproterenol (10(-6) M). A third group of muscles was frozen after incubation in low (46.5 mM) Na+ solution to Ca2+ load the cells. Pyruvate dehydrogenase activity was measured in each of the muscles. Isoproterenol caused a 39% increase in force and a 43% increase in pyruvate dehydrogenase activity but no change in mitochondrial Ca2+ (0.46 +/- 0.19 (S.E.) mmol of Ca2+/kg, dry weight) compared with control (0.54 +/- 0.12). In contrast, low Na+ increased pyruvate dehydrogenase activity by 56% and also elevated mitochondrial Ca2+ to 1.28 +/- 0.31 (p less than 0.02). These results demonstrate that mitochondrial Ca2+ is not elevated after inotropic stimulation of cardiac muscle by beta-adrenergic agonists although pyruvate dehydrogenase activity is increased. We conclude that Ca2+ uptake by mitochondria is not a requirement for activation of mitochondrial respiration after increased energy demand. PMID:1544913

  5. Angiotensin I-converting enzyme inhibitors potentiate bradykinin's inotropic effects independently of blocking its inactivation.

    PubMed

    Minshall, R D; Erdös, E G; Vogel, S M

    1997-08-01

    The positive inotropic effects of bradykinin (BK) and 2 analogs resistant to angiotensin I-converting enzyme (ACE) were potentiated on isolated guinea pig atrial preparations by enalaprilat. The stable BK analogs, dextran-BK and [Hyp3-Tyr(Me)8]-BK, were as active as BK. Pretreatment for 5 min with enalaprilat augmented the maximal positive inotropic effect of [Hyp3-Tyr(Me)8]-BK 2.8-fold, from 19% to 53% and that of BK from 28% to 42% over baseline; inotropic responses to dextran-BK (1 microM) were similarly increased. The activity of atrial ACE, a zinc-requiring enzyme, was completely inhibited by 8-hydroxyquinoline-5-sulfonic acid (QSA, 10 mM), which raised the maximal inotropic effect of BK to 39% above baseline. This value rose to 67% when in addition to QSA, 1 microM enalaprilat was added; enalaprilat thus, potentiated the effects of BK independently of enzyme inhibition. The positive inotropic effects to BK and its analogs decline with time in the presence of these agonists. After 10 min of exposure, the response to 1 microM [Hyp3-Tyr(Me)8]-BK decreased to about half, and after 20 min, to 0. Enalaprilat, when present in the tissue bath, prevented the decline in inotropy; even after tachyphylaxis occurred, it reversed this decrease in activity when added. The effects of 1 microM [Hyp3-Tyr(Me)8]-BK, in the absence or presence of enalaprilat, were abolished by the BK B2 receptor antagonist icatibant (0.75 microM). The results indicate that ACE inhibitors, by potentiating the BK effects and blocking BK B2-receptor desensitization, may contribute to the beneficial cardiac effects of BK independently of blocking its inactivation.

  6. Studies on the positive inotropic effect of phenylephrine: a comparison with isoprenaline.

    PubMed

    Ledda, F; Marchetti, P; Mugelli, A

    1975-05-01

    1. The effects of phenylephrine and isoprenaline on the isometric contraction of guinea-pig ventricle were compared over the whole range of their respective dose-response curves. 2. In preparations driven at 2.5 Hz the increase in contractile force induced by either isoprenaline of phenylephrine was linearly correlated to an increase in maximum velocity of force development. The relaxation time was shortened by isoprenaline but not by phenylephrine. 3. The negative inotropic effect induced by delta [N-(3,4-dimethoxyphenethyl)-N-methyl-amino]-alpha-(3,4,5-trimethoxyphenyl)alpha-isopropylvaleronitrile hydrochloride (D(600)) was reversed by isoprenaline, but little influenced by phenylephrine. 4. The study of the interval-force relationship shows that the increase in contractile force induced by phenylephrine (3 X 10(-5) M) was relatively greater at low frequencies of stimulation, and that the maximum effect was reached at the frequency of 1 Hz. 5. The positive inotropic effect of phenylephrine (10-4 M) was significantly higher at a frequency of 1 Hz than at 2.5 Hz; the effect of isoprenaline (3 x 10-8 M) was not significantly different at the two driving frequencies. 6. In preparations driven at 1 Hz the inotropic effect of the lower concentrations of phenylephrine was due to an increase in the time to peak tension without any change of the maximum velocity of force development; however an increase of this parameter became evident only after higher concentrations of the amine (10-5 M or more), associated with a progressive shortening of the time to peak. 7. A correlation between mechanical and electrophysiological effects of phenylephrine is attempted; the suggestion is advanced that the prolongation of the action potential and of the active state duration may be an important factor in the inotropic effect of phenylephrine.

  7. 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.

  8. Safety of dietary supplements: chronotropic and inotropic effects on isolated rat atria.

    PubMed

    Kubota, Yoko; Umegaki, Keizo; Tanaka, Naoko; Mizuno, Hideya; Nakamura, Kazuki; Kunitomo, Masaru; Shinozuka, Kazumasa

    2002-02-01

    We investigated the effects of dietary supplements on atria isolated from male Wistar rats. The examined supplements, which are increasingly used in Japan, those were Ginkgo biloba extract (GBE), catechins, isoflavones, sodium iron chlorophyllin and sodium copper chlorophyllin. GBE at 100-1000 microg/ml significantly increased the beat rate and the contractile force. Catechins at 1-100 microg/ml significantly potentiated the contractile force but did not effect the beat rates. However, isoflavones, sodium iron and sodium copper chlorophyllins did not change the contractile force or the beat rates. To identify the active ingredient of GBE, ginkgolide B, quercetin and amentoflavone on the atria were tested. Ginkgolide B weakened the contractile force. Quercetin potentiated the contractile force at only 30 microg/ml. Amentoflavone significantly increased the beat rate. From these findings, amentoflavone and quercetin were considered to be the principal ingredients of GBE producing the positive chronotropic and inotropic actions, respectively. In the case of catechins, (-)-epigallocatechin gallate (EGCg), one of the principal ingredients, produced inotropic actions. These findings suggest that there are some dietary supplements which affect cardiac function, such GBE and catechins.

  9. The action of creatinol 0-phosphate on the inotropic effect of isoprenaline in isolated rat atria and in anesthetized dogs.

    PubMed

    Godfraind, T; Ghysel-Burton, J; Merlo, L

    1979-01-01

    The action of N-methyl-N-(beta-hydroxyethyl) guanidine O-phosphate (creatinol O-phosphate) was studied on the contractility of the heart, in vitro on isolated rat atria and in vivo in anesthetized dogs. Creatinol O-phosphate was without action on the contractility of isolated rat atria stimulated by isoprenaline 10(-8) mol/l which produced the maximum inotropic effect. Creatinol O-phosphate increased the contractility evoked by isoprenaline 10(-5) mol/l and impaired the development of the negative inotropic effect evoked by prolonged treatment with this large dose. In dogs, creatinol O-phosphate pretreatment increased the positive inotropic effect of large but not low isoprenaline dosages. It is concluded that creatinol O-phosphate increased the contractile efficiency of the heart submitted to the action of an excess of catecholamines.

  10. Frequency-dependence of the positive inotropic effect of methoxamine and naphazoline mediated by alpha-Adrenoceptors in the isolated rabbit papillary muscle.

    PubMed

    Endoh, M; Schümann, H J

    1975-01-01

    Under the conditions of different stimulation frequencies the inotropic effects of the alpha-adrenoceptor stimulationg agents, methoxamine, naphazoling and oxymetazoline were studied on the isolated rabbit papillary muscle. 1. On the papillary muscle stimulated at 0.5 Hz methoxamine in concentrations from 10(-5)M caused a significant and dose-dependent positive inotropic effect. At 10(-3)M methoxamine decreased the developed tension. With increasing frequency of stimulation (0.5--1--1.5Hz), the positive inotropic effect became smaller, while the negative inotropic one was more pronounced. The time course of the disappearance of the negative inotropic effect of methoxamine by washout differed from that of the positive inotropic effect: the negative component disappeared within 30 min, whereas the positive one lasted for about 100 min. The positive inotropic effect of noradrenaline (10(-6)M), in contrast ot that of methoxamine, was not influenced by the frequency under the same conditions of stimulation. Also naphazoline (10(-5)M) caused a significant positive inotropic effect on the papillary muscle stimulated at 0.5 Hz, while oxymetazoline induced exclusively a negative inotropic effect. 2. The positive inotropic effect of metoxamine (10(-4)M) as well as of naphazoline (10(-5)M) evoked at a frequency of 0.5 Hz was abolished by phentolamine (10(-6)M). Methoxamine (10(-4)M) induced a significant negative inotropic effect in the presence of phentolamine. Phentolamine antagonized the positive inotropic effect of methoxamine in a non-competitive manner: the pD2-value was 7.76. 3. In the presence of methoxamine (10(-4)M) the developed tension in the lower range (0.05--1 Hz) of the frequency-force relationship was enhanced, while that in the higher range (greater that 1.5 Hz) was decreased. The enhancement was abolished by phentolamine (10(-6)M). 4. Papaverine (2x10(-5)M) did not affect the positive inotropic effect of methoxamine. 5. The present results show that

  11. Species differences in the negative inotropic effect of acetylcholine and soman in rat, guinea pig, and rabbit hearts. (Reannouncement with new availability information)

    SciTech Connect

    Maxwell, D.M.; Thomsen, R.H.; Baskin, S.I.

    1991-12-31

    Acetylcholine reduced atrial contractions by 82.5% in guinea pig, 50.8% in rat, and 41.5% in rabbit. 2. The EC50, values for the negative inotropic effect of acetylcholine were 3.3 x 10(-7) M in rat and guinea pig atria and 4.1 x 10(-6) M in rabbit atria. 3. There was no correlation between the species differences in the negative inotropic effect of acetylcholine in atria and the density or affinity of acetylcholinesterase or muscarinic receptors. 4. Inhibition of atrial acetylcholinesterase with soman reduced the EC50 of acetylcholine three-fold in all species, but did not change the maximal inotropic effect of acetylcholine. 5. Species differences in the negative inotropic effect of acetylcholine may be caused by differences in the coupling between myocardial muscarinic receptors and the ion channels that mediate negative inotropy. Acetylcholine, cardiovascular response, species variation negative inotropic response.

  12. [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

  13. [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

  14. Cyclic GMP-dependent protein kinase activation in the absence of negative inotropic effects in the rat ventricle

    PubMed Central

    MacDonell, Karen L; Diamond, Jack

    1997-01-01

    It has been suggested that activation of cyclic GMP-dependent protein kinase (PKG) is a necessary step in the chain of events leading to the production of negative inotropy by muscarinic receptor agonists in mammalian ventricles, and that some cyclic GMP-elevating agents, such as sodium nitroprusside (SNP), fail to exert a negative inotropic effect because they elevate cyclic GMP levels in a pool that does not activate the kinase. This hypothesis was tested in the present study by monitoring the effects of carbachol, SNP and atrial natriuretic peptide (ANP) on contractility, cyclic GMP content and PKG activity in rat intact ventricular preparations and freshly isolated ventricular cardiomyocytes.The presence of PKG in both the intact vehicle and in isolated ventricular cardiomyocytes was confirmed by MonoQ anion exchange chromatography and Western blotting. The elution profile indicated that the conditions of the PKG assay were selective for measuring PKG activity.Carbachol induced a marked negative inotropic effect in intact, perfused hearts and ventricular strips in the presence of isoproterenol. The negative inotropic effect of carbachol was not associated with significant changes in cyclic GMP content or PKG activity in intact ventricular tissue, or in PKG activity in isolated cardiomyocytes.SNP and ANP significantly increased cyclic GMP levels and activated PKG in intact ventricular preparations. Both drugs also activated PKG in isolated cardiomyocytes. However, neither drug had any negative inotropic effect in isoprenaline-stimulated perfused hearts and ANP did not change the contractility of isoprenaline-stimulated isolated cardiomyocytes.The results of this study demonstrate that the negative inotropic effects of muscarinic receptor agonists can occur in the absence of significant activation of PKG. Conversely, marked increases in ventricular cyclic GMP content and PKG activity caused by SNP or ANP were not accompanied by a negative inotropic effect

  15. Positive inotropic effects mediated by alpha 1 adrenoceptors in intact human subjects.

    PubMed

    Curiel, R; Pérez-González, J; Brito, N; Zerpa, R; Téllez, D; Cabrera, J; Curiel, C; Cubeddu, L

    1989-10-01

    The role of alpha 1-adrenergic receptors (adrenoceptors) on cardiac contractility was investigated in human subjects. The effect of methoxamine, a selective alpha-adrenoceptor agonist, and angiotensin II, on cardiac contractility was determined by means of noninvasive assessment of the slope of the end-systolic pressure (ESP)/end-systolic dimension (ESD) relationship. The slope (m) of this ratio was significantly higher with methoxamine (17.0; SD = 9.0 mm Hg/mm) than with angiotensin II (4.8; SD = 1.9 mm Hg/mm) (p less than 0.05). Slopes with methoxamine were higher when heart rates (HRs) were reflexly reduced, and were significantly diminished when reflex bradycardia was prevented by atropine (p less than 0.05) or atrial pacing (p less than 0.01). Previous treatment with propranolol did not modify m values with methoxamine (m = 15.5; SD = 4.4 mm Hg/mm). Phentolamine, given at peak methoxamine effect, did not consistently modify m values, resulting in an average slope not significantly different from that obtained with methoxamine alone. However, the addition of phentolamine did not cause an increase in ESDs at each level of ESP with respect to methoxamine. In the same subjects, infusion of phentolamine after angiotensin did not modify ESDs at comparable ESP levels. These findings suggest the existence of a positive inotropic effect mediated by alpha 1 adrenoceptors in the intact human heart.

  16. 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

  17. Inhibition of phosphodiesterase-3 by levosimendan is sufficient to account for its inotropic effect in failing human heart

    PubMed Central

    Ørstavik, Ø; Ata, S H; Riise, J; Dahl, C P; Andersen, G Ø; Levy, F O; Skomedal, T; Osnes, J-B; Qvigstad, E

    2014-01-01

    Background and Purpose Levosimendan is known as a calcium sensitizer, although it is also known to inhibit PDE3. We aimed to isolate each component and estimate their contribution to the increased cardiac contractility induced by levosimendan. Experimental Approach Contractile force was measured in electrically stimulated ventricular strips from explanted failing human hearts and left ventricular strips from normal male Wistar rats. PDE activity was measured in a two-step PDE activity assay on failing human ventricle. Key Results Levosimendan exerted a positive inotropic effect (PIE) reaching maximum at 10−5 M in ventricular strips from failing human hearts. In the presence of the selective PDE3 inhibitor cilostamide, the PIE of levosimendan was abolished. During treatment with a PDE4 inhibitor and a supra-threshold concentration of isoprenaline, levosimendan generated an amplified inotropic response. This effect was reversed by β-adrenoceptor blockade and undetectable in strips pretreated with cilostamide. Levosimendan (10−6 M) increased the potency of β-adrenoceptor agonists by 0.5 log units in failing human myocardium, but not in the presence of cilostamide. Every inotropic response to levosimendan was associated with a lusitropic response. Levosimendan did not affect the concentration–response curve to calcium in rat ventricular strips, in contrast to the effects of a known calcium sensitizer, EMD57033 [5-(1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydroquinolin-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-one]. PDE activity assays confirmed that levosimendan inhibited PDE3 as effectively as cilostamide. Conclusions and Implications Our results indicate that the PDE3-inhibitory property of levosimendan was enough to account for its inotropic effect, leaving a minor, if any, effect to a calcium-sensitizing component. PMID:24547784

  18. [The characteristics of the inotropic effects of Levosimendan on the isolated myocardium of patients with ischemic heart disease].

    PubMed

    Afanas'ev, S A; Timofeev, V Iu

    2000-01-01

    Effect of the new cardiotonic agent levosimendan on the inotropic response parameters of the isolated myocardium of patients suffering from ischemic heart disease (IHD) was studied in the 0.01-1.00 mumole/liter concentration range. The inotropic response was measured in an isometric mode (electric stimulation frequency, 0.5 Hz; temperature, 37 degrees C; perfusion rate, 10 ml/min). In the range of 0.01-0.1 mumole/liter, levosimendan preferentially increases the rate of stress growth (+dP/dt) and the relaxation time. At concentrations above 0.1 mumole/liter, levosimendan produces a pronounced increase (56%) in the amplitude of single contraction. The mechanogram exhibits the signs of Ca-overload, including the post-contraction wave. It is concluded that the inotropic action of levosimendan on the isolated myocardium of IHD patients is related predominantly to increasing calcium uptake from outside. This circumstance is apparently caused by the initially high calcium content in sarcoplasmic reticulum, which is an important feature of the entire intracellular homeostasis in cardiomyocytes during IHD.

  19. Acidosis counteracts the negative inotropic effect of K+ on ventricular muscle strips from the toad Bufo marinus.

    PubMed

    Andersen, Johnnie Bremholm; Gesser, Hans; Wang, Tobias

    2004-01-01

    Strenuous activity is associated with acidosis, increased extracellular potassium concentration ([K+]o), and elevated levels of circulating catecholamines. Acidosis and elevated [K+]o are normally considered harmful to cardiac function, and a high sympathetic tone on the heart may lead to arrhythmia. During activity, however, the heart must be able to increase rate and strength of contraction. While the individual effects of [K+]o, acidosis, and adrenaline on contractile properties of cardiac muscle have been characterized for some ectothermic species, less information is available on their interactions. Here we examine the isolated and combined effects of [K+]o, acidosis, and adrenaline on ventricular muscle strips from the toad Bufo marinus. This study showed that increased [K+]o significantly reduced twitch force, while lactic acid significantly increased twitch force and more than counteracted the negative inotropic effects of elevated [K+]o. There was no inotropic effect of Na-lactate (neutralized lactic acid), which suggests that lactic acid stimulated twitch force through reduced pH and not by serving as a substrate. Adrenaline had a positive effect on twitch force in all preparations. Irrespective of treatment, twitch force decreased as stimulation rate increased. During high [K+]o, there was a severe reduction in maximal frequency of toad ventricular strips that was not alleviated by lactic acidosis and/or adrenaline, which suggests that high [K+]o influences twitch force and maximal rate by different mechanisms. In vivo levels of lactic acid, [K+]o, adrenaline, and heart rate previously observed during forced activity in Bufo did not significantly affect the contractile properties of heart muscle strips in vitro. Thus, although [K+]o significantly decreased twitch force, this detrimental effect was more than counteracted by the positive inotropic effect of lactic acid and adrenaline.

  20. Mechanical and electrophysiological studies on the positive inotropic effect of 2-phenyl-4-oxo-hydroquinoline in rat cardiac tissues.

    PubMed Central

    Su, M. J.; Chang, G. J.; Kuo, S. C.

    1993-01-01

    1. The pharmacological and electrophysiological effect of 2-phenyl-4-oxo-hydroquinoline (YT-1), a new synthetic agent, were determined in rat isolated cardiac tissues and ventricular myocytes. 2. YT-1 was found to have a positive inotropic effect in both atria and ventricular muscles but did not cause significant increases in the spontaneously beating rate of right atria. 3. The positive inotropic effect of YT-1 was antagonized neither by beta-nor by alpha-adrenoceptor antagonists but was partially antagonized by a Ca2+ channel blocker (verapamil) and a K+ channel blocker (4-AP). 4. The action potential duration and amplitude of ventricular cells were progressively increased as the concentration of YT-1 was increased from 3 to 30 microM. 5. A voltage clamp study revealed that the prolongation of action potential duration by YT-1 was associated with a prominent inhibition of 4-AP-sensitive transient outward current (I(to)). At potentials negative to the reversal potential of K1-channels, the inward current through these channels was partially reduced by YT-1. At potentials positive to the reversal potential, the outward current through these channels was affected very little. 6. Although YT-1 blocked the amplitude of I(to), the voltage-dependence of the steady-state inactivation of I(to), was unaffected. 7. Apart from the inhibition of K+ currents, YT-1 also inhibited the sodium inward current. 8. The evidence suggests that YT-1 increases the slow inward Ca2+ current (ICa) significantly. 9. It is concluded that the positive inotropic effect of YT-1 is due predominantly to the increase of ICa and inhibition of I(to).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8106106

  1. What are the hemodynamic and respiratory effects of passive limb exercise for mechanically ventilated patients receiving low-dose vasopressor/inotropic support?

    PubMed

    Genc, Arzu; Koca, Ugur; Gunerli, Ali

    2014-01-01

    Passive limb exercises (PLEs) are used widely in the management of unconscious patients and an early start is recommended. The aim of this study was to determine the effects of PLEs on hemodynamic and respiratory parameters in mechanically ventilated critically ill patients receiving low-dose vasopressor/inotropic support. The charts of 120 mechanically ventilated patients who underwent PLEs were evaluated retrospectively between January 2000 and July 2002. Patients were grouped on the basis of administration of vasopressor/inotropic support. Thirty-eight patients did not get vasopressor/inotropic support (group 1) and 82 patients received low-dose vasopressor/inotropic support (dopamine <10 μg/kg/min, noradrenaline/adrenaline <0.1; group 2). Central venous pressure, heart rate, mean arterial pressure, and oxygen saturation were recorded before and immediately after PLEs. After PLEs in group 1 patients, central venous pressure and mean arterial pressure values increased significantly, and in group 2 patients, central venous pressure increased significantly (P < .05). No statistically significant difference was observed in the rate of change of hemodynamic or respiratory parameters between the 2 groups after the PLEs (P > .05). This retrospective study confirmed that PLEs result in similar hemodynamic and respiratory changes in critically ill patients who received low-dose vasopressor/inotropic support versus those who do not.

  2. Cardiac inotropic effects of leukotriene C4 and prostaglandin I2 in the unanesthetized American bullfrog, Rana catesbeiana.

    PubMed

    Robleto, D O; Reitmeyer, S T; Herman, C A

    1988-03-01

    The cardiovascular effects of leukotriene (LT) C4 and prostaglandin (PG) I2 were compared in the unanesthetized American bullfrog, Rana catesbeiana. Bullfrogs were instrumented to measure mean arterial pressure, peak ventricular pressure, its derivative (VP + dP/dt), and heart rate. Two hours after recovery from anesthesia, intravenous injections of LTC4 or PGI2 were tested over a dose range from 0.003 to 3 micrograms/kg body weight (bw). Both eicosanoids decreased mean arterial pressure, systolic ventricular pressure, and its derivative (VP + dP/dt). The effects of LTC4 and PGI2 on all parameters were similar at doses below 3 micrograms/kg bw. However, at 3 micrograms/kg bw, LTC4 had more potent negative inotropic effects than PGI2. Both compounds increased heart rate at 0.3 microgram/kg bw, but at 3 micrograms/kg bw PGI2 caused greater increases than LTC4. The hypotensive and negative inotropic effects of LTC4 were blunted in animals pretreated with indomethacin (4 mg/kg bw) to prevent endogenous prostaglandin and thromboxane synthesis, whereas the cardiovascular effects of PGI2 were unaffected by the blockade. The data show that both eicosanoids have similar qualitative effects on blood pressure and cardiac performance. However, the effects of LTC4 may be partially mediated by release of endogenous cyclooxygenase products, possibly PGI2. These results suggest that the bullfrog, an animal with no coronary arteries, is a useful model for comparative studies of cardiac actions of eicosanoids which are independent of effects mediated by changes in coronary vascular resistance.

  3. 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.

  4. 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

  5. Pharmacokinetics and pharmacodynamics of intravenous inotropic agents.

    PubMed

    Lehtonen, Lasse A; Antila, Saila; Pentikäinen, Pertti J

    2004-01-01

    Positive inotropic drugs have various mechanisms of action. Long-term use of cyclic adenosine monophosphate (cAMP)-dependent drugs has adverse effects on the prognosis of heart failure patients, whereas digoxin has neutral effect on mortality. There are, however, little data on the effects of intravenous inotropic drugs on the outcome of patients. Intravenous inotropic agents are used to treat cardiac emergencies and refractory heart failure. beta-Adrenergic agonists are rapid acting and easy to titrate, with short elimination half-life. However, they increase myocardial oxygen consumption and are thus hazardous during myocardial ischaemia. Furthermore they may promote myocyte apoptosis. Phosphodiesterase (PDE) III inhibiting drugs (amrinone, milrinone and enoximone) increase contractility by reducing the degradation of cAMP. In addition, they reduce both preload and afterload via vasodilation. Short-term use of intravenous milrinone is not associated with increased mortality, and some symptomatic benefit may be obtained when it is used in refractory heart failure. Furthermore, PDE III inhibitors facilitate weaning from the cardiopulmonary bypass machine after cardiac surgery. Levosimendan belongs to a new group of positive inotropic drugs, the calcium sensitisers. It has complex pharmacokinetics and long-lasting haemodynamic effects as a result of its active metabolites. In comparative trials, it has been better tolerated than the most widely used beta-agonist inotropic drug, dobutamine. The pharmacokinetics of the intravenous inotropic drugs might sometimes greatly modify and prolong the response to the therapy, for example because of long-acting active metabolites. These drugs display considerable differences in their pharmacokinetics and pharmacodynamics, and the selection of the most appropriate inotropic drug for each patient should be based on careful consideration of the clinical status of the patient and on the pharmacology of the drug.

  6. [THE EFFECT OF SEROTONIN ON THE INOTROPIC FUNCTION OF MYOCARDIUM OF THE LEFT VENTRICLE OF IMMATURE SPONTANEOUSLY HYPERTENSIVE RATS].

    PubMed

    Aflyatumova, G N; Nedorezova, R S; Nigmatullina, R R; Sadykova, D I; Mateeva, V L; Chibireva, M D

    2015-08-01

    The mechanisms of the serotonin effect on the inotropic function of the myocardium of the left ventricle of immature spontaneously hypertensive rats (SHR) are unexplored. It was found that systolic arterial blood pressure of 5-6 weeks SHR rats is 147.5 mm Hg, which is statistically significantly higher (more than 25 mm Hg) than in the same age of normotensive control Wistar- Kyoto rats. The weight of the heart, of the left ventricle myocardium, of the ventricular septum, of the aorta and the force of contraction of the left ventricle of 5-6-week-old SHR rats are increased significantly compared with the control. 0.1 pM serotonin increases and 1.0 pM and 10.0 AM serotonin reduce the force of contraction of the left ventricular myocardium in hypertensive rats, but there is a dose-dependent increase of the force of contraction in the control. Serotonin reduces the time of contraction of the myocardium of the left ventricular of SHR rats, these reactions are less pronounced as compared to the control.

  7. The influence of endogenously generated reactive oxygen species on the inotropic and chronotropic effects of adrenoceptor and ET-receptor stimulation.

    PubMed

    Sand, Carsten; Peters, Stephan L M; Pfaffendorf, Martin; van Zwieten, Pieter A

    2003-06-01

    Reactive oxygen species (ROS) play a role in cardiovascular diseases such as heart failure and hypertension. Furthermore, increasing evidence has accumulated suggesting that ROS can also be formed subsequent to the stimulation of various receptors, thus functioning as second messengers. The objective of the present study was to elucidate the role of intracellular-generated ROS in the inotropic and chronotropic effects of the alpha1- and beta-adrenoceptor and the ET-receptor stimulation in isolated rat atria. In addition, we investigated whether the MAPKerk pathway is involved in the ROS-provoked rise of contractile force. For this purpose hydrogen peroxide was applied, which is known to serve several endogenous functions as a second messenger. Moreover, hydrogen peroxide readily crosses cell membranes, which thus allows to mimic the intracellular formation. Preincubation of atria with EUK 8 (400 microM), a cell permeable superoxide dismutase- and catalase-mimetic, reduced the positive inotropic effect upon alpha1-adrenoceptor and ET-receptor stimulation. The responsiveness to beta-adrenoceptor stimulation remained unaffected by this pretreatment. The chronotropic effects were not altered by preincubation with EUK 8. In contrast to the MAPK(p38) inhibitor SB203580 (2 and 10 microM), the two MKKmek inhibitors PD98059 (30 and 100 microM) and U0126 (10 microM) significantly attenuated the positive inotropic response to hydrogen peroxide in isolated rat left atria. In addition, inhibition of the Na+/H+ exchange (NHE) by cariporide (1 microM) counteracted ROS-provoked increase of contractile force. From the present study we conclude that the inotropic responses to alpha1-adrenoceptor and ET-receptor stimulation are, at least partially, caused by intracellular-formed ROS, that subsequently may activate the MAPKerk pathway and the NHE.

  8. Cardiac myosin light chain phosphorylation and inotropic effects of a biased ligand, TRV120023, in a dilated cardiomyopathy model

    PubMed Central

    Tarigopula, Madhusudhan; Davis, Robert T.; Mungai, Paul T.; Ryba, David M.; Wieczorek, David F.; Cowan, Conrad L.; Violin, Jonathan D.; Wolska, Beata M.; Solaro, R. John

    2015-01-01

    Aims Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca2+-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that biased G-protein-coupled receptor ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca2+ transients. Our previous work demonstrated that the biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca2+ response. Methods and results We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unbiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure–volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation. Conclusion Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM. PMID:26045475

  9. 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.

  10. Inotropic effects of ethanol and dihydropyridines on the guinea pig heart atrial muscle

    SciTech Connect

    Salvatici, R.P. ); Gallardo-Carpentier, A.; Carpentier, R.G. ); Isaacson, R.L. )

    1990-01-01

    The effects of ethanol and/or dihydropyridines (DHPs) on force of contraction of atrial muscle were studied. Guinea pig atrial strips superfused with Tyrode's solution were driven while recording muscle tension. Bay K 8644 (BAYK) increased, while nimodipine or ethanol reduced, the peak tension developed and the maximum velocity of development of tension. The effects of ethanol were readily reversible, but those of the DHPs were not. The combined actions of ethanol and DHPs were the result of the synergism or antagonism of the drugs tested. The shorter duration of the action of ethanol resulted in the effect of DHPs being still evident well after the exposure to the drugs ended. In summary, ethanol and nimodipine exerted depressant actions on atrial contractile force, while BAYK had opposite effects. The different mechanisms of action may explain the different duration of the effects of ethanol and DHPs.

  11. Effects of different inotropes with antioxidant properties on acute regional myocardial ischemia in isolated rabbit hearts.

    PubMed

    Rump, A F; Schüssler, M; Acar, D; Cordes, A; Ratke, R; Theisohn, M; Rösen, R; Klaus, W; Fricke, U

    1995-05-01

    1. The antiischemic properties of the flavonoids acetylvitexin-rhamnoside (AVR) and luteolin-7-glucoside-(LUT), combining phosphodiesterase (PDE)-inhibitory and antioxidant properties, were studied in comparison to amrinone (AMR) or superoxide dismutase (SOD). The effects of the new dihydropyridine-type calcium-agonist Bay T 5006 were studied in comparison to Bay K 8644. 2. In isolated Langendorff-rabbit hearts perfused at constant pressure, acute regional ischemia (MI) was induced by coronary artery occlusion (CAO) and quantitated from epicardial NADH-fluorescence photography. Drugs were applied either before or after CAO (pre-treatment or treatment) to permit distinguishing the influence of functional and direct cytoprotective actions in the poorly collateralized rabbit hearts. 3. SOD did not affect left ventricular pressure (LVP) or coronary flow (CF) and reduced MI only if applied before CAO. LVP and CF were enhanced by LUT or AMR but not by AVR. MI was reduced to a similar extent in hearts treated with either drug. Cardioprotection by LUT was not improved by starting drug application before CAO. 4. Bay K 8644 reduced LVP and particularly CF, whereas Bay T 5006 did not affect functional parameters. MI was enlarged by Bay K 8644 and remained unaffected by treatment or pretreatment with Bay T 5006. 5. AMR, LUT and AVR possess antiischemic properties related to an improvement of myocardial perfusion. Although oxygen free radicals contribute to ischemic tissue injury, as shown by the cardioprotective effectiveness of SOD, antioxidant properties of the flavonoids LUT and AVR do not seem to be relevant for the antiischemic effects. Our findings also give no evidence for antioxidant properties of dihydropyridines relevant for cardioprotection. PMID:7789735

  12. Inotropic responses to human gene 2 (B29) relaxin in a rat model of myocardial infarction (MI): effect of pertussis toxin

    PubMed Central

    Kompa, Andrew R; Samuel, Chrishan S; Summers, Roger J

    2002-01-01

    Relaxin produces powerful inotropic and chronotropic responses in isolated atria. The effect of relaxin has been examined in a rat model of cardiac failure, induced by myocardial infarction (MI). Maximum inotropic responses to isoprenaline (sham 5.4±0.3 mN; MI 2.6±0.3 mN; P<0.001) and relaxin (sham 5.1±0.6 mN; MI 2.8±0.5 mN; P=0.013) were reduced in left atria following MI. No change in chronotropic responsiveness was observed in right atria. Pertussis toxin (PTX) treatment restored inotropic responses to isoprenaline (sham 5.5±1.3 mN; MI 5.8±1.0 mN; P=0.850) but not to relaxin. Instead, PTX reduced inotropic responses to relaxin in sham animals to the same level seen in the MI group (sham 3.2±1.7 mN; MI 2.8±0.6 mN; P=0.847). In right atria, PTX treatment did not affect the maximum chronotropic response to isoprenaline, but reduced responses to relaxin in both sham and MI animals. R3 relaxin and relaxin receptor (LGR7) mRNA was present in atria and left ventricle (LV) from sham and MI animals. R3 relaxin mRNA expression was increased in atria but not LV from MI animals. LGR7 mRNA expression was reduced in atria and LV from MI animals. PTX treatment in unoperated rats increased chronotropic responses (vehicle 184.3±5.3 beats min−1; PTX 211.3±9.5 beats min−1; P=0.029) and produced a rightward shift in the concentration-response curve to isoprenaline in left atria. PTX reduced inotropic (vehicle 3.3±0.7 mN; PTX 0.8±0.2 mN; P=0.005) and chronotropic (vehicle 130.2±8.1 beats min−1; PTX 90.6±11.1 beats min−1; P=0.012) responses to relaxin. In left atria, relaxin produced a small increase in cAMP compared to those produced by isoprenaline and forskolin. However, PTX treatment significantly reduced relaxin-, isoprenaline- and forskolin-stimulated cAMP accumulation. Cardiac failure in MI animals caused a reduced inotropic response to both relaxin and (−)-isoprenaline. In non-MI animals, PTX treatment also reduced inotropic responses to relaxin

  13. Effects of prostaglandin E1 (PGE1) on the positive inotropic response of heart muscle to elevation of external Ca++-concentration, to increased driving frequency, and to paired stimulation.

    PubMed

    Hadházy, P

    1976-01-01

    Electrically driven left guinea pig atria were exposed to positive inotropic stimuli which are thought to be related to the turnover of calcium ions. For increasing contractibility, the following procedures were used: a) varying the concentration of CaCl2 in the bath fluid; b) stimulation at frequencies from 1.0 to 3.0 Hz; c) paired stimulation. Positive inotropic responses to the increase of the rate of stimulation and to paired stimulation were not affected by 0.1 microgram/ml tetrodotoxin (TTX). This excludes the adrenergic contribution to the positive inotropic effects observed. Actions of the positive inotropic stimuli were studied both in the absence and in the presence of 0.1--1.0--10.0--1000.0 ng/ml of PGE1-PGE1 in the highest concentration used increased contractile force. The inotropic stimulus-response curves were not affected by PGE1 at any concentration. This finding suggests there is no interaction between Ca ions and PGE1 in the contractile mechanism of the guinea pig heart muscle.

  14. 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

  15. Home Inotropes and Other Palliative Care.

    PubMed

    Ginwalla, Mahazarin

    2016-07-01

    Heart failure is a leading case of morbidity and mortality worldwide, and patients with advanced heart failure have limited options without any available cure. These options mainly include cardiac transplantation or mechanical circulatory support device implantation. Chronic home inotropes are an option in these patients for a variety of indications. This report discusses the use of chronic home inotropes in palliated heart failure patients and reviews the role of palliative care management in end-stage heart failure. PMID:27371519

  16. Calcium sensitizer agents: a new class of inotropic agents in the treatment of decompensated heart failure.

    PubMed

    Perrone, Sergio V; Kaplinsky, Edgardo J

    2005-09-01

    The clinician's primary objective in treating a patient with decompensated heart failure is rapid and effective stabilization. This goal often is achieved through the use of inotropic support. Classic inotropic agents (beta-adrenergic agonists and phosphodiesterase III inhibitors) can provide short-term hemodynamic benefits, but their long-term use has been correlated with poor survival rates. Calcium sensitizers comprise a new drug class that offers hemodynamic and symptomatic improvements without increasing cAMP and intracellular calcium concentrations. These agents enhance contractility without a concurrent increase in the risk of cardiac events and thus represent a significant improvement over classic positive inotropic agents. Levosimendan is the most potent calcium sensitizer to date, exhibiting a unique dual mechanism of action that combines a positive inotropic action mediated via calcium sensitization and a vasodilator property via ATP-dependent potassium channels. Available clinical data suggest that calcium sensitizer agents represent a promising class of inotropic agents in a field that has seen few advances in recent decades.

  17. 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.

  18. Intraventricular and interventricular cellular heterogeneity of inotropic responses to α(1)-adrenergic stimulation.

    PubMed

    Chu, Charles; Thai, Kevin; Park, Ki Wan; Wang, Paul; Makwana, Om; Lovett, David H; Simpson, Paul C; Baker, Anthony J

    2013-04-01

    α1-Adrenergic receptors (α1-ARs) elicit a negative inotropic effect (NIE) in the mouse right ventricular (RV) myocardium but a positive inotropic effect (PIE) in the left ventricular (LV) myocardium. Effects on myofilament Ca(2+) sensitivity play a role, but effects on Ca(2+) handling could also contribute. We monitored the effects of α1-AR stimulation on contraction and Ca(2+) transients using single myocytes isolated from the RV or LV. Interestingly, for both the RV and LV, we found heterogeneous myocyte inotropic responses. α1-ARs mediated either a PIE or NIE, although RV myocytes had a greater proportion of cells manifesting a NIE (68%) compared with LV myocytes (36%). Stimulation of a single α1-AR subtype (α1A-ARs) with a subtype-selective agonist also elicited heterogeneous inotropic responses, suggesting that the heterogeneity arose from events downstream of the α1A-AR subtype. For RV and LV myocytes, an α1-AR-mediated PIE was associated with an increased Ca(2+) transient and a NIE was associated with a decreased Ca(2+) transient, suggesting a key role for Ca(2+) handling. For RV and LV myocytes, α1-AR-mediated decreases in the Ca(2+) transient were associated with increased Ca(2+) export from the cell and decreased Ca(2+) content of the sarcoplasmic reticulum. In contrast, for myocytes with α1-AR-induced increased Ca(2+) transients, sarcoplasmic reticulum Ca(2+) content was not increased, suggesting that other mechanisms contributed to the increased Ca(2+) transients. This study demonstrates the marked heterogeneity of LV and RV cellular inotropic responses to stimulation of α1-ARs and reveals a new aspect of biological heterogeneity among myocytes in the regulation of contraction.

  19. 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.

  20. Inotropic response to endothelin-1, isoprenaline and calcium in cardiomyocytes isolated from endotoxin treated rats: effects of ethyl-isothiourea and dexamethasone.

    PubMed

    Spiers, J P; Kelso, E J; Allen, J D; Silke, B; McDermott, B J

    2000-07-01

    1. The contractile effects of endothelin-1, isoprenaline and extracellular calcium were assessed on ventricular cardiomyocytes isolated from lipopolysaccharide-treated rats. The involvement of nitric oxide was investigated using dexamethasone (in vivo) and ethyl isothiourea (in vitro). 2. Male Wistar rats (n=70) were injected with either saline (1 ml kg(-1)) or lipopolysaccharide (LPS; 5 mg kg(-1)) alone, or following pre-treatment with dexamethasone (DEX+LPS; 5 mg kg(-1)). Ventricular cell shortening was recorded using a video edge detection system, and concentration-response relationships were established for endothelin-1, isoprenaline and calcium, in the absence or presence of ethyl isothiourea (ETU; 10 microM). iNOS expression was assessed using reverse transcription-polymerase chain reaction. 3. iNOS mRNA expression was greater (P<0.001) in the LPS (iNOS/GAPDH ratio: 0.90+/-0.09) treated group compared to saline (iNOS/GAPDH ratio: 0.36+/-0.02). Baseline contractile amplitude was reduced (P<0.05) in the LPS (7.3+/-0.2 microm) and DEX+LPS groups (6.7+/-0.3 microm) compared to saline (8. 0+/-0.2 microm). 4. The concentration-dependent contractile response to endothelin-1 was attenuated (P<0.05) in the LPS group compared to saline (maximum change: 0.45+/-0.2 vs 1.8+/-0.2 microm). Neither ETU nor dexamethasone improved contractile function in the LPS-treated animals. 5. The concentration-dependent increase in the contractile response to isoprenaline was attenuated in the LPS-treated group compared to saline (P<0.05; maximum change: 1.7+/-0.4 vs 3.1+/-0.4 microm). This effect was reversed by ETU (maximum change: 3.7+/-0.6 microm). Pre-treatment with dexamethasone prevented a significant fall in contraction amplitude (maximum change: 2.4+/-0.4 microm). 6. The contractile response to calcium was reduced (P<0.05) in the LPS group compared to saline (maximum change: 8.7+/-0.6 vs 10.7+/-0.8 microm). Neither ETU nor dexamethasone restored contractile function in the LPS

  1. Bench-to-bedside review: Inotropic drug therapy after adult cardiac surgery – a systematic literature review

    PubMed Central

    Gillies, Michael; Bellomo, Rinaldo; Doolan, Laurie; Buxton, Brian

    2005-01-01

    Many adult patients require temporary inotropic support after cardiac surgery. We reviewed the literature systematically to establish, present and classify the evidence regarding choice of inotropic drugs. The available evidence, while limited in quality and scope, supports the following observations; although all β-agonists can increase cardiac output, the best studied β-agonist and the one with the most favourable side-effect profile appears to be dobutamine. Dobutamine and phosphodiesterase inhibitors (PDIs) are efficacious inotropic drugs for management of the low cardiac output syndrome. Dobutamine is associated with a greater incidence of tachycardia and tachyarrhythmias, whereas PDIs often require the administration of vasoconstrictors. Other catecholamines have no clear advantages over dobutamine. PDIs increase the likelihood of successful weaning from cardiopulmonary bypass as compared with placebo. There is insufficient evidence that inotropic drugs should be selected for their effects on regional perfusion. PDIs also increase flow through arterial grafts, reduce mean pulmonary artery pressure and improve right heart performance in pulmonary hypertension. Insufficient data exist to allow selection of a specific inotropic agent in preference over another in adult cardiac surgery patients. Multicentre randomized controlled trials focusing on clinical rather than physiological outcomes are needed. PMID:15987381

  2. Chronic inotropic therapy in end-stage heart failure

    PubMed Central

    Hauptman, Paul J.; Mikolajczak, Peter; George, Anil; Mohr, Clinton J.; Hoover, Robert; Swindle, Jason; Schnitzler, Mark A.

    2010-01-01

    Background Interventions in advanced heart failure that provide symptom relief and decrease hospital readmission are important. Chronic intravenous inotropic therapy represents a pharmacologic approach that has been advocated for palliative treatment. However, little is known about associated mortality and cost. Therefore, we sought to describe the impact of chronic infusions on resource use and survival. Methods Data were reviewed for a 17-state Medicare region from 1995 to 2002. We obtained hospital and outpatient expenditures accrued up to 180 days before and after the initiation of chronic infusions. Health care use was defined by dollars reimbursed for drug and hospitalizations per beneficiary. Average accumulated cost curves were generated for dollars reimbursed for drug and for hospitalizations by days at risk. Results The mean age of the cohort (n = 331) was 69.1 ± 11.3 years. Mortality exceeded 40% at 6 months. Reductions in hospital days were observed at all time points. The amounts reimbursed at 30 and 60 days before and after initiation of inotrope favor drug therapy; however, at six months, the amounts reimbursed were greater due to the cost of milrinone. Conclusions Chronic intravenous inotrope use was associated with a high mortality. The cost for milrinone was significant, but there was a decrease in expenditures for subsequent hospitalizations. In the absence of appropriately designed clinical trials, the data suggest that the decision to use inotropes, the choice of inotrope, and the duration of treatment should reflect the impact on resource use. PMID:17161059

  3. A pharmacological, crystallographic, and quantum chemical study of new inotropic agents.

    PubMed

    Dorigo, P; Gaion, R M; Belluco, P; Fraccarollo, D; Maragno, I; Bombieri, G; Benetollo, F; Mosti, L; Orsini, F

    1993-08-20

    The cardiac activity of a series of milrinone analogues, 2-substituted 3-acyl-1,6-dihydro-6-oxo-5-pyridinecarbonitriles, 1,6,3,2,11,12-hexahydro-6,3-dioxo-5-quinolinecarbonitriles, the correlated carboxylic acids, 2-substituted 3-acyl-6(1H)-pyridones, and 7,8-dihydro-2,5(1H,6H)-quinolinediones, was evaluated in spontaneously beating and in electrically driven atria from reserpine-treated guinea pigs. Their effects were compared with those induced by amrinone and milrinone in both the atria preparations. Compounds SF28 (3-acetyl-1,6-dihydro-2-methyl-6-oxo-5-pyridinecarbonitrile) and SF40 (7,8-dihydro-7-methyl-2,5(1H,6H)-quinolinedione) were the most effective positive inotropic agents. An inhibition of the negative influence exerted by endogenous adenosine on heart preparations seems to be involved in their contractile activity. SF38 (3-benzoyl-2-phenyl-6(1H)-pyridinone), on the contrary, reduced the contractile force and the frequency rate of guinea pig atria with a mechanism not related to an activation of cholinergic or purinergic inhibitory receptors on the heart. X-ray analysis carried out on the three model compounds, SF28, SF40 (positive inotropic agents), and SF38 (negative inotropic agent), and molecular modeling evidenced that the change from phenyl (SF38) to methyl (SF28) or the introduction of a side cyclic aliphatic chain (SF40) results in a variation of conformational preference and topography which may address the different molecules toward distinct receptor pockets according to the resulting inotropic effect.

  4. Potentiating paired stimulation of cardiac muscle in vitro as positive inotropic reference standard.

    PubMed

    Scholtysik, G

    1986-07-01

    In rabbit papillary muscles, potentiating paired stimulation was used as a standard positive inotropic intervention. Pairs of depolarizing electrical stimuli were applied, equal in strength and with a coupling interval of the functional refractory period plus 10 msec. After five successive pairs at a basic driving rate of 0.5 Hz, maximum potentiation amounting to a two- to threefold increase in the contraction amplitude was reached. The potentiating paired stimulation was rapid in both onset and reversibility and was reproducible. Potentiating paired stimulation is sparing since internal Ca2+ pools are utilized to increase force. Using potentiating paired stimulation-induced increase in force of contraction as a new reference, the following order of potency of positive inotropic agents was obtained: ouabain greater than DPI 201-106 greater than IBMX greater than APP 201-533. Effects of these drugs on rested-state contractions and frequency-force relationship were also investigated.

  5. 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.

  6. New inotropic concepts: rationale for and differences between calcium sensitizers and phosphodiesterase inhibitors.

    PubMed

    Holubarsch, C

    1997-01-01

    Positive inotropic compounds may be harmful in the long-term treatment of chronic congestive heart failure because they may induce a calcium overload, unwanted changes in cross-bridge kinetics and an acceleration in heart rate. As a result of all three alterations, energy consumption would be increased. Different pharmacological modes of action may have different effects on the molecular mechanisms underlying the positive inotropic effect, and hence on myocardial energy consumption. Therefore, we studied the effects of a variety of cardiotonic agents on the heat released from small guinea pig papillary muscles contracting isometrically at an experimental temperature of 21 degrees C and a stimulation frequency of 12 per minute using rapid antimony-bismuth thermopiles. We were able to define the economy of muscle contraction, which was lowest with phosphodiesterase inhibitors and highest with calcium sensitizers. Compared with an increase in extracellular calcium concentration, beta 1-adrenoceptor stimulators and phosphodiesterase inhibitors profoundly decrease the economy of myocardial contraction, and calcium-sensitizers (pimobendan and EMD-53998) slightly increase myocardial economy, whereas ouabain and the calcium channel agonist BAY K 8644 have no effect on this parameter. In addition, we provide evidence that acceleration of heart rate may be harmful not only from an energetic point of view: an increase in heart rate may also decrease the contractility of the failing human myocardium (inverse force-frequency relationship). Taking these observations into consideration, an "optimal' positive inotropic compound should have no, or even negative, chronotropic effects, should not be mediated by increases in calcium transients, and should decelerate, rather than accelerate, cross-bridge kinetics.

  7. 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. PMID:26126142

  8. Acute heart failure with low cardiac output: can we develop a short-term inotropic agent that does not increase adverse events?

    PubMed

    Campia, Umberto; Nodari, Savina; Gheorghiade, Mihai

    2010-09-01

    Acute heart failure represents an increasingly common cause of hospitalization, and may require the use of inotropic drugs in patients with low cardiac output and evidence of organ hypoperfusion. However, currently available therapies may have deleterious effects and increase mortality. An ideal inotropic drug should restore effective tissue perfusion by enhancing myocardial contractility without causing adverse effects. Such a drug is not available yet. New agents with different biological targets are under clinical development. In particular, istaroxime seems to dissociate the inotropic effect exerted by digitalis (inhibition of the membrane sodium/potassium adenosine triphosphatase) from the arrhythmic effect and to ameliorate diastolic dysfunction (via sarcoendoplasmic reticulum calcium adenosine triphosphatase activation). Additionally, the myosin activator omecamtiv mecarbil appears to have promising characteristics, while genetic therapy has been explored in animal studies only. Further investigations are needed to confirm and expand the effectiveness and safety of these agents in patients with acute heart failure and low cardiac output.

  9. Reduced inotropic heart response in selenium-deficient mice relates with inducible nitric oxide synthase.

    PubMed

    Gomez, Ricardo M; Levander, Orville A; Sterin-Borda, Leonor

    2003-02-01

    Atria from mice fed a selenium-deficient (Se(-)) diet have a diminished beta-adrenoceptor-inotropic cardiac response to isoproterenol or norepinephrine compared with atria from mice fed the same diet supplemented with 0.2 mg/kg Se as sodium selenite (Se(+)). This diminished response could be reversed by feeding Se(-) mice the Se(+) diet for 1 wk or by pretreatment with nitric oxide synthase (NOS) inhibitors such as N(G)-monomethyl-l-arginine or aminopyridine. Elevated serum concentrations of nitrite/nitrate as well as a threefold increase in the atrial NOS activity were seen in the Se(-) versus Se(+) mice. Western blotting and indirect immunofluorescence indicated an enhanced expression of inducible NOS in hearts from Se(-) mice. Increased expression and activity of NOS and increased nitrite/nitrate levels from Se(-) mice correlated with an impaired response to beta-adrenoceptor inotropic cardiac stimulation. Elevated nitric oxide levels may account for some of the pathophysiological effects of Se deficiency on the heart. PMID:12529255

  10. 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

  11. Negative inotropic actions of nitric oxide require high doses in rat cardiac muscle.

    PubMed

    Wyeth, R P; Temma, K; Seifen, E; Kennedy, R H

    1996-08-01

    Initial experiments were designed to determine if vasoactive concentrations of nitric oxide (NO) alter contractility in rat heart. Contractile function was monitored in left atrial and papillary muscles (30 degrees C; paced at 0.5 Hz) during cumulative addition of 3-morpholino-sydnonimine-HCl(SIN-1), an agent that releases NO. At concentrations between 10(-7) and 10(-4) M (NO concentrations of approximately 10(-8)- 3 x 10(-7) M), SIN-1 did not affect contractility in either tissue. Similarly, 10(-4) M SIN-1 did not alter the positive inotropic responses to isoproterenol or increasing extracellular [Ca+2] ([Ca+2]o). To obtain higher concentrations of NO, additional studies were conducted using authentic NO. NO-saturated stock solutions and a corresponding control solvent were adjusted to pH 1.6 with HCl. Dose-dependent effects of NO were examined by adding aliquots of the stock solutions (or control solvent) to the bathing solution. At final concentrations of 1 x 10(-5)- 5 x 10(-4) M, NO produced transient, concentration-dependent decreases in contractility that were paralleled by reductions in buffer pH. Control solvent elicited similar reductions in pHo and transient decreases in contractility; however, the negative inotropic action elicited by the NO-containing solution was approximately 20% greater than that observed in control conditions. These data demonstrate that only high concentrations of NO depress contractility in isolated rat cardiac muscle, and suggest that this effect is mediated by both acidosis and a pHo-independent mechanism.

  12. S100A1 DNA-based Inotropic Therapy Protects Against Proarrhythmogenic Ryanodine Receptor 2 Dysfunction

    PubMed Central

    Ritterhoff, Julia; Völkers, Mirko; Seitz, Andreas; Spaich, Kristin; Gao, Erhe; Peppel, Karsten; Pleger, Sven T; Zimmermann, Wolfram H; Friedrich, Oliver; Fink, Rainer H A; Koch, Walter J; Katus, Hugo A; Most, Patrick

    2015-01-01

    Restoring expression levels of the EF-hand calcium (Ca2+) sensor protein S100A1 has emerged as a key factor in reconstituting normal Ca2+ handling in failing myocardium. Improved sarcoplasmic reticulum (SR) function with enhanced Ca2+ resequestration appears critical for S100A1's cyclic adenosine monophosphate-independent inotropic effects but raises concerns about potential diastolic SR Ca2+ leakage that might trigger fatal arrhythmias. This study shows for the first time a diminished interaction between S100A1 and ryanodine receptors (RyR2s) in experimental HF. Restoring this link in failing cardiomyocytes, engineered heart tissue and mouse hearts, respectively, by means of adenoviral and adeno-associated viral S100A1 cDNA delivery normalizes diastolic RyR2 function and protects against Ca2+- and β-adrenergic receptor-triggered proarrhythmogenic SR Ca2+ leakage in vitro and in vivo. S100A1 inhibits diastolic SR Ca2+ leakage despite aberrant RyR2 phosphorylation via protein kinase A and calmodulin-dependent kinase II and stoichiometry with accessory modulators such as calmodulin, FKBP12.6 or sorcin. Our findings demonstrate that S100A1 is a regulator of diastolic RyR2 activity and beneficially modulates diastolic RyR2 dysfunction. S100A1 interaction with the RyR2 is sufficient to protect against basal and catecholamine-triggered arrhythmic SR Ca2+ leak in HF, combining antiarrhythmic potency with chronic inotropic actions. PMID:26005840

  13. Increased Long-Term Mortality among Black CABG Patients Receiving Preoperative Inotropic Agents

    PubMed Central

    Efird, Jimmy T.; Griffin, William F.; Sarpong, Daniel F.; Davies, Stephen W.; Vann, Iulia; Koutlas, Nathaniel T.; Anderson, Ethan J.; Crane, Patricia B.; Landrine, Hope; Kindell, Linda; Iqbal, Zahra J.; Ferguson, T. Bruce; Chitwood, W. Randolph; Kypson, Alan P.

    2015-01-01

    The aim of this study was to examine racial differences in long-term mortality after coronary artery bypass grafting (CABG), stratified by preoperative use of inotropic agents. Black and white patients who required preoperative inotropic support prior to undergoing CABG procedures between 1992 and 2011 were compared. Mortality probabilities were computed using the Kaplan-Meier product-limit method. Hazard ratios (HR) and 95% confidence intervals (CI) were computed using a Cox regression model. A total of 15,765 patients underwent CABG, of whom 211 received preoperative inotropic agents within 48 hours of surgery. Long-term mortality differed by race (black versus white) among preoperative inotropic category (inotropes: adjusted HR = 1.6, 95% CI = 1.009–2.4; no inotropes: adjusted HR = 1.15, 95% CI = 1.08–1.2; Pinteraction < 0.0001). Our study identified an independent preoperative risk-factor for long-term mortality among blacks receiving CABG. This outcome provides information that may be useful for surgeons, primary care providers, and their patients. PMID:26154656

  14. 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.

  15. 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

  16. 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.

  17. Deletion of thioredoxin-interacting protein improves cardiac inotropic reserve in the streptozotocin-induced diabetic heart.

    PubMed

    Myers, Ronald B; Fomovsky, Gregory M; Lee, Samuel; Tan, Max; Wang, Bing F; Patwari, Parth; Yoshioka, Jun

    2016-06-01

    Although the precise pathogenesis of diabetic cardiac damage remains unclear, potential mechanisms include increased oxidative stress, autonomic nervous dysfunction, and altered cardiac metabolism. Thioredoxin-interacting protein (Txnip) was initially identified as an inhibitor of the antioxidant thioredoxin but is now recognized as a member of the arrestin superfamily of adaptor proteins that classically regulate G protein-coupled receptor signaling. Here we show that Txnip plays a key role in diabetic cardiomyopathy. High glucose levels induced Txnip expression in rat cardiomyocytes in vitro and in the myocardium of streptozotocin-induced diabetic mice in vivo. While hyperglycemia did not induce cardiac dysfunction at baseline, β-adrenergic challenge revealed a blunted myocardial inotropic response in diabetic animals (24-wk-old male and female C57BL/6;129Sv mice). Interestingly, diabetic mice with cardiomyocyte-specific deletion of Txnip retained a greater cardiac response to β-adrenergic stimulation than wild-type mice. This benefit in Txnip-knockout hearts was not related to the level of thioredoxin activity or oxidative stress. Unlike the β-arrestins, Txnip did not interact with β-adrenergic receptors to desensitize downstream signaling. However, our proteomic and functional analyses demonstrated that Txnip inhibits glucose transport through direct binding to glucose transporter 1 (GLUT1). An ex vivo analysis of perfused hearts further demonstrated that the enhanced functional reserve afforded by deletion of Txnip was associated with myocardial glucose utilization during β-adrenergic stimulation. These data provide novel evidence that hyperglycemia-induced Txnip is responsible for impaired cardiac inotropic reserve by direct regulation of insulin-independent glucose uptake through GLUT1 and plays a role in the development of diabetic cardiomyopathy. PMID:27037370

  18. Dobutamine-tagged MRI for inotropic reserve assessment in severe CAD: relationship with PET findings.

    PubMed

    Mazzadi, Alejandro N; Janier, Marc F; Brossier, Benjamin; André-Fouët, Xavier; McFadden, Eugene; Revel, Didier; Croisille, Pierre

    2004-05-01

    The impact of blood flow reductions on the intramyocardial inotropic reserve has not yet been established in coronary artery disease (CAD). We therefore evaluated in severe CAD the relationship between positron emission tomography (PET) patterns of perfusion and glucose uptake and the corresponding tagged magnetic resonance imaging (tagged MRI) values of midmyocardial strains under low-dose dobutamine. Eighteen patients underwent tagged MRI (at rest, with dobutamine) and H2(15)O/18F-fluorodeoxyglucose PET. Regional midmyocardial circumferential shortening (Ecc) and PET patterns (normal, match viable, mismatch viable, and infarcted) were assessed in three tagged MRI/PET short-axis slices. Regional Ecc at rest correlated with both perfusion (r = 0.49) and glucose uptake (r = 0.58). The presence of the inotropic reserve was similar in normal, match viable, and infarcted (approximately 40% of regions vs. 52% in mismatch viable, P < 0.05), but the extent of the increase after dobutamine was lower in infarcted regions (P = 0.06). Within each PET pattern, regions were grouped according to their Ecc values at rest into three categories (high, intermediate, and low contractile performance). In mismatch viable (hibernation), the inotropic reserve was similar among the three categories, but in the other PET patterns the presence and extent of the inotropic reserve was higher in those regions with lowest Ecc (without significant differences in perfusion). In severe CAD, the presence of the inotropic reserve assessed by midmyocardial changes under dobutamine does not relate to resting perfusion. At a similar level of perfusion, the presence of the inotropic reserve is inversely related to contractile performance at rest, but our results suggest that it may not be true for hibernating myocardium. PMID:14726299

  19. Specific binding of (/sup 3/H)LY186126, an analogue of indolidan (LY195115), to cardiac membranes enriched in sarcoplasmic reticulum vesicles

    SciTech Connect

    Kauffman, R.F.; Utterback, B.G.; Robertson, D.W.

    1989-05-01

    LY186126 was found to be a potent inhibitor of type IV cyclic AMP phosphodiesterase located in the sarcoplasmic reticulum of canine cardiac muscle. This compound, a close structural analogue of indolidan (LY195115), was prepared in high specific activity, tritiated form to study the positive inotropic receptor(s) for cardiotonic phosphodiesterase inhibitors such as indolidan and milrinone. A high-affinity binding site for (/sup 3/H)LY186126 was observed (Kd = 4 nM) in purified preparations of canine cardiac sarcoplasmic reticulum vesicles. Binding was proportional to vesicle protein, was inactivated by subjecting membranes to proteolysis or boiling, and was dependent on added Mg2+. Scatchard analysis suggested the presence of a single class of binding sites in the membrane preparation. Indolidan, milrinone, and LY186126 (all at 1 microM) produced essentially complete displacement of bound (/sup 3/H)LY186126, while nifedipine, propranolol, and prazosin had little or no effect at this concentration. This represents the first reported use of a radioactive analogue to label the inotropic receptor for cardiotonic phosphodiesterase inhibitors. The results suggest that (/sup 3/H)LY186126 is a useful radioligand for examining the subcellular site(s) responsible for positive inotropic effects of these drugs.

  20. Negative inotropic drugs alter indexes of cytosolic [Ca(2+)]-left ventricular pressure relationships after ischemia.

    PubMed

    Camara, Amadou K S; Chen, Qun; Rhodes, Samhita S; Riess, Matthias L; Stowe, David F

    2004-08-01

    Negative inotropic agents may differentially modulate indexes of cytosolic [Ca(2+)]-left ventricular (LV) pressure (LVP) relationships when given before and after ischemia. We measured and calculated [Ca(2+)], LVP, velocity ratios [[(d[Ca(2+)]/dt(max))/(dLVP/dt(max)); VR(max)] and [(d[Ca(2+)]/dt(min))/(dLVP/dt(min)); VR(min)

  1. Should dopamine be the first line inotrope in the treatment of neonatal hypotension? Review of the evidence

    PubMed Central

    Bhayat, Sadaf I; Gowda, Harsha M S; Eisenhut, Michael

    2016-01-01

    AIM: To determine if dopamine is effective in treating neonatal hypotension and safe to use comparing to other inotropes. METHODS: This is a review of evidence on inotropic treatment of neonatal hypotension. Databases searched were MEDLINE and the Cochrane Library, a total of 134 studies were identified. Only studies with high quality evidence (level 1a and b and 2a) were included. After review, only eight studies were included in the final analysis. Pooled risk ratios derived for each outcome [Mantel-Haenzel (M-H) fixed effect] with CI, as reported in the Cochrane reviews were plotted in forest plot form. RESULTS: Eight articles met inclusion criteria, which all included treatment in preterm infants. Dopamine increased mean arterial blood pressure (BP) (n = 163; r = 0.88, 95%CI: 0.76 to 0.94) and systolic BP (n = 142; r = 0.81, 95%CI: 0.42 to 0.94) comparing to placebo. Dopamine has been shown overall to be statistically more effective in increasing BP than dobutamine (n = 251, r = 0.26, 95%CI: 0.20-0.32). However there were no differences in short term outcomes (periventricular leucomalacia, periventricular haemorrhage) and mortality between both drugs. There is no statistical evidence of dopamine being more effective than adrenaline or corticosteroids. There was no difference in morbidity and mortality outcomes when dopamine was compared to hydrocortisone (RR 1.81, 95%CI: 0.18 to 18.39) or adrenaline. CONCLUSION: In preterms, dopamine is the most studied drug, and we suggest it could be used as first line treatment in hypotension. PMID:27170932

  2. No inotropic action of enkephalins or enkephalin derivatives on electrically-stimulated atria isolated from lean and obese rats.

    PubMed Central

    Saunders, W. S.; Thornhill, J. A.

    1985-01-01

    Inotropic actions of the endogenous enkephalins, leucine enkephalin [( Leu] enkephalin) and methionine enkephalin [( Met] enkephalin), and derivatives, [D-Ala2-methionine] enkephalinamide (DAMEA) and [D Ala2-leucine]enkephalinamide (DALEA) were tested, alone or in combination with noradrenaline (NA), (+/-)-isoprenaline or carbachol, on electrically-stimulated atria excised from Sprague-Dawley, fatty, Zucker (fa/fa) and lean, hooded heterozygous (Fa/fa) rats. [Met] enkephalin, [Leu] enkephalin, DAMEA and DALEA (4 X 10(-7)M to 4 X 10(-4)M) caused no significant changes in atrial tension in any group compared to pre-injection control values or those following the infusion of Krebs-Henseleit control solution. NA and isoprenaline (10(-7) to 10(-6)M) caused significant, dose-related increases in atrial tension in each of the three strains of rats tested with the Fa/fa group showing the greatest change and fastest rate of tension development. [Met] enkephalin, [Leu] enkephalin, DAMEA or DALEA (4 X 10(-6)M) infused concurrently with NA or isoprenaline (10(-6)M) evoked atrial tension changes within each group that were not different from those observed when NA or isoprenaline was administered alone. Carbachol (10(-9) and 10(-8)M) caused a dose-related decrease (10% and 30-40%, respectively, from pre-injection control values) in atrial tension in auricles excised from all three groups. Again, infusion of [Met] enkephalin, [Leu] enkephalin, DAMEA or DALEA (4 X 10(-6)M) together with carbachol (10(-8)M) did not affect atrial tension changes of auricles isolated from any group compared to when carbachol was given alone. The results indicate that the endogenous pentapeptides, [( Met] or [Leu] enkephalin), or derivatives (DAMEA and DALEA) do not affect atrial tension of electrically-stimulated auricles isolated from Sprague-Dawley, fa/fa or Fa/fa rats. In addition, these pentapeptides do not modify the positive inotropic actions of NA or isoprenaline or the negative inotropic

  3. Dominance of the forward compression wave in determining pulsatile components of blood pressure: similarities between inotropic stimulation and essential hypertension.

    PubMed

    Fok, Henry; Guilcher, Antoine; Brett, Sally; Jiang, Benyu; Li, Ye; Epstein, Sally; Alastruey, Jordi; Clapp, Brian; Chowienczyk, Phil

    2014-11-01

    Pulsatile components of blood pressure may arise from forward (ventricular generated) or backward wave travel in the arterial tree. The objective of this study was to determine the relative contributions of forward and backward waves to pulsatility. We used wave intensity and wave separation analysis to determine pulsatile components of blood pressure during inotropic and vasopressor stimulation by dobutamine and norepinephrine in normotensive subjects and compared pulse pressure components in hypertensive (mean±SD, 48.8±11.3 years; 165±26.6/99±14.2 mm Hg) and normotensive subjects (52.2±12.6 years; 120±14.2/71±8.2 mm Hg). Dobutamine (7.5 μg/kg per minute) increased the forward compression wave generated by the ventricle and increased pulse pressure from 36.8±3.7 to 59.0±3.4 mm Hg (mean±SE) but had no significant effect on mean arterial pressure or the midsystolic backward compression wave. By contrast, norepinephrine (50 ng/kg per minute) had no significant effect on the forward compression wave but increased the midsystolic backward compression wave. Despite this increase in the backward compression wave, and an increase in mean arterial pressure, norepinephrine increased central pulse pressure less than dobutamine (increases of 22.1±3.8 and 7.2±2.8 mm Hg for dobutamine and norepinephrine, respectively; P<0.02). An elevated forward wave component (mean±SE, 50.4±3.4 versus 35.2±1.8 mm Hg, in hypertensive and normotensive subjects, respectively; P<0.001) accounted for approximately two thirds of the total difference in central pulse pressures between hypertensive and normotensive subjects. Increased central pulse pressure during inotropic stimulation and in essential hypertension results primarily from the forward compression wave.

  4. Vasoactive Inotrope Score as a tool for clinical care in children post cardiac surgery

    PubMed Central

    Kumar, Maneesh; Sharma, Rajesh; Sethi, Sidharth Kumar; Bazaz, Subeeta; Sharma, Prerna; Bhan, Anil; Kher, Vijay

    2014-01-01

    Background: Neonates and infants undergoing heart surgery on cardiopulmonary bypass (CPB) are at high risk for significant post-operative morbidity and mortality. Hence, there is a need to identify and quantify clinical factors during the early post-operative period that are indicative of short-term as well as long-term outcomes. Multiple inotrope scores have been used in practice to quantify the amount of cardiovascular support received by neonates. Aims: The goal of this study was to determine the association between inotropic/vasoactive support and clinical outcomes in children after open cardiac surgery. Materials and Methods: This is a retrospective analysis of the 208 patients who underwent cardiac surgery for congenital heart disease at a tertiary pediatric cardiac surgery Intensive Care Unit (ICU) from January 2012 to March 2013. Multiple demographic, intra-operative and post-operative variables were recorded, including the Vasoactive Inotrope Score (VIS). Results: A total of 208 patients underwent cardiac surgery for congenital heart disease in the study period. The mean age and weight in the study were 66.94 months and 16.31 kg, respectively. Statistically significant associations were found in the various variables and VIS, including infancy, weight < 10 kg, CPB time, pump failure and post-operative variables like sepsis, hematological complications, hepatic dysfunction, acute kidney injury during admission, mortality, prolonged ventilator requirement, CPB time (in min) and hospital stay. Conclusions: Inotrope score and its adaptations are an excellent tool to measure illness severity, deciding interventions and during parental counseling in the pediatric cardiac surgery ICUs. PMID:25316975

  5. New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response

    PubMed Central

    Taniguchi, Masaya; Okamoto, Ryuji; Ito, Masaaki; Goto, Itaru; Fujita, Satoshi; Konishi, Katsuhisa; Mizutani, Hideo; Dohi, Kaoru; Hartshorne, David J.; Itoh, Takeo

    2015-01-01

    Background & Aims Cardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated. Methods and Results By Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15–30%. Conclusions We identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain. PMID:26512720

  6. Calcium Binding to Calmodulin by Molecular Dynamics with Effective Polarization.

    PubMed

    Kohagen, Miriam; Lepšík, Martin; Jungwirth, Pavel

    2014-11-20

    Calcium represents a key biological signaling ion with the EF-hand loops being its most prevalent binding motif in proteins. We show using molecular dynamics simulations with umbrella sampling that including electronic polarization effects via ionic charge rescaling dramatically improves agreements with experiment in terms of the strength of calcium binding and structures of the calmodulin binding sites. The present study thus opens way to accurate calculations of interactions of calcium and other computationally difficult high-charge-density ions in biological contexts.

  7. Effect of Detergents on Galactoside Binding by Melibiose Permeases.

    PubMed

    Amin, Anowarul; Hariharan, Parameswaran; Chae, Pil Seok; Guan, Lan

    2015-09-29

    The effect of various detergents on the stability and function of the melibiose permeases of Escherichia coli (MelBEc) and Salmonella typhimurium (MelBSt) was studied. In n-dodecyl-β-d-maltoside (DDM) or n-undecyl-β-d-maltoside (UDM), WT MelBSt binds melibiose with an affinity similar to that in the membrane. However, with WT MelBEc or MelBSt mutants (Arg141 → Cys, Arg295 → Cys, or Arg363 → Cys), galactoside binding is not detected in these detergents, but binding to the phosphotransferase protein IIA(Glc) is maintained. In the amphiphiles lauryl maltose neopentyl glycol (MNG-3) or glyco-diosgenin (GDN), galactoside binding with all of the MelB proteins is observed, with slightly reduced affinities. MelBSt is more thermostable than MelBEc, and the thermostability of either MelB is largely increased in MNG-3 or GDN. Therefore, the functional defect with DDM or UDM likely results from the relative instability of the sensitive MelB proteins, and stability, as well as galactoside binding, is retained in MNG-3 or GDN. Furthermore, isothermal titration calorimetry of melibiose binding with MelBSt shows that the favorable entropic contribution to the binding free energy is decreased in MNG-3, indicating that the conformational dynamics of MelB is restricted in this detergent.

  8. Effect of Detergents on Galactoside Binding by Melibiose Permeases.

    PubMed

    Amin, Anowarul; Hariharan, Parameswaran; Chae, Pil Seok; Guan, Lan

    2015-09-29

    The effect of various detergents on the stability and function of the melibiose permeases of Escherichia coli (MelBEc) and Salmonella typhimurium (MelBSt) was studied. In n-dodecyl-β-d-maltoside (DDM) or n-undecyl-β-d-maltoside (UDM), WT MelBSt binds melibiose with an affinity similar to that in the membrane. However, with WT MelBEc or MelBSt mutants (Arg141 → Cys, Arg295 → Cys, or Arg363 → Cys), galactoside binding is not detected in these detergents, but binding to the phosphotransferase protein IIA(Glc) is maintained. In the amphiphiles lauryl maltose neopentyl glycol (MNG-3) or glyco-diosgenin (GDN), galactoside binding with all of the MelB proteins is observed, with slightly reduced affinities. MelBSt is more thermostable than MelBEc, and the thermostability of either MelB is largely increased in MNG-3 or GDN. Therefore, the functional defect with DDM or UDM likely results from the relative instability of the sensitive MelB proteins, and stability, as well as galactoside binding, is retained in MNG-3 or GDN. Furthermore, isothermal titration calorimetry of melibiose binding with MelBSt shows that the favorable entropic contribution to the binding free energy is decreased in MNG-3, indicating that the conformational dynamics of MelB is restricted in this detergent. PMID:26352464

  9. Effect of detergents on galactoside binding by melibiose permeases

    PubMed Central

    Amin, Anowarul; Hariharan, Parameswaran; Chae, Pil Seok; Guan, Lan

    2015-01-01

    The effect of various detergents on the stability and function of melibiose permeases of Escherichia coli (MelBEc) or Salmonella typhimurium (MelBSt) were studied. In n-dodecyl-β-d-maltoside (DDM) or n-undecyl-β-d-maltoside (UDM), WT MelBSt binds melibiose with an affinity similar to that in the membrane. However, with WT MelBEc or MelBSt mutants (Arg141→Cys, Arg295→Cys or Arg363→Cys), galactoside binding is not detected in these detergents, but binding to the phosphotransferase protein IIAGlc is maintained. In the amphiphiles lauryl maltose neopentyl glycol (MNG-3) or glyco-diosgenin (GDN), galactoside binding with all the MelB proteins is observed, with slightly reduced affinities. MelBSt is more thermostable than MelBEc, and the thermostability of either MelB is largely increased in MNG-3 or GDN. Therefore, the functional defect with DDM or UDM likely results from relative instability of the sensitive MelB proteins, and stability, as well as galactoside binding, is retained in MNG-3 or GDN. Furthermore, isothermal titration calorimetry of melibiose binding with MelBSt shows that the favorable entropic contribution to the binding free energy is decreased in MNG-3, indicating that the conformational dynamics of MelB is restricted in this detergent. PMID:26352464

  10. 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

  11. Action-effect binding by observational learning.

    PubMed

    Paulus, Markus; van Dam, Wessel; Hunnius, Sabine; Lindemann, Oliver; Bekkering, Harold

    2011-10-01

    The acquisition of bidirectional action-effect associations plays a central role in the ability to intentionally control actions. Humans learn about actions not only through active experience, but also through observing the actions of others. In Experiment 1, we examined whether action-effect associations can be acquired by observational learning. To this end, participants observed how a model repeatedly pressed two buttons during an observation phase. Each of the buttonpresses led to a specific tone (action effect). In a subsequent test phase, the tones served as target stimuli to which the participants had to respond with buttonpresses. Reaction times were shorter if the stimulus-response mapping in the test phase was compatible with the action-effect association in the observation phase. Experiment 2 excluded the possibility that the impact of perceived action effects on own actions was driven merely by an association of spatial features with the particular tones. Furthermore, we demonstrated that the presence of an agent is necessary to acquire novel action-effect associations through observation. Altogether, the study provides evidence for the claim that bidirectional action-effect associations can be acquired by observational learning. Our findings are discussed in the context of the idea that the acquisition of action-effect associations through observation is an important cognitive mechanism subserving the human ability for social learning.

  12. 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

  13. The negative inotropic action of canrenone is mediated by L-type calcium current blockade and reduced intracellular calcium transients

    PubMed Central

    Costa, AR; Torres, LB; Medei, E; Ricardo, RA; França, JP; Smaili, S; Nascimento, JHM; Oshiro, MEM; Bassani, JWM; Ferreira, AT; Tucci, PJF

    2009-01-01

    Background and purpose: Adding spironolactone to standard therapy in heart failure reduces morbidity and mortality, but the underlying mechanisms are not fully understood. We analysed the effect of canrenone, the major active metabolite of spironolactone, on myocardial contractility and intracellular calcium homeostasis. Experimental approach: Left ventricular papillary muscles and cardiomyocytes were isolated from male Wistar rats. Contractility of papillary muscles was assessed with force transducers, Ca2+ transients by fluorescence and Ca2+ fluxes by electrophysiological techniques. Key results: Canrenone (300–600 µmol·L−1) reduced developed tension, maximum rate of tension increase and maximum rate of tension decay of papillary muscles. In cardiomyocytes, canrenone (50 µmol·L−1) reduced cell shortening and L-type Ca2+ channel current, whereas steady-state activation and inactivation, and reactivation curves were unchanged. Canrenone also decreased the Ca2+ content of the sarcoplasmic reticulum, intracellular Ca2+ transient amplitude and intracellular diastolic Ca2+ concentration. However, the time course of [Ca2+]i decline during transients evoked by caffeine was not affected by canrenone. Conclusion and implications: Canrenone reduced L-type Ca2+ channel current, amplitude of intracellular Ca2+ transients and Ca2+ content of sarcoplasmic reticulum in cardiomyocytes. These changes are likely to underlie the negative inotropic effect of canrenone. PMID:19663883

  14. Tagged MRI and PET in severe CAD: discrepancy between preoperative inotropic reserve and intramyocardial functional outcome after revascularization.

    PubMed

    Mazzadi, Alejandro N; Janier, Marc F; Brossier, Benjamin; André-Fouët, Xavier; Revel, Didier; Croisille, Pierre

    2004-11-01

    In severe coronary artery disease (CAD), it has been shown that intramyocardial inotropic reserve as assessed with tagged magnetic resonance imaging (MRI) is uniformly distributed among positron emission tomography (PET) patterns reflecting normal or concomitant reductions in perfusion and glucose metabolism. This preliminary study aimed to delineate the relationship between preoperative values of intramyocardial inotropic reserve (in different PET patterns of perfusion and glucose uptake) and intramyocardial functional outcome after surgical revascularization in severe CAD. Twelve patients underwent preoperative tagged MRI (baseline, 10 microg.kg(-1).min(-1) of dobutamine), H2 15O/[18F]fluorodeoxyglucose PET imaging, and postoperative resting tagged MRI. Regional midmyocardial circumferential shortening (Ecc, in %) and PET patterns (normal, match viable, mismatch viable, and infarcted) were assessed in three tagged MRI/PET short-axis slices. Ecc at baseline ranged from 12 +/- 6 to 8 +/- 5 and 4 +/- 4% in normal, match-viable, and infarcted regions, respectively (P <0.05) and was 8 +/- 5% in mismatch-viable regions. Of the 429 regions studied, 187 showed preoperative inotropic reserve with dobutamine, but 238 showed postoperative functional improvement. Postoperative functional improvement was less common in infarcted regions (41 vs. approximately 60% in the other PET patterns), but the extent of improvement was similar among PET patterns (approximately 6%). Postoperative functional improvement occurred in 53% of all (normal, match viable, and mismatch viable) regions without inotropic reserve. In severe CAD, revascularization affords greater intramyocardial functional benefit than expected from the evaluation of intramyocardial inotropic reserve with low-dose dobutamine. Postoperative functional improvement in PET-viable regions without inotropic reserve suggests that factors other than regionally enhanced perfusion contribute to such functional improvement. PMID

  15. On the influence of reward on action-effect binding.

    PubMed

    Muhle-Karbe, Paul S; Krebs, Ruth M

    2012-01-01

    Ideomotor theory states that the formation of anticipatory representations about the perceptual consequences of an action [i.e., action-effect (A-E) binding] provides the functional basis of voluntary action control. A host of studies have demonstrated that A-E binding occurs fast and effortlessly, yet little is known about cognitive and affective factors that influence this learning process. In the present study, we sought to test whether the motivational value of an action modulates the acquisition of A-E associations. To this end, we linked specific actions with monetary incentives during the acquisition of novel A-E mappings. In a subsequent test phase, the degree of binding was assessed by presenting the former effect stimuli as task-irrelevant response primes in a forced-choice response task, absent reward. Binding, as indexed by response priming through the former action-effects, was only found for reward-related A-E mappings. Moreover, the degree to which reward associations modulated the binding strength was predicted by individuals' trait sensitivity to reward. These observations indicate that the association of actions and their immediate outcomes depends on the motivational value of the action during learning, as well as on the motivational disposition of the individual. On a larger scale, these findings also highlight the link between ideomotor theories and reinforcement-learning theories, providing an interesting perspective for future research on anticipatory regulation of behavior.

  16. On the influence of reward on action-effect binding.

    PubMed

    Muhle-Karbe, Paul S; Krebs, Ruth M

    2012-01-01

    Ideomotor theory states that the formation of anticipatory representations about the perceptual consequences of an action [i.e., action-effect (A-E) binding] provides the functional basis of voluntary action control. A host of studies have demonstrated that A-E binding occurs fast and effortlessly, yet little is known about cognitive and affective factors that influence this learning process. In the present study, we sought to test whether the motivational value of an action modulates the acquisition of A-E associations. To this end, we linked specific actions with monetary incentives during the acquisition of novel A-E mappings. In a subsequent test phase, the degree of binding was assessed by presenting the former effect stimuli as task-irrelevant response primes in a forced-choice response task, absent reward. Binding, as indexed by response priming through the former action-effects, was only found for reward-related A-E mappings. Moreover, the degree to which reward associations modulated the binding strength was predicted by individuals' trait sensitivity to reward. These observations indicate that the association of actions and their immediate outcomes depends on the motivational value of the action during learning, as well as on the motivational disposition of the individual. On a larger scale, these findings also highlight the link between ideomotor theories and reinforcement-learning theories, providing an interesting perspective for future research on anticipatory regulation of behavior. PMID:23130005

  17. 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.

  18. 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.

  19. Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

    PubMed

    Foti, M; Omichinski, J G; Stahl, S; Maloney, D; West, J; Schweitzer, B I

    1999-02-01

    We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site.

  20. Effects of nucleoside analog incorporation on DNA binding to the DNA binding domain of the GATA-1 erythroid transcription factor.

    PubMed

    Foti, M; Omichinski, J G; Stahl, S; Maloney, D; West, J; Schweitzer, B I

    1999-02-01

    We investigate here the effects of the incorporation of the nucleoside analogs araC (1-beta-D-arabinofuranosylcytosine) and ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl] guanine) into the DNA binding recognition sequence for the GATA-1 erythroid transcription factor. A 10-fold decrease in binding affinity was observed for the ganciclovir-substituted DNA complex in comparison to an unmodified DNA of the same sequence composition. AraC substitution did not result in any changes in binding affinity. 1H-15N HSQC and NOESY NMR experiments revealed a number of chemical shift changes in both DNA and protein in the ganciclovir-modified DNA-protein complex when compared to the unmodified DNA-protein complex. These changes in chemical shift and binding affinity suggest a change in the binding mode of the complex when ganciclovir is incorporated into the GATA DNA binding site. PMID:10037146

  1. Enthalpy/entropy compensation effects from cavity desolvation underpin broad ligand binding selectivity for rat odorant binding protein 3.

    PubMed

    Portman, Katherine L; Long, Jed; Carr, Stephen; Briand, Loïc; Winzor, Donald J; Searle, Mark S; Scott, David J

    2014-04-15

    Evolution has produced proteins with exquisite ligand binding specificity, and manipulating this effect has been the basis for much of modern rational drug design. However, there are general classes of proteins with broader ligand selectivity linked to function, the origin of which is poorly understood. The odorant binding proteins (OBPs) sequester volatile molecules for transportation to the olfactory receptors. Rat OBP3, which we characterize by X-ray crystallography and NMR, binds a homologous series of aliphatic γ-lactones within its aromatic-rich hydrophobic pocket with remarkably little variation in affinity but extensive enthalpy/entropy compensation effects. We show that the binding energetics are modulated by two desolvation processes with quite different thermodynamic signatures. Ligand desolvation follows the classical hydrophobic effect; however, cavity desolvation is consistent with the liberation of "high energy" water molecules back into bulk solvent with a strong, but compensated, enthalpic contribution, which together underpin the origins of broad ligand binding selectivity.

  2. Alloimmune IgG binds and modulates cardiac beta-adrenoceptor activity.

    PubMed Central

    Sterin-Borda, L; Cremaschi, G; Pascual, J; Genaro, A; Borda, E

    1984-01-01

    Purified IgG from murine alloimmune sera directed against class I products from the major histocompatibility complex of the mouse, could bind to the beta-adrenoceptors and stimulate contractile activity of myocardium. Immune IgG inhibited the binding of (-) 3H-DHA to beta-adrenoceptors of mouse myocardial membranes behaving as a competitive inhibitor. Moreover, immune IgG induced positive inotropic and chronotropic effects on isolated mouse atria. These effects could be blocked by beta-adrenoceptors antagonists. Data prove that immune IgG directed against specific alloantigens are able to recognize the beta-adrenoceptors and mimic the stimulation of the beta-adrenoceptor agonist. PMID:6090043

  3. Effect of hydrostatic pressure on ligand binding to hemoglobin.

    PubMed

    Carey, F G; Knowles, F; Gibson, Q H

    1977-06-25

    Increase in hydrostatic pressure to 1000 atm increased the affinity of human and menhaden (Brevoortia tyrannus) hemoglobins for oxygen. With necessary assumptions about the form of the equilibrium curve, and after correction for changes in pH and volume due to pressure, the increase in affinity is about 2-fold for both hemoglobins. At pH 6.5, Hill's n for menhaden hemoglobin is near 1, and it is believed to remain in the T state, whereas human hemoglobin undergoes a T to R transition. This suggests that the R-T equilibrium is not disturbed by pressure. In direct experiments the binding of a fluorescent effector (8 hydroxy-1,3,6-pyrene (trisulfonic acid) to deoxyhemoglobin was not changed by pressure. The binding of n-butylisocyanide to hemoglobin and to myoglobin is also greater at high pressures, similarly suggesting that the R-T transition is not involved in the pressure effect. PMID:16924

  4. Hypothermia During Cardiopulmonary Bypass Increases Need for Inotropic Support but Does Not Impact Inflammation in Children Undergoing Surgical Ventricular Septal Defect Closure.

    PubMed

    Schmitt, Katharina Rose Luise; Fedarava, Katsiaryna; Justus, Georgia; Redlin, Mathias; Böttcher, Wolfgang; Delmo Walter, Eva Maria; Hetzer, Roland; Berger, Felix; Miera, Oliver

    2016-05-01

    Minimizing the systemic inflammatory response caused by cardiopulmonary bypass is a major concern. It has been suggested that the perfusion temperature affects the inflammatory response. The aim of this prospective study was to compare the effects of moderate hypothermia (32°C) and normothermia (36°C) during cardiopulmonary bypass on markers of the inflammatory response and clinical outcomes (time on ventilator) after surgical closure of ventricular septal defects. During surgical closure of ventricular septal defects under cardiopulmonary bypass, 20 children (median age 4.9 months, range 2.3-38 months; median weight 7.2 kg, range 5.2-11.7 kg) were randomized to a perfusion temperature of either 32°C (Group 1, n = 10) or 36°C (Group 2, n = 10). The clinical data and blood samples were collected before cardiopulmonary bypass, directly after aortic cross-clamp release, and 4 and 24 h after weaning from cardiopulmonary bypass. Time on ventilation as primary outcome did not differ between the two groups. Other clinical outcome parameters like fluid balance or length of stay in the intensive care were also similar in the two groups. Compared with Group 2, Group 1 needed significantly higher and longer inotropic support (P < 0.001). In Group 1, two infants had junctional ectopic tachycardia, and another had a pulmonary hypertensive crisis. Perfusion temperature did not influence cytokine release, organ injury, or coagulation. Cardiopulmonary bypass temperature does not influence time on ventilation or inflammatory marker release. However, in the present study, with a small patient cohort, patients operated under hypothermic bypass needed higher and longer inotropic support. The use of hypothermic cardiopulmonary bypass in infants and children should be approached with care.

  5. 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.

  6. 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.

  7. Binding profiles and cytokine-inducing effects of fish rhamnose-binding lectins on Burkitt's lymphoma Raji cells.

    PubMed

    Hosono, Masahiro; Sugawara, Shigeki; Matsuda, Atsushi; Tatsuta, Takeo; Koide, Yasuhiro; Hasan, Imtiaj; Hasan, Imtiaji; Ozeki, Yasuhiro; Nitta, Kazuo

    2014-10-01

    Rhamnose-binding lectin (RBL) is one of the animal lectin categories which take part in the innate immune responses of fish. Osmerus lanceolatus lectin (OLL) from shishamo smelt eggs is an RBL composed of two tandem-repeated domains, both of which are considered to be a carbohydrate-recognition domain. SAL, catfish (Silurus asotus) egg RBL composed of three domains, binds to Burkitt's lymphoma Raji cells through globotriaosylceramide (Gb3) carbohydrate chain and to reduce cell size and growth by altering membrane composition without causing cell death. In this experiment, we tried to compare the binding effects of these two RBLs on Raji cells. Flow cytometric and fluorescence microscopic analyses revealed that OLL also directly bound to and shrunk Raji cells with ten times less reactivity than SAL but reduced cell growth with decreasing cell viability. Anti-Gb3 antibody completely blocked the binding of SAL to Raji cells but not that of OLL. In addition, the direct bindings of OLL and SAL to Raji cells were comparably inhibited by melibiose, but lactose was more effective inhibitor for the binding of OLL than that of SAL. These results suggest that OLL has slightly different cell-binding property compared with SAL and binds not only to Gb3 but also to the other carbohydrate receptor-bearing β-galactoside chains. The quantitative RT-PCR analysis revealed that SAL induced the expression of TNF-α but not of IFN-γ, IL-1β, and IL-10. Thus, SAL-induced cytostatic effect on Raji cells might be partially caused by TNF-α-mediated signaling pathway. PMID:24861899

  8. The effect of posture and abdominal binding on respiratory pressures.

    PubMed

    Koulouris, N; Mulvey, D A; Laroche, C M; Goldstone, J; Moxham, J; Green, M

    1989-11-01

    We examined the effect of posture on the generation of respiratory pressures in 6 highly trained subjects. Transdiaphragmatic pressure was measured at FRC during bilateral percutaneous phrenic nerve stimulation (twitch Pdi) and maximal sniffs (sniff Pdi), with the abdomen bound and unbound. Maximum static inspiratory (PImax) and expiratory (PEmax) mouth pressures were measured with the abdomen unbound. Three postures were examined: seated (Se), semi-supine (30s), and supine (Su). Changes of posture did not significantly alter twitch Pdi. By contrast, sniff Pdi and static mouth pressures were significantly reduced in the Su posture. Abdominal binding significantly increased twitch Pdi only. We conclude that voluntary respiratory manoeuvres requiring activation, recruitment and coordination of different muscle groups are performed better in the Se position. We suggest that posture be standardised for serial comparative measurements of voluntary respiratory pressures in a given subject.

  9. [Effects of La3+ on calcium binding to erythrocyte cytoskeleton].

    PubMed

    Kravtsov, G M; Postnov, Iu V

    1992-02-01

    When the whole erythrocytes were exposed to LaCl3, A--23187, ionomycin, orthovanadate and saponin, there was Ca2+ binding only following La3+ treatment of the cells. The binding was evident at a wide range (0.1 microM--1.OmM) of La3+ concentrations. Iodoacetamide-induced (incubation for 3 hours, 37 degrees C) decrease in erythrocyte ATP levels was found to result in a 3-fold reduction in Ca2+ binding to the cytoskeleton. La(3+)-induced Ca2+ binding enhanced the incorporation of 14C-glucose and/or its metabolites into the red cell skeleton. Thus, the detected new type of Ca2+ binding to the cytoskeleton of human and rat erythrocytes is likely to be due to the cumulative process: direct binding of La3+ to the outer surface of a membrane and the metal-induced trigger of nucleotide--dependent intracellular process.

  10. Intentional binding effect in children: insights from a new paradigm.

    PubMed

    Cavazzana, Annachiara; Begliomini, Chiara; Bisiacchi, Patrizia S

    2014-01-01

    Intentional binding (IB) refers to the temporal attraction between a voluntary action and its sensory consequence. Since its discovery in 2002, it has been considered to be a valid implicit measure of sense of agency (SoA), since it only occurs in the context of voluntary actions. The vast majority of studies considering IB have recruited young adults as participants, while neglecting possible age-related differences. The aim of the present work is to study the development of IB in 10-year-old children. In place of Libet's classical clock method, we decided to implement a new and more suitable paradigm in order to study IB, since children could have some difficulties in dealing with reading clocks. A stream of unpredictable letters was therefore used: participants had to remember which letter was on the screen when they made a voluntary action, heard a sound, or felt their right index finger moved down passively. In Experiment I, a group of young adults was tested in order to replicate the IB effect with this new paradigm. In Experiment II, the same paradigm was then administered to children in order to investigate whether such an effect has already emerged at this age. The data from Experiment I showed the presence of the IB effect in adults. However, Experiment II demonstrated a clear reduction of IB. The comparison of the two groups revealed that the young adult group differed from the children, showing a significantly stronger linkage between actions and their consequences. The results indicate a developmental trend in the IB effect. This finding is discussed in light of the maturation process of the frontal cortical network. PMID:25202256

  11. Salt effects on polyelectrolyte-ligand binding: comparison of Poisson-Boltzmann, and limiting law/counterion binding models.

    PubMed

    Sharp, K A; Friedman, R A; Misra, V; Hecht, J; Honig, B

    1995-08-01

    The theory for salt dependence of the free energy, entropy, and enthalpy of a polyelectrolyte in the PB (PB) model is extended to treat the nonspecific salt dependence of polyelectrolyte-ligand binding reactions. The salt dependence of the binding constant (K) is given by the difference in osmotic pressure terms between the reactants and products. For simple 1-1 salts it is shown that this treatment is equivalent to the general preferential interaction model for the salt dependence of binding [C. Anderson and M. Record (1993) Journal of Physical Chemistry, Vol. 97, pp. 7116-7126]. The salt dependence, entropy, and enthalpy are compared for the PB model and one specific form of the preferential interaction coefficient model that uses counterion condensation/limiting law (LL) behavior. The PB and LL models are applied to three ligand-polyelectrolyte systems with the same net ligand charge: a model sphere-cylinder binding reaction, a drug-DNA binding reaction, and a protein-DNA binding reaction. For the small ligands both the PB and limiting law models give (In K vs. In[salt]) slopes close in magnitude to the net ligand charge. However, the enthalpy/entropy breakdown of the salt dependence is quite different. In the PB model there are considerable contributions from electrostatic enthalpy and dielectric (water reorientation) entropy, compared to the predominant ion cratic (release) entropy in the limiting law model. The relative contributions of these three terms in the PB model depends on the ligand: For the protein, ion release entropy is the smallest contribution to the salt dependence of binding. The effect of three approximations made in the LL model is examined: These approximations are (1) the ligand behaves ideally, (2) the preferential interaction coefficient of the polyelectrolyte is unchanged upon ligand binding, and (3) the polyelectrolyte preferential interaction coefficient is given by the limiting law/counterion-condensation value. Analysis of the PB

  12. 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.

  13. EFFECTS OF SOG ON DPP-RECEPTOR BINDING*

    PubMed Central

    LOU, YUAN; NIE, QING; WAN, FREDERIC Y. M.

    2007-01-01

    Concentration gradients of morphogens are known to be instrumental in cell signaling and tissue patterning. Of interest here is how the presence of a competitor of BMP ligands affects cell signaling. The effects of Sog on the binding of Dpp with cell receptors are analyzed for dorsal-ventral morphogen gradient formation in vertebrate and Drosophila embryos. This prototype system includes diffusing ligands, degradation of morphogens, and cleavage of Dpp-Sog complexes by Tolloid to free up Dpp. Simple and biologically meaningful necessary and sufficient conditions for the existence of a steady state gradient configuration are established, and existence theorems are proved. For high Sog production rates (relative to the Dpp production rate), it is found that the steady state configuration exhibits a more intense Dpp-receptor concentration near the dorsal midline. Numerical simulations of the evolution of the system show that, beyond some threshold Sog production rate, the transient Dpp-receptor concentration at the dorsal midline would become more intense than that of the steady state, before subsiding and approaching a nonuniform steady state of lower magnitude. The magnitude of the transient concentration has been found to increase by several fold with increasing Sog production rate. The highly intense Dpp activity at and around the dorsal midline is consistent with available experimental observations and other analytical studies. PMID:17377624

  14. 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.

  15. Computational Investigation of Glycosylation Effects on a Family 1 Carbohydrate-binding Module*

    PubMed Central

    Taylor, Courtney B.; Talib, M. Faiz; McCabe, Clare; Bu, Lintao; Adney, William S.; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

    2012-01-01

    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. PMID:22147693

  16. 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

    2015-07-01

    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.

  17. 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.

  18. Effect of ethanol administration and withdrawal on GABA receptor binding in rat cerebral cortex

    SciTech Connect

    Volicer, L.; Biagioni, T.M.

    1982-01-01

    Sodium independent GABA receptor binding was measured in synaptosomes prepared from cerebral cortex of rats made ethanol dependent by three daily ethanol administrations. In rats sacrificed 1 hour after the last ethanol dose there was a lower number of low affinity binding sites and lower affinity of the high affinity binding than in controls. The decreased affinity was present only in rats who showed symptoms of ethanol withdrawal during the course of ethanol administration. In rats sacrificed during ethanol withdrawal the affinity of the high affinity binding was lower than in controls and other binding characteristics were unchanged. This decreased binding was normalized by repeated Triton X-100 incubations indicating involvement of an endogenous inhibitor in this ethanol effect. Acute ethanol administration did not change GABA receptor binding.

  19. A novel steroid-like compound F90927 exerting positive-inotropic effects in cardiac muscle

    PubMed Central

    Pignier, Christophe; Keller, Markus; Vié, Bruno; Vacher, Bernard; Santelli, Maurice; Niggli, Ernst; Egger, Marcel; Le Grand, Bruno

    2006-01-01

    Here we report a novel steroid-like compound F90363, exhibiting positive inotropy in vivo and in vitro in various cardiac muscle preparations. F90363 is a racemic mixture composed of the stereoisomers (−)-F90926 and (+)-F90927. Only F90927 exerted positive inotropy, while F90926 induced a weak negative inotropy, but only at concentrations 103 times higher than F90927 and most likely resulting from an unspecific interaction. The rapid time course of the action of F90927 suggested a direct interaction with a cellular target rather than a genomic alteration. We could identify the L-type Ca2+ current ICa(L) as a main target of F90927, while excluding other components of cardiac Ca2+ signalling as potential contributors. In addition, several other signaling pathways known to lead to positive inotropy (e.g. α- and β-adrenergic stimulation, cAMP pathways) could be excluded as targets of F90927. However, vessel contraction and stiffening of the cardiac muscle at high doses (>30 μM, 0.36 mg kg−1, respectively) prevent the use of F90927 as a candidate for drug development. Since the compound may still find valuable applications in research, the aim of the present study was to identify the cellular target and the mechanism of inotropy of F90927. PMID:16474419

  20. 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.

  1. Inhibitory effect of midkine-binding peptide on tumor proliferation and migration

    PubMed Central

    Huang, Hui-Lian; Shen, Jian-Fen; Min, Li-Shan; Ping, Jin-Liang; Lu, Yong-Liang; Dai, Li-Cheng

    2015-01-01

    Background: To investigate the inhibitory effect of midkine-binding peptides on human umbilical vein endothelial cells (HUVECs) proliferation and angiogenesis of xenograft tumor. Methods: The midkine-binding peptides were panned by Ph.D.-7™ Phage Display Peptide Library Kit, and the specific binding activities of positive clones to target protein were examined by phage ELISA. The effect of midkine-binding peptides on proliferation of HUVECs was confirmed by MTT test. The xenograft tumor model was formed in BALB/c mice with the murine hepatocarcinoma cells H22 (H22). Microvessel density (MVD) was analyzed by immunohistochemistry of factor VIII staining. Results: Midkine-binding peptides have the inhibitory effects on tumor angiogenesis, a proliferation assay using human umbilical vein endothelial cells (HUVECs) indicated that particular midkine-binding peptides significantly inhibited the proliferation of the HUVECs. Midkine-binding peptides were also observed to efficiently suppress angiogenesis induced by murine hepatocarcinoma H22 cells in BALB/c nude mice. Conclusion: The midkine-binding peptides can inhibit solid tumor growth by retarding the formation of new blood vessels. The results indicate that midkine-binding peptides may represent potent anti-angiogenesis agents in vivo. PMID:26191241

  2. 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.

  3. The effect of antenatal dexamethasone on maternal and fetal retinol-binding protein.

    PubMed

    Hustead, V A; Zachman, R D

    1986-01-01

    Sixteen rhesus monkeys received 0.1 to 15 mg/kg of antenatal dexamethasone at 132 days' gestation; seven control animals received placebo. At 135 days' gestation they underwent cesarean section, and maternal and fetal serum was assayed for retinol-binding protein. Fetal and maternal concentrations of retinol-binding protein increased after dexamethasone (p less than 0.05) and there was a trend for fetal levels of retinol-binding protein to increase with increasing dosage (p less than 0.01). Whether the elevation of retinol-binding protein in response to antenatal dexamethasone is a desirable side effect is not clear at this time. PMID:3946495

  4. Metal ion binding with carbon nanotubes and graphene: Effect of chirality and curvature

    NASA Astrophysics Data System (ADS)

    Umadevi, Deivasigamani; Sastry, G. Narahari

    2012-10-01

    First principles calculations have been used to comprehensively study the binding of a series alkali (Li+, Na+, K+) and alkaline earth (Be2+, Mg2+, Ca2+) metal ions with carbon nanotubes (CNTs) and graphene. It is interesting to note that the mono-cationic systems prefer binding to armchair CNTs over zigzag CNTs, while the preference for the di-cationic systems is exactly opposite. We have also observed significant changes in the HOMO-LUMO energy gap of the CNTs on metal ion binding and these results indicate that the fine tuning of energy gap of the CNTs can be effected through metal ion binding.

  5. Metal replacement in "zinc finger" and its effect on DNA binding.

    PubMed Central

    Predki, P F; Sarkar, B

    1994-01-01

    Metal replacement studies were used to investigate the metal requirement of a bacterially expressed polypeptide encoding the zinc finger DNA binding domain of the estrogen receptor. Apopolypeptide was generated by dialysis of native polypeptide against low-pH buffer under reducing conditions. Specific DNA binding can be restored by refolding the apopolypeptide in the presence of ionic zinc, cadmium, or cobalt. However, refolding in the presence of copper or nickel fails to regenerate DNA binding activity. While cobalt-reconstituted polypeptide has a reduced affinity for its AGGTCA-binding site compared to zinc- or cadmium-polypeptide, it has the surprising property of increased cooperative DNA binding. Our work indicates that metal substitution results in a range of effects upon DNA binding in vitro. The potential biological significance of metal substitution in vivo is discussed. Images Figure 1. Figure 2. Figure 3. Figure 4. PMID:7843097

  6. Effects of membrane physical parameters on hematoporphyrin-derivative binding to liposomes: a spectroscopic study

    SciTech Connect

    Gross, E.; Malik, Z.; Ehrenberg, B.

    1987-01-01

    Physical parameters of membrane bilayers were studied for their effect on the binding of hematoporphyrin derivative (Hpd), which is used as a sensitizer in photodynamic therapy of cancerous tissues. The purpose of this study was to clarify which parameters were relevant, under physiological conditions, to the selectivity of Hpd binding to cancer cells. Fluorescence spectroscopy was used to measure the relative partitioning of the dye between the lipid and aqueous media. Increasing the microviscosity of the liposomes' membranes by various bilayer additives results in a strong reduction of Hpd binding, to an extent independent of the specific additive. The effect of temperature near the physiological value as well as the effect of cross membrane potential are small. Surface potential does not affect the binding constant, indicating that the binding species does not carry a net electric charge.

  7. Palmitate uptake by hepatocyte monolayers. Effect of albumin binding.

    PubMed Central

    Fleischer, A B; Shurmantine, W O; Luxon, B A; Forker, E L

    1986-01-01

    The uptake of 14C-palmitate by rat liver cell monolayers is depressed by binding of the fatty acid to albumin. When the uptake flux is divided by the concentration of free palmitate in the bathing medium, however, the resulting clearance is approximately 14 times greater in the presence of albumin than in its absence. These findings are not accounted for by the different diffusion rates of free and bound palmitate across an unstirred fluid layer, nor attributable to nonequilibrium binding. Instead we argue that the most plausible explanation is accelerated dissociation of albumin-palmitate complexes mediated by the cell surface--an interpretation that also explains the uptake kinetics of other albumin-bound organic anions by perfused rat liver. Images PMID:3949983

  8. Effects of spermine binding on Taxol-stabilized microtubules

    NASA Astrophysics Data System (ADS)

    Cheng, Shengfeng; Regmi, Chola

    Previous studies have shown that polyamines such as spermine present in cells at physiological concentrations can facilitate the polymerization of tubulins into microtubules (MTs). A recent experiment demonstrates that in the presence of high-concentration spermine, Taxol-stabilized MTs undergo a shape transformation into inverted tubulin tubules (ITTs), the outside surface of which corresponds to the inside surface of a regular MT. However, the molecular mechanism underlying the shape transformation of MTs into ITTs is unclear. We perform all atom molecular dynamics simulations on Taxol-stabilized MT sheets containing two protofilaments surrounded by spermine ions. The spermine concentration is varied from 0 to 25mM to match the range probed experimentally. We identify important spermine binding regions on the MT surface and the influence of the spermine binding on the structure and dynamics of MTs. In contrast to Taxol, our results show that spermine binding seems to decrease the flexibility of tubulin proteins, resulting in weaker tubulin-tubulin contacts and promoting the bending of protofilaments into curved protofilaments, inverted rings, and eventually inverted tubules.

  9. MutaBind estimates and interprets the effects of sequence variants on protein-protein interactions.

    PubMed

    Li, Minghui; Simonetti, Franco L; Goncearenco, Alexander; Panchenko, Anna R

    2016-07-01

    Proteins engage in highly selective interactions with their macromolecular partners. Sequence variants that alter protein binding affinity may cause significant perturbations or complete abolishment of function, potentially leading to diseases. There exists a persistent need to develop a mechanistic understanding of impacts of variants on proteins. To address this need we introduce a new computational method MutaBind to evaluate the effects of sequence variants and disease mutations on protein interactions and calculate the quantitative changes in binding affinity. The MutaBind method uses molecular mechanics force fields, statistical potentials and fast side-chain optimization algorithms. The MutaBind server maps mutations on a structural protein complex, calculates the associated changes in binding affinity, determines the deleterious effect of a mutation, estimates the confidence of this prediction and produces a mutant structural model for download. MutaBind can be applied to a large number of problems, including determination of potential driver mutations in cancer and other diseases, elucidation of the effects of sequence variants on protein fitness in evolution and protein design. MutaBind is available at http://www.ncbi.nlm.nih.gov/projects/mutabind/. PMID:27150810

  10. When moving without volition: implied self-causation enhances binding strength between involuntary actions and effects.

    PubMed

    Dogge, Myrthel; Schaap, Marloes; Custers, Ruud; Wegner, Daniel M; Aarts, Henk

    2012-03-01

    The conscious awareness of voluntary action is associated with systematic changes in time perception: The interval between actions and outcomes is experienced as compressed in time. Although this temporal binding is thought to result from voluntary movement and provides a window to the sense of agency, recent studies challenge this idea by demonstrating binding in involuntary movement. We offer a potential account for these findings by proposing that binding between involuntary actions and effects can occur when self-causation is implied. Participants made temporal judgements concerning a key press and a tone, while they learned to consider themselves as the cause of the effect or not. Results showed that implied self-causation (vs. no implied self-causation) increased temporal binding. Since intrinsic motor cues of movement were absent, these results suggest that sensory evidence about the key press caused binding in retrospect and in line with the participant's sense of being an agent.

  11. Therapeutic safety of high myocardial expression levels of the molecular inotrope S100A1 in a preclinical heart failure model

    PubMed Central

    Weber, C; Neacsu, I; Krautz, B; Schlegel, P; Sauer, S; Raake, P; Ritterhoff, J; Jungmann, A; Remppis, AB; Stangassinger, M; Koch, WJ; Katus, HA; Müller, OJ; Most, P; Pleger, ST

    2014-01-01

    Low levels of the molecular inotrope S100A1 are sufficient to rescue post-ischemic heart failure (HF). As a prerequisite to clinical application and to determine the safety of myocardial S100A1 DNA-based therapy, we investigated the effects of high myocardial S100A1 expression levels on the cardiac contractile function and occurrence of arrhythmia in a preclinical large animal HF model. At 2 weeks after myocardial infarction domestic pigs presented significant left ventricular (LV) contractile dysfunction. Retrograde application of AAV6-S100A1 (1.5 × 1013 tvp) via the anterior cardiac vein (ACV) resulted in high-level myocardial S100A1 protein peak expression of up to 95-fold above control. At 14 weeks, pigs with high-level myocardial S100A1 protein overexpression did not show abnormalities in the electrocardiogram. Electrophysiological right ventricular stimulation ruled out an increased susceptibility to monomorphic ventricular arrhythmia. High-level S100A1 protein overexpression in the LV myocardium resulted in a significant increase in LV ejection fraction (LVEF), albeit to a lesser extent than previously reported with low S100A1 protein overexpression. Cardiac remodeling was, however, equally reversed. High myocardial S100A1 protein overexpression neither increases the occurrence of cardiac arrhythmia nor causes detrimental effects on myocardial contractile function in vivo. In contrast, this study demonstrates a broad therapeutic range of S100A1 gene therapy in post-ischemic HF using a preclinical large animal model. PMID:24305416

  12. Binding and functional characterization of the cardioselective muscarinic antagonist methoctramine.

    PubMed

    Giraldo, E; Micheletti, R; Montagna, E; Giachetti, A; Viganò, M A; Ladinsky, H; Melchiorre, C

    1988-03-01

    The antimuscarinic properties of the newly synthetized polymethylene tetramine derivative, methoctramine, were investigated in binding and functional assays. Methoctramine displaced the specific binding of [3H]-N-methylscopolamine [( 3H]NMS) and [3H] pirenzepine from membranes of rat tissues with the following order of affinities: heart = cerebellum greater than cortex greater than submandibular glands, the ratio of the affinities of the compound for the heart and the glands amounting to about 130. Computer fits of binding curves generated in cardiac and cortical membranes were compatible with an interaction at one binding site, whereas those in submandibular glands and cerebellum had slopes significantly lower than 1. Experiments performed in cardiac membranes to investigate the effect of methoctramine on the dissociation kinetics of [3H]-NMS showed that concentrations of compound up to 1 microM did not affect the dissociation of [3H]-NMS elicited by an excess of NMS. At greater concentrations (10-100 microM), methoctramine dose dependently inhibited [3H]-NMS dissociation, thus revealing an allosteric interaction. In in vitro functional assays, methoctramine displayed more than 100 times greater affinity for the muscarinic receptors mediating negative inotropic and chronotropic effects in guinea pig atria than for those responsible for tracheal contraction. Similarly, the compound was a more potent antagonist of the bradycardial response to bethanechol than of the bladder tonus increase, saliva secretion and hypotension induced by the muscarinic agonist in anesthetized cats. Finally, in the pithed rat, methoctramine preferentially inhibited cardiac M2 (vagal bradycardia) over ganglionic M1 (McN-A-343-induced hypertension) responses. The evidence appears to characterize methoctramine as being the most selective M2 muscarinic antagonist described to date.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3252019

  13. 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.

  14. Double binding energy differences: Mean-field or pairing effect?

    NASA Astrophysics Data System (ADS)

    Qi, Chong

    2012-10-01

    In this Letter we present a systematic analysis on the average interaction between the last protons and neutrons in atomic nuclei, which can be extracted from the double differences of nuclear binding energies. The empirical average proton-neutron interaction Vpn thus derived from experimental data can be described in a very simple form as the interplay of the nuclear mean field and the pairing interaction. It is found that the smooth behavior as well as the local fluctuations of the Vpn in even-even nuclei with N ≠ Z are dominated by the contribution from the proton-neutron monopole interactions. A strong additional contribution from the isoscalar monopole interaction and isovector proton-neutron pairing interaction is seen in the Vpn for even-even N = Z nuclei and for the adjacent odd-A nuclei with one neutron or proton being subtracted.

  15. Quantification of the Effects of Ionic Strength, Viscosity, and Hydrophobicity on Protein–Ligand Binding Affinity

    PubMed Central

    2014-01-01

    In order to quantify the interactions between molecules of biological interest, the determination of the dissociation constant (Kd) is essential. Estimation of the binding affinity in this way is routinely performed in “favorable” conditions for macromolecules. Crucial data for ligand–protein binding elucidation is mainly derived from techniques (e.g., macromolecular crystallography) that require the addition of high concentration of salts and/or other additives. In this study we have evaluated the effect of temperature, ionic strength, viscosity, and hydrophobicity on the Kd of three previously characterized protein–ligand systems, based on variation in their binding sites, in order to provide insight into how these often overlooked unconventional circumstances impact binding affinity. Our conclusions are as follows: (1) increasing solvent viscosity in general is detrimental to ligand binding, (2) moderate increases in temperature have marginal effects on the dissociation constant, and (3) the degree of hydrophobicity of the ligand and the binding site determines the extent of the influence of cosolvents and salt concentration on ligand binding affinity. PMID:25147617

  16. Elucidation of Nonadditive Effects in Protein-Ligand Binding Energies: Thrombin as a Case Study.

    PubMed

    Calabrò, Gaetano; Woods, Christopher J; Powlesland, Francis; Mey, Antonia S J S; Mulholland, Adrian J; Michel, Julien

    2016-06-23

    Accurate predictions of free energies of binding of ligands to proteins are challenging partly because of the nonadditivity of protein-ligand interactions; i.e., the free energy of binding is the sum of numerous enthalpic and entropic contributions that cannot be separated into functional group contributions. In principle, molecular simulations methodologies that compute free energies of binding do capture nonadditivity of protein-ligand interactions, but efficient protocols are necessary to compute well-converged free energies of binding that clearly resolve nonadditive effects. To this end, an efficient GPU-accelerated implementation of alchemical free energy calculations has been developed and applied to two congeneric series of ligands of the enzyme thrombin. The results show that accurate binding affinities are computed across the two congeneric series and positive coupling between nonpolar R(1) substituents and a X = NH3(+) substituent is reproduced, albeit with a weaker trend than experimentally observed. By contrast, a docking methodology completely fails to capture nonadditive effects. Further analysis shows that the nonadditive effects are partly due to variations in the strength of a hydrogen-bond between the X = NH3(+) ligands family and thrombin residue Gly216. However, other partially compensating interactions occur across the entire binding site, and no single interaction dictates the magnitude of the nonadditive effects for all the analyzed protein-ligand complexes. PMID:27248478

  17. 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.

  18. 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

  19. Polar solvent effects on tartaric acid binding by aromatic oligoamide foldamer capsules.

    PubMed

    Chandramouli, Nagula; El-Behairy, Mohammed Farrag; Lautrette, Guillaume; Ferrand, Yann; Huc, Ivan

    2016-02-28

    Aromatic oligoamide sequences able to fold into single helical capsules were functionalized with two types of side chains to make them soluble in various solvents such as chloroform, methanol or water and their propensity to recognize tartaric acid was evaluated. The binding affinities to tartaric acid and binding thermodynamics in different media were investigated by variable temperature (1)H NMR and ITC experiments, the two methods giving consistent results. We show that tartaric acid binding mainly rests on enthalpically favourable polar interactions that were found to be sufficiently strong to be effective in the presence of a polar aprotic solvent (DMSO) and even in pure methanol. Binding in water was very weak. The stronger binding interactions were found to be more susceptible to the effect of competitive solvents and compensated by unfavourable entropic effects. Thus, the best host in a less polar medium eventually was found to be the worst host in protic solvents. An interesting case of entropically driven binding was evidenced in methanol.

  20. Effects of cell volume regulating osmolytes on glycerol 3-phosphate binding to triosephosphate isomerase.

    PubMed

    Gulotta, Miriam; Qiu, Linlin; Desamero, Ruel; Rösgen, Jörg; Bolen, D Wayne; Callender, Robert

    2007-09-01

    During cell volume regulation, intracellular concentration changes occur in both inorganic and organic osmolytes in order to balance the extracellular osmotic stress and maintain cell volume homeostasis. Generally, salt and urea increase the Km's of enzymes and trimethylamine N-oxide (TMAO) counteracts these effects by decreasing Km's. The hypothesis to account for these effects is that urea and salt shift the native state ensemble of the enzyme toward conformers that are substrate-binding incompetent (BI), while TMAO shifts the ensemble toward binding competent (BC) species. Km's are often complex assemblies of rate constants involving several elementary steps in catalysis, so to better understand osmolyte effects we have focused on a single elementary event, substrate binding. We test the conformational shift hypothesis by evaluating the effects of salt, urea, and TMAO on the mechanism of binding glycerol 3-phosphate, a substrate analogue, to yeast triosephosphate isomerase. Temperature-jump kinetic measurements promote a mechanism consistent with osmolyte-induced shifts in the [BI]/[BC] ratio of enzyme conformers. Importantly, salt significantly affects the binding constant through its effect on the activity coefficients of substrate, enzyme, and enzyme-substrate complex, and it is likely that TMAO and urea affect activity coefficients as well. Results indicate that the conformational shift hypothesis alone does not account for the effects of osmolytes on Km's. PMID:17696453

  1. 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. PMID:25330347

  2. 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.

  3. Binding of comminuted meat: Effect of high pressure.

    PubMed

    Macfarlane, J J; McKenzie, I J; Turner, R H; Jones, P N

    1984-01-01

    Patties prepared from comminuted meat were pressure-treated at up to 150 MPa at 0-3°C and the cohesion between meat particles in the cooked patty investigated from tensile strength measurements. Pressure treatment increased tensile strength, the magnitude of the increase depending upon the intensity and duration of pressure treatment, the concentration of salt in the patty and pH value. The effect was most pronounced in patties of pH 5 to 6 and with 1% salt in the aqueous phase. Under these conditions cooking losses were reduced. When compared with the effect of addition of 0·5% tetrasodium pyrophosphate in a patty with 1% salt, pressure treatment retained its effect at lower pH values.

  4. 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. ...

  5. Prevention of the expression of inducible nitric oxide synthase by a novel positive inotropic agent, YS 49, in rat vascular smooth muscle and RAW 264.7 macrophages

    PubMed Central

    Kang, Young Jin; Koo, Eui Bon; Lee, Young Soo; Yun-Choi, Hye Sook; Chang, Ki Churl

    1999-01-01

    The effects of a novel positive inotropic isoquinoline compound, YS 49, on NO production and iNOS protein expression were investigated in cultured rat aortic vascular smooth muscle cells (RAVSMC) and RAW 264.7 cells exposed to lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). In addition, the effects of YS 49 on vascular hyporeactivity in vitro and ex vivo, and on survival rate (mice) and serum NOx (rat) levels, were also investigated in LPS-treated animals.Pre- or co-treatment of YS 49 with LPS plus IFN-γ, concentration-dependently reduced NO production in RAVSMC and RAW 264.7 cells (IC50 values, 22 and 30 μM, respectively). Although the inhibitory effect on NO production was reduced when YS 49 was applied 2 and 4 h after cytokine in RAW 264.7 cells, it was still statistically significant (P<0.05).YS 49 reduced iNOS mRNA expression in LPS-treated rat aorta in vitro, an effect which was associated with restoration of contractility to the vasoconstrictor, phenylephrine (PE), and reduction in L-arginine-induced relaxation.Serum NOx levels were significantly (P<0.01) reduced by YS 49 (5 mg kg−1, i.p.) in LPS-treated rats (10 mg kg−1, i.p.). Administration of YS 49 (10 and 20 mg kg−1) 30 min prior to LPS (10 mg kg−1) also significantly (P<0.01) increased the subsequent survival rates in mice.Finally, expression of iNOS protein induced by LPS plus IFN-γ in RAVSMC and RAW 264.7 cells was suppressed by YS 49, in a concentration-dependent manner.These data strongly suggest that YS 49 suppresses iNOS gene expression induced by LPS and/or cytokines in RAVSMC and RAW 264.7 cells at the transcriptional level. YS 49 could therefore be beneficial in septic shock and other diseases associated with iNOS over-expression. PMID:10510445

  6. Effects of extracts of ginkgo, onion, and celery on the contractility of isolated rat atria.

    PubMed

    Radenković, Mirjana; Kitić, Dušanka; Kostić, Milica; Mrkaić, Ana; Pavlović, Dragana; Miladinović, Bojana; Branković, Suzana

    2013-01-01

    The acute negative inotropic and chronotropic effects of the commercial extract of ginkgo leaf (Gingium(®)) and ethanolic extracts of onion bulbs and celery root on spontaneous and noradrenalin-stimulated contractions of the Wistar rat atria were investigated. All analyzed extracts exerted negative inotropic effects on the isolated rat atria. Among the tested extracts, the extract of ginkgo showed the strongest negative inotropic effects on rat atria, either in spontaneous or noradrenalin-induced contractions, being more potent than the onion and celery extracts, respectively.

  7. 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

  8. Effective tight-binding model for transition metal dichalcogenides

    NASA Astrophysics Data System (ADS)

    Ho, Yen-Hung; Cazalilla, Miguel; Ochoa, Hector

    For transition metal dichalcogenides, various band models have been developed to describe the novel subband features. In this work, we propose a new effective minimum-band model by preforming a canonical transformation on the full-band Hamiltonian. We found that, depending on the form of transformation, both the Γ- and K-valley electrons can be well captured, including the frequency and band effective mass. And, for the full-band parameters used, starting from Wannier function basis set leads to a better result than from Slater-Koster basis set. A close inspection of the transformation projection also enables us to extract the modification on the site energy, as well as the orbital hopping between several nearest neighboring atoms. Instead of pure empirical fitting, our effective models preserve rich orbital physics inside, which is shown to be versatile in studying a variety of fundamental physical properties. Ministry of Science and Technology of Taiwan (NSC 102-2112-M-007-024-MY5).

  9. Statistical Thermodynamics for Actin-Myosin Binding: The Crucial Importance of Hydration Effects.

    PubMed

    Oshima, Hiraku; Hayashi, Tomohiko; Kinoshita, Masahiro

    2016-06-01

    Actomyosin is an important molecular motor, and the binding of actin and myosin is an essential research target in biophysics. Nevertheless, the physical factors driving or opposing the binding are still unclear. Here, we investigate the role of water in actin-myosin binding using the most reliable statistical-mechanical method currently available for assessing biomolecules immersed in water. This method is characterized as follows: water is treated not as a dielectric continuum but as an ensemble of molecules; the polyatomic structures of proteins are taken into consideration; and the binding free energy is decomposed into physically insightful entropic and energetic components by accounting for the hydration effect to its full extent. We find that the actin-myosin binding brings large gains of electrostatic and Lennard-Jones attractive interactions. However, these gains are accompanied by even larger losses of actin-water and myosin-water electrostatic and LJ attractive interactions. Although roughly half of the energy increase due to the losses is cancelled out by the energy decrease arising from structural reorganization of the water released upon binding, the remaining energy increase is still larger than the energy decrease brought by the gains mentioned above. Hence, the net change in system energy is positive, which opposes binding. Importantly, the binding is driven by a large gain of configurational entropy of water, which surpasses the positive change in system energy and the conformational entropy loss occurring for actin and myosin. The principal physical origin of the large water-entropy gain is as follows: the actin-myosin interface is closely packed with the achievement of high shape complementarity on the atomic level, leading to a large increase in the total volume available to the translational displacement of water molecules in the system and a resultant reduction of water crowding (i.e., entropic correlations among water molecules). PMID

  10. 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.

  11. Specific binding and biological effects of tumor promoting phorbol esters on sponges.

    PubMed

    Mazzorana, M; Garrone, R; Martel, N; Yamasaki, H

    1984-01-01

    Sponges grown in the presence of 12-O-tetradecanoyl phorbol-13-acetate (TPA) show deep alterations of their structure and development. Their aquiferous system (flagellated cells and canals) is largely altered and the tissues show an unusually high cell density. This focalized effect of TPA on the aquiferous system seems specific and is reversible at low concentrations (100 ng/ml). A toxic, non-specific effect is also noted, particularly at high concentrations (5000 ng/ml). Using 3H-phorbol-12, 13-dibutyrate (3H-PDBu), we demonstrate a class of specific binding sites for phorbol esters in the homogenates of sponges. These binding sites have high affinity (Kd = 26.0 nM) for PDBu and at saturation about 20 pmoles of 3H-PDBu is bound per mg protein of sponge homogenates. The binding of 3H-PDBu was inhibited by other phorbol esters and their congeners, and there was a good correlation between their potency in binding inhibition and their tumor promoting activity. It is concluded that sponges have a class of specific saturable and high affinity receptors for phorbol esters and that there is a very high conservation of these receptors during evolution. Such specific binding may be responsible for subsequent biological effect of TPA on sponges.

  12. 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

  13. Binding effect of polychlorinated compounds and environmental carcinogens on rice bran fiber.

    PubMed

    Sera, Nobuyuki; Morita, Kunimasa; Nagasoe, Masami; Tokieda, Hisako; Kitaura, Taeko; Tokiwa, Hiroshi

    2005-01-01

    To accelerate the fecal excretion of polycyclic biphenyl (PCB), polychlorinated dibenzofurans (PCDFs), polychlorinated-p-dioxines (PCDDs) and various mutagens and carcinogens, their binding effect on rice bran fiber (RBF) was investigated for nine heterocyclic amines, six nitroarenes, 4-nitroquinoline-N-oxide, benzo[a]pyrene, furylfuramide, two kinds of flavonoid compounds and formaldehyde and ascorbic acid. PCBs, PCDFs and PCDDs suspended in nonane were incubated with RBF (10 mg/ml) at 37 degrees C and after centrifugation, unbound chemicals in the supernatant were analyzed by high-performance liquid chromatography (HPLC) and gas chromatography (GC). The binding effects on RBF were enhanced more than other dietary fibers (DFs), which were tested including corn, wheat bran, spinach, Hijiki (a kind of seaweed), sweet potatoes and burdock fibers. It was found that the binding effects were related to lignin contents. Binding of 3-amino-1(or 1,4)-dimethyl-5H-pyrido[4,3-b]indole (Trp-p-1 and Trp-p-2), food-derived carcinogens and 1-nitropyrene (1-NP), suspended in methanol, to RBF occurred within 10 min of incubation at 37 degrees C at pH 5-7, and decreased below pH 4; binding of food-derived carcinogens was pH dependent. The binding effects to RBF and pulp lignin were obtained at ratio of over 90%, while corn fiber and cellulose were at ratios of 4-30%. Polycyclic aromatic compounds were related to the number of rings, showing high binding effects to chemical structures with triple rings. Binding of 1-NP and PCB to RBF was not influenced in any aerobic and anaerobic bacterial cultures. It was also found that RBF was capable of binding even conjugates containing mutagens such as glucuronides and sulfates, as well as metabolites in urine. It was suggested, therefore, that mutagens and carcinogens were available for the fecal excretion of residual chemicals and their metabolites, and also for the fecal excretion of PCBs, PCDFs and related compound residues in patients of

  14. Unconjugated bilirubin effect on 3H-ouabain binding to human fetal red cells.

    PubMed

    Corchs, J L; Corchs, M J; Serrani, R E

    1994-03-01

    Human fetal red cells show heterogeneity of 3H-ouabain binding sites. These cells were chosen as a model to look into unconjugated bilirubin effects on the primary active Na(+)-K+ transport mechanism. Evidences are presented suggesting that unconjugated bilirubin affects 3H-ouabain binding but not through a direct effect. This is supported by the fact that the "low affinity" subgroup sites of the last mentioned ligand persists after unconjugated bilirubin treatment of cells, whereas the "high-affinity" subgroup disappears.

  15. 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.

  16. Cooperative protein-DNA interactions: effects of KCl on lambda cI binding to OR.

    PubMed

    Koblan, K S; Ackers, G K

    1991-08-01

    The effects of monovalent salt activity on the site-specific and cooperative interactions of cI repressor with its three operator sites OR were studied by using quantitative DNase I footprint titration methods. Individual-site binding isotherms were obtained for binding repressor dimers to each site of wild-type OR and to mutant operator templates in which binding to one or two sites has been eliminated. The standard Gibbs energies for intrinsic binding, delta G1, delta G2, and delta G3, and cooperative interactions, delta G12 and delta G23, were determined at each condition (range 50-200 mM KCl). It is found that the dimer affinity for each of the three sites increases as [KCl] decreases, a striking result given that the monomer-dimer equilibrium shifts toward monomer formation under identical solution conditions [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry (preceding paper in this issue)]. The magnitudes of ion-linked effects are found to differ at the three operator sites, while the intrinsic interaction binding free energies for sites OR1 and OR3 change in parallel over the entire range of [KCl]. The KCl dependencies at OR1 and OR3 represent the average release of 3.7 +/- 0.6 and 3.8 +/- 0.6 apparent ions, respectively. By contrast, the KCl dependency of OR2 binding corresponds to the displacement of 5.2 +/- 0.7 apparent ions. The ability of cI repressor to discriminate between the three operator sites thus appears linked to ion binding/release reactions.

  17. Effect of surface binding on heterogeneous DNA melting equilibria: a Monte Carlo simulation study.

    PubMed

    Allen, John H; Schoch, Emily T; Stubbs, John M

    2011-02-24

    Deoxyribonucleic acid (DNA) microarrays are constructed with a surface-immobilized single-stranded probe sequence that hydrogen bonds with its complementary target strand from solution and is subsequently detected, making their hybridization equilibrium of central importance. Unexpectedly, the effect of surface immobilization is that if the sequences of probe and target are exchanged, the hybridization equilibrium shifts. Here, configurational-bias Monte Carlo simulations using a coarse-grained model for DNA were carried out for an undecamer double helix both in solution and bound to a surface to determine dissociation equilibria. Four possible surface binding orientations were independently investigated. Analysis shows that the effect of surface binding is to destabilize hydrogen-bonding interactions of bases proximal to the binding site and enhance those of distal bases due to the double helix lying flat on the surface. Results have implications for predicting surface-bound DNA hybridization equilibria.

  18. 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.

  19. 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.

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

    PubMed

    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

  1. 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

  2. Effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes

    SciTech Connect

    Brissette, L.; Nol, S.P.

    1986-05-25

    Upon incubation with rat liver membranes, radioiodinated rat intermediate density lipoproteins (IDL) interacted with at least two binding sites having a low and a high affinity as demonstrated by the curvilinear Scatchard plots obtained from the specific binding data. The purpose of our work was to identify the nature of these binding sites. Human low density lipoproteins (LDL), contain apolipoprotein B only, and human high density lipoproteins (HDL3), containing neither apolipoprotein B nor E, were both capable of decreasing the specific binding of rat /sup 125/I-IDL. The Scatchard analysis clearly revealed that only the low affinity component was affected by the addition of these human lipoproteins. In fact, the low affinity binding component gradually decreased as the amount of human LDL or HDL3 increased in the binding assay. At a 200-fold excess of human LDL or HDL3, the low affinity binding was totally masked, and the Scatchard plot of the specific /sup 125/I-IDL binding became linear. Only the high affinity binding component was left, enabling a precise measurement of its binding parameters. In a series of competitive displacement experiments in which the binding assay contained a 200-fold excess of human LDL or HDL3, only unlabeled rat IDL effectively displaced the binding of rat /sup 125/I-IDL. We conclude that the low affinity binding of rat IDL to rat liver membranes is due to weak interactions with unspecified lipoprotein binding sites. The camouflage of these sites by human lipoproteins makes possible the study of IDL binding to the high affinity component which likely represents the combined effect of IDL binding to both the remnant and the LDL receptors.

  3. Binding of recombinant annexin V to endothelial cells: effect of annexin V binding on endothelial-cell-mediated thrombin formation.

    PubMed Central

    van Heerde, W L; Poort, S; van 't Veer, C; Reutelingsperger, C P; de Groot, P G

    1994-01-01

    Annexin V binds with high affinity to procoagulant phospholipid vesicles and thereby inhibits the procoagulant reactions catalysed by these surfaces in vitro. In vivo, vascular endothelial cells are known to catalyse the formation of thrombin by the expression of binding sites at which procoagulant complexes can assemble. Here, we have studied the binding capacity of recombinant annexin V (rANV) to quiescent, phorbol 12-myristate 13-acetate (PMA)- and tumour necrosis factor alpha (TNF-alpha)-stimulated cultured human umbilical-vein endothelial cells (HUVEC). The dissociation constant (Kd) was 15.5 +/- 3.3 nM and the number of binding sites was 8.8 (+/- 3.9) x 10(6)/cell. These binding parameters did not change significantly during a 30 h incubation period with PMA or TNF-alpha. rANV inhibited HUVEC-mediated factor Xa formation via the extrinsic as well as the intrinsic route. Activation of factor X by the tissue factor-factor VII-factor X complex and tenase complex was inhibited with IC50 values of 43 +/- 30 nM and 33 +/- 24 nM respectively. Endothelial-cell-mediated generation of thrombin by the prothrombinase complex was inhibited by rANV with an IC50 of 16 +/- 12 nM. Preincubation of rANV with the endothelial cells did not significantly influence the IC50 values. These results show that rANV binds to the same extent to quiescent, PMA- and TNF-stimulated HUVEC, and, as a result of this binding, rANV efficiently inhibits endothelial-cell-mediated thrombin formation. PMID:8068019

  4. Dopamine transporter binding in social anxiety disorder: the effect of treatment with escitalopram.

    PubMed

    Warwick, J M; Carey, P D; Cassimjee, N; Lochner, C; Hemmings, S; Moolman-Smook, H; Beetge, E; Dupont, P; Stein, D J

    2012-06-01

    Social anxiety disorder (SAD) is characterised by fear of social or performance situations where the individual is exposed to unfamiliar people or to possible scrutiny by others. The literature on dopamine ligands and dopamine genotypes in SAD is however inconsistent. In this study we measured the effects of SSRI pharmacotherapy on dopamine transporter (DAT) binding in patients with SAD, also addressing variability in DAT genotype. Adult subjects meeting DSM-IV criteria for generalised SAD were studied before and after 12 weeks of pharmacotherapy with the selective serotonin reuptake inhibitor (SSRI) escitalopram. DAT single photon emission computed tomography (SPECT) using (123)I-FP-CIT was performed at baseline, and repeated at 12 weeks. Striatal DAT binding was analysed for changes following therapy, and for correlations with clinical efficacy, in the whole group as well as for a subgroup with the A10/A10 DAT genotype. The study included 14 subjects (9 male, 5 female) with a mean (SD) age of 41 (±13) years. The subjects' Liebowitz Social Anxiety Scale (LSAS) score was significantly decreased following pharmacotherapy. In the combined group the left caudate and left putamen showed clusters of increased DAT binding after therapy. The left caudate changes were also observed in the subgroup of 9 A10/A10 homozygotes. However no correlation was found between improved symptoms and DAT binding. The changes found in DAT binding in the caudate and putamen may be due to serotonergic activation of dopamine function by SSRI therapy. This is consistent with previous work indicating decreased DAT binding in SAD, and increased DAT binding after SSRI administration.

  5. Effects of boiling on the IgE-binding properties of tropomyosin of shrimp (Litopenaeus vannamei).

    PubMed

    Liu, Guang-Ming; Cheng, Hsiaopo; Nesbit, Jacqueline B; Su, Wen-Jin; Cao, Min-Jie; Maleki, Soheila J

    2010-01-01

    The thermal stability and IgE binding of raw and boiled shrimp extracts and the tropomyosins (TM) have not been reported. In this study, we compare the stability of raw and boiled shrimp TM of Litopenaeus vannamei and evaluate how boiling may alter the allergenicity of L. vannamei. Extracts were prepared from raw and boiled shrimp and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis. The IgE-binding of the extracts was determined by western-blot and competitive inhibition enzyme-linked immunosorbent assay (iELISA). The TM was then purified from raw and boiled shrimp, the secondary structures analyzed by circular dichroism (CD) spectroscopy, and the IgE binding compared by slot blot analysis. The soluble protein content decreased and the higher molecular weight proteins increased in the extracts from boiled versus raw shrimp. Similar IgE binding characteristics were seen by extracts when using western blot analysis. Although iELISA results showed that extracts from raw shrimp bound higher IgE than extracts from boiled shrimp, dot-blot assay demonstrates higher IgE binding to purified TM from boiled shrimp than raw shrimp. The purified TM had a typical alpha-helical secondary structure and the stability of boiled TM was lower than that of raw TM. Extracts from boiled shrimp produce lower IgE binding than extracts from raw shrimp, which suggest that boiling can be used as a tool in attempting to reduce shrimp allergenicity. However, the purified TM from boiled shrimp, which shows enhanced IgE binding over that of raw shrimp, may be a more effective antigen in diagnosing shrimp allergy through immunoassay.

  6. Improving the binding efficiency of quartz crystal microbalance biosensors by applying the electrothermal effect

    PubMed Central

    Huang, Yao-Hung; Chang, Jeng-Shian; Chao, Sheng D.; Wu, Kuang-Chong; Huang, Long-Sun

    2014-01-01

    A quartz crystal microbalance (QCM) serving as a biosensor to detect the target biomolecules (analytes) often suffers from the time consuming process, especially in the case of diffusion-limited reaction. In this experimental work, we modify the reaction chamber of a conventional QCM by integrating into the multi-microelectrodes to produce electrothermal vortex flow which can efficiently drive the analytes moving toward the sensor surface, where the analytes were captured by the immobilized ligands. The microelectrodes are placed on the top surface of the chamber opposite to the sensor, which is located on the bottom of the chamber. Besides, the height of reaction chamber is reduced to assure that the suspended analytes in the fluid can be effectively drived to the sensor surface by induced electrothermal vortex flow, and also the sample costs are saved. A series of frequency shift measurements associated with the adding mass due to the specific binding of the analytes in the fluid flow and the immobilized ligands on the QCM sensor surface are performed with or without applying electrothermal effect (ETE). The experimental results show that electrothermal vortex flow does effectively accelerate the specific binding and make the frequency shift measurement more sensible. In addition, the images of the binding surfaces of the sensors with or without applying electrothermal effect are taken through the scanning electron microscopy. By comparing the images, it also clearly indicates that ETE does raise the specific binding of the analytes and ligands and efficiently improves the performance of the QCM sensor. PMID:25538808

  7. 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...

  8. Biophysical studies of a ruthenium(II) polypyridyl complex binding to DNA and RNA prove that nucleic acid structure has significant effects on binding behaviors.

    PubMed

    Xu, Hong; Liang, Yi; Zhang, Peng; Du, Fen; Zhou, Bing-Rui; Wu, Jun; Liu, Jian-Hong; Liu, Zhi-Gang; Ji, Liang-Nian

    2005-08-01

    The interactions of a metal complex [Ru(phen)(2)PMIP](2+) {Ru=ruthenium, phen=1,10-phenanthroline, PMIP=2-(4-methylphenyl)imidazo[4,5-f]1,10-phenanthroline} with yeast tRNA and calf thymus DNA (CT DNA) have been investigated comparatively by UV-vis spectroscopy, fluorescence spectroscopy, viscosity measurements, isothermal titration calorimetry (ITC), as well as equilibrium dialysis and circular dichroism (CD). Spectroscopic studies together with ITC and viscosity measurements indicate that both binding modes of the Ru(II) polypyridyl complex to yeast tRNA and CT DNA are intercalation and yeast tRNA binding of the complex is stronger than CT DNA binding. ITC experiments show that the interaction of the complex with yeast tRNA is driven by a moderately favorable enthalpy decrease in combination with a moderately favorable entropy increase, while the binding of the complex to CT DNA is driven by a large favorable enthalpy decrease with a less favorable entropy increase. The results from equilibrium dialysis and CD suggest that both interactions are enantioselective and the Delta enantiomer of the complex may bind more favorably to both yeast tRNA and CT DNA than the Lambda enantiomer does, and that the complex is a better candidate for an enantioselective binder to yeast tRNA than to CT DNA. Taken together, these results indicate that the structures of nucleic acids have significant effects on the binding behaviors of metal complexes.

  9. 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.

  10. 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.

  11. 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.

  12. (/sup 3/H)-8-cyclopentyl-1,3-dipropylxanthine binding to A1 adenosine receptors of intact rat ventricular myocytes

    SciTech Connect

    Martens, D.; Lohse, M.J.; Schwabe, U.

    1988-09-01

    The purpose of the present study was the identification of A1 adenosine receptors in intact rat ventricular myocytes, which are thought to mediate the negative inotropic effects of adenosine. The adenosine receptor antagonist (/sup 3/H)-8-cyclopentyl-1,3-dipropylxanthine was used as radioligand. Binding of the radioligand to intact myocytes was rapid, reversible, and saturable with a binding capacity of 40,000 binding sites per cell. The dissociation constant of the radioligand was 0.48 nM. The adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener, and theophylline were competitive inhibitors with affinities in agreement with results obtained for A1 receptors in other tissues. Competition experiments using the adenosine receptor agonists R-N(6)-phenylisopropyladenosine, 5'-N-ethylcarboxamidoadenosine, and S-N(6)-phenylisopropyladenosine gave monophasic displacement curves with Ki values of 50 nM, 440 nM, and 4,300 nM, which agreed well with the GTP-inducible low affinity state in cardiac membranes. The low affinity for agonists was not due to agonist-induced desensitization, and correlated well with the corresponding IC50 values for the inhibition of cyclic AMP accumulation by isoprenaline. It is suggested that only a low affinity state of A1 receptors can be detected in intact rat myocytes due to the presence of high concentrations of guanine nucleotides in intact cells.

  13. Effect of mutations of the human serpin protein corticosteroid-binding globulin on cortisol-binding, thermal and protease sensitivity.

    PubMed

    Braun, Beate C; Meyer, Hellmuth-A; Reetz, Antje; Fuhrmann, Ulrike; Köhrle, Josef

    2010-05-01

    Corticosteroid-binding globulin (CBG, transcortin) belongs to the serpin family of serine protease inhibitors (SERPINA6) and is mainly secreted by the liver. The negative acute phase protein CBG regulates free cortisol levels in the blood and distributes cortisol to its target tissues. So far no CBG serpin partner protease has been identified. However, its cleavage by human neutrophil elastase destroys ligand binding capacity and supposedly liberates cortisol at sites of inflammation. Here we report on the recombinant expression and secretion of human wild-type CBG and several novel mutants by human 293-EBNA cells. Functional characterization of wild-type and mutant CBG revealed distinct differences in ligand binding sensitivity to heat or elastase. Certain mutants are almost devoid of cortisol binding activity (Q232R and CBG Lyon), some display higher sensitivity for heat inactivation (G335V, Q232R and CBG Lyon) or for elastase cleavage (G335V). CBG mutant T342A is more resistant to elastase cleavage. Our data support the validity of the serpin structural concept. The expression system used provides functionally active human recombinant transcortin for further functional characterization of wild-type and human CBG mutant variants, which have been associated with altered serum free cortisol levels or pathophysiological constellations such as increased body weight, fatigue or hypotension.

  14. 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.

  15. 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. PMID:27541005

  16. 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.

  17. Effect of BET Missense Mutations on Bromodomain Function, Inhibitor Binding and Stability

    PubMed Central

    Lori, Laura; Pasquo, Alessandra; Lori, Clorinda; Petrosino, Maria; Chiaraluce, Roberta; Tallant, Cynthia; Knapp, Stefan; Consalvi, Valerio

    2016-01-01

    Lysine acetylation is an important epigenetic mark regulating gene transcription and chromatin structure. Acetylated lysine residues are specifically recognized by bromodomains, small protein interaction modules that read these modification in a sequence and acetylation dependent way regulating the recruitment of transcriptional regulators and chromatin remodelling enzymes to acetylated sites in chromatin. Recent studies revealed that bromodomains are highly druggable protein interaction domains resulting in the development of a large number of bromodomain inhibitors. BET bromodomain inhibitors received a lot of attention in the oncology field resulting in the rapid translation of early BET bromodomain inhibitors into clinical studies. Here we investigated the effects of mutations present as polymorphism or found in cancer on BET bromodomain function and stability and the influence of these mutants on inhibitor binding. We found that most BET missense mutations localize to peripheral residues in the two terminal helices. Crystal structures showed that the three dimensional structure is not compromised by these mutations but mutations located in close proximity to the acetyl-lysine binding site modulate acetyl-lysine and inhibitor binding. Most mutations affect significantly protein stability and tertiary structure in solution, suggesting new interactions and an alternative network of protein-protein interconnection as a consequence of single amino acid substitution. To our knowledge this is the first report studying the effect of mutations on bromodomain function and inhibitor binding. PMID:27403962

  18. 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.

  19. Epitope-distal effects accompany the binding of two distinct antibodies to hepatitis B virus capsids.

    PubMed

    Bereszczak, Jessica Z; Rose, Rebecca J; van Duijn, Esther; Watts, Norman R; Wingfield, Paul T; Steven, Alasdair C; Heck, Albert J R

    2013-05-01

    Infection of humans by hepatitis B virus (HBV) induces the copious production of antibodies directed against the capsid protein (Cp). A large variety of anticapsid antibodies have been identified that differ in their epitopes. These data, and the status of the capsid as a major clinical antigen, motivate studies to achieve a more detailed understanding of their interactions. In this study, we focused on the Fab fragments of two monoclonal antibodies, E1 and 3120. E1 has been shown to bind to the side of outward-protruding spikes whereas 3120 binds to the "floor" region of the capsid, between spikes. We used hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the effects on HBV capsids of binding these antibodies. Conventionally, capsids loaded with saturating amounts of Fabs would be too massive to be readily amenable to HDX-MS. However, by focusing on the Cp protein, we were able to acquire deuterium uptake profiles covering the entire 149-residue sequence and reveal, in localized detail, changes in H/D exchange rates accompanying antibody binding. We find increased protection of the known E1 and 3120 epitopes on the capsid upon binding and show that regions distant from the epitopes are also affected. In particular, the α2a helix (residues 24-34) and the mobile C-terminus (residues 141-149) become substantially less solvent-exposed. Our data indicate that even at substoichiometric antibody binding an overall increase in the rigidity of the capsid is elicited, as well as a general dampening of its breathing motions. PMID:23597076

  20. Neural control of left ventricular contractility in the dog heart: synaptic interactions of negative inotropic vagal preganglionic neurons in the nucleus ambiguus with tyrosine hydroxylase immunoreactive terminals.

    PubMed

    Massari, V J; Dickerson, L W; Gray, A L; Lauenstein, J M; Blinder, K J; Newsome, J T; Rodak, D J; Fleming, T J; Gatti, P J; Gillis, R A

    1998-08-17

    Recent physiological evidence indicates that vagal postganglionic control of left ventricular contractility is mediated by neurons found in a ventricular epicardial fat pad ganglion. In the dog this region has been referred to as the cranial medial ventricular (CMV) ganglion [J.L. Ardell, Structure and function of mammalian intrinsic cardiac neurons, in: J.A. Armour, J.L. Ardell (Eds.). Neurocardiology, Oxford Univ. Press, New York, 1994, pp. 95-114; B.X. Yuan, J.L. Ardell, D.A. Hopkins, A.M. Losier, J.A. Armour, Gross and microscopic anatomy of the canine intrinsic cardiac nervous system, Anat. Rec., 239 (1994) 75-87]. Since activation of the vagal neuronal input to the CMV ganglion reduces left ventricular contractility without influencing cardiac rate or AV conduction, this ganglion contains a functionally selective pool of negative inotropic parasympathetic postganglionic neurons. In the present report we have defined the light microscopic distribution of preganglionic negative inotropic neurons in the CNS which are retrogradely labeled from the CMV ganglion. Some tissues were also processed for the simultaneous immunocytochemical visualization of tyrosine hydroxylase (TH: a marker for catecholaminergic neurons) and examined with both light microscopic and electron microscopic methods. Histochemically visualized neurons were observed in a long slender column in the ventrolateral nucleus ambiguus (NA-VL). The greatest number of retrogradely labeled neurons were observed just rostral to the level of the area postrema. TH perikarya and dendrites were commonly observed interspersed with vagal motoneurons in the NA-VL. TH nerve terminals formed axo-dendritic synapses upon negative inotropic vagal motoneurons, however the origin of these terminals remains to be determined. We conclude that synaptic interactions exist which would permit the parasympathetic preganglionic vagal control of left ventricular contractility to be modulated monosynaptically by

  1. 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

  2. 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.

  3. Growth hormone (GH) binding and effects of GH analogs in transgenic mice

    SciTech Connect

    Bartke, A.; Steger, R.W.; Turyn, D.

    1994-12-31

    Overexpression of human (h) or bovine (b) growth hormone (GH) in transgenic mice is associated with marked (2- to 12-fold) and significant increase in hepatic binding of GH and prolactin (PRL). This is due to an increase in the number of GH and PRL receptors (GHR, PRLR) per mg of microsomal protein without changes in binding affinity. Comparison of results obtained in transgenic animals expressing bGH with a mouse metallothionein (MT) or a rat phosphoenolpyruvate carboxykinase (PEPCK) promoter suggests that effects of bGH on hepatic GHR and PRLR do not require GH overexpression during fetal life and, within the dose range tested, the effects on PRLR are not dose dependent. The increase in hepatic GHR was accompanied by significant increases in plasma GH-binding protein (GHBP) and in mean residence time of injected GH. Thus life-long elevation of peripheral GH levels alters the availability of both free GH and GHR. Site-directed in vitro mutagenesis was used to produce hGH and bGH analogs mutated within one of the sites involved in binding to GHR and PRLR. Mutating hGH to produce amino acid identity with bGH at Position 11, 18 (within Helix 1), 57, or 60 (within the loop between Helix 1 and 2) did not affect binding to GHR in vitro, or somatotropic activity in transgenic mice in vivo but reduced lactogenic activity in Nb{sub 2} cells by 22%-45%. Mutations of bGH designed to produce amino acid identity with hGH at one to four of the corresponding positions in the bGH molecule did not interfere with binding to GHR or somatotropic activity in vivo, and failed to produce significant binding to PRLR but resulted in alterations in the effects on the hypothalamic and anterior pituitary function in transgenic mice. Apparently region(s) outside the domains examined are essential for lactogenic activity of hGH, and different portions of the GH molecule are responsible for its diverse actions in vivo. 35 refs.

  4. The effect of 75% glycerol on the oxygen binding properties of carp hemoglobin.

    PubMed

    Kwiatkowski, L D; Noble, R W

    1993-09-30

    At pH 6 in the presence of inositol hexaphosphate, IHP, conditions where ligand-saturated carp hemoglobin is already in the low affinity T state, the addition of glycerol has little effect on affinity and ligand binding remains noncooperative. At all other pH values examined, with and without IHP, the effect of glycerol is to lower oxygen affinity possibly by shifting the equilibrium between the T state and the high affinity R state in the direction of the T state. Although glycerol does not appear to have an appreciable effect on the T state itself, a small effect on the R state cannot be excluded by our data.

  5. Effects of intracoronary infusions of acetylcholine and nicotine on the dog heart in vivo.

    PubMed

    Ross, G

    1973-08-01

    1. In anaesthetized dogs intracoronary infusions of high doses of nicotine and acetylcholine increased myocardial contractile force and this could be prevented by pre-treatment with desmethylimipramine or phenoxybenzamine.2. The inotropic effect of nicotine was brief and subsided during the continuing infusion of the drug. The infusion of nicotine did not reduce the inotropic effects of cardiac sympathetic nerve stimulation.3. The motropic effect of intracoronary acetylcholine often fluctuated during prolonged infusions and was not altered by pretreatment with atropine. Acetylcholine infusions reduced the inotropic responses produced by cardiac sympathetic nerve stimulation and led to a substantial transient reduction in the associated pressor responses. Intracoronary acetylcholine also reduced the pressor and inotropic effect of intravenous noradrenaline. The attenuation of these adrenergic cardiovascular responses by acetylcholine was prevented by atropine.

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

    PubMed Central

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

    2016-01-01

    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. PMID:27030165

  7. 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.

  8. 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.

  9. Effects of copper ions on DNA binding and cytotoxic activity of a chiral salicylidene Schiff base.

    PubMed

    Fei, Bao-Li; Xu, Wu-Shuang; Tao, Hui-Wen; Li, Wen; Zhang, Yu; Long, Jian-Ying; Liu, Qing-Bo; Xia, Bing; Sun, Wei-Yin

    2014-03-01

    A chiral Schiff base HL N-(5-bromo-salicylaldehyde)dehydroabietylamine (1) and its chiral dinuclear copper complex [Cu2L4]·4DMF (2) have been synthesized and fully characterized. The interactions of 1 and 2 with salmon sperm DNA have been investigated by viscosity measurements, UV, fluorescence and circular dichroism (CD) spectroscopic techniques. Absorption spectral (Kb=3.30 × 10(5)M(-)(1) (1), 6.63 × 10(5)M(-)(1)(2)), emission spectral (Ksv=7.58 × 10(3)M(-)(1) (1), 1.52 × 10(4)M(-)(1) (2)), and viscosity measurements reveal that 1 and 2 interact with DNA through intercalation and 2 exhibits a higher DNA binding ability. In addition, CD study indicates 2 cause a more evident perturbation on the base stacking and helicity of B-DNA upon binding to it. In fluorimetric studies, the enthalpy (ΔH>0) and entropy (ΔS>0) changes of the reactions between the compounds with DNA demonstrate hydrophobic interactions. 1 and 2 were also screened for their cytotoxic ability and 2 demonstrates higher growth inhibition of the selected cancer cells at concentration of 50 μM, this result is identical with their DNA binding ability order. All the experimental results show that the involvement of Cu (II) centers has some interesting effect on DNA binding ability and cytotoxicity of the chiral Schiff base.

  10. 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.

  11. 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.

  12. 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.

  13. Estramustine binding protein and anti-proliferative effect of estramustine in human glioma cell lines.

    PubMed Central

    von Schoultz, E.; Lundblad, D.; Bergh, J.; Grankvist, K.; Henriksson, R.

    1988-01-01

    Four human cell lines derived from malignant gliomas were immunohistochemically examined for their content of estramustine-binding protein (EMBP). EMBP was detected in a large amount in all glioma cells during the entire cell cycle. EMBP has previously been demonstrated to be the major receptor protein in prostatic cancers for the cytostatic drug estramustine-phosphate (EMP). EMP caused a dose-dependent inhibition of exponentially growing cells by increasing the number of cells in G2/M stage of the cell cycle as monitored by flow cytofluorometry. The effect may be coupled to arrest of the glioma cells at metaphase. The presence of EMBP may suggest a selective binding and effect of EMP in glioma cells. Images Figure 2 PMID:3052561

  14. O2-sulfonylethyl protected isopropylamine diazen-1-ium-1,2-diolates as nitroxyl (HNO) donors: synthesis, β-elimination fragmentation, HNO release, positive inotropic properties, and blood pressure lowering studies.

    PubMed

    Huang, Zhangjian; Kaur, Jatinder; Bhardwaj, Atul; Alsaleh, Nasser; Reisz, Julie A; DuMond, Jenna F; King, S Bruce; Seubert, John M; Zhang, Yihua; Knaus, Edward E

    2012-11-26

    New types of nonexplosive O(2)-sulfonylethyl protected (-CH(2)CH(2)SO(2)R; R = OMe, NHOMe, NHOBn, Me) derivatives of isopropylamine diazen-1-ium-1,2-diolate (IPA/NO) (2-5) were developed that are designed to act as novel HNO donors. These compounds, with suitable half-lives (6.6-17.1 h) at pH 7.4, undergo a base-induced β-elimination reaction that releases a methyl vinyl sulfone product and the parent IPA/NO anion which subsequently preferentially releases HNO (46-61% range). Importantly, the O(2)-methylsulfonylethyl compound 5 exhibited a significant in vitro inotropic effect up to 283% of the baseline value and increased the rates of contraction and relaxation but did not induce a chronotropic effect. Furthermore, compound 5 (22.5 mg/kg po dose) provided a significant reduction in blood pressure up to 6 h after drug administration. All these data suggest that O(2)-sulfonylethyl protected derivatives of IPA/NO, which are efficient HNO donors, could have potential applications to treat cardiovascular disease(s) such as congestive heart failure.

  15. Exciton Binding energies and effective masses in Organo-lead Tri-Halide Perovskites

    NASA Astrophysics Data System (ADS)

    Portugall, Oliver; Miyata, Atsuhiko; Mitioglu, Anatol; Plochocka, Paulina; Wang, Jacob Tse-Wei; Stranks, Samuel; Snaith, Henry; Nicholas, Robin; Lncmi Toulouse Team; Oxford University Team

    2015-03-01

    Solid-state perovskite-based solar cells have made a dramatic impact on emerging PV research with efficiencies of over 17% already achieved. However, to date the basic electronic properties of the perovskites such as the electron and hole effective masses and the exciton binding energy are not well known. We have measured both for methyl ammonium lead tri-iodide using magneto absorption in very high magnetic fields up to 150T showing that the exciton binding energy at low temperatures is only 16 meV, a value three times smaller than previously thought and sufficiently small to completely transform the way in which the devices must operate. Landau level spectroscopy shows that the reduced effective mass of 0.104 me is also smaller than previously thought. In addition by using a fast pulse 150T magnet we measure the band structure change due to the structural phase transition that occurs in this system at around 160K. We also observe Landau levels in the high temperature phase as used for device production, which has a very similar effective mass and the analysis suggests an exciton binding energy which is even smaller than in the low temperature phase.

  16. [Transthyretin-binding activity of hexabromocyclododecanes (HBCDs) and its thyroid hormone disrupting effects after developmental exposure].

    PubMed

    Ji, Xiu-Ling; Liu, Yang; Liu, Fang; Lu, Yue; Zhong, Gao-Ren

    2010-09-01

    In vivo and in vitro research approaches were carried out to survey the potential health risk of environmental exposure by hexabromocyclododecanes (HBCDs). Transthyretin-binding assay was designed to test for the potency of HBCDs to compete with thyroxine (T4) for binding to the transport protein. The results showed that the binding of 25I-T4 and T4 was only slightly inhabited even at the highest competitive concentration of HBCDs (75.08%, 80 micromol x L(-1)), indicating the marginally interfere potency of HBCDs in the transportation of T4. Sprague-Dawley rats of 3-days old were exposed to 0.2 mg/kg and 1 mg/kg HBCDs for 21 d to examine the thyroid hormones (THs) disrupting effects of HBCDs after developmental exposure. Compared with the controls, levels of total 3,3',5-triiodothyronine (TT3), free 3,3',5-triiodothyronine (FT3), increased significantly (p < 0.05, p < 0.05) in low- and high-dose exposures, thyroid stimulating hormone (TSH) also increased slightly while the total thyroxine (TT4), free thyroxine (FT4) had a decline about two-fold inversely. Combined both the in vivo and in vitro results, the possible mode of action of HBCDs on THs disruption may through the synergy or substitution effect of T3. The findings support further investigation of the potential THs disrupting effects of HBCDs on public health, especially on children during brain development. PMID:21072945

  17. Effects of inorganic polyphosphate on the proteolytic and DNA-binding activities of Lon in Escherichia coli.

    PubMed

    Nomura, Kazutaka; Kato, Junichi; Takiguchi, Noboru; Ohtake, Hisao; Kuroda, Akio

    2004-08-13

    Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift. Previously, we revealed that inorganic polyphosphate (polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon. In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon. An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins. A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain. Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon. In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA. Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein. PMID:15187082

  18. Distinct effects of Cu2+-binding on oligomerization of human and rabbit prion proteins.

    PubMed

    Lin, Kejiang; Yu, Ziyao; Yu, Yuanhui; Liao, Xinli; Huang, Pei; Guo, Chenyun; Lin, Donghai

    2015-10-01

    The cellular prion protein (PrP(C)) is a kind of cell-surface Cu(2+)-binding glycoprotein. The oligomerization of PrP(C) is highly related to transmissible spongiform encephalopathies (TSEs). Cu(2+) plays a vital role in the oligomerization of PrP(C), and participates in the pathogenic process of TSE diseases. It is expected that Cu(2+)-binding has different effects on the oligomerization of TSE-sensitive human PrP(C) (HuPrP(C)) and TSE-resistant rabbit PrP(C) (RaPrP(C)). However, the details of the distinct effects remain unclear. In the present study, we measured the interactions of Cu(2+) with HuPrP(C) (91-230) and RaPrP(C) (91-228) by isothermal titration calorimetry, and compared the effects of Cu(2+)-binding on the oligomerization of both PrPs. The measured dissociation constants (Kd) of Cu(2+) were 11.1 ± 2.1 μM for HuPrP(C) and 21.1 ± 3.1 μM for RaPrP(C). Cu(2+)-binding promoted the oligomerization of HuPrP(C) more significantly than that of RaPrP(C). The far-ultraviolet circular dichroism spectroscopy experiments showed that Cu(2+)-binding induced more significant secondary structure change and increased more β-sheet content for HuPrP(C) compared with RaPrP(C). Moreover, the urea-induced unfolding transition experiments indicated that Cu(2+)-binding decreased the conformational stability of HuPrP(C) more distinctly than that of RaPrP(C). These results suggest that RaPrP(C) possesses a low susceptibility to Cu(2+), potentially weakening the risk of Cu(2+)-induced TSE diseases. Our work sheds light on the Cu(2+)-promoted oligomerization of PrP(C), and may be helpful for further understanding the TSE-resistance of rabbits. PMID:26350098

  19. Effects of detergent on the binding of solubilized sodium channels to immobilized wheat germ agglutinin: structural implications.

    PubMed

    Weiner, J S; Rudy, B

    1988-10-20

    The binding of the solubilized voltage-dependent sodium channel from rat brain to immobilized wheat germ agglutinin (WGA) is detergent-dependent. When solubilized in sodium cholate, only 11% of total recovered channels bound to a WGA-Sepharose column. When solubilized in Triton X-100 or CHAPS, however, 80% and 60% could bind, respectively. The effect of cholate on sodium channel binding is relatively specific: the major rat brain glycoproteins which bind to immobilized WGA are roughly the same in either Triton or cholate, as analyzed by SDS gel electrophoresis. The structural implications for the channel are discussed.

  20. Allosteric effects of the antipsychotic drug trifluoperazine on the energetics of calcium binding by calmodulin.

    PubMed

    Feldkamp, Michael D; O'Donnell, Susan E; Yu, Liping; Shea, Madeline A

    2010-08-01

    Trifluoperazine (TFP; Stelazine) is an antagonist of calmodulin (CaM), an essential regulator of calcium-dependent signal transduction. Reports differ regarding whether, or where, TFP binds to apo CaM. Three crystallographic structures (1CTR, 1A29, and 1LIN) show TFP bound to (Ca(2+))(4)-CaM in ratios of 1, 2, or 4 TFP per CaM. In all of these, CaM domains adopt the "open" conformation seen in CaM-kinase complexes having increased calcium affinity. Most reports suggest TFP also increases calcium affinity of CaM. To compare TFP binding to apo CaM and (Ca(2+))(4)-CaM and explore differential effects on the N- and C-domains of CaM, stoichiometric TFP titrations of CaM were monitored by (15)N-HSQC NMR. Two TFP bound to apo CaM, whereas four bound to (Ca(2+))(4)-CaM. In both cases, the preferred site was in the C-domain. During the titrations, biphasic responses for some resonances suggested intersite interactions. TFP-binding sites in apo CaM appeared distinct from those in (Ca(2+))(4)-CaM. In equilibrium calcium titrations at defined ratios of TFP:CaM, TFP reduced calcium affinity at most levels tested; this is similar to the effect of many IQ-motifs on CaM. However, at the highest level tested, TFP raised the calcium affinity of the N-domain of CaM. A model of conformational switching is proposed to explain how TFP can exert opposing allosteric effects on calcium affinity by binding to different sites in the "closed," "semi-open," and "open" domains of CaM. In physiological processes, apo CaM, as well as (Ca(2+))(4)-CaM, needs to be considered a potential target of drug action.

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

    PubMed

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

    2014-07-01

    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.

  2. 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…

  3. 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...

  4. Binding energies of exciton complexes in transition metal dichalcogenide monolayers and effect of dielectric environment

    NASA Astrophysics Data System (ADS)

    Kylänpää, Ilkka; Komsa, Hannu-Pekka

    2015-11-01

    Excitons, trions, biexcitons, and exciton-trion complexes in two-dimensional transition metal dichalcogenide sheets of MoS2, MoSe2, MoTe2, WS2, and WSe2 are studied by means of density functional theory and path-integral Monte Carlo method in order to accurately account for the particle-particle correlations. In addition, the effect of dielectric environment on the properties of these exciton complexes is studied by modifying the effective interaction potential between particles. Calculated exciton and trion binding energies are consistent with previous experimental and computational studies, and larger systems such as biexciton and exciton-trion complex are found highly stable. Binding energies of biexcitons are similar to or higher than those of trions, but the binding energy of the trion depends significantly stronger on the dielectric environment than that of biexciton. Therefore, as a function of an increasing dielectric constant of the environment the exciton-trion complex "dissociates" to a biexciton rather than to an exciton and a trion.

  5. Arsenic and 17-β-estradiol bind to each other and neutralize each other's signaling effects.

    PubMed

    Kumar, Sukhdeep; Mukherjee, Tapan K; Guptasarma, Purnananda

    2016-09-01

    We report that arsenic trioxide (ATO) and 17-beta-estradiol (E2) abolish each other's independent cell signaling effects in respect of cell survival and proliferation/migration of breast cancer (MCF-7) cells. The possibility that this is due to binding of ATO to E2 was confirmed through difference absorption spectroscopy, chromatography-coupled voltammometry and 1-D (1)H and (13)C NMR spectroscopy. Binding leads to attenuation of E2's hydroxyl (1)H peaks at its C17 and C3 carbon positions. The results suggest that ATO and E2 can titrate each other's levels, potentially explaining why sustained arsenic exposure tends to be associated with delays in age of menarche, advanced age of menopause, poorer sperm quality, higher overall morbidity in men, and lower incidences of breast cancer in women in some arsenic-contaminated areas. PMID:27346132

  6. In vitro assessment of the effects of vedolizumab binding on peripheral blood lymphocytes.

    PubMed

    Wyant, Timothy; Yang, Lili; Fedyk, Eric

    2013-01-01

    Vedolizumab (VDZ) is a humanized monoclonal antibody in development for the treatment of inflammatory bowel disease. VDZ binds to the α4β7 integrin complex and inhibits its binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1), thus preventing lymphocyte extravasation to gut mucosal tissues. To understand whether VDZ has additional effects that may affect its overall safety as a therapeutic molecule, we examined other potential actions of VDZ. In vitro assays with human peripheral blood lymphocytes demonstrated that VDZ fails to elicit cytotoxicity, lymphocyte activation, and cytokine production from memory T lymphocytes and does not interfere with the suppressive ability of regulatory T cells. Furthermore, we demonstrated that VDZ induces internalization of α4β7 and that the integrin is rapidly re-expressed and fully functional after VDZ withdrawal. These studies provide insight into the mechanisms underlying the observed safety profile of VDZ in clinical trials. PMID:24492340

  7. 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.

  8. Effect of protein binding on the in vitro activity and pharmacodynamics of faropenem.

    PubMed

    Boswell, F J; Ashby, J P; Andrews, J M; Wise, R

    2002-10-01

    The influence of protein binding upon different aspects of the in vitro activity of faropenem on recently isolated Staphylococcus aureus and respiratory pathogens was determined. The protein binding of faropenem was investigated in inactivated human serum and albumin by ultrafiltration. The effect of the presence of inactivated human serum and albumin on the in vitro activity of faropenem and amoxicillin was established and the influence of protein binding on the pharmacodynamic properties of faropenem and amoxicillin was compared. The protein binding of faropenem was 96% and 95% in pooled inactivated human serum and 99% and 98% in 45 mg/L human albumin, at 8 and 25 mg/L, respectively. The presence of inactivated human serum (20% and 70%) increased the mean faropenem MICs by two dilution steps and albumin increased the mean faropenem MICs by three dilution steps. The mean amoxicillin MICs were less affected than faropenem by the presence of either inactivated human serum or albumin. Faropenem and amoxicillin exhibited similar time-dependent kinetics. Faropenem was bacteriostatic on Moraxella catarrhalis, Haemophilus influenzae and group A streptococci, and bactericidal for Streptococcus pneumoniae (after 4 h with concentrations equivalent to 5 x and 10 x MIC) in Iso-Sensitest broth. In 70% inactivated human serum faropenem was slowly bactericidal against M. catarrhalis, H. influenzae (one strain) and S. pneumoniae (one strain) but not group A streptococci and the other S. pneumoniae strain. A significant inoculum effect was observed with all strains except S. pneumoniae. Both faropenem and amoxicillin appeared more active in 70% inactivated human serum than in Iso-Sensitest broth. PMID:12356797

  9. Effect of factor H-binding protein sequence variation on factor H binding and survival of Neisseria meningitidis in human blood.

    PubMed

    Dunphy, Kathleen Y; Beernink, Peter T; Brogioni, Barbara; Granoff, Dan M

    2011-01-01

    Binding of the complement inhibitor factor H (fH) to the surface of Neisseria meningitidis is critical for evasion of innate host defenses. The meningococcal vaccine candidate factor H-binding protein (fHbp) serves as an fH ligand. We prepared 16 recombinant fHbp natural sequence variants. By enzyme-linked immunosorbent assay (ELISA), the variants from a New Zealand epidemic strain (fHbp ID 14) and from an endemic United Kingdom strain (ID 15) showed 10-fold lower fH binding than a reference fHbp from an epidemic Norwegian strain (ID 1). By surface plasmon resonance, association rate constants (k(a)) for fHbp ID 14 and 15 were similar to those for ID 1, but dissociation rate constants (k(d)) were 4- to 10-fold higher than those for ID 1. To determine the effect of fH affinity on fHbp fitness, we prepared isogenic mutants of strain H44/76 that expressed fHbp ID 1, 14, or 15. By flow cytometry, mutants expressing fHbp ID 14 or 15 had lower fH binding than ID 1. When incubated in plasma or blood of nonimmune donors, all three mutants showed similar increases in CFU/ml. In contrast, an isogenic fHbp knockout mutant, which grew well in broth, was rapidly killed in plasma or blood. Thus, although fHbp expression was required for survival of strain H44/76 in blood or plasma, expression of two natural fHbp sequence variants with lower fH affinity had minimal or no effect on nonimmune clearance. One reason may be the high fH concentrations in normal serum, which favor saturation of fH binding to fHbp, even when dissociation rates varied over 10-fold.

  10. Effects of Supplemental Calcium or Calcium-binding Agents on Staphylococcal Bacteriophage Proliferation in Skim Milk.

    PubMed

    Das, N K; Marshall, R T

    1967-11-01

    Additions of 0.0005 N calcium borogluconate to Trypticase Soy Broth (TSB) produced an increase in phage titer about 1 million-fold, whereas its addition to skim milk resulted in about a 100-fold decrease in the maximal titer. Supplemental calcium had a stimulatory influence on bacterial growth in TSB but not in skim milk. Studies were made of the effect of binding of calcium of skim milk on the proliferation of staphylococcal bacteriophage. Sequestering the calcium with 2% phosphate mixture inactivated the phages without affecting the bacterial growth. However, chelation of calcium by 0.012% ethylenediaminetetraacetic acid produced an inhibitory effect on both the phages and the bacteria.

  11. 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.

  12. Effects of some beta lactam antibiotics on (/sup 3/H)-methyl-yohimbine binding to intact human platelets

    SciTech Connect

    Borst, S.E.; Hui, K.K.; Conolly, M.E.

    1985-05-01

    Several antibiotics have been reported to cause a bleeding diathesis in man, characterized by reduced platelet aggregation. The authors investigated the effects of several of the penicillins and of moxalactam on the binding of (/sup 3/H)-methyl-yohimbine to intact human platelets. The (/sup 3/H)-methyl-yohimbine binding met the criteria for interaction at an alpha2 adrenergic binding site and showed low interindividual variability. Penicillin G, ticarcillin, carbenicillin, piperacillin and moxalactam all inhibited (/sup 3/H)-methyl-yohimbine binding, but at concentrations far in excess of clinically achievable plasma levels. They conclude that these compounds exert their antiplatelet effects by a mechanism other than competitive inhibition of catecholamine binding.

  13. Cellular effects of phosphotyrosine-binding domain inhibitors on insulin receptor signaling and trafficking.

    PubMed Central

    Giorgetti-Peraldi, S; Ottinger, E; Wolf, G; Ye, B; Burke, T R; Shoelson, S E

    1997-01-01

    Shc and insulin receptor substrate 1 (IRS-1) are cytoplasmic substrates of tyrosine kinase receptors that engage, localize, and activate downstream SH2 enzymes. Each contains a phosphotyrosine-binding (PTB) domain that is structurally unrelated to SH2 domains. We have designed high-affinity, cellular inhibitors of the Shc PTB domain by incorporating nonnatural, phosphatase-resistant amino acids into short peptides. None of the inhibitors bind the IRS-1 PTB domain, consistent with distinct specificities for domains. The best inhibitor of the Shc domain was introduced by electroporation into Rat1 fibroblasts that express human insulin receptors. Insulin-stimulated phosphorylation of Shc was inhibited, with no effect on IRS-1, and downstream effects on mitogen-activated protein kinase and DNA synthesis were both inhibited. The PTB domain inhibitor had less influence on epidermal growth factor-induced effects and essentially no impact on serum- or phorbol ester-induced effects. The inhibitor did not affect insulin internalization and its degradation. We conclude that the PTB domain of Shc is critical for its phosphorylation by the insulin receptor, that Shc is an important mediator of insulin's mitogenic effects, and that Shc is not central to insulin receptor cycling in these cells. PTB domains can be inhibited selectively in cells and represent potential targets for drug discovery. PMID:9032245

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

    PubMed

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

    2013-08-14

    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. PMID:23947845

  15. 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

  16. Charging effect in Au nanoparticle memory device with biomolecule binding mechanism.

    PubMed

    Jung, Sung Mok; Kim, Hyung-Jun; Kim, Bong-Jin; Yoon, Tae-Sik; Kim, Yong-Sang; Lee, Hyun Ho

    2011-07-01

    Organic memory device having gold nanoparticle (Au NPs) has been introduced in the structure of metal-pentacene-insulator-silicon (MPIS) capacitor device, where the Au NPs layer was formed by a new bonding method. Biomolecule binding mechanism between streptavidin and biotin was used as a strong binding method for the formation of monolayered Au NPs on polymeric dielectric of poly vinyl alcohol (PVA). The self-assembled Au NPs was functioned to show storages of charge in the MPIS device. The binding by streptavidin and biotin was confirmed by AFM and UV-VIS. The UV-VIS absorption of the Au NPs was varied at 515 nm and 525 nm depending on the coating of streptavidin. The AFM image showed no formation of multi-stacked layers of the streptavidin-capped Au NPs on biotin-NHS layer. Capacitance-voltage (C-V) performance of the memory device was measured to investigate the charging effect from Au NPs. In addition, charge retention by the Au NPs storage was tested to show 10,000 s in the C-V curve.

  17. Autoradiographical imaging of PPARgamma agonist effects on PBR/TSPO binding in TASTPM mice.

    PubMed

    Roberts, Jennifer C; Friel, Sarah L; Roman, Shilina; Perren, Marion; Harper, Alex; Davis, John B; Richardson, Jill C; Virley, David; Medhurst, Andrew D

    2009-04-01

    Chronic inflammation is known to occur in the brains of Alzheimer's Disease (AD) patients, including the presence of activated microglia close to amyloid plaques. We utilised real time autoradiography and immunohistochemistry to investigate microglial activation and the potential anti-inflammatory effects of PPARgamma agonists in the Thy-1 APP695swe/Thy-1 PS-1.M146V (TASTPM) overexpressing transgenic mouse model of AD. An age dependent increase in specific [3H](R)-PK11195 binding to peripheral benzodiazepine receptors (PBR)/translocator protein (18 kDa) (TSPO) was observed in the cortex of TASTPM mice compared to wild type mice, indicative of microglial activation. This was consistent with immunohistochemical data showing age-dependent increases in CD68 immunoreactivity co-localised with amyloid beta (Abeta) deposits. In 10 month old TASTPM mice, pioglitazone (20 mg/kg) and ciglitazone (50 mg/kg) significantly reduced [3H](R)-PK11195 and [3H]DPA-713 binding in cortex and hippocampus, indicative of reduced microglial activation. In AD brain, significant [3H](R)-PK11195 and [3H]DPA-713 binding was observed across all stages of the disease. These results support the use of PBR/TSPO autoradiography in TASTPM mice as a functional readout of microglial activation to assess anti-inflammatory drugs prior to evaluation in AD patients.

  18. Modification of position-effect variegation by competition for binding to Drosophila satellites.

    PubMed

    Monod, Caroline; Aulner, Nathalie; Cuvier, Olivier; Käs, Emmanuel

    2002-08-01

    White-mottled (w(m4)) position-effect variegation (PEV) arises by translocation of the white gene near the pericentric AT-rich 1.688 g/cm3 satellite III (SATIII) repeats of the X chromosome of Drosophila. The natural and artificial A*T-hook proteins D1 and MATH20 modify w(m4) PEV in opposite ways. D1 binds SATIII repeats and enhances PEV, presumably via a recruitment of protein partners, whereas MATH20 suppresses it. We show that D1 and MATH20 compete for binding to identical sites of SATIII repeats in vitro and that conditional MATH20 expression results in a displacement of D1 from pericentric heterochromatin in vivo. In the presence of intermediate levels of MATH20, we show that this displacement becomes selective for SATIII repeats. These results strongly suggest that the suppression of w(m4) PEV by MATH20 is due to a displacement of D1 from its preferred binding sites and provide additional support for a direct role of D1 in the assembly of AT-rich heterochromatin.

  19. 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.

  20. EF-hand Ca 2+-binding bioluminescent proteins: effects of mutations and alternative divalent cations

    NASA Astrophysics Data System (ADS)

    Rowe, Laura; Ensor, Mark; Daunert, Sylvia

    2007-02-01

    Bioluminescent photoproteins, such as aequorin and obelin, are proteins that emit light upon binding calcium. Aequorin and obelin contain four EF-hand domains arranged into a globular structure. The loop region of these EF-hand domains binds calcium by coordinating it in a pentagonal bipyramidal structure with oxygen atoms. The binding of calcium to these EF-hands causes a slight conformational change in the protein, which leads to the oxidation of the internally sequestered chromophore, coelenterazine, producing coelenteramide and CO II. The excited coelenteramide then relaxes radiatively, emitting bioluminescence at 471 nm in aequorin or 491 nm in obelin. Although calcium is the traditional, and generally the most powerful, triggering ligand in this bioluminescence reaction, alternative di- and trivalent cations can also bind to the EF-hand loops and stimulate luminescence. Species capable of this cross-reactivity include: Cd 2+, Ba 2+, Mn 2+, Sr 2+, Mg 2+, and several lanthanides. Magnesium is also known to modulate the bioluminescence of wild-type aequorin, increase its stability, and decrease its aggregation tendency. Both wild-type aequorin and wild-type obelin contain several cysteine residues, aequorin has three and obelin has five. It is believed that these cysteine residues play an important, but as of yet unknown, role in the bioluminescence of these proteins, since mutating most of these residues causes significant loss in bioluminescent activity. In order to explore whether or not these cysteine residues contributed to the specificity of the EF-hand domains for cations we generated four aequorin and obelin mutants and observed their luminescent intensity and decay kinetics by stimulation with calcium, barium, and magnesium. It was found that the cysteine mutations do appear to alter the effects that alternative divalent cations have on the bioluminescence of both aequorin and obelin.

  1. 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.

  2. Asynchronous ligand binding and proton release in a root effect hemoglobin.

    PubMed

    Saffran, W A; Gibson, Q H

    1981-05-10

    CO binding to the Root effect hemoglobin of menhaden, Brevoortia tyrannus, has been studied by flash photolysis and equilibrium measurements in [bis(2-hydroxyethyl)amino]Tris(hydroxymethyl)methane and Tris buffers, containing 0.2 M NaCl, between pH 6.0 and 8.0. The equilibrium and kinetic data were analyzed according to the two-state model, extended to include chain differences. The calculated value of the allosteric constant, L, varied from 3 X 10(6) at pH 6.0 to 20 at pH 8.0, lower at each pH value than that computed for phosphate buffer. In addition, the intrinsic rate constants of both T and R states were found to vary with pH. The kinetics of CO binding and of proton release, followed by absorbance changes in the pH indicator dye phenol red, were observed in 0.2 M NaCl, at pH values ranging from 6.3 to 7.8. Proton release lags behind CO binding across this pH range, the larger lags occurring at lower pH; this suggests that some proton release is associated with quaternary conformational change. The CO binding progress curves in unbuffered solution were simulated by the two-state model; in these calculations the value of L was systematically changed during the course of the reaction. The time courses of reaction intermediates, obtained from these computations, were then used to represent the kinetics of proton release. A simple model, assuming that proton release accompanies quaternary conformational transition but a modified model, incorporating pH dependence of the intrinsic T and R state affinities, describes proton release across the pH range studied. PMID:7217097

  3. 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.

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

    PubMed

    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. PMID:26374001

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

    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.

  6. CCCTC-binding Factor Mediates Effects of Glucose On Beta Cell Survival

    PubMed Central

    Tsui, Shanli; Dai, Wei; Lu, Luo

    2013-01-01

    Objectives Pancreatic islet β-cell survival is important in regulating insulin activities and maintaining glucose homeostasis. Recently, Pax6 has been shown to be essential for many vital functions in β-cells, though the molecular mechanisms of its regulation in β-cells remain unclear. The present study investigates the novel effects of glucose- and insulin-induced CTCF activity on Pax6 gene expression as well as the subsequent effects of insulin-activated signaling pathways on β-cell proliferation. Material and methods Pancreatic β-TC-1-6 cells were cultured in DMEM medium and stimulated with high concentrations of glucose (5 to 125 mM) and cell viability was assessed by MTT assays. The effect of CTCF on Pax6 was evaluated in high glucose-induced and CCCTC-binding Factor (CTCF)/Erk suppressed cells by promoter reporter and Western analyses. Results Increases in glucose and insulin concentrations up-regulated CTCF and consequently down-regulated Pax6 in β-cell survival and proliferation. Knocking-down CTCF directly affected Pax6 transcription through CTCF binding and blocked the response to glucose. Altered Erk activity mediated the effects of CTCF on controlling Pax6 expression, which partially regulates β-cell proliferation. Conclusions CTCF functions as a molecular mediator between insulin-induced upstream Erk signaling and Pax6 expression in pancreatic β-cells. This pathway may contribute to regulation of β-cell survival and proliferation. PMID:24354619

  7. Co-amoxiclav Effects on the Structural and Binding Properties of Human Serum Albumin

    PubMed Central

    Hesami Takallu, Saeed; Rezaei Tavirani, Mostafa; Kalantari, Shiva; Amir Bakhtiarvand, Mahrooz; Mahdavi, Sayed Mohammad

    2010-01-01

    Human serum albumin (HSA) is the most abundant plasma protein in the human body. HSA plays an important role in drug transport and metabolism. This protein has a high affinity to a very wide range of materials, including metals such as Cu2+ and Zn2+, fatty acids, amino acids and metabolites such as bilirubin and many drug compounds. In this study, we investigated the effects of co-amoxiclav, as a drug which could be carried by this protein, on HSA structure and binding properties via spectroscopy and electrochemistry techniques. Based on this study, it was found that a therapeutic dose of co-amoxiclav as well as doses 4 to 8 folds higher than the therapeutic dose has no considerable effect on the HSA tertiary structure at 37oC. However, a dose 2 folds that of the therapeutic dose has a slight effect, but higher doses of the drug has a mild effect in pathological temperature (42oC). In addition, charge density of HSA surface is decreased at 42oC, compared to 37oC. Hence, this finding suggests a reduced role of HSA in regulation of osmotic pressure in the fever conditions, compared to the physiological conditions. Co-amoxiclav reduces the charge surface density of HSA at physiological and pathological temperatures and therefore alters its binding properties, which could be important in drug interference and complications. PMID:24363734

  8. A circulating IgG in Chagas' disease which binds to beta-adrenoceptors of myocardium and modulates their activity.

    PubMed Central

    Borda, E; Pascual, J; Cossio, P; De La Vega, M; Arana, R; Sterin-Borda, L

    1984-01-01

    It has been shown that sera from chagasic patients with positive EVI serology could act in co-operation with complement or normal human lymphocytes as a partial beta-adrenoceptor agonist increasing the contractile tension and frequency of isolated rat atria, as occurs with IgG purified from chagasic serum. In this paper we demonstrated that IgG present in chagasic patients sera could bind to the beta-adrenoceptors of the heart and stimulate contractile activity of myocardium. The positive inotropic and chronotropic effect could be blocked by the specific beta 1-adrenoceptor antagonist but not by the beta 2-adrenoceptor antagonist. Chagasic IgG inhibited the binding of (-) 3H-DHA to beta-adrenoceptors of purified rat myocardial membranes behaving as non-competitive inhibitors. The reactivity of chagasic serum or IgG with beta 1-adrenoceptor was lost after absorptions with turkey red blood cells. In contrast, guinea-pig red blood cells were unable to remove the beta 1 reactivity of chagasic serum or chagasic IgG. This supports the specificity of beta 1-adrenoceptors of the chagasic IgG and the independence of beta 1-adrenoceptor reactivity in relation to the EVI system. Clinical specificity of the beta 1-adrenoceptor reactivity seems rather high in Chagas' disease since it was lacking in 14 individuals with other cardiopathies, such as ischaemic and rheumatic heart disease, even after heart surgery. PMID:6088139

  9. The Effect of Cigarette Smoke Exposure on Developing Folate Binding Protein-2 Null Mice

    PubMed Central

    Horn, Kristin H.; Esposito, Emily R.; Greene, Robert M.; Pisano, M. Michele

    2008-01-01

    Environmental tobacco smoke exposures have been linked to adverse health effects. Folate is essential for normal development, with deficiencies often causing fetal growth restriction. Mice lacking the folate binding protein-2 receptor (Folr2) exhibit increased susceptibility to teratogens. The purpose of the current study was to determine if the loss of Folr2 would increase sensitivity to cigarette smoke-induced effects on development. Pregnant Folr2−/−, Folr2+/+, and C57BL/6J mice were exposed to sidestream cigarette smoke during gestation. Exposure to sidestream smoke on gd 6–9 had no adverse effects on fetal outcomes. However, cigarette smoke exposure on gd 6–18 increased the number of fetal resorptions (Folr2−/− cohort) and decreased crown-rump length (Folr2+/+ fetuses). These data confirm an association between sidestream smoke exposure and fetal growth restriction, but do not suggest that loss of Folr2 increased susceptibility to these effects. PMID:18992323

  10. 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.

  11. 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

  12. 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.

  13. 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. PMID:27247422

  14. The effect of oxime reactivators on muscarinic receptors: functional and binding examinations.

    PubMed

    Soukup, O; Kumar, U K; Proska, J; Bratova, L; Adem, A; Jun, D; Fusek, J; Kuca, K; Tobin, G

    2011-05-01

    The antidotal treatment of organophosphorus poisoning is still a problematic issue since no versatile antidote has been developed yet. In our study, we focused on an interesting property, which does not relate to the reactivation of inhibited acetylcholinesterase (AChE) of some oximes, but refers to their anti-muscarinic effects which may contribute considerably to their treatment efficacy. One standard reactivator (HI-6) and two new compounds (K027 and K203) have been investigated for their antimuscarinic properties. Anti-muscarinic effects were studies by means of an in vitro stimulated atrium preparation (functional test), the [(3)H]-QNB binding assay and G-protein coupled receptor assay (GPCR, beta-Arrestin Assay). Based on the functional data HI-6 demonstrates the highest anti-muscarinic effect. However, only when comparing [(3)H]-QNB binding results and GPCR data, K203 shows a very promising compound with regard to anti-muscarinic potency. The therapeutic impact of these findings has been discussed.

  15. 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

  16. 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

    2006-02-01

    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

  17. 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.

  18. From Binding-Induced Dynamic Effects in SH3 Structures to Evolutionary Conserved Sectors

    PubMed Central

    Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic

    2016-01-01

    Src Homology 3 domains are ubiquitous small interaction modules known to act as docking sites and regulatory elements in a wide range of proteins. Prior experimental NMR work on the SH3 domain of Src showed that ligand binding induces long-range dynamic changes consistent with an induced fit mechanism. The identification of the residues that participate in this mechanism produces a chart that allows for the exploration of the regulatory role of such domains in the activity of the encompassing protein. Here we show that a computational approach focusing on the changes in side chain dynamics through ligand binding identifies equivalent long-range effects in the Src SH3 domain. Mutation of a subset of the predicted residues elicits long-range effects on the binding energetics, emphasizing the relevance of these positions in the definition of intramolecular cooperative networks of signal transduction in this domain. We find further support for this mechanism through the analysis of seven other publically available SH3 domain structures of which the sequences represent diverse SH3 classes. By comparing the eight predictions, we find that, in addition to a dynamic pathway that is relatively conserved throughout all SH3 domains, there are dynamic aspects specific to each domain and homologous subgroups. Our work shows for the first time from a structural perspective, which transduction mechanisms are common between a subset of closely related and distal SH3 domains, while at the same time highlighting the differences in signal transduction that make each family member unique. These results resolve the missing link between structural predictions of dynamic changes and the domain sectors recently identified for SH3 domains through sequence analysis. PMID:27213566

  19. Pyrrolic tripodal receptors effectively recognizing monosaccharides. Affinity assessment through a generalized binding descriptor.

    PubMed

    Nativi, Cristina; Cacciarini, Martina; Francesconi, Oscar; Vacca, Alberto; Moneti, Gloriano; Ienco, Andrea; Roelens, Stefano

    2007-04-11

    Pyrrolic and imino (3) or amino (4) H-bonding ligands were incorporated into a benzene-based tripodal scaffold to develop a new generation of receptors for molecular recognition of carbohydrates. Receptors 3 and 4 effectively bound a set of octylglycosides of biologically relevant monosaccharides, including glucose (Glc), galactose (Gal), mannose (Man), and N-acetyl-glucosamine (GlcNAc), showing micromolar affinities in CDCl3 and millimolar affinities in CD3CN by NMR titrations. Both receptors selectively recognized Glc among the investigated monosaccharides, with 3 generally less effective than 4 but showing selectivities for the all-equatorial beta-glycosides of Glc and GlcNAc among the largest reported for H-bonding synthetic receptors. Selectivities in CDCl3 spanned a range of nearly 250-fold for 3 and over 30-fold for 4. Affinities and selectivities were univocally assessed through the BC50 descriptor, for which a generalized treatment is described that extends the scope of the descriptor to include any two-reagent host-guest system featuring any number of binding constants. ITC titrations of betaGlc in acetonitrile evidenced, for both receptors, a strong enthalpic contribution to the binding interaction, suggesting multiple H bonding. Selectivity trends toward alphaGlc and betaGlc analogous to those obtained in solution were also observed in the gas phase for 3 and 4 by collision-induced dissociation experiments. From comparison with appropriate reference compounds, a substantial contribution to carbohydrate binding emerged for both the imino/amino and the pyrrolic H-bonding groups but not for the amidic group. This previously undocumented behavior, supported by crystallographic evidence, has been discussed in terms of geometric, functional, and coordinative complementarity between H-bonding groups and glycosidic hydroxyls and opens the way to a new designer strategy of H-bonding receptors for carbohydrates.

  20. Neural mechanisms of context effects on face recognition: automatic binding and context shift decrements.

    PubMed

    Hayes, Scott M; Baena, Elsa; Truong, Trong-Kha; Cabeza, Roberto

    2010-11-01

    Although people do not normally try to remember associations between faces and physical contexts, these associations are established automatically, as indicated by the difficulty of recognizing familiar faces in different contexts ("butcher-on-the-bus" phenomenon). The present fMRI study investigated the automatic binding of faces and scenes. In the face-face (F-F) condition, faces were presented alone during both encoding and retrieval, whereas in the face/scene-face (FS-F) condition, they were presented overlaid on scenes during encoding but alone during retrieval (context change). Although participants were instructed to focus only on the faces during both encoding and retrieval, recognition performance was worse in the FS-F than in the F-F condition ("context shift decrement" [CSD]), confirming automatic face-scene binding during encoding. This binding was mediated by the hippocampus as indicated by greater subsequent memory effects (remembered > forgotten) in this region for the FS-F than the F-F condition. Scene memory was mediated by right parahippocampal cortex, which was reactivated during successful retrieval when the faces were associated with a scene during encoding (FS-F condition). Analyses using the CSD as a regressor yielded a clear hemispheric asymmetry in medial temporal lobe activity during encoding: Left hippocampal and parahippocampal activity was associated with a smaller CSD, indicating more flexible memory representations immune to context changes, whereas right hippocampal/rhinal activity was associated with a larger CSD, indicating less flexible representations sensitive to context change. Taken together, the results clarify the neural mechanisms of context effects on face recognition.

  1. 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.

  2. 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.

  3. Enhancing ligand-protein binding in affinity thermoprecipitation: elucidation of spacer effects

    PubMed

    Vaidya; Lele; Kulkarni; Mashelkar

    1999-08-20

    Copolymers of N-isopropylacrylamide and N-acryloyl amino acid spacers of varying chain length were synthesized. p-Aminobenzamidine (PABA) was chemically linked to the pendant carboxyl groups of these polymers to obtain thermoprecipitating affinity polymers. The inhibition constant (Ki) of these polymers for trypsin decreased, i. e., the efficiency of PABA-trypsin binding increased with increase in the spacer chain length. The polymer to which PABA was linked through a spacer of five methylene groups exhibited eleven times lower Ki than that of the polymer containing PABA without a spacer. Investigations on model inhibitors N-acyl-p-aminobenzamidines showed that this enhancement in trypsin binding by the polymers was due to the spacer as well as to microenvironmental effects. Recovery and specific activity of the trypsin recovered increased with the spacer chain length. Separation of trypsin from a mixture of trypsin and chymotrypsin was also enhanced with the spacer chain length. The inhibition constants of these affinity polymers were not adversely affected by the crowding effect. Copyright 1999 John Wiley & Sons, Inc. PMID:10397880

  4. Neurotransmitters, neuropeptides and binding sites in the rat mediobasal hypothalamus: effects of monosodium glutamate (MSG) lesions.

    PubMed

    Meister, B; Ceccatelli, S; Hökfelt, T; Andén, N E; Andén, M; Theodorsson, E

    1989-01-01

    Indirect immunofluorescence histochemistry and receptor autoradiography were used to study the localization of transmitter-/peptide-containing neurons and peptide binding sites in the mediobasal hypothalamus in normal rats and in rats treated neonatally with repeated doses of the neurotoxin monosodium-glutamate (MSG). In the arcuate nucleus, the results showed a virtually complete loss of cell bodies containing immunoreactivity for growth hormone-releasing factor (GRF), galanin (GAL), dynorphin (DYN), enkephalin (ENK), corticotropin-like intermediate peptide (CLIP), neuropeptide Y (NPY), and neuropeptide K (NPK). Tyrosine hydroxylase(TH)-glutamic acid decarboxylase(GAD)-, neurotensin(NT)- and somatostatin(SOM)-immunoreactive (IR) cells were, however, always detected in the ventrally dislocated, dorsomedial division of the arcuate nucleus. In the median eminence, marked decreases in numbers of GAD-, NT-, GAL-, GRF-, DYN-, and ENK-IR fibers were observed. The numbers of TH-, SOM- and NPY-IR fibers were in contrast not or only affected to a very small extent, as revealed with the immunofluorescence technique. Biochemical analysis showed a tendency for MSG to reduce dopamine levels in the median eminence of female rats, whereas no effect was observed in male rats. Autoradiographic studies showed high to moderate NT binding sites, including strong binding over presumably dorsomedial dopamine cells. In MSG-treated rats, there was a marked reduction in GAL binding in the ventromedial nucleus. The findings implicate that most neurons in the ventrolateral and ventromedial arcuate nucleus are sensitive to the toxic effects of MSG, whereas a subpopulation of cells in the dorsomedial division of the arcuate nucleus, including dopamine neurons, are not susceptible to MSG-neurotoxicity. The results indicate, moreover that the very dense TH-IR fiber network in the median eminence predominantly arises from the dorsomedial TH-IR arcuate cells, whereas the GAD-, NT-, GAL-, GRF- and

  5. Ion-pair binding: is binding both binding better?

    PubMed

    Roelens, Stefano; Vacca, Alberto; Francesconi, Oscar; Venturi, Chiara

    2009-08-17

    It is often tempting to explain chemical phenomena on the basis of intuitive principles, but this practice can frequently lead to biased analysis of data and incorrect conclusions. One such intuitive principle is brought into play in the binding of salts by synthetic receptors. Following the heuristic concept that "binding both is binding better", it is widely believed that ditopic receptors capable of binding both ionic partners of a salt are more effective than monotopic receptors because of a cooperative effect. Using a newly designed ditopic receptor and a generalized binding descriptor, we show here that, when the problem is correctly formulated and the appropriate algorithm is derived, the cooperativity principle is neither general nor predictable, and that competition between ion binding and ion pairing may even lead to inhibition rather than enhancement of the binding of an ion to a ditopic receptor.

  6. 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.

  7. Inhibitory Effect of Bridged Nucleosides on Thermus aquaticus DNA Polymerase and Insight into the Binding Interactions

    PubMed Central

    Kim, Sung-Kun; Castro, Aaron; Kim, Edward S.; Dinkel, Austin P.; Liu, Xiaoyun; Castro, Miguel

    2016-01-01

    Modified nucleosides have the potential to inhibit DNA polymerases for the treatment of viral infections and cancer. With the hope of developing potent drug candidates by the modification of the 2’,4’-position of the ribose with the inclusion of a bridge, efforts were focused on the inhibition of Taq DNA polymerase using quantitative real time PCR, and the results revealed the significant inhibitory effects of 2’,4’-bridged thymidine nucleoside on the polymerase. Study on the mode of inhibition revealed the competitive mechanism with which the 2’,4’-bridged thymidine operates. With a Ki value of 9.7 ± 1.1 μM, the 2’,4’-bridged thymidine proved to be a very promising inhibitor. Additionally, docking analysis showed that all the nucleosides including 2’,4’-bridged thymidine were able to dock in the active site, indicating that the substrate analogs reflect a structural complementarity to the enzyme active site. The analysis also provided evidence that Asp610 was a key binding site for 2’,4’-bridged thymidine. Molecular dynamics (MD) simulations were performed to further understand the conformational variations of the binding. The root-mean-square deviation (RMSD) values for the peptide backbone of the enzyme and the nitrogenous base of the inhibitor stabilized within 0.8 and 0.2 ns, respectively. Furthermore, the MD analysis indicates substantial conformational change in the ligand (inhibitor) as the nitrogenous base rotated anticlockwise with respect to the sugar moiety, complemented by the formation of several new hydrogen bonds where Arg587 served as a pivot axis for binding formation. In conclusion, the active site inhibition of Taq DNA polymerase by 2’,4’-bridged thymidine suggests the potential of bridged nucleosides as drug candidates. PMID:26820310

  8. 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

  9. Opposite effects on human colon cancer cell proliferation of two dietary Thomsen-Friedenreich antigen-binding lectins.

    PubMed

    Yu, L G; Milton, J D; Fernig, D G; Rhodes, J M

    2001-02-01

    Increased cell surface expression of the Thomsen-Friedenreich antigen (TF antigen, Galbeta1-3GalNAcalpha-) is a common feature in malignant and pre-malignant epithelia. Our previous studies have shown that dietary TF-binding lectins from peanut (Arachis hypogea) and edible mushroom (Agaricus bisporus) produce marked but different effects on human intestinal epithelial cell proliferation. This study investigates the proliferative effects of the other two known dietary TF-binding lectins: jacalin (Artocarpus integrifolia, JAC) and amaranth lectin (Amaranthus caudatus, ACA). JAC produced dose-dependent and non-cytotoxic inhibition of proliferation in HT29 human colon cancer cells with maximal effects of 46 +/- 4% at 20 microg/ml, whereas ACA produced dose-dependent stimulation of proliferation with maximal effects of 22 +/- 3% at 20 microg/ml when assessed both by incorporation of [3H]thymidine into DNA and by cell counting. The lectin-mediated effects were inhibitable by the presence of appropriate Galbeta1-3GalNAc-expressing glycoproteins but differences existed between JAC and ACA in their patterns of inhibition by such substances. Ligand binding equilibrium studies using iodinated lectins revealed different Kd of the two lectins for HT29 cell surface glycoproteins. Lectin blots of cell membrane extracts showed different binding patterns in all the four TF-binding lectins. These results provide further evidence that dietary TF-binding lectins can have marked effects on the proliferation of human malignant gastro-intestinal epithelial cells and hence may play a role in intestinal cancer development, and also show that the biological effects of dietary lectins cannot be predicted solely from their carbohydrate binding properties.

  10. The effects of sex hormone binding globulin (SHBG) on testosterone transport into the cerebrospinal fluid.

    PubMed

    Hobbs, C J; Jones, R E; Plymate, S R

    1992-07-01

    The movement of testosterone (T) from blood across the blood-brain barrier (BBB) is thought to reflect the combined effects of T's lipid solubility and the presence of circulating binding proteins for T such as albumin or sex hormone binding globulin (SHBG). Since the adult rat lacks a circulating specific high affinity sex steroid binding protein, examination of the disappearance from serum and uptake into cerebrospinal fluid (CSF) of [3H]T before and after SHBG or albumin infusion should provide insight into the function of these two proteins with respect T transport. Three groups of adult male Sprague-Dawley rats were cannulated at the femoral vein and cisterna magna. In a control group (n = 8), [3H]T was given as an intravenous bolus beginning at time zero; multiple serum and CSF collections were assayed for counts per min (cpm) during the subsequent 45 min. Data from these animals were then compared to those seen in animals that received either purified human SHBG (hSHBG) (n = 7) or human albumin (hALB) (n = 6) 10 min prior to the [3H]T infusion. High performance liquid chromatography was used to monitor the metabolic fate of the steroid infusate at the end of each study period. Infusion of hSHBG increased serum concentrations from undetectable to 93.8 nM/l (mean +/- SEM, n = 6). Administration of hALB significantly increased (25.0 +/- 1.2 g/l at baseline, 33.4 +/- 1.6 g/l post-infusion, mean +/- SEM, P less than 0.03, n = 5) the circulating albumin concentration. Comparison of data from each group of animals demonstrated that (1) following an i.v. injection of radiolabeled T, the initial decline in serum [3H]T was significantly reduced (P less than 0.03) in the presence of hSHBG, (2) hALB did not affect the movement of [3H]T out of serum, (3) the time to peak appearance of [3H]T in the CSF was significantly delayed (P less than 0.02) by the presence of circulating hSHBG, and (4) the net quantity of [3H]T found in the cSF under steady-state conditions was not

  11. Effect of counterion binding efficiency on structure and dynamics of wormlike micelles.

    PubMed

    Oelschlaeger, C; Suwita, P; Willenbacher, N

    2010-05-18

    We have studied the effect of counterion binding efficiency on the linear viscoelastic properties of wormlike micelles formed from hexadecyltrimethylammonium bromide (CTAB) in the presence of different nonpenetrating inorganic salts: potassium bromide (KBr), sodium nitrate (NaNO(3)), and sodium chlorate (NaClO(3)). We have varied the salt/surfactant ratio R at fixed surfactant concentration of 350 mM. Results are compared to data for the system cetylpyridinium chloride (CPyCl) and the penetrating counterion sodium salicylate (NaSal) (Oelschlaeger, C.; Schopferer, M.; Scheffold, F.; Willenbacher, N. Langmuir 2009, 25, 716-723). Mechanical high-frequency rheology and diffusing wave spectroscopy (DWS) based tracer microrheology are used to determine the shear moduli G' and G'' in the frequency range from 0.1 Hz up to 1 MHz (Willenbacher, N.; Oelschlaeger, C.; Schopferer, M.; Fischer, P.; Cardinaux, F.; Scheffold, F. Phys. Rev. Lett. 2007, 99, 068302, 1-4). This enables us to determine the plateau modulus G(0), which is related to the cross-link density or mesh size of the entanglement network, the bending stiffness kappa (also expressed as persistence length l(p) = kappa/k(B)T) corresponding to the semiflexible nature of the micelles, and the scission energy E(sciss), which is related to their contour length. The viscosity maximum shifts to higher R values, and the variation of viscosity with R is less pronounced as the binding strength decreases. The plateau modulus increases with R at low ionic strength and is constant around the viscosity maximum; the increase in G(0) at high R, which is presumably due to branching, is weak compared to the system with penetrating counterion. The scission energy E(sciss) approximately = 20 k(B)T is independent of counterion binding efficiency irrespective of R and is slightly higher compared to the system CPyCl/NaSal, indicating that branching may be significant already at the viscosity maximum in this latter case. The micellar

  12. 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.

  13. Substituent Effects on the Binding of Halides by Neutral and Dicationic Bis(triazolium) Receptors.

    PubMed

    Nepal, Binod; Scheiner, Steve

    2015-12-31

    The effects of substituent and overall charge upon the binding of a halide anion by a bis(triazolium) receptor are studied by M06-2X DFT calculations, with the aug-cc-pVDZ basis set. Comparison is also made between a receptor that engages in H-bonds, with a halogen-bonding species. Fluoride is clearly most strongly bound, followed by Cl(-), Br(-), and I(-) in that order. The dicationic receptor engages in stronger complexes, but not by a very wide margin compared to its neutral counterpart. The binding is enhanced as the substituent on the two triazolium rings becomes progressively more electron-withdrawing. Halogen-substituted receptors, whether neutral or cationic, display a greater sensitivity to substituent than do their H-bonding counterparts. Both Coulombic and charge transfer factors obey the latter trends but do not correctly reproduce the stronger halogen vs hydrogen bonding. Both H-bonds and halogen bonds are nearly linear within the complexes, due in part to bond rotations within the receptor that bring the two triazole rings closer to coplanarity with the central benzene ring. PMID:26645536

  14. 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.

  15. The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes.

    PubMed

    Nomura, Kaoru; Corzo, Gerardo

    2006-09-01

    Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and (31)P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (DeltaH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were -18.1 kcal/mol and -15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K(p)) was found to be 3.9x10(3) M(-1). (31)P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. (31)P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, (31)P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.

  16. Nonphosphate-binding effects of sevelamer--are they of clinical relevance?

    PubMed

    Marangon, Nicola; Lindholm, Bengt; Stenvinkel, Peter

    2008-01-01

    Sevelamer is an ion-exchanging resin that binds phosphate in the gut. Because it does so without increasing the calcium load, treatment with sevelamer may lead to less vascular calcification and better survival in chronic kidney disease patients. However, the results of available clinical studies have not been consistent; recent observations challenge the hypothesis that the extra calcium load inherent in calcium-based phosphate binder therapy increases cardiovascular mortality by accelerating vascular calcification. This reemphasizes the fact that we still lack detailed understanding on the complex relationships between vascular calcification, bone metabolism, vascular disease and outcome in the context of uremia. Thus, the role of phosphate binders may be more complex than initially anticipated and not limited to the extra calcium load. Even if detailed mechanisms of action for sevelamer are not yet clearly established (except for its lipid-lowering action), sevelamer may have a number of additional nonphosphate-lowering actions (including lipid lowering as well as improvement in endothelial function, modulation of inflammation and oxidative stress and binding of uremic toxin absorption). Whether these potentially very interesting pleiotropic effects of sevelamer may be translated into significant clinical benefits remains to be established.

  17. Effects of NaCl and sultopride on striatal [(3)H]spiperone binding in neonatal, adult and senescent rats.

    PubMed

    Makihata, J; Nomura, Y

    1984-01-01

    Effects of NaCl, (+)-and (-)-sultopride on striatal [(3)H]spiperone binding was investigated in 7-day, 70-day and 2-year-old rats. The amount of specific [(3)H]spiperone binding was the highest at 70 days and the value at adult stage was significantly (P < 0.001) higher than those at 7 days and 2 years. NaCl (100 mM) significantly increased [(3)H]spiperone binding in neonatal (P < 0.01), adult (P < 0.05) and senescent (P < 0.05) animals. Scatchard analysis showed that the Bmax of low-affinity [(3)H]spiperone binding was significantly elevated by 100 mM NaCl compared to the value in control of adult animals. More potent inhibition of (-)-sultopride for [(3)H]spiperone binding than that of the (+)-enantiomer at adult stage was also observed at neonatal and senescent stages. NaCl (100 mM) significantly enhanced inhibitory activities of (+)- and (-)-sultopride at every stage. It is suggested that stabilizing effect of Na(+) on dopamine (DA) receptor complexes and increasing effect of Na(+) on binding affinity of benzamide to DA2 receptors keep functions through development and aging.

  18. 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).

  19. Ligand interactions with lactose repressor protein and the repressor-operator complex: the effects of ionization and oligomerization on binding.

    PubMed

    Wilson, Corey J; Zhan, Hongli; Swint-Kruse, Liskin; Matthews, Kathleen S

    2007-03-01

    Specific interactions between proteins and ligands that modify their functions are crucial in biology. Here, we examine sugars that bind the lactose repressor protein (LacI) and modify repressor affinity for operator DNA using isothermal titration calorimetry and equilibrium DNA binding experiments. High affinity binding of the commonly-used inducer isopropyl-beta,D-thiogalactoside is strongly driven by enthalpic forces, whereas inducer 2-phenylethyl-beta,D-galactoside has weaker affinity with low enthalpic contributions. Perturbing the dimer interface with either pH or oligomeric state shows that weak inducer binding is sensitive to changes in this distant region. Effects of the neutral compound o-nitrophenyl-beta,D-galactoside are sensitive to oligomerization, and at elevated pH this compound converts to an anti-inducer ligand with slightly enhanced enthalpic contributions to the binding energy. Anti-inducer o-nitrophenyl-beta,D-fucoside exhibits slightly enhanced affinity and increased enthalpic contributions at elevated pH. Collectively, these results both demonstrate the range of energetic consequences that occur with LacI binding to structurally-similar ligands and expand our insight into the link between effector binding and structural changes at the subunit interface. PMID:16860458

  20. 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). PMID:26414950

  1. 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

  2. GM1 Ganglioside in Parkinson’s Disease: Pilot Study of Effects on Dopamine Transporter Binding

    PubMed Central

    Schneider, Jay S.; Cambi, Franca; Gollomp, Stephen M.; Kuwabara, Hiroto; Brašić, James R.; Leiby, Benjamin; Sendek, Stephanie; Wong, Dean F.

    2015-01-01

    Objective GM1 ganglioside has been suggested as a treatment for Parkinson’s disease (PD), potentially having symptomatic and disease modifying effects. The current pilot imaging study was performed to examine effects of GM1 on dopamine transporter binding, as a surrogate measure of disease progression, studied longitudinally. Methods Positron emission tomography (PET) imaging data were obtained from a subset of subjects enrolled in a delayed start clinical trial of GM1 in PD1: 15 Early-start (ES) subjects, 14 Delayed-start (DS) subjects, and 11 Comparison (standard-of-care) subjects. Treatment subjects were studied over a 2.5 year period while Comparison subjects were studied over 2 years. Dynamic PET scans were performed over 90 minutes following injection of [11C]methylphenidate. Regional values of binding potential (BPND) were analyzed for several striatal volumes of interest. Results Clinical results for this subset of subjects were similar to those previously reported for the larger study group. ES subjects showed early symptomatic improvement and slow symptom progression over the study period. DS and Comparison subjects were initially on the same symptom progression trajectory but diverged once DS subjects received GM1 treatment. Imaging results showed significant slowing of BPND loss in several striatal regions in GM1-treated subjects and in some cases, an increased BPND in some striatal regions was detected after GM1 use. Interpretation Results of this pilot imaging study provide additional data to suggest a potential disease modifying effect of GM1 on PD. These results need to be confirmed in a larger number of subjects. PMID:26099170

  3. 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.

  4. Effects of a Variety of Food Extracts and Juices on the Specific Binding Ability of Norovirus GII.4 P Particles

    PubMed Central

    LI, DAN; BAERT, LEEN; XIA, MING; ZHONG, WEIMING; JIANG, XI; UYTTENDAELE, MIEKE

    2014-01-01

    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. PMID:22980024

  5. Calix[4]arene-linked bisporphyrin hosts for fullerenes: binding strength, solvation effects, and porphyrin-fullerene charge transfer bands.

    PubMed

    Hosseini, Ali; Taylor, Steven; Accorsi, Gianluca; Armaroli, Nicola; Reed, Christopher A; Boyd, Peter D W

    2006-12-13

    A calix[4]arene scaffolding has been used to construct bisporphyrin ("jaws" porphyrin) hosts for supramolecular binding of fullerene guests. Fullerene affinities were optimized by varying the nature of the covalent linkage of the porphyrins to the calixarenes. Binding constants for C60 and C70 in toluene were explored as a function of substituents at the periphery of the porphyrin, and 3,5-di-tert-butylphenyl groups gave rise to the highest fullerene affinities (26,000 M(-1) for C60). The origin of this high fullerene affinity has been traced to differential solvation effects rather than to electronic effects. Studies of binding constants as a function of solvent (toluene < benzonitrile < dichloromethane < cyclohexane) correlate inversely with fullerene solubility, indicating that desolvation of the fullerene is a major factor determining the magnitude of binding constants. The energetics of fullerene binding have been determined in terms of DelatH and DeltaS and are consistent with an enthalpy-driven, solvation-dependent process. A direct relationship between supramolecular binding of a fullerene guest to a bisporphyrin host and the appearance of a broad NIR absorption band have been established. The energy of this band moves in a predictable manner as a function of the electronic structure of the porphyrin, thereby establishing its origin in porphyrin-to-fullerene charge transfer.

  6. 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.

  7. Effect of sodium chloride on the binding of polyaromatic hydrocarbon guests with sodium cholate aggregates.

    PubMed

    Fuentealba, Denis; Thurber, Katie; Bovero, Enrico; Pace, Tamara C S; Bohne, Cornelia

    2011-09-01

    Sodium cholate aggregates are adaptable host systems. The effect of changing the ionic strength with the addition of NaCl on the properties for guest binding to sodium cholate aggregates was investigated by using pyrene, perylene and 1-ethylnaphthalene as guests. Fluorescence, anisotropy and laser flash photolysis studies provided information on the protection efficiency of the aggregate bound guest, and provided information on the dynamics and correlation times for the host-guest system. Different trends for the protection efficiency of the bound guests were observed when the NaCl concentration was raised depending on the charge of the aqueous solubilized quencher. The increase in ionic strength was also shown to lengthen the correlation time of the aggregate bound guest and led to faster dynamics for the host-guest complex. These results show that the properties of sodium cholate aggregates as a supramolecular host system are significantly altered with changes in the ionic strength of the medium. PMID:21472184

  8. Effects of carbon doping on the electronic properties of boron nitride nanotubes: Tight binding calculation

    NASA Astrophysics Data System (ADS)

    Chegel, Raad

    2016-10-01

    The electronic properties of pure and carbon doped zigzag and armchair Boron Nitride Nanotubes (BNNTs) have been investigated based on tight binding formalism. It was found that the band gap is reduced due to substitution of Boron or Nitrogen atoms by carbon atoms and the doping effects of B- and N-substituted BNNTs are different. The applied electric field converts the carbon doped BNNTs from semiconductor to metal. The gap energy reduction shows an identical dependence to electric field and doping for both armchair and zigzag carbon doped BNNTs. Our results indicate that the band gap of carbon doped BNNTs is a function of the Impurity concentration, electric field strength and the direction between the electric field and dopant location. The band gap for C-doped BNNTs with four carbon atoms decreases linearly but for two carbon atoms, it is constant at first then decreases linearly.

  9. Effect of the mannose-binding Artocarpus integer lectin on the cellular proliferation of murine lymphocytes.

    PubMed

    Lim, S B; Kanthimathi, M S; Hashim, O H

    1998-12-01

    The effect of the mannose-binding champedak (Artocarpus integer) lectin-M on the cellular proliferation of murine lymphocytes was investigated in this study. Our data demonstrated that the lectin was the main mitogenic component in the crude extract of the champedak seeds. It stimulated the proliferation of murine T cells at an optimal concentration of 2.5 microg/ml in a 3 day culture. Lectin-M appeared to be a T-cell mitogen as it does not induce significant DNA synthesis when cultured with spleen cells from the nude mouse. In the absence of T cells, the lectin was incapable of inducing resting B cells to differentiate into immunoglobulin secreting plasma cells.

  10. 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

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

    PubMed Central

    Brenner, T; Beyth, Y; Abramsky, O

    1980-01-01

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

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

    PubMed Central

    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

  13. 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

  14. 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

  15. Effect of four-alpha-helix bundle cavity size on volatile anesthetic binding energetics.

    PubMed

    Manderson, Gavin A; Michalsky, Stuart J; Johansson, Jonas S

    2003-09-30

    Currently, it is thought that inhalational anesthetics cause anesthesia by binding to ligand-gated ion channels. This is being investigated using four-alpha-helix bundles, small water-soluble analogues of the transmembrane domains of the "natural" receptor proteins. The study presented here specifically investigates how multiple alanine-to-valine substitutions (which each decrease the volume of the internal binding cavity by 38 A(3)) affect structure, stability, and anesthetic binding affinity of the four-alpha-helix bundles. Structure remains essentially unchanged when up to four alanine residues are changed to valine. However, stability increases as the number of these substitutions is increased. Anesthetic binding affinities are also affected. Halothane binds to the four-alpha-helix bundle variants with 0, 1, and 2 substitutions with equivalent affinities but binds to the variants with 3 and 4 more tightly. The same order of binding affinities was observed for chloroform, although for a particular variant, chloroform was bound less tightly. The observed differences in binding affinities may be explained in terms of a modulation of van der Waals and hydrophobic interactions between ligand and receptor. These, in turn, could result from increased four-alpha-helix bundle binding cavity hydrophobicity, a decrease in cavity size, or improved ligand/receptor shape complementarity.

  16. 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

  17. Effects of lectins with different carbohydrate-binding specificities on hepatoma, choriocarcinoma, melanoma and osteosarcoma cell lines.

    PubMed

    Wang, H; Ng, T B; Ooi, V E; Liu, W K

    2000-03-01

    The effects of lectins with different carbohydrate-binding specificities on human hepatoma (H3B), human choriocarcinoma (JAr), mouse melanoma (B16) and rat osteosarcoma (ROS) cell lines were investigated. Cell viability was estimated by uptake of crystal violet. Wheat germ lectin was the lectin with the most deleterious effect on the viability of H3B, JAr and ROS cell lines. The cytotoxicity of lectins with similar sugar-binding specificity to wheat germ lectin, including Maackia amurensis lectin and Solanum tuberosum lectin, was weaker than that of wheat germ lectin. N-acetylgalactosamine-and galactose-binding Tricholoma mongolicum lectin ranked third, after wheat germ lectin and Maackia amurensis lectin, with regard to its effect on H3B, and ranked, together with Maackia amurensis lectin, as the lectins with the second most pronounced effects on ROS. However, the cytotoxic effects of Tricholoma mongolicum lectin on JAr were much weaker than those of Maackia amurensis lectin, Solanum tuberosum lectin and Anguilla anguilla lectin. Artocarpus integrifolia lectin, Lens culinaris lectin and Anguilla anguilla lectin possessed milder cytotoxicity than the remaining lectins. which were approximately equipotent. The mannose-binding Narcissus pseudonarcissus and Lens culinaris lectins were only weakly cytotoxic, the exception being a stronger effect on H3B. The N-acetylgalactosamine-binding Glycine max lectin and methylgalactose-binding Artocarpus integrifolia lectin similarly exhibited low cytotoxicity. It can thus be concluded that in general the ranking was wheat germ lectin > Maackia amurensis lectin approximately Trichloma mongolicum lectins > other aforementioned lectins in cytotoxicity. A particular lectin may manifest more conspicuous toxicity on certain cell lines and less on others.

  18. Protein binding and its potential for eliciting minimal systemic side effects with a novel inhaled corticosteroid, ciclesonide.

    PubMed

    Rohatagi, Shashank; Luo, Yongyi; Shen, Liduo; Guo, Zuyu; Schemm, Christina; Huang, Yongqing; Chen, Kelly; David, Michael; Nave, Ruediger; King, S Peter

    2005-01-01

    Freely circulating, protein unbound, active inhaled corticosteroid (ICS) can cause systemic adverse effects. Desisobutyryl-ciclesonide (des-CIC) is the active metabolite of ciclesonide, an effective, novel ICS for persistent asthma. This study examines the free fraction of ciclesonide and des-CIC and determines whether the presence of other agents or disease states affects protein binding. Protein binding of des-CIC (0.5, 5.0, 25, 100, and 500 ng/mL) was determined, using both equilibrium dialysis and ultrafiltration, in plasma from humans (healthy and either renally or hepatically impaired) and several animal species and in the presence of either salicylates or warfarin. Dialyzed samples were analyzed by liquid chromatography with tandem mass spectroscopy to determine both free and bound concentrations of des-CIC. After ultrafiltration, spiked plasma plus H-des-CIC was separated into free and bound fractions by centrifugation and quantified by scintillation counting. Additionally, in another study, protein binding of ciclesonide was determined by equilibrium dialysis. For equilibrium dialysis, the mean percentages of des-CIC (0.5-500 ng/mL) plasma protein binding across species were high, approximately 99%, and no apparent saturation of protein binding was observed. Results were similar for ultrafiltration analysis. Protein binding of des-CIC did not change in the presence of warfarin or salicylates or in the plasma of renally or hepatically impaired patients. The protein binding of ciclesonide was 99.4% in human serum. The very low fraction of unbound des-CIC in the systemic circulation suggests minimal systemic exposure of unbound des-CIC, thus suggesting a low potential for systemic adverse effects after administration of inhaled ciclesonide. PMID:15891262

  19. 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.

  20. Probing Zn2+-binding effects on the zinc-ribbon domain of human general transcription factor TFIIB.

    PubMed Central

    Ghosh, Mahua; Elsby, Laura M; Mal, Tapas K; Gooding, Jane M; Roberts, Stefan G E; Ikura, Mitsuhiko

    2004-01-01

    The general transcription factor, TFIIB, plays an important role in the assembly of the pre-initiation complex. The N-terminal domain (NTD) of TFIIB contains a zinc-ribbon motif, which is responsible for the recruitment of RNA polymerase II and TFIIF to the core promoter region. Although zinc-ribbon motif structures of eukaryotic and archaeal TFIIBs have been reported previously, the structural role of Zn2 binding to TFIIB remains to be determined. In the present paper, we report NMR and biochemical studies of human TFIIB NTD, which characterize the structure and dynamics of the TFIIB Zn2-binding domain in both Zn2-bound and -free states. The NMR data show that, whereas the backbone fold of NTD is pre-formed in the apo state, Zn2 binding reduces backbone mobility in the b-turn (Arg28-Gly30), induces enhanced structural rigidity of the charged-cluster domain in the central linker region of TFIIB and appends a positive surface charge within the Zn2-binding site. V8 protease-sensitivity assays of full-length TFIIB support the Zn2-dependent structural changes. These structural effects of Zn2 binding on TFIIB may have a critical role in interactions with its binding partners, such as the Rpb1 subunit of RNA polymerase II. PMID:14641108

  1. Effect of caffeine on the plasma protein binding and the disposition of ceftriaxone.

    PubMed

    Kwon, K I; Bourne, D W; Ho, P C

    1985-11-01

    The effects of caffeine on the in-vitro protein binding and the pharmacokinetics of ceftriaxone (a highly protein bound cephalosporin) were investigated. Caffeine failed to decrease in-vitro protein binding of ceftriaxone. Rabbit plasma concentrations of ceftriaxone (30 mg kg-1 i.v.) were elevated significantly (P less than 0.05 at 0.3, 0.6 and 1 h after injection) when caffeine 5 or 10 mg kg-1 i.v. was co-administered compared with ceftriaxone given alone. Caffeine increased the volume of distribution of the central compartment (V1) for ceftriaxone significantly from 49 +/- 38 ml kg-1 (mean +/- s.d., n = 6) to 97 +/- 33 ml kg-1 (caffeine 5 mg kg-1, P less than 0.05), and 94 +/- 8 ml kg-1 (caffeine 10 mg kg-1, P less than 0.05) and decreased the volume of distribution of the peripheral compartment (V2) from 145 +/- 106 ml kg-1 (mean +/- s.d., n = 6) to 31 +/- 18 ml kg-1 (caffeine 5 mg kg-1, P less than 0.5) and 36 +/- 31 ml kg-1 (caffeine 10 mg kg-1, P less than 0.1). The rate of transfer of ceftriaxone to the peripheral compartment (k12) was also decreased significantly (P less than 0.05) after caffeine. The elevated plasma concentration of ceftriaxone, increased V1 value and the decreased V2 and k12 values are probably the result of caffeine altering the distribution of ceftriaxone to the central and the peripheral compartments. PMID:2867172

  2. Differences between horse and human haemoglobins in effects of organic and inorganic anions on oxygen binding.

    PubMed Central

    Giardina, B; Brix, O; Clementi, M E; Scatena, R; Nicoletti, B; Cicchetti, R; Argentin, G; Condo, S G

    1990-01-01

    Despite the fact that the horse is one of the more common domesticated animals, there are few reports dealing with the properties of its blood, and no comprehensive study has been performed on the reactivity of horse haemoglobin towards organic and inorganic ions. Here we report data on the effects of the organic phosphates D-glycerate-2,3-bisphosphate (2,3-DPG) and InsP6, and of chloride on the properties of horse haemoglobin. Thus the effect of saturating concentrations of 2,3-DPG on the oxygen affinity of horse haemoglobin is about 60% lower than with human adult haemoglobin under the same experimental conditions. The same applies also to InsP6, whose effect on oxygen binding to horse haemoglobin is decreased by about 55% compared with human adult haemoglobin. On the whole, horse haemoglobin appears to be much less sensitive to organic phosphates than previously believed. These results are discussed in the light of the primary structure of the molecule. PMID:2327974

  3. 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

  4. Effects of an EGFR-binding affibody molecule on intracellular signaling pathways.

    PubMed

    Nordberg, E; Ekerljung, L; Sahlberg, S H; Carlsson, J; Lennartsson, J; Glimelius, B

    2010-04-01

    Effects on intracellular signaling were studied in cells treated with the affibody molecule (ZEGFR:955)2 that targets the epithelial growth factor receptor (EGFR). EGFR is overexpressed in many types of cancers and plays a fundamental role in cell signaling and it is of interest to find targeting agents capable of blocking the receptor. The clinically approved antibody cetuximab (Erbitux) and the natural ligand EGF were included as reference molecules. Two EGFR-rich cell lines, A-431 and U-343, were exposed to the three targeting agents and lysed. The cell lysates were immunoprecipitated with the receptors, or directly separated by SDS-Page. Autophosphorylation of the receptors and phosphorylation of the downstream signaling proteins Erk and Akt, were evaluated by Western blotting. Although the three different agents compete for the same binding site on EGFR, they influenced the signaling differently. The affibody molecule did not induce autophosphorylation of EGFR or any other receptor in the EGFR-family but, in spite of this, induced phosphorylation of Erk in both cell lines and Akt in the A-431 cells. Thus, the results suggest that the signaling pattern induced by (ZEGFR:955)2 is only partly similar to that induced by cetuximab. This makes the affibody molecule a potentially interesting alternative to cetuximab for EGFR-targeted therapy since it might give different therapy-related effects on tumor cells and different side effects on normal tissues. PMID:20198342

  5. Biological effect of varying peptide binding affinity to the BoLA-DRB3*2703 allele

    PubMed Central

    2003-01-01

    MHC class I and II molecules are immunoregulatory cell surface glycoproteins, which selectively bind to and present antigenic peptides to T-lymphocytes. Murine and human studies show that variable peptide binding affinity to MHC II molecules influences Th1/Th2 responses by inducing distinctive cytokine expression. To examine the biological effects of peptide binding affinity to bovine MHC (BoLA), various self peptides (BoLA-DQ and fibrinogen fragments) and non-self peptides from ovalbumin (OVA), as well as VP2 and VP4 peptides from foot and mouth disease virus (FMD-V) were used to (1) determine binding affinities to the BoLA-DRB3*2703 allele, previously associated with mastitis susceptibility and (2) determine whether peptide binding affinity influences T-lymphocyte function. Peptide binding affinity was determined by a competitive assay using high affinity biotinylated self-peptide incubated with purified BoLA-DRB3*2703 in the presence of various concentrations of competing peptides. The concentrations of non-self peptide required to inhibit self-peptide binding by 50% (IC50) were variable, ranging from 26.92 to > 320 μM. Peptide-specific T-lymphocyte function was determined by measuring DNA synthesis, cell division, and IFN-γ production in cultures of mononuclear cells from a BoLA-DRB3*2703 homozygous cow. When compared to non-stimulated control cultures, differences in lymphocyte function were observed for all of the assessed parameters; however, peptide-binding affinity did not always account for the observed differences in lymphocyte function. PMID:12927080

  6. Albumin-binding of diclofenac and the effect of a site II inhibitor in the aqueous humor of cataract patients with the instillation of diclofenac.

    PubMed

    Osaki, Takashi; Ozaki, Mineo; Takamura, Norito; Ogata, Kenji; Tokunaga, Jin; Setoguchi, Nao; Arimori, Kazuhiko

    2014-05-01

    Diclofenac instillation has been used widely in cataract surgery to prevent postoperative inflammation. Since diclofenac binds strongly to albumin in the circulation, it does not have a sufficient effect on patients in whom diclofenac binds strongly to albumin in the aqueous humor. A decrease in diclofenac binding and an increase in free diclofenac levels are necessary in these patients. The binding of diclofenac to albumin was investigated in the aqueous humor. In a diclofenac binding assay with albumin in the aqueous humor of individual patients, diclofenac was extracted from aliquots of the aqueous humor, and its total levels were measured using ultra high performance liquid chromatography (UHPLC). Free diclofenac levels were measured using ultrafiltration and UHPLC. The albumin-binding fraction of diclofenac was 0.8 or higher in the aqueous humor of some patients. Ibuprofen significantly inhibited diclofenac binding to site II of albumin in mimic aqueous humor, but not in pooled aqueous humor. This difference may have been due to the weak binding of diclofenac to site II in the pooled aqueous humor. Flurbiprofen was used instead of diclofenac. Flurbiprofen has been shown to bind more strongly than diclofenac to the same site of albumin. Thus, the inhibitory effect of ibuprofen on the binding of flurbiprofen to albumin was investigated in pooled aqueous humor. The results indicated that ibuprofen significantly inhibited the flurbiprofen binding. An effective diclofenac administration method may be established for clinical application by the instillation of an appropriate inhibitor of binding to the albumin site II.

  7. In vivo effect of mutations at the PRPP binding site of the Bacillus subtilis purine repressor.

    PubMed

    Rappu, Pekka; Pullinen, Terhi; Mäntsälä, Pekka

    2003-11-01

    The Bacillus subtilis PurR mediates adenine repression and guanosine induction of purA. PRPP inhibits binding of PurR to DNA in vitro. Mutations in the PRPP binding motif of PurR caused strong repression regardless of purine exclusions or additions, establishing the role of PRPP as regulator of PurR.

  8. The Effect of Calcium on the Binding of Calmodulin to Calcium/Calmodulin Protein Kinase II.

    ERIC Educational Resources Information Center

    Porta, Angela R.

    2000-01-01

    Introduces a follow-up laboratory experiment demonstrating the formation change when calcium binds to calmodulin. This conformation change allows this complex to bind to a target protein. Presents the necessary information to conduct the experiment and discusses the results. (YDS)

  9. 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

  10. 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.

  11. The inhibitory effects of Escherichia coli maltose binding protein on β-amyloid aggregation and cytotoxicity.

    PubMed

    Sharoar, Md Golam; Shahnawaz, Md; Islam, Md Imamul; Ramasamy, Vijay Sankar; Shin, Song Yub; Park, Il-Seon

    2013-10-01

    The aggregation of β-amyloid (Aβ) peptide from its monomeric to its fibrillar form importantly contributes to the development of Alzheimer's disease. Here, we investigated the effects of Escherichia coli maltose binding protein (MBP), which has been previously used as a fusion protein, on Aβ42 fibrillization, in order to improve understanding of the self-assembly process and the cytotoxic mechanism of Aβ42. MBP, at a sub-stoichiometric ratio with respect to Aβ42, was found to have chaperone-like inhibitory effects on β-sheet fibril formation, due to the accumulation of Aβ42 aggregates by sequestration of active Aβ42 species as Aβ42-MBP complexes. Furthermore, MBP increased the lag time of Aβ42 polymerization, decreased the growth rate of fibril extension, and suppressed Aβ42 mediated toxicity in human neuroblastoma SH-SY5Y cells. It appears that MBP decreases the active concentration of Aβ42 by sequestering it as Aβ42-MBP complex, and that this sequestration suppresses ongoing nucleation and retards the growth rate of Aβ42 species required for fibril formation. We speculate that inhibition of the growth rate of potent Aβ42 species by MBP suppresses Aβ42-mediated toxicity in SH-SY5Y cells. PMID:23948569

  12. Effects of iron deficiency on iron binding and internalization into acidic vacuoles in Dunaliella salina.

    PubMed

    Paz, Yakov; Shimoni, Eyal; Weiss, Meira; Pick, Uri

    2007-07-01

    Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe(3+) ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability. PMID:17513481

  13. 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

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

    PubMed

    Newton, Claire L; 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

  15. Multivariate Analysis of Side Effects of Drug Molecules Based on Knowledge of Protein Bindings and ProteinProtein Interactions.

    PubMed

    Hasegawa, Kiyoshi; Funatsu, Kimito

    2014-12-01

    Here, we examined the relationships between 969 side effects associated with 658 drugs and their 1368 human protein targets using our hybrid approaches. Firstly, L-shaped PLS (LPLS) was used to construct a multivariate model of side effects and protein bindings of drug molecules. LPLS is an extension of standard PLS regression, where, in addition to the response matrix Y and the regressor matrix X, an extra data matrix Z is constructed that summarizes the background information of X. X and Y are matrices comprising drugs-target proteins, and drugs-side effects, respectively. The Z matrix is the proteinprotein interaction data. From the loading plot of Y, we could identify two remarkable side effects (urinary incontinence and increased salivation) From the corresponding loading plot of X, the responsible protein targets causing each side effect could be estimated (sodium channels and gamma-aminobutyric acid (GABA) receptors). The loading plot of the Z matrix indicated that the GABA receptors interact with each other and they heavily influence the side effect of increased salivation. Secondly, Bayesian classifier methods were separately applied to the cases of the two side effects. That is, the Bayesian classifier method was used to classify drug molecules as binding or not binding to the responsible protein targets associated with each side effect. Using atom-coloring techniques, it was possible to estimate which fragments on the drug molecule might cause the specific side effects. This information is valuable for drug design to avoid specific side effects.

  16. M13 phage peptide ZL4 exerts its targeted binding effect on schistosoma japonicum via alkaline phosphatase.

    PubMed

    Liu, Yan; Yang, Shenghui; Xiao, Jianhua; Yu, Liang; Chen, Li; Zou, Ju; Wang, Kegeng; Tan, Sijie; Yu, Zhengyang; Zeng, Qingren

    2015-01-01

    The present study was to determine the targeting effect of M13 phage peptide ZL4 (MppZL4) on Schistosoma japonicum (S.j). Mice infected with S.j were injected with MppZL4. Real-time PCR was used to detect the distribution and metabolism of MppZL4 in the livers and lungs of mice. In vivo refusion test was performed to detect the targeting of MppZL4. Western blotting was employed to determine the expression of MppZL4. Live imaging was used to detect the distribution of oligopeptide MppZL4. Immunohistochemistry was employed to determine MppZL4 location on adult S.j body surface. Gomori method was employed to detect the influence of oligopeptide MppZL4 on alkaline phosphatase activity. The distribution and metabolism of MppZL4 and M13KE are not significantly different from each other at each time point. The abundance of MppZL4 is changed as S.j migrates in mice. The targeted binding effect of MppZL4 varies at different stages. ZL4 oligopeptide targets S.j in mice. The specific binding sites of MppZL4 on S.j body are mainly located in syncytial cells. The binding sites of MppZL4 on S.j body surface might be ALP or ALP-related proteins. MppZL4 had targeted binding effect on S.j with its binding site being associated with proteins related to S.j alkaline phosphatase. S.j tegument had a specifically binding site with exogenous peptides, offering new means to explore the interactions between hosts and parasites. Additionally, MppZL4 can possibly be used as targeting molecules in worm-resistant drugs or as tracing molecules in imaging diagnosis technologies.

  17. M13 phage peptide ZL4 exerts its targeted binding effect on schistosoma japonicum via alkaline phosphatase

    PubMed Central

    Liu, Yan; Yang, Shenghui; Xiao, Jianhua; Yu, Liang; Chen, Li; Zou, Ju; Wang, Kegeng; Tan, Sijie; Yu, Zhengyang; Zeng, Qingren

    2015-01-01

    The present study was to determine the targeting effect of M13 phage peptide ZL4 (MppZL4) on Schistosoma japonicum (S.j). Mice infected with S.j were injected with MppZL4. Real-time PCR was used to detect the distribution and metabolism of MppZL4 in the livers and lungs of mice. In vivo refusion test was performed to detect the targeting of MppZL4. Western blotting was employed to determine the expression of MppZL4. Live imaging was used to detect the distribution of oligopeptide MppZL4. Immunohistochemistry was employed to determine MppZL4 location on adult S.j body surface. Gomori method was employed to detect the influence of oligopeptide MppZL4 on alkaline phosphatase activity. The distribution and metabolism of MppZL4 and M13KE are not significantly different from each other at each time point. The abundance of MppZL4 is changed as S.j migrates in mice. The targeted binding effect of MppZL4 varies at different stages. ZL4 oligopeptide targets S.j in mice. The specific binding sites of MppZL4 on S.j body are mainly located in syncytial cells. The binding sites of MppZL4 on S.j body surface might be ALP or ALP-related proteins. MppZL4 had targeted binding effect on S.j with its binding site being associated with proteins related to S.j alkaline phosphatase. S.j tegument had a specifically binding site with exogenous peptides, offering new means to explore the interactions between hosts and parasites. Additionally, MppZL4 can possibly be used as targeting molecules in worm-resistant drugs or as tracing molecules in imaging diagnosis technologies. PMID:25973009

  18. 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.

  19. 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. PMID:27444879

  20. 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.

  1. Selective and effective binding of pillar[5,6]arenes toward secondary ammonium salts with a weakly coordinating counteranion.

    PubMed

    Li, Chunju; Shu, Xiaoyan; Li, Jian; Fan, Jiazeng; Chen, Zhenxia; Weng, Linhong; Jia, Xueshun

    2012-08-17

    The selective and effective binding of secondary ammoniums with a weakly coordinating tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (BArF) counteranion by per-ethylated pillar[5,6]arenes is reported. The construction of a first pillararene-based self-sorting system consisting of two wheels and two axles is also described. PMID:22866893

  2. In vitro assessment of the effectiveness of non-nutritive sorbent materials as binding agents for boar taint compounds.

    PubMed

    Jen, K; Squires, E J

    2011-09-01

    Boar taint, an off-odor and an off-flavor in the meat from some uncastrated male pigs, is due to high levels of the testicular steroid hormone, androstenone, and the indole, skatole. Thus far, there are no known methods for controlling both androstenone and skatole through dietary means. We tested the adsorbent agents, cholestyramine (CH), activated carbon (AC), tween-60 (Tween), bentonite (BNT) and polyvinylpolypyrrolidone (PVPP) for binding androstenone, estrone (E(1)), estrone sulfate (E(1)S) and skatole from buffer solutions in an in vitro system. The goal was to determine the potential utility of these binding agents as feed additives to control boar taint. Michaelis-Menten analysis was utilized to determine the effectiveness of the adsorbents. At pH 7.4, E(1)S was bound to AC and CH with the highest B(max) (maximum binding), whereas Tween and AC had the greatest B(max) for E(1). The B(max) for skatole at pH 7.4 was highest for AC, CH and PVPP. AC had a higher B(max) for androstenone than CH and Tween. The B(max) values at pH 3.0 with E(1)S for AC and CH were essentially 100%, whereas the binding of Tween to E(1)S at pH 3.0 decreased by 49.5% from binding at pH 7.4 (P < 0.05). The Ad(int) values, which represent efficiency of binding, illustrated that AC bound E(1), androstenone and skatole with greater efficiency than the other binding agents at pH 7.4, whereas AC bound E(1)S as efficiently as CH. We conclude that AC was the most effective adsorbent agent for binding E(1), E(1)S, androstenone and skatole in vitro, followed by CH, Tween, PVPP and lastly BNT. These adsorbent agents may be useful for binding boar taint compounds in in vivo studies to decrease the risk of boar taint.

  3. 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.

  4. Effects of a hydrated sodium calcium aluminosilicate (T-Bind) on mycotoxicosis in young broiler chickens.

    PubMed

    Kubena, L F; Harvey, R B; Bailey, R H; Buckley, S A; Rottinghaus, G E

    1998-10-01

    Experiments were conducted to determine the ability of a hydrated sodium calcium aluminosilicate (T-Bind) sorbent to reduce the toxicity of aflatoxins (AF) or T-2 toxin in male broiler chickens from day of hatch to 21 d of age. In Experiment 1, the sorbent was added at 0.250 or 0.375% to diets containing AF at 5 or T-2 toxin at 8 mg/kg of diet. When compared with controls, AF reduced BW gain by 27% and T-2 toxin reduced BW gain by 17%. The addition of the sorbent at 0.250 or 0.375%, in the absence of added mycotoxins, did not alter the performance of the chicks. The sorbent reduced the toxic effects of 5 mg AF/kg of diet on BW gain by 43% but did not significantly diminish the toxic effects of 8 mg T-2 toxin/kg of diet. The decreased efficiency of feed utilization and the increased relative organ weights caused by AF were significantly diminished to differing degrees by the sorbent. Oral lesions caused by T-2 toxin were not affected by the sorbent. In Experiment 2, the sorbent was added at 0.80% to a diet containing 8 mg T-2 toxin/kg of diet. The sorbent did not diminish the toxic effects of T-2 toxin when added at 0.80% of the diet. These data demonstrate that this specific sorbent can provide protection against the toxicity of AF, but not T-2 toxin, in young broiler chicks.

  5. Effect of carboxymethylation conditions on the water-binding capacity of chitosan-based superabsorbents.

    PubMed

    Bidgoli, Hosein; Zamani, Akram; Taherzadeh, Mohammad J

    2010-12-10

    A superabsorbent polymer (SAP) from chitosan was provided via carboxymethylation of chitosan, followed by cross-linking with glutaraldehyde and freeze-drying. This work was focused on an investigation of the effects of monochloroacetic acid (MCAA), sodium hydroxide, and reaction time on preparation of carboxymethyl chitosan (CMCS). The CMCS products were characterized using FTIR spectroscopy, and their degrees of substitution (DS) were measured using conductimetry and FTIR analysis. The highest DS value was obtained when the carboxymethylation reaction was carried out using 1.75g MCAA and 1.75g NaOH per g of chitosan in 4h. The water solubilities of the CMCS products at various pHs were also evaluated, and the results indicated a significant impact of the reaction parameters on the solubility of CMCS. The CMCSs with the highest DS value resulted in SAPs having the highest water-binding capacity (WBC). The WBC of the best SAP measured after 10min exposure in distilled water, 0.9% NaCl solution, synthetic urine, and artificial blood was 104, 33, 30, and 57g/g, respectively. The WBC of this SAP at pH 2-9 passed a maximum at pH 6.

  6. 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.

  7. Solubilization of phenol and benzene in cationic micelles: Binding sites and effect on structure

    SciTech Connect

    Kandori, K.; McGreevy, R.J.; Schechter, R.S. )

    1989-02-23

    The effect of phenol and benzene additives on micellar structure in aqueous solutions of dodecyltrimethylammonium bromide has been studied by means of various experimental measurements. The solution properties studied include additive solubilities, tracer diffusion coefficients, electrical conductivity, viscosity, and ultraviolet absorbance. From the trace diffusion measurements the degree of partitioning of phenol into the micelles was calculated as a function of phenol concentration. A procedure for determining surfactant aggregation number and micelle concentration in ionic surfactant systems by means of diffusion and electrical conductivity data is presented. The solubilization of phenol and benzene in this system causes the micelles to swell, and it was observed that phenol addition leads to a greater increase in the size of aggregates than addition of benzene. Ultraviolet absorbance measurements revealed that the site of solubilization within the micelles is different for the two additives: Benzene solubilizes in the central core, while at low concentrations phenol is taken up in the outer palisade layer. However, the site of phenol solubilization, the shape of the micelles, and the physical properties of DTAB/phenol solutions change at a concentration of 1 mol of phenol solubilizate per mole of surfactant. Due to the saturation of the palisade layer with surfactant and additive molecules, phenol added to the DTAB system beyond this transition point most likely binds to the exterior of the micelles.

  8. Effect of binding to carbon black on the dynamics of 1,4-polybutadiene.

    PubMed

    Roh, J H; Tyagi, M; Hogan, T E; Roland, C M

    2013-10-01

    The nature of the interactions of polymers at the surface of nanoparticles is crucial to understanding the dynamics and their relation to mechanical properties. The effect of binding (both chemical attachment and physical adsorption) on the local and global dynamics of chain molecules remains a controversial subject. Using neutron scattering and dynamic mechanical spectroscopies, we measured the slow conformational and terminal relaxations, as well as the fast local dynamics, of 1,4-polybutadiene (PBD) containing carbon black (CB) particles. We observed a substantial decrease in the flexibility of bound segments at temperatures through the glass transition temperature, T(g). The longer range motions of the PBD become more suppressed and cooperative as temperature decreases, while the relaxation time of the fast local dynamics is little affected by the CB particles. The mobile fraction of PBD is less sensitive to temperature when bound. Mechanical spectroscopy indicates that both the local segmental dynamics and the global chain modes are slowed by the filler. These results are consistent with transient structural arrest of the slow dynamics of atoms adjacent to the particles. PMID:24116583

  9. Effect of binding to carbon black on the dynamics of 1,4-polybutadiene

    NASA Astrophysics Data System (ADS)

    Roh, J. H.; Tyagi, M.; Hogan, T. E.; Roland, C. M.

    2013-10-01

    The nature of the interactions of polymers at the surface of nanoparticles is crucial to understanding the dynamics and their relation to mechanical properties. The effect of binding (both chemical attachment and physical adsorption) on the local and global dynamics of chain molecules remains a controversial subject. Using neutron scattering and dynamic mechanical spectroscopies, we measured the slow conformational and terminal relaxations, as well as the fast local dynamics, of 1,4-polybutadiene (PBD) containing carbon black (CB) particles. We observed a substantial decrease in the flexibility of bound segments at temperatures through the glass transition temperature, Tg. The longer range motions of the PBD become more suppressed and cooperative as temperature decreases, while the relaxation time of the fast local dynamics is little affected by the CB particles. The mobile fraction of PBD is less sensitive to temperature when bound. Mechanical spectroscopy indicates that both the local segmental dynamics and the global chain modes are slowed by the filler. These results are consistent with transient structural arrest of the slow dynamics of atoms adjacent to the particles.

  10. Ouabain binding in tadpole ventral skin I. Kinetics and effect on intracellular ions

    SciTech Connect

    Robinson, D.H.; Mills, J.W. )

    1987-09-01

    To determine the Na pump sensitivity to ouabain, the kinetics of ({sup 3}H)-ouabain binding in tadpole epidermis and the effect of ouabain on intracellular Na and K were tested. In tadpole epidermis a high-affinity dissociation constant (K{sub D}) for ouabain of 1.21 {plus minus} 0.20 {times} 10{sup {minus}7} M was found, which is similar to the K{sub D} in frog skin. Incubation of tadpole epidermis in ouabain for 1 h increased intracellular Na concentration and decreased K concentration. Two hours of ouabain exposure increased intracellular Na concentration and decreased K concentration. Regression analysis of the change in Na and K concentrations reveals a slope of 14.7 {plus minus} 2.5 mM/h for the Na gain, which is similar in magnitude to the rate of K decrease. It is hypothesized that the residual short-circuit current seen in tadpole skin after ouabain exposure is a result of current being carried through the tissue by a slow gain of Na across the apical membrane and an equivalent loss of K across the basolateral membrane.

  11. Contribution of the Myosin Binding Protein C Motif to Functional Effects in Permeabilized Rat Trabeculae

    PubMed Central

    Razumova, Maria V.; Bezold, Kristina L.; Tu, An-Yue; Regnier, Michael; Harris, Samantha P.

    2008-01-01

    Myosin binding protein C (MyBP-C) is a thick-filament protein that limits cross-bridge cycling rates and reduces myocyte power output. To investigate mechanisms by which MyBP-C affects contraction, we assessed effects of recombinant N-terminal domains of cardiac MyBP-C (cMyBP-C) on contractile properties of permeabilized rat cardiac trabeculae. Here, we show that N-terminal fragments of cMyBP-C that contained the first three immunoglobulin domains of cMyBP-C (i.e., C0, C1, and C2) plus the unique linker sequence termed the MyBP-C “motif” or “m-domain” increased Ca2+ sensitivity of tension and increased rates of tension redevelopment (i.e., ktr) at submaximal levels of Ca2+. At concentrations ≥20 μM, recombinant proteins also activated force in the absence of Ca2+ and inhibited maximum Ca2+-activated force. Recombinant proteins that lacked the combination of C1 and the motif did not affect contractile properties. These results suggest that the C1 domain plus the motif constitute a functional unit of MyBP-C that can activate the thin filament. PMID:18955596

  12. Effects of IGF-binding protein 5 in dysregulating the shape of human hair.

    PubMed

    Sriwiriyanont, Penkanok; Hachiya, Akira; Pickens, William L; Moriwaki, Shigeru; Kitahara, Takashi; Visscher, Marty O; Kitzmiller, William J; Bello, Alexander; Takema, Yoshinori; Kobinger, Gary P

    2011-02-01

    The hair follicle has a unique dynamic property to cyclically regenerate throughout life. Despite significant progress in hair structure and hair shape determination using animal models, the mechanisms controlling the architecture and the shape of the human hair remain largely unexplored. In this study, comparison of the genetic expression of several human genes, especially those involved in growth, development, and differentiation, between Caucasian curly hair and naturally straight hair was performed. Thereafter, analyses using human recombinant and lentiviral vector technologies were conducted to further dissect and elucidate a molecular mechanism that regulates hair growth and development, particularly in controlling the shape of the hair shaft. Overexpression of IGF-binding protein 5 (IGFBP-5) in the human hair xenografts obtained from straight- and curly-haired individuals was found to result in the decreased expression of several extracellular matrix proteins and disassembly of adhesional junctions, resulting in twisted hair shafts as well as an unusual deposition of hair cuticle that may be derived from the disturbance of normal proliferation and differentiation. This study provides evidence that IGFBP-5 has an effect on human hair shape, and that lentiviral transduction regimen can be used for functional analysis of genes involved in human hair morphogenesis.

  13. Effects of mono- and divalent metal ions on DNA binding and catalysis of human apurinic/apyrimidinic endonuclease 1.

    PubMed

    Miroshnikova, Anastasia D; Kuznetsova, Alexandra A; Vorobjev, Yuri N; Kuznetsov, Nikita A; Fedorova, Olga S

    2016-05-26

    Here, we used stopped-flow fluorescence techniques to conduct a comparative kinetic analysis of the conformational transitions in human apurinic/apyrimidinic endonuclease 1 (APE1) and in DNA containing an abasic site in the course of their interaction. Effects of monovalent (K(+)) and divalent (Mg(2+), Mn(2+), Ca(2+), Zn(2+), Cu(2+), and Ni(2+)) metal ions on DNA binding and catalytic stages were studied. It was shown that the first step of substrate binding (corresponding to formation of a primary enzyme-substrate complex) does not depend on the concentration (0.05-5.0 mM) or the nature of divalent metal ions. In contrast, the initial DNA binding efficiency significantly decreased at a high concentration (5-250 mM) of monovalent K(+) ions, indicating the involvement of electrostatic interactions in this stage. It was also shown that Cu(2+) ions abrogated the DNA binding ability of APE1, possibly, due to a strong interaction with DNA bases and the sugar-phosphate backbone. In the case of Ca(2+) ions, the catalytic activity of APE1 was lost completely with retention of binding potential. Thus, the enzymatic activity of APE1 is increased in the order Zn(2+) < Ni(2+) < Mn(2+) < Mg(2+). Circular dichroism spectra and calculation of the contact area between APE1 and DNA reveal that Mg(2+) ions stabilize the protein structure and the enzyme-substrate complex. PMID:27063150

  14. Binding Motifs in Bacterial Gene Promoters Modulate Transcriptional Effects of Global Regulators CRP and ArcA

    PubMed Central

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

    2012-01-01

    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. PMID:22701314

  15. 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.

  16. Effects of extracellular acetylcholine on muscarinic receptor binding assessed by [125I]dexetimide and a simple probe.

    PubMed

    Sánchez-Roa, P M; Wagner, H N; Villemagne, V L; London, E D; Lever, J R

    1998-10-01

    New pharmacologic approaches to enhance brain cholinergic function focus on increasing intrasynaptic acetylcholine. We examined the usefulness of a simple probe and [125I]dexetimide to evaluate in vivo the effects of extracellular acetylcholine on muscarinic receptor binding in the mouse brain. After radiotracer injection continuous time/activity curves were generated over 330 min. [125I]Dexetimide reached a plateau at 90 min post-injection. To increase extracellular acetylcholine, the anticholinesterase physostigmine was administered at 120 min, producing a reversible decrease in [125I]dexetimide specific binding (23%) for 30 min. These findings demonstrate that dynamic changes in extracellular acetylcholine can be evaluated by displacement of [125I]dexetimide binding in vivo using a simple probe system. PMID:9822886

  17. Electrical and mechanical effects of new aminosteroids on guinea-pig isolated ventricular muscle.

    PubMed Central

    Adamantidis, M. M.; Honoré, E. R.; Dupuis, B. A.

    1988-01-01

    1. LND 623 and LND 796 are two aminosteroid derivatives which exert similar positive inotropic effects to digitalis. Their electrophysiological, toxic and inotropic effects were investigated in both normal and partially K+-depolarized ventricular muscle. 2. In guinea-pig myocardial fibres, LND 623 and LND 796 required tenfold higher concentrations than digoxin to induce the same signs of toxicity; e.g. triggered activities generated from delayed afterdepolarizations, leading to the marked depression of action potential characteristics and inexcitability. These abnormal rhythms and delayed afterdepolarizations were abolished by 1 mM caffeine. The toxic effects were reversed by washout, particularly in the case of LND 796. 3. In normal-K+ solution, LND 623 and LND 796 exhibited concentration-dependent positive inotropic effects on guinea-pig papillary muscle and increased concomitantly resting membrane potential and action potential amplitude. The range of active concentrations (0.1 to 1 microM) of LND 623 was larger than that of digoxin (0.3 to 1 microM). Like digoxin, LND 796 exerted negative inotropic effects at the lowest concentrations (0.01 to 0.03 microM) and positive inotropic effects at high concentrations (1 and 3 microM). 4. In partially K+-depolarized papillary muscle, in the presence of 2 microM histamine, LND 623 (3 and 10 microM) and LND 796 (10 and 30 microM) enhanced the two components P1 and P2 of the contraction and increased slow action potential amplitude, resting potential and maximal rate of depolarization. Low concentrations (0.03 to 0.3 microM) of LND 796 induced negative inotropic effects. beta-Adrenoceptor blockade with atenolol (1 microM) did not modify the activity of LND 623 but significantly enhanced the negative inotropic effect on P2 induced by 1 and 3 microM LND 796 and reduced the positive inotropic effect on P1 and P2 of the highest concentration (30 microM) studied. 5. In the presence of either caffeine (1 mM) or Ca2+-free, Sr2

  18. Molecular dynamics studies on the effect of trifluoperazine in the Ca 2+ binding process of calmodulin

    NASA Astrophysics Data System (ADS)

    Kim, Eun Sung; Kang, Nam Sook; Jhon, Mu Shik

    1996-06-01

    A detailed investigation of the binding of trifluoperazine (TFP) to calmodulin (CAM) can give information on the loop-selectivity of the first Ca 2+ binding and determination of Ca 2+ cooperativity in the N-terminal domain. This information has been obtained by carrying out molecular dynamics simulations. By using interactive computer graphics, seven models for CAM are constructed according to sequential Ca 2+ occupancy and TFP occupancy. Analysis of the resulting structure shows that the binding site of the first Ca 2+ in the N-terminal domain is EFII rather than EFI. Electrostatic interactions between each Ca 2+ and its opposite loop play an important role in the cooperativity. TFP increases the Ca 2+ affinity and Ca 2+ cooperativity, and TFP prefers to bind to CAM with three rather than four bound Ca 2+ ions.

  19. 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...

  20. Dissecting Hofmeister Effects: Direct Anion-Amide Interactions Are Weaker than Cation-Amide Binding.

    PubMed

    Balos, Vasileios; Kim, Heejae; Bonn, Mischa; Hunger, Johannes

    2016-07-01

    Whereas there is increasing evidence for ion-induced protein destabilization through direct ion-protein interactions, the strength of the binding of anions to proteins relative to cation-protein binding has remained elusive. In this work, the rotational mobility of a model amide in aqueous solution was used as a reporter for the interactions of different anions with the amide group. Protein-stabilizing salts such as KCl and KNO3 do not affect the rotational mobility of the amide. Conversely, protein denaturants such as KSCN and KI markedly reduce the orientational freedom of the amide group. Thus these results provide evidence for a direct denaturation mechanism through ion-protein interactions. Comparing the present findings with results for cations shows that in contrast to common belief, anion-amide binding is weaker than cation-amide binding. PMID:27237055

  1. 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.

  2. 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.

  3. 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.

  4. Effects of chronic delta-9-tetrahydrocannabinol (THC) administration on neurotransmitter concentrations and receptor binding in the rat brain.

    PubMed

    Ali, S F; Newport, G D; Scallet, A C; Gee, K W; Paule, M G; Brown, R M; Slikker, W

    1989-01-01

    THC is the major psychoactive constituent of marijuana and is also known as an hallucinogenic compound. Numerous reports have shown that large doses of THC produce significant alterations in various neurotransmitter systems. The present study was designed to determine whether chronic exposure to THC produces significant alterations in selected neurotransmitter systems (dopamine, serotonin, acetylcholine, GABAergic, benzodiazepine, and opiate) in the rat brain. In Experiment 1, male Sprague-Dawley rats were gavaged with vehicle, 10 or 20 mg THC/kg body weight daily, 5 days/week for 90 days. Animals were killed either 24 hours or two months after the last dose. Brains were dissected into different regions for neurochemical analyses. Two months after the cessation of chronic administration, there was a significant decrease in GABA receptor binding in the hippocampus of animals in the high dose group. However, no other significant changes were found in neurotransmitter receptor binding characteristics in the hippocampus or in neurotransmitter concentrations in the caudate nucleus, hypothalamus or septum after chronic THC administration. In an attempt to replicate the GABA receptor binding changes and also to determine the [35S]TBPS binding in hippocampus, we designed Experiment 2. In this experiment, we dosed the animals by gavage with 0, 5, 10 or 20 mg THC/kg daily, 5 days/week or with 20 mg THC/kg Monday through Thursday and 60 mg/kg on Friday for 90 days. Results from this experiment failed to replicate the dose-dependent effect of THC on GABA receptor binding in hippocampus. Modulation of [35S]TBPS binding by GABA or 3 alpha-OH-DHP or inhibition by cold TBPS in frontal cortex did not show any significant dose-related effects. Results from these experiments suggest that chronic exposure to THC does not produce significant alterations in catecholamine or indoleamine neurotransmitter systems or in opiate or GABA receptor systems in the rat brain.

  5. Cellular or viral protein binding to a cytomegalovirus promoter transcription initiation site: effects on transcription.

    PubMed Central

    Macias, M P; Huang, L; Lashmit, P E; Stinski, M F

    1996-01-01

    We have previously shown that the IE2 protein of human cytomegalovirus (CMV) represses its own synthesis by binding to the major immediate-early promoter (M. P. Macias and M. F. Stinski, Proc. Natl. Acad. Sci. USA 90:707-711, 1993). The binding of a viral protein (IE2) and a cellular protein in the region of the transcription start site was investigated by site-specific mutational analysis and electrophoretic mobility shift assay. The viral protein and the cellular protein require different but adjacent core DNA sequence elements for binding. In situ chemical footprinting analysis of DNA-protein interactions with purified CMV IE2 protein or HeLa cell nuclear extracts demonstrated binding sites that overlap the transcription start site. The IE2 protein footprint was between bp -15 and +2, relative to the transcription start site, and the cellular protein was between bp -16 and +7. The ability of the unknown human cellular protein of approximately 150 kDa to bind the CMV major immediate-early promoter correlates with an increase in the level of transcription efficiency. Mutations in the core DNA sequence element for cellular protein binding significantly reduced the level of in vitro transcription efficiency. Mutations upstream and downstream of the core sequence moderately reduced the transcription efficiency level. Negative autoregulation of the CMV promoter by the viral IE2 protein may involve both binding to the DNA template and interference with the function of a cellular protein that binds to the transcription start site and enhances transcription efficiency. PMID:8648697

  6. Functional Analysis of a Bacterial Antifreeze Protein Indicates a Cooperative Effect between Its Two Ice-Binding Domains.

    PubMed

    Wang, Chen; Oliver, Erin E; Christner, Brent C; Luo, Bing-Hao

    2016-07-19

    Antifreeze proteins make up a class of ice-binding proteins (IBPs) that are possessed and expressed by certain cold-adapted organisms to enhance their freezing tolerance. Here we report the biophysical and functional characterization of an IBP discovered in a bacterium recovered from a deep glacial ice core drilled at Vostok Station, Antarctica (IBPv). Our study showed that the recombinant protein rIBPv exhibited a thermal hysteresis of 2 °C at concentrations of >50 μM, effectively inhibited ice recrystallization, and enhanced bacterial viability during freeze-thaw cycling. Circular dichroism scans indicated that rIBPv mainly consists of β strands, and its denaturing temperature was 53.5 °C. Multiple-sequence alignment of homologous IBPs predicted that IBPv contains two ice-binding domains, a feature unique among known IBPs. To examine functional differences between the IBPv domains, each domain was cloned, expressed, and purified. The second domain (domain B) expressed greater ice binding activity. Data from thermal hysteresis and gel filtration assays supported the idea that the two domains cooperate to achieve a higher ice binding effect by forming heterodimers. However, physical linkage of the domains was not required for this effect. PMID:27359086

  7. Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

    SciTech Connect

    Ponec, M.; Weerheim, A. ); Havekes, L. ); Boonstra, J. )

    1987-08-01

    The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.

  8. ATP binds to proteasomal ATPases in pairs with distinct functional effects implying an ordered reaction cycle

    PubMed Central

    Smith, David M.; Fraga, Hugo; Reis, Christian; Kafri, Galit; Goldberg, Alfred L.

    2011-01-01

    In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits, and in archaea by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules, or two ATPγS plus two ADP molecules it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate-opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and “wobble” on top of the heptameric 20S proteasome. PMID:21335235

  9. Computational investigation of stoichiometric effects, binding site heterogeneities, and selectivities of molecularly imprinted polymers.

    PubMed

    Terracina, Jacob J; Bergkvist, Magnus; Sharfstein, Susan T

    2016-06-01

    A series of quantum mechanical (QM) computational optimizations of molecularly imprinted polymer (MIP) systems were used to determine optimal monomer-to-target ratios. Imidazole- and xanthine-derived target molecules were studied. The investigation included both small-scale models (3-7 molecules) and larger-scale models (15-35 molecules). The optimal ratios differed between the small and larger scales. For the larger models containing multiple targets, binding-site surface area analysis was used to quantify the heterogeneity of these sites. The more fully surrounded sites had greater binding energies. No discretization of binding modes was seen, furthering arguments for continuous affinity distribution models. Molecular mechanical (MM) docking was then used to measure the selectivities of the QM-optimized binding sites. Selectivity was also shown to improve as binding sites become more fully encased by the monomers. For internal sites, docking consistently showed selectivity favoring the molecules that had been imprinted via QM geometry optimizations. The computationally imprinted sites were shown to exhibit size-, shape-, and polarity-based selectivity. Here we present a novel approach to investigate the selectivity and heterogeneity of imprinted polymer binding sites, by applying the rapid orientation screening of MM docking to the highly accurate QM-optimized geometries. Modeling schemes were designed such that no computing clusters or other specialized modeling equipment would be required. Improving the in silico analysis of MIP system properties will ultimately allow for the production of more sensitive and selective polymers. PMID:27207254

  10. The effect of ageing on recollection: the role of the binding updating process.

    PubMed

    Boujut, Arnaud; Clarys, David

    2016-10-01

    The aim of this study was to highlight the underlying process responsible for the age-related deficit in recollection. Through two experiments using the Remember-Know-Guess procedure (Gardiner, J. M., & Richardson-Klavehn, A. [2000]. Remembering and knowing. In The Oxford handbook of memory (pp. 229-244). New York, NY: Oxford University Press) in recognition, we manipulated the opportunity to update bindings between target items and their encoding context, in young and older adults. In the first experiment we impaired the binding updating process during the encoding of items, while in the second we supported this process. The results indicated that the "Remember" responses in the younger group were specifically reduced by the impairment of the binding updating process (Exp. 1), suggesting that this ability is useful for them to encode a specific episode. Conversely, only the "Remember" responses in the older group were improved in accuracy by supporting the binding updating process (Exp. 2), suggesting that their weakness in this ability is the source of their failure to improve the accuracy of their memories. The overall results support the hypothesis that the age-related decline in episodic memory is partly due to a greater vulnerability to interference on bindings, impairing the ability to update content-context bindings as and when events occur. PMID:27560656

  11. 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.

  12. 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.

  13. Lysozyme binding ability toward psychoactive stimulant drugs: Modulatory effect of colloidal metal nanoparticles.

    PubMed

    Sonu, Vikash K; Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Mitra, Sivaprasad

    2016-10-01

    The interaction and binding behavior of the well-known psychoactive stimulant drugs theophylline (THP) and theobromine (THB) with lysozyme (LYS) was monitored by in-vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of the drugs is due to the formation of protein-drug complex in the ground state in both the cases. However, the binding interaction is almost three orders of magnitude stronger in THP, which involves mostly hydrogen bonding interaction in comparison with THB where hydrophobic binding plays the predominant role. The mechanism of fluorescence quenching (static type) remains same also in presence of gold and silver nanoparticles (NPs); however, the binding capacity of LYS with the drugs changes drastically in comparison with that in aqueous buffer medium. While the binding affinity of LYS to THB increases ca. 100 times in presence of both the NPs, it is seen to decrease drastically (by almost 1000 fold) for THP. This significant modulation in binding behavior indicates that the drug transportation capacity of LYS can be controlled significantly with the formation protein-NP noncovalent assembly system as an efficient delivery channel. PMID:27419646

  14. 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.

  15. Technical note: effects of forage protein-binding polyphenols on chemistry of dairy excreta.

    PubMed

    Powell, J M; Broderick, G A; Grabber, J H; Hymes-Fecht, U C

    2009-04-01

    Forage chemistry can affect intake, digestion, milk production, and manure excretion. Although information is available on the effects of forage protein-binding polyphenols on small ruminant production and manure excretion, little information is available for dairy cattle. The objective of this study was to compare fecal and urinary N excretion of diets formulated with alfalfa (Medicago sativa L.) silage versus condensed tannin-containing birdsfoot trefoil (Lotus corniculatus) or o-quinone-containing red clover (Trifolium pratense L.) silages. Significantly higher concentrations of N were excreted in urine by lactating Holstein dairy cows fed red clover and low-tannin birdsfoot trefoil (8.2 g/L) than by cows fed high-tannin birdsfoot trefoil or alfalfa (7.1 g/L). Fecal N concentrations were similar (33.6 g/kg) among all diets. Dairy cows fed red clover had lower rates of urinary N excretion (5.0 g/h) compared with other forages (6.6 g/h). Fecal N excretion rates were lowest for red clover (4.1 g/h), intermediate for alfalfa (5.8 g/h), and greatest for cows fed high- and low-tannin birdsfoot trefoil (6.4 g/h). The ratio of fecal N to urinary N was highest for high-tannin trefoil, lowest for alfalfa and red clover, and higher in excreta collected in morning than evening. Concentrations of neutral detergent fiber (NDF) in feces, of N in NDF (NDIN) and acid detergent fiber (ADIN), and relative amounts of NDIN and ADIN excreted in feces were significantly higher from cows fed high-tannin birdsfoot trefoil than the other silage types. Study results imply that collection of excreta for environmental studies needs to consider forage polyphenol and diurnal effects on chemistry of dairy excreta.

  16. Effects of corticosterone pellets on baseline and stress-induced corticosterone and corticosteroid-binding-globulin.

    PubMed

    Müller, Claudia; Almasi, Bettina; Roulin, Alexandre; Breuner, Creagh W; Jenni-Eiermann, Susanne; Jenni, Lukas

    2009-01-01

    Exogenous administration of glucocorticoids is a widely used and efficient tool to investigate the effects of elevated concentrations of these hormones in field studies. Because the effects of corticosterone are dose and duration-dependent, the exact course of plasma corticosterone levels after exogenous administration needs to be known. We tested the performance of self-degradable corticosterone pellets (implanted under the skin) in elevating plasma corticosterone levels. We monitored baseline (sampled within 3min after capture) total corticosterone levels and investigated potential interactions with corticosteroid-binding-globulin (CBG) capacity and the endogenous corticosterone response to handling in Eurasian kestrel Falco tinnunculus and barn owl Tyto alba nestlings. Corticosterone pellets designed for a 7-day-release in rodents elevated circulating baseline total corticosterone during only 2-3 days compared to placebo-nestlings. Highest levels occurred 1-2days after implantation and levels decreased strongly thereafter. CBG capacity was also increased, resulting in a smaller, but still significant, increase in baseline free corticosterone levels. The release of endogenous corticosterone as a response to handling was strong in placebo-nestlings, but absent 2 and 8 days after corticosterone pellet implantation. This indicates a potential shut-down of the hypothalamo-pituitary-adrenal axis after the 2-3 days of elevated baseline corticosterone levels. 20 days after pellet implantation, the endogenous corticosterone response to handling of nestlings implanted with corticosterone pellets attained similar levels as in placebo-nestlings. Self-degradable pellets proved to be an efficient tool to artificially elevate circulating baseline corticosterone especially in field studies, requiring only one intervention. The resulting peak-like elevation of circulating corticosterone, the concomitant elevation of CBG capacity, and the absence of an endogenous corticosterone

  17. Effects of corticosterone pellets on baseline and stress-induced corticosterone and corticosteroid-binding-globulin.

    PubMed

    Müller, Claudia; Almasi, Bettina; Roulin, Alexandre; Breuner, Creagh W; Jenni-Eiermann, Susanne; Jenni, Lukas

    2009-01-01

    Exogenous administration of glucocorticoids is a widely used and efficient tool to investigate the effects of elevated concentrations of these hormones in field studies. Because the effects of corticosterone are dose and duration-dependent, the exact course of plasma corticosterone levels after exogenous administration needs to be known. We tested the performance of self-degradable corticosterone pellets (implanted under the skin) in elevating plasma corticosterone levels. We monitored baseline (sampled within 3min after capture) total corticosterone levels and investigated potential interactions with corticosteroid-binding-globulin (CBG) capacity and the endogenous corticosterone response to handling in Eurasian kestrel Falco tinnunculus and barn owl Tyto alba nestlings. Corticosterone pellets designed for a 7-day-release in rodents elevated circulating baseline total corticosterone during only 2-3 days compared to placebo-nestlings. Highest levels occurred 1-2days after implantation and levels decreased strongly thereafter. CBG capacity was also increased, resulting in a smaller, but still significant, increase in baseline free corticosterone levels. The release of endogenous corticosterone as a response to handling was strong in placebo-nestlings, but absent 2 and 8 days after corticosterone pellet implantation. This indicates a potential shut-down of the hypothalamo-pituitary-adrenal axis after the 2-3 days of elevated baseline corticosterone levels. 20 days after pellet implantation, the endogenous corticosterone response to handling of nestlings implanted with corticosterone pellets attained similar levels as in placebo-nestlings. Self-degradable pellets proved to be an efficient tool to artificially elevate circulating baseline corticosterone especially in field studies, requiring only one intervention. The resulting peak-like elevation of circulating corticosterone, the concomitant elevation of CBG capacity, and the absence of an endogenous corticosterone

  18. The mitomycin C (MMC)-binding protein from MMC-producing microorganisms protects from the lethal effect of bleomycin: crystallographic analysis to elucidate the binding mode of the antibiotic to the protein.

    PubMed

    Danshiitsoodol, Narandalai; de Pinho, Catherine Azzariti; Matoba, Yasuyuki; Kumagai, Takanori; Sugiyama, Masanori

    2006-07-01

    Antibiotic-producing microorganisms must be protected from the lethal effect of their own antibiotic. We have previously determined the X-ray crystal structure of the bleomycin (Bm)-binding protein, designated BLMA, as a self-resistance determinant from Bm-producing Streptomyces verticillus, which suggests that the binding of the first Bm to one of two pockets formed in the BLMA homodimer induces the cooperative binding of the second Bm to the other pocket. In the present study, we noticed that the X-ray crystallographic structure of a self-resistance determinant from a mitomycin C-producing microorganism, designated MRDP, reveals similarity to the folding pattern on the BLMA, although no sequence homology exists. To clarify the hypothesis that MRDP may function as a resistance determinant to Bm, we characterized and determined the crystal structure of MRDP complexed with the Cu(II)-bound form of BmA(2) grouped into the Bm family of antibiotics. The biochemical and structural studies for Bm binding provide evidence that the first Bm binds anti-cooperatively to a pocket of MRDP with binding affinity of the nanomolar order, whereas the second Bm binds to the other pocket, which has binding affinity of the micromolar order. The invisibility of the second Bm in the structure agrees with the observation that Escherichia coli-expressing MRDP displays lower resistance to Bm than that expressing BLMA. The structure of MRDP, which is complexed with the Cu(II)-bound BmA(2), revealed that the gamma-aminopropyldimethylsulphonium moiety of the antibiotic is sandwiched between the peripheral residues of the binding pocket and that its positively charged sulphonium head is accommodated completely in the negatively charged region of the MRDP pocket. Furthermore, the Cu(II)-bound BmA(2) has a very compact structure, in which the bithiazole ring of BmA(2) is folded back to the metal-binding domain. PMID:16756991

  19. Effect of enzymatic deamidation of soy protein by protein-glutaminase on the flavor-binding properties of the protein under aqueous conditions.

    PubMed

    Suppavorasatit, Inthawoot; Cadwallader, Keith R

    2012-08-15

    The effect of the enzymatic deamidation by protein-glutaminase (PG) on flavor-binding properties of soy protein isolate (SPI) under aqueous conditions was evaluated by a modified equilibrium dialysis (ultrafiltration) technique. Binding parameters, such as number of binding sites (n) and binding constants (K), were derived from Klotz plots. The partial deamidation of SPI by PG (43.7% degree of deamidation) decreased overall flavor-binding affinity (nK) at 25 °C for both vanillin and maltol by approximately 9- and 4-fold, respectively. The thermodynamic parameters of binding indicated that the flavor-protein interactions were spontaneous (negative ΔG°) and that the driving force of the interactions shifted from entropy to enthalpy driven as a result of deamidation. Deamidation of soy protein caused a change in the mechanism of binding from hydrophobic interactions or covalent bonding (Schiff base formation) to weaker van der Waals forces or hydrogen bonding. PMID:22831747

  20. Effect of repeated benzene inhalation exposures on benzene metabolism, binding to hemoglobin, and induction of micronuclei.

    PubMed

    Sabourin, P J; Sun, J D; MacGregor, J T; Wehr, C M; Birnbaum, L S; Lucier, G; Henderson, R F

    1990-05-01

    Metabolism of benzene is thought to be necessary to produce the toxic effects, including carcinogenicity, associated with benzene exposure. To extrapolate from the results of rodent studies to potential health risks in man, one must know how benzene metabolism is affected by species, dose, dose rate, and repeated versus single exposures. The purpose of our studies was to determine the effect of repeated inhalation exposures on the metabolism of [14C]benzene by rodents. Benzene metabolism was assessed by characterizing and quantitating urinary metabolites, and by quantitating 14C bound to hemoglobin and micronuclei induction. F344/N rats and B6C3F1 mice were exposed, nose-only, to 600 ppm benzene or to air (control) for 6 hr/day, 5 days/week for 3 weeks. On the last day, both benzene-pretreated and control animals were exposed to 600 ppm, 14C-labeled benzene for 6 hr. Individual benzene metabolites in urine collected for 24 hr after the exposure were analyzed. There was a significant decrease in the respiratory rate of mice (but not rats) pretreated with benzene which resulted in lower levels of urinary [14C]benzene metabolites. The analyses indicated that the only effects of benzene pretreatment on the metabolite profile in rat or mouse urine were a slight shift from glucuronidation to sulfation in mice and a shift from sulfation to glucuronidation in rats. Benzene pretreatment also had no effect, in either species, on formation of [14C]benzene-derived hemoglobin adducts. Mice and rats had similar levels of hemoglobin adduct binding, despite the higher metabolism of benzene by mice. This indicates that hemoglobin adduct formation occurs with higher efficiency in rats. After 1 week of exposure to 600 ppm benzene, the frequency of micronucleated, polychromatic erythrocytes (PCEs) in mice was significantly increased. Exposure to the same level of benzene for an additional 2 weeks did not further increase the frequency of micronuclei in PCEs. These results indicate

  1. 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

  2. The Effect of Environment on the Recognition and Binding of Vancomycin to Native and Resistant Forms of Lipid II

    PubMed Central

    Jia, ZhiGuang; O'Mara, Megan L.; Zuegg, Johannes; Cooper, Matthew A.; Mark, Alan E.

    2011-01-01

    Molecular dynamics simulations and free energy calculations have been used to examine in detail the mechanism by which a receptor molecule (the glycopeptide antibiotic vancomycin) recognizes and binds to a target molecule (lipid II) embedded within a membrane environment. The simulations show that the direct interaction of vancomycin with lipid II, as opposed to initial binding to the membrane, leads most readily to the formation of a stable complex. The recognition of lipid II by vancomycin occurred via the N-terminal amine group of vancomycin and the C-terminal carboxyl group of lipid II. Despite lying at the membrane-water interface, the interaction of vancomycin with lipid II was found to be essentially identical to that of soluble tripeptide analogs of lipid II (Ac-d-Ala-d-Ala; root mean-square deviation 0.11 nm). Free energy calculations also suggest that the relative binding affinity of vancomycin for native, resistant, and synthetic forms of membrane-bound lipid II was unaffected by the membrane environment. The effect of the dimerization of vancomycin on the binding of lipid II, the position of lipid II within a biological membrane, and the effect of the isoamylene tail of lipid II on membrane fluidity have also been examined. PMID:22261057

  3. 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. PMID:19585467

  4. 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.

  5. Packing density of glycolipid biosurfactant monolayers give a significant effect on their binding affinity toward immunoglobulin G.

    PubMed

    Imura, Tomohiro; Masuda, Yuma; Ito, Seya; Worakitkanchanakul, Wannasiri; Morita, Tomotake; Fukuoka, Tokuma; Sakai, Hideki; Abe, Masahiko; Kitamoto, Dai

    2008-01-01

    Mannosylerythritol lipid-A (MEL-A) is one of the most promising glycolipid biosurfactants, and abundantly produced by Pseudozyma yeasts. MEL-A gives not only excellent self-assembling properties but also a high binding affinity toward human immunoglobulin G (HIgG). In this study, three kinds of MEL-A were prepared from methyl myristate [MEL-A (m)], olive oil [MEL-A (o)], and soybean oil [MEL-A (s)], and the effect of interfacial properties of each MEL-A monolayer on the binding affinity toward HIgG was investigated using surface plasmon resonance (SPR) and the measurement of surface pressure (pi)-area (A) isotherms. Based on GC-MS analysis, the main fatty acids were C(8) and C(10) acids in all MEL-A, and the content of unsaturated fatty acids was 0% for MEL-A (m), 9.1% for MEL-A (o), 46.3% for MEL-A (s), respectively. Interestingly, the acid content significantly influenced on their binding affinity, and the monolayer of MEL-A (o) gave a higher binding affinity than that of MEL-A (m) and MEL-A (s). Moreover, the mixed MEL-A (o)/ MEL-A (s) monolayer prepared from 1/1 molar ratio, which comprised of 27.8% of unsaturated fatty acids, indicated the highest binding affinity. At the air/water interface, MEL-A (o) monolayer exhibited a phase transition at 13 degrees C from a liquid condensed monolayer to a liquid expanded monolayer, and the area per molecule significantly expanded above 13 degrees C, while the amount of HIgG bound to the liquid expanded monolayer was much higher than that bound to liquid condensed monolayer. The binding affinity of MEL-A toward HIgG is thus likely to closely relate to the monolayer packing density, and may be partly controlled by temperature. PMID:18622124

  6. 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

  7. 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

  8. 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.

  9. 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. PMID:27267136

  10. The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.

    PubMed

    Denisov, Ilia G; Mak, Piotr J; Grinkova, Yelena V; Bastien, Dominic; Bérubé, Gervais; Sligar, Stephen G; Kincaid, James R

    2016-05-01

    Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4. PMID:26774838

  11. Quantitative Analysis of Multivalent Ligand Presentation on Gold Glyconanoparticles and Their Effects on Protein Binding

    NASA Astrophysics Data System (ADS)

    Wang, Xin; Ramström, Olof; Yan, Mingdi

    2010-03-01

    Bio-functionalized nanomaterials, which combine functions of biological ligands and unique properties of nano-sized building blocks, have exhibited increased potential applications in biosensing, therapeutics, and diagnostics. Glyconanoparitcles carrying a monolayer of carbohydrate ligands on nanoparticles provide an excellent platform for sensitive protein recognitions. Using Au nanoparticles as the scaffold, multivalent interactions between glycan ligands and proteins have been demonstrated. However, quantitative analysis especially the binding affinity of the resulting glyconanoparticles is challenging to determine. Here we present a new characterization technique, based on fluorescent competition binding assays, for measuring dissociation constants for glyconanoparticles-protein interactions. Au nanoparticles coupled with a series of un-derivatized carbohydrates were prepared by a photocoupling chemistry. Dramatic binding affinity enhancement was observed due to the high ligand density on nanoparticles, which was highly relevant to ligand display, controlled by the linker type, chain length, ligand size and density.

  12. Substituent effects on fluoride binding by lanthanide complexes of DOTA-tetraamides.

    PubMed

    Blackburn, Octavia A; Routledge, Jack D; Jennings, Laura B; Rees, Nicholas H; Kenwright, Alan M; Beer, Paul D; Faulkner, Stephen

    2016-02-21

    Fluoride binding by a series of europium and ytterbium complexes of DOTA-tetraamide ligands derived from primary, secondary and tertiary amides has been studied by NMR and luminescence spectroscopies. In all the systems studied, fluoride binding results in a change in the nature of the magnetic anisotropy at the metal centre from an easy axis, to an easy plane anisotropy. This results in reversal of the peaks in the NMR spectra, and in changes to the fine structure of the luminescence spectra. Furthermore, changes to the periphery of the binding cavity are implicated in determining the affinity constant for fluoride. There are clear differences in the entropic contribution to the free energy of activation between systems with benzylic amides and those with methylamides. PMID:26765788

  13. Effect of carbohydrate modifications of factor VIII/von Willebrand factor on binding to platelets.

    PubMed

    Goudemand, J; Mazurier, C; Samor, B; Bouquelet, S; Montreuil, J; Goudemand, M

    1985-06-24

    This study compares the ability of unmodified and carbohydrate-modified forms of factor VIII/von Willebrand factor (FVIII/vWF) protein to bind to platelets in the presence of ristocetin or thrombin. Treatment of intact FVIII/vWF with alpha-D-neuraminidase results in more than 95% desialylation. Asialo FVIII/vWF retains total activity in ristocetin- and thrombin-mediated binding to platelets as demonstrated by direct and competitive binding assays. Examination of its multimeric pattern by sodium dodecyl sulfate-agarose electrophoresis reveals a normal multimeric structure. Treatment of intact FVIII/vWF with beta-D-galactosidase results in the removal of 20% of galactose (agalacto FVIII/vWF) whereas 55% of galactose is released from asialo FVIII/vWF (asialo agalacto FVIII/vWF). Agalacto and asialo-agalacto FVIII/vWF are both unable to bind to platelets in the presence of ristocetin. In contrast, they still bind to thrombin-stimulated human (except thrombasthenic) platelets. Removal of either ultimate (agalacto FVIII/vWF) or ultimate and penultimate (asialo-agalacto FVIII/vWF) galactose results in the same loss of the larger molecular weight multimers and in an increase of smaller multimers. These results suggest (1) that sialic acid does not play a significant role in ristocetin- or thrombin-mediated FVIII/vWF-platelets interactions and multimeric structure of FVIII/vWF (2) that ultimate beta-linked galactose residues are essential for the maintenance of a normal multimer organization (3) that ristocetin- and thrombin-mediated binding of FVIII/vWF to platelets differ in FVIII/vWF galactose requirement.

  14. Comparison of Maximum Vasoactive Inotropic Score and Low Cardiac Output Syndrome As Markers of Early Postoperative Outcomes After Neonatal Cardiac Surgery

    PubMed Central

    Butts, Ryan J.; Scheurer, Mark A.; Atz, Andrew M.; Zyblewski, Sinai C.; Hulsey, Thomas C.; Bradley, Scott M.; Graham, Eric M.

    2014-01-01

    Low cardiac output syndrome (LCOS) and maximum vasoactive inotropic score (VIS) have been used as surrogate markers for early postoperative outcomes in pediatric cardiac surgery. The objective of this study was to determine the associations between LCOS and maximum VIS with clinical outcomes in neonatal cardiac surgery. This was a secondary retrospective analysis of a prospective randomized trial, and the setting was a pediatric cardiac intensive care unit in a tertiary care children's hospital. Neonates (n = 76) undergoing corrective or palliative cardiac operations requiring cardiopulmonary bypass were prospectively enrolled. LCOS was defined by a standardized clinical criteria. VIS values were calculated by a standard formula during the first 36 postoperative hours, and the maximum score was recorded. Postoperative outcomes included hospital mortality, duration of mechanical ventilation, intensive care unit (ICU) and hospital lengths of stay (LOS), as well as total hospital charges. At surgery, the median age was 7 days and weight was 3.2 kg. LCOS occurred in 32 of 76 (42%) subjects. Median maximum VIS was 15 (range 5–33). LCOS was not associated with duration of mechanical ventilation, ICU LOS, hospital LOS, and hospital charges. Greater VIS was moderately associated with a longer duration of mechanical ventilation (p = 0.001, r = 0.36), longer ICU LOS (p = 0.02, r = 0.27), and greater total hospital costs (p = 0.05, r = 0.22) but not hospital LOS (p = 0.52). LCOS was not associated with early postoperative outcomes. Maximum VIS has only modest correlation with duration of mechanical ventilation, ICU LOS, and total hospital charges. PMID:22349666

  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. 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.

  17. 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.

  18. The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

    PubMed

    Beharry, S; Bragg, P D

    1991-04-01

    Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.

  19. Intersubjective action-effect binding: eye contact modulates acquisition of bidirectional association between our and others' actions.

    PubMed

    Sato, Atsushi; Itakura, Shoji

    2013-06-01

    In everyday social life, we predict others' actions in response to our own actions. Subsequently, on the basis of these predictions, we control our actions to attain desired social outcomes and/or adjust our actions to accommodate the anticipated actions of the others. Representation of the bidirectional association between our and others' actions, that is, intersubjective action-effect binding, could make such intersubjective action control easier and smoother. The present study investigated not only whether or not intersubjective action-effect binding was acquired but also whether or not eye contact modulated it. Experiment 1 showed that after a repeated experience during which participants' finger movements triggered a target female individual's mouth gesture, observing the target's mouth gestures came to automatically trigger the participants' finger movements. Experiments 2 and 3 revealed that this effect was not observed when the target's gaze direction was averted (Experiment 2) or when the target's eyes were closed (Experiment 3) throughout the acquisition phase. These results indicate that intersubjective action-effect binding occurs and that an ostensive signal, that is, eye contact modulates it.

  20. 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

    2014-11-01

    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.

  1. 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

  2. A gel electrophoresis study of the competitive effects of monovalent counterion on the extent of divalent counterions binding to DNA.

    PubMed Central

    Li, A Z; Huang, H; Re, X; Qi, L J; Marx, K A

    1998-01-01

    The behavior of alkaline earth metal cations (Mg2+ and Ca2+) and transition metal cations (Zn2+ and Cu2+) interacting with lambda-DNA-HindIII fragments ranging from 2,027 to 23,130 bp in Tris-borate-EDTA buffer solutions was investigated. The divalent counterions competed with Tris+ and Na+ for binding to polyion DNA, and the competition binding situations were investigated by measuring the reduction of the DNA mobility, by pulsed- or constant-field gel electrophoresis. The interaction of Mg2+ with DNA was intensively studied over a wide range of Mg2+ concentrations. In addition, we examined the competition binding as a function of ionic strength and DNA size. To compare valence effects, we studied Co(NH3)6(3+) interaction with DNA fragments under conditions similar to that of Mg2+. At relatively low Mg2+ concentration, the normalized titration curves of DNA mobility were well fit by Manning's two-variable counterion condensation (CC) theory. The agreement between the predicted value (total charge neutralization fraction theta) from Manning's CC theory and the data based on our measured DNA electrophoretic mobility reduction was consistent under our experimental conditions. In contrast to alkaline earth metal cations (Mg2+ and Ca2+), different binding behaviors were observed for the transition metal cations (Zn2+ and Cu2+). These differences highlight the usefulness of our reduced DNA electrophoretic mobility measurement approach to describing cation interactions with polyelectrolyte DNA. PMID:9533707

  3. A gel electrophoresis study of the competitive effects of monovalent counterion on the extent of divalent counterions binding to DNA.

    PubMed

    Li, A Z; Huang, H; Re, X; Qi, L J; Marx, K A

    1998-02-01

    The behavior of alkaline earth metal cations (Mg2+ and Ca2+) and transition metal cations (Zn2+ and Cu2+) interacting with lambda-DNA-HindIII fragments ranging from 2,027 to 23,130 bp in Tris-borate-EDTA buffer solutions was investigated. The divalent counterions competed with Tris+ and Na+ for binding to polyion DNA, and the competition binding situations were investigated by measuring the reduction of the DNA mobility, by pulsed- or constant-field gel electrophoresis. The interaction of Mg2+ with DNA was intensively studied over a wide range of Mg2+ concentrations. In addition, we examined the competition binding as a function of ionic strength and DNA size. To compare valence effects, we studied Co(NH3)6(3+) interaction with DNA fragments under conditions similar to that of Mg2+. At relatively low Mg2+ concentration, the normalized titration curves of DNA mobility were well fit by Manning's two-variable counterion condensation (CC) theory. The agreement between the predicted value (total charge neutralization fraction theta) from Manning's CC theory and the data based on our measured DNA electrophoretic mobility reduction was consistent under our experimental conditions. In contrast to alkaline earth metal cations (Mg2+ and Ca2+), different binding behaviors were observed for the transition metal cations (Zn2+ and Cu2+). These differences highlight the usefulness of our reduced DNA electrophoretic mobility measurement approach to describing cation interactions with polyelectrolyte DNA.

  4. Chromatin binding and polymerization of the endogenous Xenopus egg lamins: the opposing effects of glycogen and ATP.

    PubMed

    Lourim, D; Krohne, G

    1998-12-18

    We have previously identified and quantitated three B-type lamin isoforms present in the nuclei of mature Xenopus laevis oocytes, and in cell-free egg extracts. As Xenopus egg extracts are frequently used to analyze nuclear envelope assembly and lamina functions, we felt it was imperative that the polymerization and chromatin-binding properties of the endogenous B-type egg lamins be investigated. While we have demonstrated that soluble B-type lamins bind to chromatin, we have also observed that the polymerization of egg lamins does not require membranes or chromatin. Lamin assembly is enhanced by the addition of glycogen/glucose, or by the depletion of ATP from the extract. Moreover, the polymerization of egg cytosol lamins and their binding to demembranated sperm or chromatin assembled from naked lambda-DNA is inhibited by an ATP regeneration system. These ATP-dependent inhibitory activities can be overcome by the coaddition of glycogen to egg cytosol. We have observed that glycogen does not alter ATP levels during cytosol incubation, but rather, as glycogen-enhanced lamin polymerization is inhibited by okadaic acid, we conclude that glycogen activates protein phosphatases. Because protein phosphatase 1 (PP1) is the only phosphatase known to be specifically regulated by glycogen our data indicate that PP1 is involved in lamin polymerization. Our results show that ATP and glycogen effect lamin polymerization and chromatin binding by separate and opposing mechanisms. PMID:9819358

  5. Binding of protein synthesis initiation factor 4E to oligoribonucleotides: effects of cap accessibility and secondary structure.

    PubMed

    Carberry, S E; Friedland, D E; Rhoads, R E; Goss, D J

    1992-02-11

    The binding of rabbit globin mRNA to the 25-kDa cap binding protein eIF-4E from human erythrocytes was found to be 5.3-fold stronger than the binding of the cap analogue m7GpppG to eIF-4E [Gross et al. (1990) Biochemistry 29, 5008-5012]. In order to investigate whether this effect is due to the longer sequence of nucleotides in globin mRNA or to other features such as cap accessibility or secondary structure, oligoribonucleotide analogues of rabbit alpha-globin mRNA were synthesized by T7 RNA polymerase from a synthetic oligodeoxynucleotide template in the presence of m7GpppG; these oligoribonucleotide analogues possess varying degrees of cap accessibility and secondary structure. Equilibrium association constants for the interaction of these oligoribonucleotides and purified human erythrocyte eIF-4E were obtained from direct fluorescence titration experiments. The data indicate that while the presence of the m7G cap is required for efficient recognition by eIF-4E, the cap need not be completely sterically accessible, since other structural features within the mRNA also influence binding.

  6. Binding of glycosaminoglycans to cyano-activated agarose membranes: kinetic and diffusional effects on yield and homogeneity.

    PubMed

    Mattern, Kristin J; Deen, William M

    2007-11-01

    Methods were developed for binding a glycosaminoglycan (GAG, a 50 kDa chondroitin sulfate) to thin agarose membranes using 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) as the activating agent. Process conditions were optimized to achieve high yields and spatially uniform concentrations of bound ligand. Yields were varied mainly by manipulating the duration and temperature of the aqueous washes prior to coupling, which affected the concentration of active sites available for subsequent GAG binding. The rate constants for degradation of the active cyanate esters in 0.1M bicarbonate solutions were 0.24+/-0.02 h(-1) at 4 degrees C and 0.08+/-0.03 h(-1) at 0 degrees C. Steric limitations in the 3% agarose gels severely restricted binding, with only about 0.1% of active sites being accessible to GAG molecules. The GAG binding occurred primarily in the outer 50-70 microm of the membranes, so that coupling was homogeneous only for thin gels. A model of GAG diffusion and reaction in the coupling step was developed to explain the observed effects of parameters such as the GAG concentration in solution and the membrane thickness. An analysis of the key time scales in the synthesis provides design principles that should be useful also for other cyanylating agents, other ligands, and for beads as well as membranes. PMID:17610855

  7. Effects of macromolecular crowding on a small lipid binding protein probed at the single-amino acid level.

    PubMed

    Pérez Santero, Silvia; Favretto, Filippo; Zanzoni, Serena; Chignola, Roberto; Assfalg, Michael; D'Onofrio, Mariapina

    2016-09-15

    Macromolecular crowding is a distinctive feature of the cellular interior, influencing the behaviour of biomacromolecules. Despite significant advancements in the description of the effects of crowding on global protein properties, the influence of cellular components on local protein attributes has received limited attention. Here, we describe a residue-level systematic interrogation of the structural, dynamic, and binding properties of the liver fatty acid binding protein (LFABP) in crowded solutions. Two-dimensional NMR spectral fingerprints and relaxation data were collected on LFABP in the presence of polymeric and biomolecular crowders. Non-interacting crowders produced minimal site-specific spectral perturbations on ligand-free and lipid-bound LFABP. Conformational adaptations upon ligand binding reproduced those observed in dilute solution, but a perturbation of the free oleate state resulted in less favorable uptake. When LFABP engaged in direct interactions with background molecules, changes in local chemical environments were detected for residues of the internal binding pocket and of the external surface. Enhanced complexity was introduced by investigating LFABP in cell lysates, and in membrane-bounded compartments. LFABP was able to capture ligands from prokaryotic and eukaryotic cell lysates, and from artificial cells (water-in-oil emulsion droplets). The data suggest that promiscuous interactions are a major factor influencing protein function in the cell. PMID:27457417

  8. Predicting Allosteric Effects from Orthosteric Binding in Hsp90-Ligand Interactions: Implications for Fragment-Based Drug Design

    PubMed Central

    Larsson, Andreas; Nordlund, Paer; Jansson, Anna; Anand, Ganesh S.

    2016-01-01

    A key question in mapping dynamics of protein-ligand interactions is to distinguish changes at binding sites from those associated with long range conformational changes upon binding at distal sites. This assumes a greater challenge when considering the interactions of low affinity ligands (dissociation constants, KD, in the μM range or lower). Amide hydrogen deuterium Exchange mass spectrometry (HDXMS) is a robust method that can provide both structural insights and dynamics information on both high affinity and transient protein-ligand interactions. In this study, an application of HDXMS for probing the dynamics of low affinity ligands to proteins is described using the N-terminal ATPase domain of Hsp90. Comparison of Hsp90 dynamics between high affinity natural inhibitors (KD ~ nM) and fragment compounds reveal that HDXMS is highly sensitive in mapping the interactions of both high and low affinity ligands. HDXMS reports on changes that reflect both orthosteric effects and allosteric changes accompanying binding. Orthosteric sites can be identified by overlaying HDXMS onto structural information of protein-ligand complexes. Regions distal to orthosteric sites indicate long range conformational changes with implications for allostery. HDXMS, thus finds powerful utility as a high throughput method for compound library screening to identify binding sites and describe allostery with important implications for fragment-based ligand discovery (FBLD). PMID:27253209

  9. Binding of glycosaminoglycans to cyano-activated agarose membranes: kinetic and diffusional effects on yield and homogeneity.

    PubMed

    Mattern, Kristin J; Deen, William M

    2007-11-01

    Methods were developed for binding a glycosaminoglycan (GAG, a 50 kDa chondroitin sulfate) to thin agarose membranes using 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) as the activating agent. Process conditions were optimized to achieve high yields and spatially uniform concentrations of bound ligand. Yields were varied mainly by manipulating the duration and temperature of the aqueous washes prior to coupling, which affected the concentration of active sites available for subsequent GAG binding. The rate constants for degradation of the active cyanate esters in 0.1M bicarbonate solutions were 0.24+/-0.02 h(-1) at 4 degrees C and 0.08+/-0.03 h(-1) at 0 degrees C. Steric limitations in the 3% agarose gels severely restricted binding, with only about 0.1% of active sites being accessible to GAG molecules. The GAG binding occurred primarily in the outer 50-70 microm of the membranes, so that coupling was homogeneous only for thin gels. A model of GAG diffusion and reaction in the coupling step was developed to explain the observed effects of parameters such as the GAG concentration in solution and the membrane thickness. An analysis of the key time scales in the synthesis provides design principles that should be useful also for other cyanylating agents, other ligands, and for beads as well as membranes.

  10. Effects of aromatic compounds on antennal responses and on the pheromone-binding proteins of the gypsy moth (Lymantria dispar).

    PubMed

    Gong, Yongmei; Plettner, Erika

    2011-03-01

    Female gypsy moths emit a pheromone, (+)-disparlure, which the males follow until they locate the emitter. The male moths' antennae are covered with innervated sensory hairs, specialized in detection of the pheromone. The neurons in these sensory hairs are bathed by a solution rich in pheromone-binding protein (PBP). PBPs are soluble proteins that bind the pheromone and other odorants reversibly with variable thermodynamic and kinetic selectivity and are essential for olfactory responses. Here, we have studied the interaction between 2 gypsy moth PBPs with aromatic compounds that modulate the responses of male moth antennae to (+)-disparlure. The aromatic compounds do not elicit responses by themselves, but when administered together with pheromone, they inhibit, enhance, or prolong the electrophysiological response to the pheromone. Three interactions between the compounds and PBPs were studied: 1) the equilibrium binding of the compounds by themselves to the PBPs, 2) the equilibrium binding of the compounds in the presence of pheromone or a fluorescent reporter ligand, and 3) the effect of the compounds on the conformation of the pheromone-PBP complex. A subset of compounds causes a prolongation of the electroantennogram response, and from this study, we conclude that these compounds follow a structure-activity pattern and stabilize a particular conformer of the PBPs that appears to activate the olfactory response.

  11. 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

  12. The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea-pig ventricular myocytes.

    PubMed Central

    Guerra, L.; Cerbai, E.; Gessi, S.; Borea, P. A.; Mugelli, A.

    1996-01-01

    1. We used electrophysiological and binding techniques to determine the effects of oxygen free radicals (OFRs) generated by dihydroxyfumaric acid (DHF, 5 mM) on calcium current and dihydropyridine binding sites in guinea-pig isolated ventricular myocytes. 2. Binding of [3H]-PN200-110 to isolated ventricular myocytes revealed one population of binding sites with a KD of 0.11 +/- 0.01 nM and Bmax of 139.1 +/- 6.9 fmol mg-1 protein (n = 24). After 15 min of exposure to DHF, the density, but not the affinity of [3H]-PN200-110 binding sites was significantly (P < 0.01) reduced to 35% of the control value (Bmax = 49.4 +/- 3.7 fmol mg-1 protein, KD = 0.11 +/- 0.01 nM, n = 15). In the presence of superoxide dismutase (SOD) and catalase (CAT) the reduction in [3H]-PN200-110 binding sites was almost completely prevented (Bmax = 120.5 +/- 7.4 in control, n = 4 and 98.8 +/- 7.4 fmol mg-1 protein in DHF plus SOD and CAT, n = 4). KD values were not modified (0.08 +/- 0.01 in control and 0.09 +/- 0.01 nM in DHF plus SOD and CAT). 3. The time-course of the reduction of [3H]-PN200-110 binding sites by OFRs was paralleled by the decrease in L-type calcium current (Ica,L) measured in patch-clamped guinea-pig ventricular myocytes either in the absence or in the presence of EGTA in the patch pipette. In the former conditions OFRs induced the appearance of calcium-dependent alterations, i.e. the transient inward current, within 10 min. After 30 min of incubation with DHF, [3H]-PN200-110 binding sites were reduced to 25% of the control value. 4. In myocytes incubated with the antilipoperoxidant agent, butylated hydroxytoluene (BHT, 50 microM), the decrease in [3H]-PN200-110 binding sites caused by DHF was partially prevented (Bmax values after 30 min exposure to DHF were 55.5 +/- 1.9 and 23.7 +/- 5.9 fmol mg-1 protein in the presence and in the absence of BHT respectively, P < 0.05). BHT did not affect the decrease in [3H]-PN200-110 binding sites during the first 15 min of exposure to

  13. 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...

  14. 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…

  15. Effect of Dioxygen on Copper(II) Binding to α-Synuclein

    PubMed Central

    Lucas, Heather R.; Lee, Jennifer C.

    2010-01-01

    Using the fluorescent amino acid tryptophan (Trp), we have characterized the copper(II) binding of F4W α-synuclein in the presence and absence of dioxygen at neutral pH. Variations in Trp fluorescence indicate that copper(II) binding is enhanced by the presence of dioxygen, with the apparent dissociation constant (Kd(app)) changing from 100 nM (anaerobic) to 10 nM (aerobic). To investigate the possible role of methionine oxidation, complementary work focused on synthetic peptide models of the N-terminal Cu(II)-α-syn site, MDV(F/W) and M*DV(F/W), where M*= methionine sulfoxide. Furthermore, we employed circular dichroism (CD) spectroscopy to demonstrate that the phenyl-to-indole (F→W) substitution does not alter copper(II) binding properties and to confirm the 1:1 metal-peptide binding stoichiometry. CD comparisons also revealed that Met1 oxidation does not affect the copper-peptide conformation and further suggested the possible existence of a CuII-Trp/Phe (cation-π) interaction. PMID:20064662

  16. Effect of dioxygen on copper(II) binding to alpha-synuclein.

    PubMed

    Lucas, Heather R; Lee, Jennifer C

    2010-03-01

    Using the fluorescent amino acid tryptophan (Trp), we have characterized the copper(II) binding of F4W alpha-synuclein in the presence and absence of dioxygen at neutral pH. Variations in Trp fluorescence indicate that copper(II) binding is enhanced by the presence of dioxygen, with the apparent dissociation constant (K(d(app))) changing from 100nM (anaerobic) to 10nM (aerobic). To investigate the possible role of methionine oxidation, complementary work focused on synthetic peptide models of the N-terminal Cu(II)-alpha-syn site, MDV(F/W) and M( *)DV(F/W), where M( *)=methionine sulfoxide. Furthermore, we employed circular dichroism (CD) spectroscopy to demonstrate that the phenyl-to-indole (F-->W) substitution does not alter copper(II) binding properties and to confirm the 1:1 metal-peptide binding stoichiometry. CD comparisons also revealed that Met1 oxidation does not affect the copper-peptide conformation and further suggested the possible existence of a Cu(II)-Trp/Phe (cation-pi) interaction. PMID:20064662

  17. Co-administration of Apelin and T4 Protects Inotropic and Chronotropic Changes Occurring in Hypothyroid Rats

    PubMed Central

    Akhondali, Zahra; Badavi, Mohammad; Dianat, Mahin; Faraji, Farzaneh

    2015-01-01

    Background One of the most important thyroid hormone targets is the cardiovascular system. Hemodynamic changes, such as decreased resting heart rate (HR), myocardial contractility, and cardiac output, and increased diastolic pressure and systemic vascular resistance, have been observed in hypothyroid patients. Moreover, in these patients, ECG changes include sinus bradycardia and low voltage complexes (P waves or QRS complexes). Objective This study aimed at evaluating the prophylactic effect of apelin on HR changes and QRS voltage that occur in propylthiouracil (PTU)-induced hypothyroid rats. Method In this study, 48 adult male Wistar rats weighing 170-235g were randomly divided into 6 groups: Control group (normal saline ip injection + tap water gavage); P group (PTU 0.05%, in drinking water); A group (apelin 200 µg.kg-1.day-1, ip); PA group [co-administration of PTU and apelin]; PT group [co-administration of PTU + T4 (0.2 mg/g per day, gavage)]; and PAT group (co-administration of PTU, apelin and T4). All experiments were performed for 28 consecutive days, and then the animals were anesthetized with an ip injection of ketamine (80 mg/kg) and xylazine (12 mg/kg). Lead II electrocardiogram was recorded to calculate HR and QRS voltage. Results Heart rate and QRS voltage increased more significantly in the hypothyroid group that consumed both apelin and T4 (201 ± 4 beat/min, 0.71 ± 0.02 mv vs. hypothyroid 145 ± 9 beat/min, 0.563 ± 0.015 mv; respectively). Conclusion The co-administration of apelin and T4 showed a protective effect on QRS voltage and HR in PTU‑induced hypothyroid rats. PMID:26247243

  18. Mechanism of the negative inotropic action of leukotrienes C4 and D4 on isolated rat heart.

    PubMed

    Bittl, J A; Pfeffer, M A; Lewis, R A; Mehrotra, M M; Corey, E J; Austen, K F

    1985-07-01

    Leukotrienes C4 and D4 (LTC4 and LTD4), possible mediators of cardiac dysfunction during inflammatory injury, may depress cardiac function by reducing coronary flow or by exerting a negative effect directly on the myocardium. We used an isovolumic rat heart preparation perfused at constant pressure and measured left ventricular developed pressure (mmHg), coronary flow (ml.min-1), oxygen extraction, and myocardial oxygen consumption and delivery (mumol O2.[gramme dry weight]-1.min-1) during infusion of five doses of angiotensin II, LTC4, LTD4 (approximately 10 to approximately 300 pmol.min-1), and noradrenaline (400 to 2000 pmol.min-1), or perfusion with medium which contained calcium at half-concentration. LTC4 and LTD4 were equipotent with angiotensin. At low effective doses, increased oxygen extraction offset the decrease in oxygen delivery, maintaining a stable level of oxygen consumption and left ventricular developed pressure. At the highest doses, angiotensin, LTC4 and LTD4 reduced coronary flow from 21 to 15, 21 to 13, and 21 to 13 ml.min-1, respectively. And, despite greater oxygen extraction of 59%, 58% and 65% for angiotensin, LTC4 and LTD4, left ventricular developed pressure fell from a baseline of 120 mmHg to 113, 106 and 92, respectively. In contrast, low calcium perfusion reduced left ventricular developed pressure (126 to 92) and oxygen extraction (46 to 30%) without changing coronary flow or oxygen delivery. These results suggest that LTC4 and LTD4 are potent coronary vasoconstricting agents which depress cardiac function by limiting oxygen delivery.

  19. The effect of anabolic-androgenic steroids on aromatase activity and androgen receptor binding in the rat preoptic area.

    PubMed

    Roselli, C E

    1998-05-11

    The level of aromatase in the preoptic area of rats is transcriptionally regulated through a specific androgen-receptor mediated mechanism and can be used as a measure of central androgenic effect. Therefore, several commonly abused anabolic-androgenic steroids (AAS) were tested for their ability to induce aromatase activity in the preoptic area of castrated rats. In addition, we determined the relative binding affinities of these compounds for the androgen receptor, as well as their ability to bind androgen receptor in vivo following subcutaneous injections. All of the AAS compounds tested significantly stimulated POA aromatase activity above castrate levels. The compounds that produced the greatest stimulation of aromatase activity were those that bound most avidly to the androgen receptor in vitro (i.e., testosterone, dihydrotestosterone and nandrolone). In contrast, the 17alpha-alkylated compounds that were tested (stanozolol, danazol, methandrostenolone) modestly stimulated aromatase and were weak competitors for the androgen receptor. The subcutaneous injection of AAS compounds increased the concentrations of occupied nuclear androgen receptors in the brain, but the magnitude of effect was not related to their potency for inducing aromatase or their relative binding affinity for the androgen receptor suggesting that androgen receptor occupancy in POA is not correlated with the action of androgen on aromatase. The present results help explain the behavioral effects of AAS compounds in rats. PMID:9593936

  20. Comparison of operator-specific and nonspecific DNA binding of the lambda cI repressor: [KCl] and pH effects.

    PubMed

    Senear, D F; Batey, R

    1991-07-01

    The effects of proton and KCl activity on the nonspecific lambda cI repressor-DNA interactions and on the site-specific repressor-O(R) interactions were compared, in order to assess their roles in site specificity. The repressor-O(R) interactions were studied by using DNase I footprint titration. The Gibbs free energy changes for binding and for cooperativity were determined between 25 and 300 mM KCl, from individual-site isotherms for the binding of repressor to O(R) and to reduced-valency mutants. The proton-linked effects on repressor-O(R) interactions have been published [Senear, D. F., & Ackers, G. K. (1990) Biochemistry 29, 6568-6577; Senear, D. F., & Bolen, D. W. (1991) Methods Enzymol. (in press)]. Nonspecific binding was studied by using a nitrocellulose filter binding assay, which proved advantageous in this case, due to the relatively weak nonspecific binding, and precipitation of repressor-DNA complexes. Filter binding provided measurements at low binding density where precipitation did not occur. The data provide estimates of the Gibbs free energy changes for nonspecific, intrinsic binding, but not for cooperativity. The KCl concentration dependencies of the intrinsic binding constants indicate that ion release plays similar roles in distinguishing between the operators and in discriminating operator from nonoperator DNA. Binding to DNA is accompanied by net proton absorption. Near neutral pH, proton linkages to operator and nonoperator binding are the same. Differences at acid and at basic pH implicate the same ionizable repressor groups in distinguishing between the operators and in discriminating operator from nonoperator DNA. The results indicate similar overall modes of operator and nonoperator binding of repressor, but implicate indirect effects of DNA sequence as important contributors to sequence recognition.

  1. Effect of Binding Pose and Modeled Structures on SVMGen and GlideScore Enrichment of Chemical Libraries.

    PubMed

    Xu, David; Meroueh, Samy O

    2016-06-27

    Virtual screening consists of docking libraries of small molecules to a target protein followed by rank-ordering of the resulting structures using scoring functions. The ability of scoring methods to distinguish between actives and inactives depends on several factors that include the accuracy of the binding pose during the docking step and the quality of the three-dimensional structure of the target. Here, we build on our previous work to introduce a new scoring approach (SVMGen) that uses machine learning trained with features from statistical pair potentials obtained from three-dimensional crystal structures. We use SVMGen and GlideScore to explore how enrichment or rank-ordering is affected by binding pose accuracy. To that end, we create a validation set that consists strictly of proteins whose crystal structure was solved in complex with their inhibitors. For the rank-ordering studies, we use crystal structures from PDBbind along with corresponding binding affinity data provided in the database. In addition to binding pose, we investigate the effect of using modeled structures for the target on the enrichment performance of SVMGen and GlideScore. To accomplish this, we generated homology models for protein kinases in DUD-E for which crystal structures are available to enable comparison of enrichment between modeled and crystal structure. We also generate homology models for kinases in SARfari for which there are many known small-molecule inhibitors but no known crystal structure. These models are used to assess the ability of SVMGen and GlideScore to distinguish between actives and decoys. We focus our work on protein kinases considering the wealth of structural and binding affinity data that exists for this family of proteins.

  2. Effects of verocytotoxin-1 on nonadherent human monocytes: binding characteristics, protein synthesis, and induction of cytokine release.

    PubMed

    van Setten, P A; Monnens, L A; Verstraten, R G; van den Heuvel, L P; van Hinsbergh, V W

    1996-07-01

    The epidemic form of the hemolytic uremic syndrome (HUS) has been associated with a verocytotoxin producing Escherichia coli infection. Endothelial cell damage of glomeruli and arterioles of the kidney plays a central role in the pathogenesis of HUS. A number of observations in vivo and in vitro indicate that inflammatory mediators contribute to this process. In this study we investigated the binding of 125I-verocytotoxin-1 (VT-1) to freshly isolated human nonadherent monocytes as well as the nature of the ligand to which VT-1 binds on monocytes. On the average, freshly isolated monocytes have 0.07 x 10(5) specific binding sites for 125I-VT-1 per cell. Preincubation of nonadherent monocytes with bacterial lipopolysaccharide (LPS) caused a 23- to 30-fold increase of specific binding sites for VT-1 as shown by Scatchard plot analysis. Thin-layer chromatography of extracted neutral glycolipids of the cells and subsequent binding of 125I-VT-1 showed that human monocytes bind VT-1 to a globotriaosylceramide (Gb3) species that is different from that found on endothelial cells, probably a short-chain fatty acyl Gb3 or an alpha-OH-Gb3. In addition, we evaluated the functional consequences of VT-1 binding to human monocytes by investigating the effects of VT-1 on the total protein synthesis and, specifically, the production of the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8. We observed that VT-1 did not inhibit overall protein synthesis, nor under basal conditions, neither after stimulation with LPS, in contrast to previous observations with endothelial cells. Furthermore, we found that VT-1 induces the synthesis of the cytokines IL-1 beta, TNF-alpha, IL-6, and IL-8 in nonstimulated monocytes by a LPS-independent cell activation. The increase in the production of cytokines was parallelled by an increase in mRNA, as was demonstrated for IL-6 by reverse transcription-polymerase chain reaction. These data suggest that

  3. Effects of adenosine triphosphate and alkaline phosphatase on solubilized 3,5,3'-triiodothyronine-binding activity.

    PubMed

    Faure, R; Dussault, J H

    1988-09-01

    The T3-binding activity of salt-extractable nuclear proteins from rat liver was affected when ATP (2-10 mM; pH 8.0) was added concomitantly with T3 in the incubation medium. Scatchard analysis revealed that the equilibrium association constant was significantly reduced [5 mM ATP, 0.3 +/- 0.1 (+/- SE) 10(10) M-1; control, 1.1 +/- 0.15 X 10(10) M-1], but the maximum binding capacity remained unchanged. Similar values of inhibition were obtained when unbound receptors were preincubated with ATP. ATP achieved its maximal effect after 45 min of incubation at 30 C. Dilution experiments indicated that the effect of ATP was reversible. The inhibiting potency of nucleoside triphosphates at pH 8.0 was in the following order: ATP = CTP greater than GTP, whereas UTP had no effect. Nonhydrolyzable analogs of ATP were also inhibitory, and HPLC fractionation showed an approximately 98% recovery of ATP after incubation with nuclear extract. The adenine ring with at least two phosphates was essential, since ADP was as potent as ATP, whereas AMP had no effect. When the pH of the incubation medium was lowered to 7.3, the T3-binding activity was inhibited by ATP in the 0.1-1 mM range. Magnesium (3 mM) greatly increases the ATP effect at pH 7.3, but not at pH 8. The T3-binding activity was also drastically reduced when calf intestine alkaline phosphatase was added concomitantly in the incubation medium. Eight micrograms per ml enzyme were necessary to inhibit the T3 specific binding by 50% (30 C for 45 min). Scatchard analysis showed that the receptor affinity for T3 was decreased (control, 1.1 +/- 0.02 x 10(10) M-1; alkaline phosphatase, 0.41 +/- 0.03 x 10(10) M-1; n = 6), whereas the maximum binding capacity remained unchanged. Incubations performed with increasing concentrations of beta-mercaphoethanol (2.5, 5, 10, and 25 mM) revealed that the phosphatase inhibitory effect is thiol dependent. The inhibition was maximal at 2.5 mM and progressively decreased at 5 and 10 mM. No

  4. Effect of bioceramic functional groups on drug binding and release kinetics

    NASA Astrophysics Data System (ADS)

    Trujillo, Christopher

    release the amount of Vanc available for release for samples P-0 and P-331 was .873 mg and 1.106 mg, respectively. These results indicated the effect of phosphate content on decreasing the drug release rate. The drug release kinetics study showed that the release of phosphate ions from the surface of Cris prior to drug loading exposed active silicate functional groups that enhanced drug binding by physisorption which in turn facilitated rapid release kinetics. On the other hand, a slower drug release rate was observed as the phosphate functional groups increased on the material surface due to chemisorption. Results from the present study indicate that it is possible to enhance the burst release stage of a bioceramic drug carrier by increasing the silicate functional groups. The sustained release profile can be engineered by controlling the phosphate content of the bioceramic drug carrier.

  5. Isolation of Prunin from the fruit shell of Bixa orellana and the effect of β-cyclodextrin on its binding with calf thymus DNA.

    PubMed

    Yousuf, Sameena; Sudha, N; Murugesan, G; Enoch, Israel V M V

    2013-01-10

    Naringenin-7-O-glucoside [Prunin (Pru)] was isolated from the fruit shell of Bixa orellana L. The binding of Pru with calf thymus DNA (ctDNA) and the influence of cyclomaltoheptaose (β-cyclodextrin, β-CD) on the binding were studied by absorption and fluorescence spectroscopic techniques. The comparison of the binding modes of Pru/β-CD and ctDNA-Pru/β-CD suggested that β-CD extracted Pru from DNA for forming inclusion complex. Molecular modeling gave added support to the above results. Fluorescence microscopy was used to visualize the effect of β-CD on the bindings. PMID:23202537

  6. 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.

  7. Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction.

    PubMed

    Risse, B; Stempfer, G; Rudolph, R; Möllering, H; Jaenicke, R

    1992-12-01

    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

  8. The effect of social stress on adrenal axis activity in horses: the importance of monitoring corticosteroid-binding globulin capacity.

    PubMed

    Alexander, S L; Irvine, C H

    1998-06-01

    Plasma cortisol is largely bound to corticosteroid-binding globulin (CBG), which regulates its bioavailability by restricting exit from capillaries. Levels of CBG may be altered by several factors including stress and this can influence the amount of cortisol reaching cells. This study investigated the effect of social instability on plasma concentrations of CBG, total and free (not protein bound) cortisol in horses. Horses new to our research herd ('newcomers') were confined in a small yard with four dominant resident horses for 3-4 h daily for 3-4 (n = 5) or 9-14 (n = 3) days. Jugular blood was collected in the mornings from newcomers before the period of stress began ('pre-stress'), and then before each day's stress. Residents were bled before stress on the first and thirteenth day. Residents always behaved aggressively towards newcomers. By the end of the stress period, all newcomers were subordinate to residents. In newcomers (n = 8) after 3-4 days of social stress, CBG binding capacity had fallen (P = 0.0025), while free cortisol concentrations had risen (P = 0.0016) from pre-stress values. In contrast, total cortisol did not change. In residents, CBG had decreased slightly but significantly (P = 0.0162) after 12 days of stress. Residents and newcomers did not differ in pre-stress CBG binding capacity, total or free cortisol concentrations. However, by the second week of stress, CBG binding capacity was lower (P = 0.015) and free cortisol higher (P = 0.030) in newcomers (n = 3) than in residents. Total cortisol did not differ between the groups. In conclusion social stress clearly affected the adrenal axis of subordinate newcomer horses, lowering the binding capacity of CBG and raising free cortisol concentrations. However, no effect of stress could be detected when only total cortisol was measured. Therefore, to assess adrenal axis status accurately in horses, it is essential to monitor the binding capacity of CBG and free cortisol concentrations in addition

  9. 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.

  10. 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.

  11. Effect of B-ring substitution pattern on binding mode of propionamide selective androgen receptor modulators.

    PubMed

    Bohl, Casey E; Wu, Zengru; Chen, Jiyun; Mohler, Michael L; Yang, Jun; Hwang, Dong Jin; Mustafa, Suni; Miller, Duane D; Bell, Charles E; Dalton, James T

    2008-10-15

    Selective androgen receptor modulators (SARMs) are essentially prostate sparing androgens, which provide therapeutic potential in osteoporosis, male hormone replacement, and muscle wasting. Herein we report crystal structures of the androgen receptor (AR) ligand-binding domain (LBD) complexed to a series of potent synthetic nonsteroidal SARMs with a substituted pendant arene referred to as the B-ring. We found that hydrophilic B-ring para-substituted analogs exhibit an additional region of hydrogen bonding not seen with steroidal compounds and that multiple halogen substitutions affect the B-ring conformation and aromatic interactions with Trp741. This information elucidates interactions important for high AR binding affinity and provides new insight for structure-based drug design.

  12. Binding of IRBIT to the IP3 receptor: determinants and functional effects.

    PubMed

    Devogelaere, Benoit; Nadif Kasri, Nael; Derua, Rita; Waelkens, Etienne; Callewaert, Geert; Missiaen, Ludwig; Parys, Jan B; De Smedt, Humbert

    2006-04-28

    IRBIT has previously been shown to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in an IP3-sensitive way. So far it remained to be elucidated whether this interaction was direct or indirect, and whether it was functionally relevant. We now show that IRBIT can directly interact with the IP3R, and that both the suppressor domain and the IP3-binding core of the IP3R are essential for a strong interaction. Moreover, we identified a PEST motif and a PDZ-ligand on IRBIT which were critical for the interaction with the IP3R. Furthermore, we identified Asp-73 as a critical residue for this interaction. Finally, we demonstrated that this interaction functionally affects the IP3R: IRBIT inhibits both IP3 binding and IP3-induced Ca2+ release.

  13. Exposure of activated sludge to nanosilver and silver ion: Inhibitory effects and binding to the fractions of extracellular polymeric substances.

    PubMed

    Geyik, Ayse Gul; Çeçen, Ferhan

    2016-07-01

    The main aim of the study was to determine the inhibitory effects of Ag(+) and AgNP (commercial and synthesized) on activated sludge by using respirometry. Along with this aim, also the changes taking place in extracellular polymeric substances (EPS) were studied. Additionally, the binding of Ag(+) or AgNP to the different fractions in EPS was assessed using voltammetry. Synthesized AgNP led to an obvious inhibition whereas commercial AgNP had no effect on activated sludge. For Ag(+) and AgNP, IC50 values were found between 2.3-3.0mg/L and 3.2-11.1mg/L, respectively. Thus, AgNP was less inhibitory than silver ion, since the release of free silver from AgNP was very small. The protein and carbohydrate content of EPS generally increased when Ag(+) was added. Both tightly- and loosely bound fractions in EPS could bind Ag(+) and AgNP. Silver binding capacity of EPS was seen to depend on the molecular weight of proteins.

  14. Effect of phosphorylation of phosphatidylinositol on myelin basic protein-mediated binding of actin filaments to lipid bilayers in vitro.

    PubMed

    Boggs, Joan M; Rangaraj, Godha; Dicko, Awa

    2012-09-01

    Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocytes and is believed to be responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also assemble actin filaments and tether them to lipid bilayers through electrostatic interactions. Here we investigate the effect of increased negative charge of the lipid bilayer due to phosphorylation of phosphatidylinositol (PI) on MBP-mediated binding of actin to the lipid bilayer, by substituting phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate for PI in phosphatidylcholine/phosphatidylglycerol lipid vesicles. Phosphorylation of PI caused dissociation of the MBP/actin complex from the lipid vesicles due to repulsion of the negatively charged complex from the negatively charged membrane surface. An effect of phosphorylation could be detected even if the inositol lipid was only 2mol% of the total lipid. Calcium-calmodulin dissociated actin from the MBP-lipid vesicles and phosphorylation of PI increased the amount dissociated. These results show that changes to the lipid composition of myelin, which could occur during signaling or other physiological events, could regulate the ability of MBP to act as a scaffolding protein and bind actin filaments to the lipid bilayer.

  15. 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

  16. Effective Binding of a Phosphatidylserine-Targeting Antibody to Ebola Virus Infected Cells and Purified Virions

    PubMed Central

    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. PMID:25815346

  17. Distinct effect of xenobiotics on the metal-binding properties of protein molecules.

    PubMed

    Novikova, Natalia; Kovalchuk, Mikhail; Stepina, Nina; Gaynutdinov, Radmir; Chukhrai, Elena; Yurieva, Eleonora

    2015-07-01

    The X-ray standing-wave method was applied to study the elemental composition and molecular organization of ordered protein films of alkaline phosphatase exposed to different xenobiotics (drug compounds, lead). Binding of metal ions from triply distilled water to protein molecules has been experimentally observed. Definite differences in the arrangement of impurity metal ions in the films have been established. The considerable enhancement of protein-metal interactions is attributed to partial rearrangement of the protein native structure, induced by xenobiotics.

  18. 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.

  19. 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. PMID:24919511

  20. A Binding Mode Hypothesis of Tiagabine Confirms Liothyronine Effect on γ-Aminobutyric Acid Transporter 1 (GAT1)

    PubMed Central

    2015-01-01

    Elevating GABA levels in the synaptic cleft by inhibiting its reuptake carrier GAT1 is an established approach for the treatment of CNS disorders like epilepsy. With the increasing availability of crystal structures of transmembrane transporters, structure-based approaches to elucidate the molecular basis of ligand–transporter interaction also become feasible. Experimental data guided docking of derivatives of the GAT1 inhibitor tiagabine into a protein homology model of GAT1 allowed derivation of a common binding mode for this class of inhibitors that is able to account for the distinct structure–activity relationship pattern of the data set. Translating essential binding features into a pharmacophore model followed by in silico screening of the DrugBank identified liothyronine as a drug potentially exerting a similar effect on GAT1. Experimental testing further confirmed the GAT1 inhibiting properties of this thyroid hormone. PMID:25679268

  1. 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.

  2. Effect of Aptamer Binding on the Electron-Transfer Properties of Redox Cofactors.

    PubMed

    Emahi, Ismaila; Gruenke, Paige R; Baum, Dana A

    2015-12-01

    In vitro selection or SELEX has allowed for the identification of functional nucleic acids (FNAs) that can potentially mimic and replace protein enzymes. These FNAs likely interact with cofactors, just like enzymes bind cofactors in their active sites. Investigating how FNA binding affects cofactor properties is important for understanding how an active site is formed and for developing useful enzyme mimics. Oxidoreductase enzymes contain cofactors in their active sites that allow the enzymes to do redox chemistry. In certain applications, these redox cofactors act as electron-transfer shuttles that transport electrons between the enzymes' active sites and electrode surfaces. Three redox cofactors commonly found in oxidoreductases are flavin adenine dinucleotide, nicotinamide adenine dinucleotide (NAD(+)), and pyrroloquinoline quinone (PQQ). We are interested in investigating how DNA aptamers that bind these cofactors influence the cofactors' redox abilities and if these aptamer-cofactor complexes could serve as redox catalysts. We employed cyclic voltammetry and amperometry to study the electrochemical properties of NAD(+) and PQQ when bound to DNA aptamers. Our results suggest that the aptamers provide a stable environment for the cofactor to participate in redox reactions, although enhanced redox activity was not observed. This work provides a foundation for the development of new FNAs capable of redox activity.

  3. 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. PMID:25528066

  4. Effects of Healthy Ageing on Precision and Binding of Object Location in Visual Short Term Memory

    PubMed Central

    2014-01-01

    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. PMID:25528066

  5. 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. PMID:26558995

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

    PubMed Central

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

    1984-01-01

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

  7. Polarization effects stabilize bacteriorhodopsin's chromophore binding pocket: a molecular dynamics study.

    PubMed

    Babitzki, G; Denschlag, R; Tavan, P

    2009-07-30

    Hybrid methods, which combine a quantum mechanical description of a chromophore by density functional theory (DFT) with a molecular mechanics (MM) model of the surrounding protein binding pocket, can enable highly accurate computations of the chromophore's in situ vibrational spectra. As a prerequisite, one needs a MM model of the chromophore-protein complex, which allows a correct sampling of its room-temperature equilibrium fluctuations by molecular dynamics (MD) simulation. Here, we show for the case of bacteriorhodopsin (BR) that MM-MD descriptions with standard nonpolarizable force fields entail a collapse of the chromophore binding pocket. As demonstrated by us, this collapse can be avoided by employing a polarized MM force field derived by DFT/MM hybrid computations. The corresponding MD simulations, which are complemented by a novel Hamiltonian replica exchange approach, then reveal a structural heterogeneity within the binding pocket of the retinal chromophore, which mainly pertains to the structure of the lysine chain covalently connecting the retinal chromophore with the protein backbone.

  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. Na+ site in blood coagulation factor IXa: effect on catalysis and factor VIIIa binding.

    PubMed

    Schmidt, Amy E; Stewart, Jonathan E; Mathur, Akash; Krishnaswamy, Sriram; Bajaj, S Paul

    2005-07-01

    During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca2+, phospholipid, and factor VIIIa (FVIIIa). The serine protease domain of FIXa contains a Ca2+ site and is predicted to contain a Na+ site. Comparative homology analysis revealed that Na+ in FIXa coordinates to the carbonyl groups of residues 184A, 185, 221A, and 224 (chymotrypsin numbering). Kinetic data obtained at several concentrations of Na+ and Ca2+ with increasing concentrations of a synthetic substrate (CH3-SO2-d-Leu-Gly-Arg-p-nitroanilide) were fit globally, assuming rapid equilibrium conditions. Occupancy by Na+ increased the affinity of FIXa for the synthetic substrate, whereas occupancy by Ca2+ decreased this affinity but increased k(cat) dramatically. Thus, Na+-FIXa-Ca2+ is catalytically more active than free FIXa. FIXa(Y225P), a Na+ site mutant, was severely impaired in Na+ potentiation of its catalytic activity and in binding to p-aminobenzamidine (S1 site probe) validating that substrate binding in FIXa is linked positively to Na+ binding. Moreover, the rate of carbamylation of NH2 of Val16, which forms a salt-bridge with Asp194 in serine proteases, was faster for FIXa(Y225P) and addition of Ca2+ overcame this impairment only partially. Further studies were aimed at delineating the role of the FIXa Na+ site in macromolecular catalysis. In the presence of Ca2+ and phospholipid, with or without saturating FVIIIa, FIXa(Y225P) activated FX with similar K(m) but threefold reduced k(cat). Further, interaction of FVIIIa:FIXa(Y225P) was impaired fourfold. Our previous data revealed that Ca2+ binding to the protease domain increases the affinity of FIXa for FVIIIa approximately 15-fold. The present data indicate that occupancy of the Na+ site further increases the affinity of FIXa for FVIIIa fourfold and k(cat) threefold. Thus, in the presence of Ca2+, phospholipid, and FVIIIa, binding of Na+ to FIXa increases its biologic activity by approximately 12-fold, implicating its role

  10. Reciprocal effects of an inhibitory factor on catalytic activity and noncatalytic cGMP binding sites of rod phosphodiesterase. [Rana catesbiana

    SciTech Connect

    Yamazaki, A.; Bartucca, F.; Ting, A.; Bitensky, M.W.

    1982-06-01

    In illuminated rod outer segment membranes, GTP and guanosine 5'-(..beta..,..gamma..-imido)triphosphate (p(NH)ppG) have reciprocal effects on cGMP phosphodiesterase (PDEase; 3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) activity and cGMP binding to noncatalytic sites on that enzyme. Two micromolar p(NH)ppG increased PDEase activity more than 2-fold while inhibiting cGMP binding more than 40%. Reduction of noncatalytic cGMP binding, which followed addition of p(NH)ppG, was not a result of PDEase activation. Both effects of p(NH)ppG were completely dependent on the presence of bleached rhodopsin. A heat-stable factor has been found to inhibit PDEase activity and also to stimulate cGMP binding to noncatalytic cGMP binding sites. Addition of p(NH)ppG reversed the effects of this factor on both PDEase activity and cGMP binding. During purification of this material, the activity peaks for both PDEase inhibition and activation of noncatalytic cGMP binding comigrated on both Blue Sepharose CL-6B column chromatography and sucrose density gradients centrifugation, suggesting that the same factor could be responsible for both inhibition of PDEase activity and enhancement of noncatalytic cGMP binding. Limited tryptic proteolysis of PDEase, which markedly reduced cGMP binding to the noncatalytic sites, and experiments using highly purified cAMP (free of cGMP) as substrate for PDEase showed that the binding of cGMP to noncatalytic sites was not required for the heat-stable inhibitory factor to inhibit PDEase activity. We discuss possible relationships between the regulation of PDEase and the binding of cGMP to noncatalytic sites.

  11. Comparative effect of lidocaine, bupivacaine and RAC 109 on myocardial conduction and contractility in the rabbit.

    PubMed

    Kariya, Nobutaka; Cosson, Claudine; Mazoit, Jean-Xavier

    2012-09-15

    Local anesthetic toxicity includes a decrease in ventricular conduction velocity and a decrease in myocardial contractile force. We tested the hypothesis that, like conduction, contraction was stereoselectively impaired by bupivacaine. We compared R(+) and S(-) bupivacaine to S(+) and R(-) RAC 109 in order to study the effects of hydrophobicity and ionization. We measured the changes in QRS duration and developed pressure in isolated perfused rabbit hearts. Binding of bupivacaine and RAC 109 to the ryanodine receptor was measured. The effect on cell shortening and relenghtening was measured on isolated rabbit cardiomyocytes. A mixed-effect pharmacodynamic model was used. The decrease in conduction velocity induced by the molecules was markedly stereospecific. All local anesthetics decreased ventricular velocity in a stereospecific manner with a RAC I(+)/II(-) and bupivacaine R(+)/S(-) potency ratio of maximum effect of 1.7 and 2.25 respectively. Contractility decreased in a dose dependent manner but this negative inotropic effect was not stereospecific with a C50 (concentration leading to half maximum effect) of 30 and 23 μM for RAC and bupivacaine respectively. The two drugs exhibited two-site binding to ryanodyne that may partly explain the observed effect. An effect on relaxation was observed only at very high concentrations. In conclusion, bupivacaine, a long acting local anesthetic, decreases ventricular conduction in a stereospecific manner, and decreases contractility non-stereospecifically.

  12. 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.

  13. 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

  14. Common whelk (Buccinum undatum) allergy: identification of IgE-binding components and effects of heating and digestive enzymes.

    PubMed

    Lee, Byung-Jae; Park, Hae-Sim

    2004-12-01

    In Korea, common whelk (Buccinum undatum) is a popular edible shellfish. The aim of this study was to observe the sensitization rate to common whelk and to characterize its allergens. We carried out skin prick test (SPT) in 1,700 patients with various allergic diseases. Specific IgE were detected by ELISA in the patient sera and ELISA inhibition tests were conducted. IgE-binding components were identified by means of SDS-PAGE and IgE-immunoblotting. The effects of digestive enzymes were evaluated in both raw and thermally treated extracts. SPT to common whelk was positive (>/=2+) in 83 (4.9%) patients studied. Twenty-four (38.7%) out of 62 SPT positive patients had high serum specific IgE to common whelk. ELISA inhibition test showed significant inhibitions by abalone as well as by common whelk. IgE-immunoblotting demonstrated three IgE-binding components (40, 71, 82 kDa), which were digested by simulated intestinal fluid and moderately digested by simulated gastric fluid, and the digestibility of allergens remained unchanged after thermal treatment. In conclusion, IgE-sensitization rate to common whelk was 4.9% in allergy patients. IgE-immunoblotting demonstrated three IgE-binding components, which were degraded by digestive enzymes. Further studies are needed to evaluate the clinical significance of the sensitized patients to common whelk.

  15. Common Whelk (Buccinum undatum) Allergy: Identification of IgE-binding Components and Effects of Heating and Digestive Enzymes

    PubMed Central

    Lee, Byung-Jae

    2004-01-01

    In Korea, common whelk (Buccinum undatum) is a popular edible shellfish. The aim of this study was to observe the sensitization rate to common whelk and to characterize its allergens. We carried out skin prick test (SPT) in 1,700 patients with various allergic diseases. Specific IgE were detected by ELISA in the patient sera and ELISA inhibition tests were conducted. IgE-binding components were identified by means of SDS-PAGE and IgE-immunoblotting. The effects of digestive enzymes were evaluated in both raw and thermally treated extracts. SPT to common whelk was positive (≥2+) in 83 (4.9%) patients studied. Twenty-four (38.7%) out of 62 SPT positive patients had high serum specific IgE to common whelk. ELISA inhibition test showed significant inhibitions by abalone as well as by common whelk. IgE-immunoblotting demonstrated three IgE-binding components (40, 71, 82 kDa), which were digested by simulated intestinal fluid and moderately digested by simulated gastric fluid, and the digestibility of allergens remained unchanged after thermal treatment. In conclusion, IgE-sensitization rate to common whelk was 4.9% in allergy patients. IgE-immunoblotting demonstrated three IgE-binding components, which were degraded by digestive enzymes. Further studies are needed to evaluate the clinical significance of the sensitized patients to common whelk. PMID:15608387

  16. Effect of inhibitory avoidance training on [3H]-glutamate binding in the hippocampus and parietal cortex of rats.

    PubMed

    Schröder, N; De-Paris, F; Roesler, R; Medina, J H; Souza, D O; Izquierdo, I

    2000-02-01

    Glutamate receptors have been implicated in memory formation. The aim of the present study was to determine the effect of inhibitory avoidance training on specific [3H]-glutamate binding to membranes obtained from the hippocampus or parietal cortex of rats. Adult male Wistar rats were trained (0.5-mA footshock) in a step-down inhibitory avoidance task and were sacrificed 0, 5, 15 or 60 min after training. Hippocampus and parietal cortex were dissected and membranes were prepared and incubated with 350 nM [3H]-glutamate (N = 4-6 per group). Inhibitory avoidance training induced a 29% increase in glutamate binding in hippocampal membranes obtained from rats sacrificed at 5 min (P<0.01), but not at 0, 15, or 60 min after training, and did not affect glutamate binding in membranes obtained from the parietal cortex. These results are consistent with previous evidence for the involvement of glutamatergic synaptic modification in the hippocampus in the early steps of memory formation.

  17. Cytotoxic effects of native and recombinant frutalin, a plant galactose-binding lectin, on HeLa cervical cancer cells.

    PubMed

    Oliveira, Carla; Nicolau, Ana; Teixeira, José A; Domingues, Lucília

    2011-01-01

    Frutalin is the α-D-galactose-binding lectin isolated from breadfruit seeds. Frutalin was obtained from two different sources: native frutalin was purified from its natural origin, and recombinant frutalin was produced and purified from Pichia pastoris. This work aimed to study and compare the effect of native and recombinant frutalin on HeLa cervical cancer cells proliferation and apoptosis. Furthermore, the interaction between frutalin and the HeLa cells was investigated by confocal microscopy. Despite having different carbohydrate-binding affinities, native and recombinant frutalin showed an identical magnitude of cytotoxicity on HeLa cells growth (IC₅₀~100 μg/mL) and equally induced cell apoptosis. The interaction studies showed that both lectins were rapidly internalised and targeted to HeLa cell's nucleus. Altogether, these results indicate that frutalin action is not dependent on its sugar-binding properties. This study provides important information about the bioactivity of frutalin and contributes to the understanding of the plant lectins cytotoxic activity.

  18. Allosteric Effects of Sodium Ion Binding on Activation of the M3 Muscarinic G-Protein-Coupled Receptor

    PubMed Central

    Miao, Yinglong; Caliman, Alisha D.; McCammon, J. Andrew

    2015-01-01

    G-protein-coupled receptors (GPCRs) are important membrane proteins that mediate cellular signaling and represent primary targets for about one-third of currently marketed drugs. Recent x-ray crystallographic studies identified distinct conformations of GPCRs in the active and inactive states. An allosteric sodium ion was found bound to a highly conserved D2.50 residue in inactive GPCRs, whereas the D2.50 allosteric pocket became collapsed in active GPCR structures. However, the dynamic mechanisms underlying these observations remain elusive. In this study, we aimed to understand the mechanistic effects of sodium ion binding on dynamic activation of the M3 muscarinic GPCR through long-timescale accelerated molecular dynamics (aMD) simulations. Results showed that with the D2.50 residue deprotonated, the M3 receptor is bound by an allosteric sodium ion and confined mostly in the inactive state with remarkably reduced flexibility. In contrast, the D2.50-protonated receptor does not exhibit sodium ion binding to the D2.50 allosteric site and samples a significantly larger conformational space. The receptor activation is captured and characterized by large-scale structural rearrangements of the transmembrane helices via dynamic hydrogen bond and salt bridge interactions. The residue motions are highly correlated during receptor activation. Further network analysis revealed that the allosteric signaling between residue D2.50 and key residues in the intracellular, extracellular, and orthosteric pockets is significantly weakened upon sodium ion binding. PMID:25863070

  19. The effects of liver and renal disease on stereoselective serum binding of flurbiprofen.

    PubMed Central

    Blouin, R; Chaudhary, I; Nishihara, K; Cox, S

    1993-01-01

    Stereoselectivity in the serum binding of flurbiprofen, a non-steroidal anti-inflammatory drug which is highly bound to albumin, was studied in patients with liver and renal disease. Subjects with renal disease or liver disease with ascites had significantly lower serum albumin concentrations than normals, resulting in higher free fractions of both the R(-) and S(+) enantiomers of flurbiprofen as determined by equilibrium dialysis. The ratio (+/- s.d.) of R/S-flurbiprofen free fractions was lower in the subjects with ascites (0.714 +/- 0.298) than in those without ascites (0.796 +/- 0.090) (P < 0.05). PMID:8448071

  20. Identification of the sAPRIL Binding Peptide and Its Growth Inhibition Effects in the Colorectal Cancer Cells

    PubMed Central

    Liu, Fang; Li, Jing; He, Mei-rong

    2015-01-01

    Background A proliferation-inducing ligand (APRIL) is a member of the tumor necrosis factor (TNF) super family. It binds to its specific receptors and is involved in multiple processes during tumorigenesis and tumor cells proliferation. High levels of APRIL expression are closely correlated to the growth, metastasis, and 5-FU drug resistance of colorectal cancer. The aim of this study was to identify a specific APRIL binding peptide (BP) able to block APRIL activity that could be used as a potential treatment for colorectal cancer. Methods A phage display library was used to identify peptides that bound selectively to soluble recombinant human APRIL (sAPRIL). The peptides with the highest binding affinity for sAPRIL were identified using ELISA. The effects of sAPRIL-BP on cell proliferation and cell cycle/apoptosis in vitro were evaluated using the CCK-8 assay and flow cytometry, respectively. An in vivo mouse model of colorectal cancer was used to determine the anti-tumor efficacy of the sAPRIL-BP. Results Three candidate peptides were characterized from eight phage clones with high binding affinity for sAPRIL. The peptide with the highest affinity was selected for further characterization. The identified sAPRIL-BP suppressed tumor cell proliferation and cell cycle progression in LOVO cells in a dose-dependent manner. In vivo in a mouse colorectal challenge model, the sAPRIL-BP reduced the growth of tumor xenografts in nude mice by inhibiting proliferation and inducing apoptosis intratumorally. Moreover, in an in vivo metastasis model, sAPRIL-BP reduced liver metastasis of colorectal cancer cells. Conclusions sAPRIL-BP significantly suppressed tumor growth in vitro and in vivo and might be a candidate for treating colorectal cancers that express high levels of APRIL. PMID:25826583

  1. Biomolecular interaction study of hydralazine with bovine serum albumin and effect of β-cyclodextrin on binding by fluorescence, 3D, synchronous, CD, and Raman spectroscopic methods.

    PubMed

    Bolattin, Mallavva B; Nandibewoor, Sharanappa T; Chimatadar, Shivamurti A

    2016-07-01

    Spectrofluoremetric technique was employed to study the binding behavior of hydralazine with bovine serum albumin (BSA) at different temperatures. Binding study of bovine serum albumin with hydralazine has been studied by ultraviolet-visible spectroscopy, fluorescence spectroscopy and confirmed by three-dimensional, synchronous, circular dichroism, and Raman spectroscopic methods. Effect of β-cyclodextrin on binding was studied. The experimental results showed a static quenching mechanism in the interaction of hydralazine with bovine serum albumin. The binding constant and the number of binding sites are calculated according to Stern-Volmer equation. The thermodynamic parameters ∆H(o) , ∆G(o) , ∆S(o) at different temperatures were calculated. These indicated that the hydrogen bonding and weak van der Waals forces played an important role in the interaction. Based on the Förster's theory of non-radiation energy transfer, the binding average distance, r, between the donor (BSA) and acceptor (hydralazine) was evaluated and found to be 3.95 nm. Spectral results showed that the binding of hydralazine to BSA induced conformational changes in BSA. The effect of common ions on the binding of hydralazine to BSA was also examined. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Effect of cadmium administration on intestinal calcium absorption and vitamin D-dependent calcium-binding protein

    SciTech Connect

    Fullmer, C.S.; Oku, T.; Wasserman, R.H.

    1980-08-01

    The effects of cadmium on intestinal calcium absorption and calcium-binding protein (CaBP) were investigated in chicks by means of the in situ ligated duodenal loop technique. Dietary cadmium, administered in the feed or by gastric intubation, resulted in significant declines in intestinal calcium absorption and mucosal calcium-binding protein concentrations. Cadmium chloride injected directly into the ligated loop of naive chicks also diminished calcium absorption and CaBP concentrations in an apparently dose-response related fashion. No adverse effects of cadmium administration on either the 25- or 1..cap alpha..-hydroxylation reactions of vitamin D were observed. While the general effect of cadmium administration was a reduction in intestinal calcium absorption, plasma calcium levels were consistently elevated in Cd-treated chicks, with the exception of those also maintained on diets low in Ca. The results indicate that cadmium toxicity exerts at least two effects on Ca metabolism, one at the intestinal level and another at the level of the bone, kidney, or both.

  3. Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with alpha-bungarotoxin-binding activity.

    PubMed

    Paulo, Joao A; Hawrot, Edward

    2009-10-01

    The molluscan acetylcholine-binding protein (AChBP) is a soluble homopentameric homolog of the extracellular domain of various ligand-gated ion channels. Previous studies have reported that AChBP, when fused to the ion pore domain of the serotonin receptor (5HT(3A)R), can form a functional ligand-gated chimeric channel only if the AChBP loop regions between beta-strands beta1 and beta2 (beta1-beta2), beta6 and beta7 (beta6-beta7), and beta8 and beta9 (beta8-beta9) are replaced with those of the 5HT(3A)R. To investigate further the potential interactions among these three important loop regions in a membrane- and detergent-free system, we designed AChBP constructs in which loops beta1-beta2, beta6-beta7, and beta8-beta9 of the AChBP were individually and combinatorially substituted in all permutations with the analogous loops of the 5HT(3A)R. These chimeras were expressed as secreted proteins using the Pichia pastoris yeast expression system. [(125)I]-alpha-Bungarotoxin-binding was detected in the culture media obtained from homologous recombinant clones expressing the wild-type AChBP, the beta1-beta2 loop-only chimera, and the chimera containing all three 5HT(3A)R loop substitutions. The remaining chimeras failed to show [(125)I]-alpha-bungarotoxin binding, and further analysis of cellular extracts allowed us to determine that these binding-negative chimeric constructs accumulated intracellularly and were not secreted into the culture medium. Our results demonstrate that coordinated interactions among loops beta1-beta2, beta6-beta7, and beta8-beta9 are essential for the formation of a functional ligand-binding site, as evidenced by [(125)I]-alpha-bungarotoxin-binding, and for efficient protein secretion. In addition, the constructs described here demonstrate the feasibility of utilizing soluble scaffolds to explore functionally important interactions within the extracellular domain of membrane-bound proteins. PMID:19427904

  4. 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.

  5. How different DNA sequences are recognized by a DNA-binding protein: effects of partial proteolysis.

    PubMed

    Supakar, P C; Zhang, X Y; Githens, S; Khan, R; Ehrlich, K C; Ehrlich, M

    1989-11-11

    MDBP is a sequence-specific DNA-binding protein from mammals that recognizes a variety of DNA sequences, all of which show much homology to a partially palindromic 14 base-pair consensus sequence. MDBP subjected to limited proteolysis and then incubated with various specific oligonucleotide duplexes yielded two types of complexes. The relative concentrations of these complexes varied greatly depending on how closely the MDBP site matched the consensus sequence. No such DNA sequence-specific differences in the types of complexes formed were seen with intact MDBP. Partial proteolysis also changed the relative affinity of MDBP for several of its binding sites. The nature of the two types of complexes formed from fragmented MDBP and DNA was studied by DNA competition assays, protein titration, site-directed mutagenesis, and dimethyl sulfate and missing base interference assays. The results suggest that, for some specific DNA sequences, half-site interactions with one MDBP subunit predominate and for others, strong interaction of two subunits with both half-sites readily occur.

  6. Tubulin assembly, taxoid site binding, and cellular effects of the microtubule-stabilizing agent dictyostatin.

    PubMed

    Madiraju, Charitha; Edler, Michael C; Hamel, Ernest; Raccor, Brianne S; Balachandran, Raghavan; Zhu, Guangyu; Giuliano, Kenneth A; Vogt, Andreas; Shin, Youseung; Fournier, Jean-Hugues; Fukui, Yoshikazu; Brückner, Arndt M; Curran, Dennis P; Day, Billy W

    2005-11-15

    (-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.

  7. Effect of simulated stages of the canine oestrous cycle on Escherichia coli binding to canine endometrium.

    PubMed

    Krekeler, N; Lodge, K M; Anderson, G A; Browning, G F; Charles, J A; Wright, P J

    2012-12-01

    Pyometra, a prevalent infectious uterine disease that affects intact middle-aged bitches, is typically associated with Escherichia coli. Our hypotheses were (i) that bacterial adhesion to canine endometrium differs between different stages of the oestrous cycle and (ii) that the adhesin FimH facilitates this adhesion. Twelve post-pubertal, ovariectomized greyhound bitches were treated with exogenous hormones to simulate different stages of the oestrous cycle. Tissue samples from each uterus were incubated with a pathogenic E. coli strain carrying the fimH gene, but no other adhesin genes (P4-wt)--or an E. coli strain in which fimH was insertionally inactivated (P4-∆fimH::kan)--or with phosphate-buffered saline as a negative control. After washing, tissue samples were homogenized for quantification of adherent bacteria. The differences in binding to canine endometrium at different stages of the oestrous cycle were not significant. However, the mean difference in binding of the P4-wt and the P4-∆fimH::kan across all stages of the simulated oestrous cycle was significant (p < 0.001 by paired t-test on geometric means). Individual differences in numbers of P4-wt bacteria bound between dogs might suggest genetic variations or epigenetic differences in FimH receptor expression by the endometrium, unrelated to the stage of the oestrous cycle. PMID:23279531

  8. Effect of Protein Binding on Unbound Atazanavir and Darunavir Cerebrospinal Fluid Concentrations

    PubMed Central

    Delille, Cecile A.; Pruett, Sarah T.; Marconi, Vincent C.; Lennox, Jeffrey L.; Armstrong, Wendy S.; Arrendale, Richard F.; Sheth, Anandi N.; Easley, Kirk A.; Acosta, Edward P.; Vunnava, Aswani; Ofotokun, Ighovwerha

    2015-01-01

    HIV-1 protease inhibitors (PIs) exhibit different protein binding affinities and achieve variable plasma and tissue concentrations. Degree of plasma protein binding may impact central nervous system penetration. This cross-sectional study assessed cerebrospinal fluid (CSF) unbound PI concentrations, HIV-1 RNA, and neopterin levels in subjects receiving either ritonavir-boosted darunavir (DRV), 95% plasma protein bound, or atazanavir (ATV), 86% bound. Unbound PI trough concentrations were measured using rapid equilibrium dialysis and liquid chromatography/tandem mass spectrometry. Plasma and CSF HIV-1 RNA and neopterin were measured by Ampliprep/COBAS® Taqman® 2.0 assay (Roche) and enzyme-linked immunosorbent assay (ALPCO), respectively. CSF/plasma unbound drug concentration ratio was higher for ATV, 0.09 [95% confidence interval (CI) 0.06–0.12] than DRV, 0.04 (95%CI 0.03–0.06). Unbound CSF concentrations were lower than protein adjusted wild-type inhibitory concentration-50 (IC50) in all ATV and 1 DRV-treated subjects (P < 0.001). CSF HIV-1 RNA was detected in 2/15 ATV and 4/15 DRV subjects (P = 0.65). CSF neopterin levels were low and similar between arms. ATV relative to DRV had higher CSF/plasma unbound drug ratio. Low CSF HIV-1 RNA and neopterin suggest that both regimens resulted in CSF virologic suppression and controlled inflammation. PMID:24691856

  9. Effects of milk proteins on sperm binding to the zona pellucida and intracellular Ca(2+) concentration in stallion sperm.

    PubMed

    Coutinho da Silva, Marco A; Seidel, George E; Squires, Edward L; Graham, James K; Carnevale, Elaine M

    2014-11-10

    Objectives were to determine the effects of extracellular Ca(2+) and milk proteins on intracellular Ca(2+) concentrations in stallion sperm; and to determine the effects of single caseins on sperm binding to the zona pellucida (ZP). In Experiment I, sperm were incubated in media containing 2 or 4mM Ca(2+) and intracellular Ca(2+) concentration was determined after ionomycin treatment and long-term incubation (3h). Extracellular Ca(2+) concentrations (2 compared with 4mM) did not affect baseline intracellular Ca(2+) concentration of sperm. However, incubating sperm in a medium containing 4 compared with 2mM Ca(2+) resulted in greater (P<0.05) influx of Ca(2+) into sperm. In Experiment II, sperm incubated in media containing 1mg/mL of native phosphocaseinate (NP) or sodium caseinate (SC) showed similar baseline intracellular Ca(2+) and influx of Ca(2+) than control (TALP). In Experiment III, sperm-ZP binding assays were performed in TALP medium containing: no additions (TALP); 1mg/mL SC; 1 or 3mg/mL of α-casein; 1 or 3mg/mL of β-casein; and 1 or 3mg/mL of κ-casein. The number of stallion sperm bound to bovine ZP was greatest (P<0.05) when SC was used. Co-incubation in media containing single caseins (α-, β- or κ-casein) resulted in similar results to TALP; however, a dose effect (P<0.05) was observed for β- and κ-caseins. In conclusion, extracellular Ca(2+) concentration and milk proteins did not affect baseline intracellular calcium in stallion sperm. It appears that β- and κ-caseins may be responsible for enhancing sperm binding to ZP, but the mechanism remains unknown.

  10. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  11. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  12. 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.

  13. Inhibition of thrombin-mediated cellular effects by triabin, a highly potent anion-binding exosite thrombin inhibitor.

    PubMed

    Glusa, E; Bretschneider, E; Daum, J; Noeske-Jungblut, C

    1997-06-01

    Triabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis is a potent thrombin inhibitor interfering with the anion-binding exosite of the enzyme. The recombinant protein, produced by the baculovirus/insect cell system, was used to study the inhibitory effect on thrombin-mediated cellular responses. The thrombin (1 nM)-stimulated aggregation of washed human platelets and the rise in cytoplasmic calcium in platelets were inhibited by triabin at nanomolar concentrations. In contrast, the rise in calcium induced by the thrombin receptor-activating peptide (10 microM) was not suppressed by triabin. In isolated porcine pulmonary arteries, preconstricted with PGF 2 alpha thrombin (2 nM) elicited an endothelium-dependent relaxation which was inhibited by triabin in the same concentration range as found for the inhibition of platelet aggregation. Higher concentrations of triabin were required to diminish the contractile response of endotheliumdenuded pulmonary vessels to thrombin (10 nM). In cultured bovine coronary smooth muscle cells, the mitogenic activity of thrombin (3 nM), measured by [3H]thymidine incorporation, was also suppressed by triabin. In all these assays, the inhibitory effect of triabin was dependent on the thrombin concentration used. These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of thrombin-mediated cellular effects. PMID:9241757

  14. Effect of a membrane-stabilizing compound on calcium binding to the plasma membrane of Acanthamoeba castellanii.

    PubMed

    Przełecka, A; Fritsch, R S; Wollweber, L; Sobota, A

    1980-01-01

    Binding of calcium ions at the plasma membrane was studied in Acanthamoeba cells pretreated with ZIMET 3164, a benzimidazole nitrogen mustard derivative, which is known to show a potent immunosuppressive action combined with a membrane-stabilizing effect in mice. For reference, 2 compounds were applied: ZIMET 3393 (Cytostasan¿), another benzimidazole mustard derivative, which exerts only a moderate membrane effect and acts as a strong cytostatic, and ZIMET 176/68, a barbituric acid derivative, which acts as an inhibitor of humoral immune responses but without membrane-stabilizing effect. Application of any of the 3 compounds does not reduce the appearance of calcium binding sites, visualized by means of ultracytochemical reaction, notwithstanding their different action in the mammalian organism. On the contrary, it was estimated by morphometric analysis that the number of Ca-dependent deposits was augmented after treatment with low doses of any of the 3 compounds, what seems to be connected with the induced metabolic disturbances in low molecular phosphates level. High doses and/or prolongation of treatment of the cells resulted in diminution of the number of deposits and induces profound disturbances in cell ultrastructure, probably due to the toxic action of the applied doses. In these cases, band-like structures crosslinking the two leaflets of the plasma membrane may be observed; it is suggested that they represent integral membrane proteins. PMID:6774578

  15. 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

  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. 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.

  18. van der Waals binding and band structure effects in graphene overlayers and graphane multilayers

    NASA Astrophysics Data System (ADS)

    Hyldgaard, Per; Rohrer, Jochen

    2011-03-01

    We study graphene formation (by selective Si evaporation) and adhesion on SiC surfaces as well as stacking and binding of graphane multilayers using a number of versions of the van der Waals Density Functional (vdW-DF) method and plane-wave density functional theory calculations. For the graphene/SiC systems and for the graphane multilayers we document that the bonding is entirely dominated by van der Waals (vdW) forces. At the same time we find that dispersive forces acting on the layers produce significant modifications in the graphene and graphane band structure. We interpret the changes and discuss a competition between wave function hybridization and interaction with the charge enhancement (between the layers) that results from density overlap. Supported by Svenska Vetenskapsrådet VR #621-2008-4346.

  19. Logic nanoparticle beacon triggered by the binding-induced effect of multiple inputs.

    PubMed

    Yang, Jing; Dong, Chen; Dong, Yafei; Liu, Shi; Pan, Linqiang; Zhang, Cheng

    2014-08-27

    Recently, the toehold-mediated DNA strand displacement reaction has been widely used in detecting molecular signals. However, traditional strand displacement, without cooperative signaling among DNA inputs, is insufficient for the design of more complicated nanodevices. In this work, a logic computing system is established using the cooperative "binding-induced" mechanism, based on the AuNP-based beacons, in which five kinds of multiple-input logic gates have been constructed. This system can recognize DNA and protein streptavidin simultaneously. Finally, the manipulations of the logic system are also demonstrated by controlling programmed conjugate DNA/AuNP clusters. This study provides the possibility of detecting multiple input signals and designing complex nanodevices that can be potentially applied to the detection of multiple molecular targets and the construction of large-scale DNA-based computation.

  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

    2016-09-01

    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. Musicians have enhanced audiovisual multisensory binding: experience-dependent effects in the double-flash illusion.

    PubMed

    Bidelman, Gavin M

    2016-10-01

    Musical training is associated with behavioral and neurophysiological enhancements in auditory processing for both musical and nonmusical sounds (e.g., speech). Yet, whether the benefits of musicianship extend beyond enhancements to auditory-specific skills and impact multisensory (e.g., audiovisual) processing has yet to be fully validated. Here, we investigated multisensory integration of auditory and visual information in musicians and nonmusicians using a double-flash illusion, whereby the presentation of multiple auditory stimuli (beeps) concurrent with a single visual object (flash) induces an illusory perception of multiple flashes. We parametrically varied the onset asynchrony between auditory and visual events (leads and lags of ±300 ms) to quantify participants' "temporal window" of integration, i.e., stimuli in which auditory and visual cues were fused into a single percept. Results show that musically trained individuals were both faster and more accurate at processing concurrent audiovisual cues than their nonmusician peers; nonmusicians had a higher susceptibility for responding to audiovisual illusions and perceived double flashes over an extended range of onset asynchronies compared to trained musicians. Moreover, temporal window estimates indicated that musicians' windows (<100 ms) were ~2-3× shorter than nonmusicians' (~200 ms), suggesting more refined multisensory integration and audiovisual binding. Collectively, findings indicate a more refined binding of auditory and visual cues in musically trained individuals. We conclude that experience-dependent plasticity of intensive musical experience extends beyond simple listening skills, improving multimodal processing and the integration of multiple sensory systems in a domain-general manner.

  2. 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

    2016-09-01

    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. PMID:27402846

  3. cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH

    SciTech Connect

    Gapstur, S.M.; Homma, S.; Dousa, T.P.

    1988-08-01

    Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-(32P)-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-(32P)cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-(32P)cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.

  4. Surface effects on core-level binding energies and valence in thulium chalcogenides

    NASA Astrophysics Data System (ADS)

    Kaindl, G.; Laubschat, C.; Reihl, B.; Pollak, R. A.; Mårtensson, N.; Holtzberg, F.; Eastman, D. E.

    1982-08-01

    Vacuum-cleaved (100) surfaces of mixed-valent TmSe, divalent TmTe, and trivalent TmS were studied by high-resolution, angle-integrated photoelectron spectroscopy with the use of synchrotron radiation in the energy range 30<=hν<=110 eV. In the topmost surface layers of TmSe and TmTe the 4f levels are found to be shifted to higher binding energies by 0.32+/-0.04 and 0.41+/-0.05 eV, respectively. In both TmSe and TmS the topmost surface layers are divalent. In the case of TmSe a separation of the Tm2+ 4f12 spectral feature into surface and bulk contributions allows a determination of the bulk mean valence v¯=2.55+/-0.05. While a shift of the Se 3d levels to lower binding energy is observed for Se atoms in the topmost surface layer of TmSe, no such shift can be resolved for the Te 4d levels of TmTe. The surface-derived divalent spectral features can be quenched completely in all three cases by exposure of the surfaces to submonolayer amounts of oxygen, resulting in the formation of trivalent surface oxides. Values for the electron mean free path l are derived from the observed surface- and bulk-spectral intensities, with l decreasing with decreasing electron kinetic energy down to ≅45 eV. Smaller singularity indices α of the Doniach-Sunjić line shapes as well as smaller extrinsic losses are observed for photoemission from the divalent surface layers as compared to the bulk.

  5. Musicians have enhanced audiovisual multisensory binding: experience-dependent effects in the double-flash illusion.

    PubMed

    Bidelman, Gavin M

    2016-10-01

    Musical training is associated with behavioral and neurophysiological enhancements in auditory processing for both musical and nonmusical sounds (e.g., speech). Yet, whether the benefits of musicianship extend beyond enhancements to auditory-specific skills and impact multisensory (e.g., audiovisual) processing has yet to be fully validated. Here, we investigated multisensory integration of auditory and visual information in musicians and nonmusicians using a double-flash illusion, whereby the presentation of multiple auditory stimuli (beeps) concurrent with a single visual object (flash) induces an illusory perception of multiple flashes. We parametrically varied the onset asynchrony between auditory and visual events (leads and lags of ±300 ms) to quantify participants' "temporal window" of integration, i.e., stimuli in which auditory and visual cues were fused into a single percept. Results show that musically trained individuals were both faster and more accurate at processing concurrent audiovisual cues than their nonmusician peers; nonmusicians had a higher susceptibility for responding to audiovisual illusions and perceived double flashes over an extended range of onset asynchronies compared to trained musicians. Moreover, temporal window estimates indicated that musicians' windows (<100 ms) were ~2-3× shorter than nonmusicians' (~200 ms), suggesting more refined multisensory integration and audiovisual binding. Collectively, findings indicate a more refined binding of auditory and visual cues in musically trained individuals. We conclude that experience-dependent plasticity of intensive musical experience extends beyond simple listening skills, improving multimodal processing and the integration of multiple sensory systems in a domain-general manner. PMID:27334887

  6. Neuroprotective effects of cold-inducible RNA-binding protein during mild hypothermia on traumatic brain injury

    PubMed Central

    Wang, Guan; Zhang, Jian-ning; Guo, Jia-kui; Cai, Ying; Sun, Hong-sheng; Dong, Kun; Wu, Cheng-gang

    2016-01-01

    Cold-inducible RNA-binding protein (CIRP), a key regulatory protein, could be facilitated by mild hypothermia in the brain, heart and liver. This study observed the effects of mild hypothermia at 31 ± 0.5°C on traumatic brain injury in rats. Results demonstrated that mild hypothermia suppressed apoptosis in the cortex, hippocampus and hypothalamus, facilitated CIRP mRNA and protein expression in these regions, especially in the hypothalamus. The anti-apoptotic effect of mild hypothermia disappeared after CIRP silencing. There was no correlation between mitogen-activated extracellular signal-regulated kinase activation and CIRP silencing. CIRP silencing inhibited extracellular signal-regulated kinase-1/2 activation. These indicate that CIRP inhibits apoptosis by affecting extracellular signal-regulated kinase-1/2 activation, and exerts a neuroprotective effect during mild hypothermia for traumatic brain injury. PMID:27335561

  7. Dlx5 Homeodomain:DNA Complex: Structure, Binding and Effect of Mutations Related to Split Hand and Foot Malformation Syndrome.

    PubMed

    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

    2016-03-27

    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 in humans. Our characterization of a Dlx5 homeodomain:(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A Q186H mutation linked to split hand and foot malformation 1 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 they likely destabilize the formation of functional complexes. PMID:26829219

  8. 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

  9. Co-solvation effect on the binding mode of the α-mangostin/β-cyclodextrin inclusion complex.

    PubMed

    Rungnim, Chompoonut; Phunpee, Sarunya; Kunaseth, Manaschai; Namuangruk, Supawadee; Rungsardthong, Kanin; Rungrotmongkol, Thanyada; Ruktanonchai, Uracha

    2015-01-01

    Cyclodextrins (CDs) have been extensively utilized as host molecules to enhance the solubility, stability and bioavailability of hydrophobic drug molecules through the formation of inclusion complexes. It was previously reported that the use of co-solvents in such studies may result in ternary (host:guest:co-solvent) complex formation. The objective of this work was to investigate the effect of ethanol as a co-solvent on the inclusion complex formation between α-mangostin (α-MGS) and β-CD, using both experimental and theoretical studies. Experimental phase-solubility studies were carried out in order to assess complex formation, with the mechanism of association being probed using a mathematical model. It was found that α-MGS was poorly soluble at low ethanol concentrations (0-10% v/v), but higher concentrations (10-40% v/v) resulted in better α-MGS solubility at all β-CD concentrations studied (0-10 mM). From the equilibrium constant calculation, the inclusion complex is still a binary complex (1:1), even in the presence of ethanol. The results from our theoretical study confirm that the binding mode is binary complex and the presence of ethanol as co-solvent enhances the solubility of α-MGS with some effects on the binding affinity with β-CD, depending on the concentration employed. PMID:26734079

  10. Co-solvation effect on the binding mode of the α-mangostin/β-cyclodextrin inclusion complex

    PubMed Central

    Rungnim, Chompoonut; Phunpee, Sarunya; Kunaseth, Manaschai; Namuangruk, Supawadee; Rungsardthong, Kanin

    2015-01-01

    Summary Cyclodextrins (CDs) have been extensively utilized as host molecules to enhance the solubility, stability and bioavailability of hydrophobic drug molecules through the formation of inclusion complexes. It was previously reported that the use of co-solvents in such studies may result in ternary (host:guest:co-solvent) complex formation. The objective of this work was to investigate the effect of ethanol as a co-solvent on the inclusion complex formation between α-mangostin (α-MGS) and β-CD, using both experimental and theoretical studies. Experimental phase-solubility studies were carried out in order to assess complex formation, with the mechanism of association being probed using a mathematical model. It was found that α-MGS was poorly soluble at low ethanol concentrations (0–10% v/v), but higher concentrations (10–40% v/v) resulted in better α-MGS solubility at all β-CD concentrations studied (0–10 mM). From the equilibrium constant calculation, the inclusion complex is still a binary complex (1:1), even in the presence of ethanol. The results from our theoretical study confirm that the binding mode is binary complex and the presence of ethanol as co-solvent enhances the solubility of α-MGS with some effects on the binding affinity with β-CD, depending on the concentration employed. PMID:26734079

  11. Co-solvation effect on the binding mode of the α-mangostin/β-cyclodextrin inclusion complex.

    PubMed

    Rungnim, Chompoonut; Phunpee, Sarunya; Kunaseth, Manaschai; Namuangruk, Supawadee; Rungsardthong, Kanin; Rungrotmongkol, Thanyada; Ruktanonchai, Uracha

    2015-01-01

    Cyclodextrins (CDs) have been extensively utilized as host molecules to enhance the solubility, stability and bioavailability of hydrophobic drug molecules through the formation of inclusion complexes. It was previously reported that the use of co-solvents in such studies may result in ternary (host:guest:co-solvent) complex formation. The objective of this work was to investigate the effect of ethanol as a co-solvent on the inclusion complex formation between α-mangostin (α-MGS) and β-CD, using both experimental and theoretical studies. Experimental phase-solubility studies were carried out in order to assess complex formation, with the mechanism of association being probed using a mathematical model. It was found that α-MGS was poorly soluble at low ethanol concentrations (0-10% v/v), but higher concentrations (10-40% v/v) resulted in better α-MGS solubility at all β-CD concentrations studied (0-10 mM). From the equilibrium constant calculation, the inclusion complex is still a binary complex (1:1), even in the presence of ethanol. The results from our theoretical study confirm that the binding mode is binary complex and the presence of ethanol as co-solvent enhances the solubility of α-MGS with some effects on the binding affinity with β-CD, depending on the concentration employed.

  12. Selection of a novel FGF23-binding peptide antagonizing the inhibitory effect of FGF23 on phosphate uptake.

    PubMed

    Huang, Tao; Lin, Xiaomian; Li, Quchou; Luo, Wu; Song, Li; Tan, Xiangpeng; Wang, Wenhui; Li, Xiaokun; Wu, Xiaoping

    2015-04-01

    Fibroblast growth factor 23 (FGF23) is a bone-derived endocrine regulator of phosphate homeostasis and has been considered as a potential therapeutic target for hypophosphatemic disorders. Herein, we isolated a novel FGF23-binding peptide by screening a phage display library with FGF23180-205, the minimal epitope of FGF23 binding to the binary fibroblast growth factor receptor (FGFR)-Klotho complex. The corresponding peptide (referred to as 23-b6) showed high homology to the immunoglobulin-like (Ig-like) domain III (D3) of FGFR1c, the predominant receptor mediating the phosphaturic activity of FGF23. The 23-b6 peptide and panning target FGF23180-205 carried opposite charges and shared similar hydrophilic profiles. Functional analysis indicated that synthetic 23-b6 peptide exhibited antagonistic effect on the inhibition of phosphate uptake by FGF23 in opossum kidney cells (OK cells). The mechanisms of 23-b6 peptide impairing the bioactivity of FGF23 involved blockade of the activation of Erk cascade and up-regulation of NaPi-2a and NaPi-2c expression in OK cells. Our results demonstrate that the 23-b6 peptide is a potent FGF23 antagonist with increased effect on phosphate uptake in kidney cells and might have therapeutic potentials in hypophosphatemic disorders characterized by an abnormally high level of FGF23. PMID:25515813

  13. 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; 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;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

  14. The Inhibitory Effects of Bioactive Compounds of Tomato Juice Binding to Hepatic HMGCR: In Vivo Study and Molecular Modelling

    PubMed Central

    Navarro-González, Inmaculada; Pérez-Sánchez, Horacio; Martín-Pozuelo, Gala; García-Alonso, Javier; Periago, Maria Jesús

    2014-01-01

    The hypocholesterolemic effect of tomato juice has been investigated in an intervention study with rats, along with the possible inhibition effect of bioactive tomato compounds binding to the HMGCR enzyme. Two experimental groups (n = 8 Sprague-Dawley rats) were fed ad libitum for five weeks, with water or tomato juice provided to the control and intervention groups, respectively. Total, LDL and HDL cholesterol, and total triglycerides were analysed in plasma, and the lycopene content and the expression and activity of the enzyme HMGCR were determined in liver samples. A computational molecular modelling was carried out to determine the interactions between HMGCR and lycopene, chlorogenic acid and naringenin. Total, LDL and HDL cholesterol were significantly lower in the intervention group after the intake of tomato juice. In addition, a significant reduction in HMGCR activity was observed, although this was not accompanied by changes in gene expression. The molecular modelling showed that components of tomato can bind to the active site of the enzyme and compete with the ligand HMGCoA. Lycopene, from tomato juice, accumulates in the liver and can inhibit the activity of the rate-limiting enzyme of cholesterol biosynthesis, HMGCR. PMID:24392102

  15. The inhibitory effects of bioactive compounds of tomato juice binding to hepatic HMGCR: in vivo study and molecular modelling.

    PubMed

    Navarro-González, Inmaculada; Pérez-Sánchez, Horacio; Martín-Pozuelo, Gala; García-Alonso, Javier; Periago, Maria Jesús

    2014-01-01

    The hypocholesterolemic effect of tomato juice has been investigated in an intervention study with rats, along with the possible inhibition effect of bioactive tomato compounds binding to the HMGCR enzyme. Two experimental groups (n = 8 Sprague-Dawley rats) were fed ad libitum for five weeks, with water or tomato juice provided to the control and intervention groups, respectively. Total, LDL and HDL cholesterol, and total triglycerides were analysed in plasma, and the lycopene content and the expression and activity of the enzyme HMGCR were determined in liver samples. A computational molecular modelling was carried out to determine the interactions between HMGCR and lycopene, chlorogenic acid and naringenin. Total, LDL and HDL cholesterol were significantly lower in the intervention group after the intake of tomato juice. In addition, a significant reduction in HMGCR activity was observed, although this was not accompanied by changes in gene expression. The molecular modelling showed that components of tomato can bind to the active site of the enzyme and compete with the ligand HMGCoA. Lycopene, from tomato juice, accumulates in the liver and can inhibit the activity of the rate-limiting enzyme of cholesterol biosynthesis, HMGCR. PMID:24392102

  16. /sup 125/I-luteinizing hormone (LH) binding to soluble receptors from the primate (Macaca mulatta) corpus luteum: effects of ethanol exposure

    SciTech Connect

    Danforth, D.R.; Stouffer, R.L.

    1988-01-01

    In the current study, we compared the effects of ethanol on gonadotropin receptors solubilized from macaque luteal membranes to those on receptors associated with the lipid bilayer. Treatment with 1% Triton X-100 for 30 min at 4C, followed by precipitation with polyethylene glycol, resulted in recovery of 50% more binding sites for /sup 125/I-human luteinizing hormone (hLH) than were available in particulate preparations. However, the soluble receptors displayed a 3-fold lower affinity for /sup 125/I-hLH. Conditions which enhanced LH binding to particulates, i.e., 1-8% ethanol at 25C, decreased specific /sup 125/I-hLH binding to soluble receptors. Steady-state LH binding to soluble receptors during incubation at 4C was half of that observed at 25C. The presence of 8% ethanol at 4C restored LH binding to levels observed in the absence of ethanol at 25C. Thus, LH binding sites in the primate corpus luteum can be effectively solubilized with Triton X-100. The different binding characteristics of particulate and soluble receptors, including the response to ethanol exposure, suggest that the lipid environment in the luteal membrane modulates the availability and affinity of gonadotropin receptors.

  17. 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).

  18. 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.

  19. The effect of annatto on insulin binding properties in the dog.

    PubMed

    Russell, K R M; Morrison, E Y St A; Ragoobirsingh, D

    2005-05-01

    Various plants are used in Caribbean folklore for the treatment of a variety of illnesses including diabetes mellitus. Preliminary investigations of several crude plant extracts have indicated that the annatto (Bixa orellana), among others, does in fact exhibit hypoglycaemic properties. This present investigation sought to isolate the hypoglycaemic principle(s) from the crude extract and to determine the mechanism of action. Purification experiments employing thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) resulted in an oil-soluble, partially purified annatto extract. The latter caused a decrease in blood glucose level of 5.62+/-0.13 (n=34) mmol/dL versus 6.31+/-0.12 (n=34) for the control (p<0.01) 1 h after administration. This hypoglycaemia persisted for an additional hour when the oral glucose tolerance test (OGTT) was performed on dogs treated with annatto and compared with the control. Plasma insulin levels measured at 1.0 h showed that there was an increase in plasma insulin levels of 59.57+/-8.3 microIU/mL for the annatto treated dogs versus 40.95+/-5.46 microIU/mL for the control (p<0.05), this elevation persisted throughout the duration of the OGTT which followed. Insulin receptor studies, using a modification of the method of Gambhir et al. done on mononuclear leucocytes and erythrocytes obtained from blood taken 1 h after administration showed that there was an increase in the percentage receptor binding when compared with the control. Insulin affinity results showed that there was an increase of 1.8+/-0.2x10(8) m-1 (n=12) in mononuclear leucocytes for the annatto treated dogs versus 1.2+/-0.2x10(8) m-1 for the control (p<0.05). In the enythrocytes, there was also an increase in affinity from 1.2+/-0.2x10(8) m-1 to 2.3+/-0.2x10(8) m-1 for the control and treated animals, respectively. In conclusion, it can be stated that annatto is responsible for the hypoglycaemic episodes seen in the dogs which was mediated by an

  20. Anti-bevacizumab idiotype antibody vaccination is effective in inducing vascular endothelial growth factor-binding response, impairing tumor outgrowth.

    PubMed

    Sanches, Jéssica de Souza; de Aguiar, Rodrigo Barbosa; Parise, Carolina Bellini; Suzuki, Juliana Mayumi; Chammas, Roger; de Moraes, Jane Zveiter

    2016-04-01

    Tumors require blood supply and, to overcome this restriction, induce angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in this process, which explains the great number of antiangiogenic therapies targeting VEGF. The research and development of targeted therapy has led to the approval of bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), in clinical settings. However, side effects have been reported, usually as a consequence of bolus-dose administration of the antibody. This limitation could be circumvented through the use of anti-idiotype (Id) antibodies. In the present study, we evaluated the efficacy of an active VEGF-binding immune response generated by an anti-bevacizumab idiotype mAb, 10.D7. The 10.D7 anti-Id mAb vaccination led to detectable levels of VEGF-binding anti-anti-Id antibodies. In order to examine whether this humoral immune response could have implications for tumor development, 10.D7-immunized mice were challenged with B16-F10 tumor cells. Mice immunized with 10.D7 anti-Id mAb revealed reduced tumor growth when compared to control groups. Histological analyses of tumor sections from 10.D7-immunized mice showed increased necrotic areas, decreased CD31-positive vascular density and reduced CD68-positive cell infiltration. Our results encourage further therapeutic studies, particularly if one considers that the anti-Id therapeutic vaccination maintains stable levels of VEGF-binding antibodies, which might be useful in the control of tumor relapse.

  1. Effect of trypsin treatments on the structure and binding capacity of volatile compounds of myosin.

    PubMed

    Lv, Tong; Wang, Ying; Pan, Daodong; Cao, Jinxuan; Zhang, Xin; Sun, Yangying; Chen, Yinji; Liu, Yuan

    2017-01-01

    In order to investigate the mechanism between flavor binding and proteins degradation during meat processing, the influence of different trypsin contents on the structure of myosin and the adsorption capacity on aldehydes and ketones was determined. The 1% treatment produced subfragment 2 (S2), light meromyosin (LMM) and decreased 18 and 16kDa light chains; 5% and 10% treatments produced 100 and 65kDa new bands and more S2, LMM and cleaned light chains. With the rising trypsin contents, β-sheet, β-turn, random coil, hydrophobicity and total sulfydryl content increased; solubility, α-helix and free percentages of aldehydes and ketones decreased. The increase of absorbing capacity could be attributed to the increased hydrophobicity and total sulphydryl and the unfolding of secondary structures by exposing reactive amino and thiol groups and hydrophobic sites; the decreased solubility was related to the increased hydrophobicity. The trypsin-dose dependent proteolysis of myosin increased the retention of volatile compounds. PMID:27507529

  2. Haem-binding-site heterogeneity and haem Cotton effects of Glycera dibranchiata monomeric haemoglobins.

    PubMed Central

    DiFeo, T J; Addison, A W

    1989-01-01

    The five major components of the monomeric haemoglobin from Glycera dibranchiata were separated and characterized by absorption spectroscopy, isoelectric focusing, azide-binding affinities and nitrosyl autoreduction kinetics. The differences found among the components are discussed in terms of haem-pocket variations. In addition, the Fourier-transform i.r. spectra of pooled monomeric haemoglobin carbonyl (HbmCO) and the major component carbonyl are reported. The c.d. spectra of the carbonyl and azide derivatives of the five components are compared and found to be similar. The c.d. spectra of myoglobin(II) carbonyl [Mb(II)CO] and of apomyoglobin (apoMb) reconstituted with a symmetric synthetic iron porphyrin carbonyl, meso-tetra-(p-carboxyphenyl)porphinatoiron(II) carbonyl [TCPPFe(II)CO], are compared with the c.d. spectra of pooled HbmCO and its TCPPFe(II)CO analogue. HbmTCPPFe(II)CO shows a negative Soret c.d. band whereas MbTCPPFe(II)CO produces both a negative and a positive Soret c.d. band. Displacement of the symmetric porphyrin by 8-anilinonaphthalene-1-sulphonate and the resulting fluorescence emission are reported. PMID:2764907

  3. 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

  4. 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

  5. Potato (Solanum tuberosum) juice exerts an anticonvulsant effect in mice through binding to GABA receptors.

    PubMed

    Muceniece, Ruta; Saleniece, Kristine; Krigere, Liga; Rumaks, Juris; Dzirkale, Zane; Mezhapuke, Rudolfs; Kviesis, Jorens; Mekss, Peteris; Klusa, Vija; Schiöth, Helgi B; Dambrova, Maija

    2008-04-01

    Naturally occurring benzodiazepines have been identified in regular food such as wheat and potato, but there is still no evidence that potato extracts can affect CNS responses in vivo. Here we found that undiluted potato juice and potato juice diluted with saline 1 : 2 administered 10 min intracisternally ( I. C.) and 30 min per os before bicuculline exerted significant anticonvulsant activity in the bicuculline-induced seizure threshold test in mice. In vitro, potato juice from different harvests at dilution series from 10 % to 0.000001 %, diluted 100,000-fold, displaced 50 % of gamma-aminobutyric acid (GABA) receptor ligand [ (3)H]GABA and diluted 40-fold displaced 50 % of [(3)H]flunitrazepam from binding sites in mice forebrain membranes. The low content of diazepam (0.04 +/- 0.01 mg/kg) determined by HPLC and mass spectrometry in the potato extracts could not sustain the anticonvulsant activity of potato juice in vivo; therefore we hypothesized that potato juice might contain GABA (A) receptor GABA-site active compounds. The findings of this study suggest that potato juice as well as potato taken as food may have the capacity of influencing brain GABA-ergic activity.

  6. Catalytic and Biocatalytic Iron Porphyrin Carbene Formation: Effects of Binding Mode, Carbene Substituent, Porphyrin Substituent, and Protein Axial Ligand

    PubMed Central

    2016-01-01

    Iron porphyrin carbenes (IPCs) are important intermediates in various chemical reactions catalyzed by iron porphyrins and engineered heme proteins, as well as in the metabolism of various xenobiotics by cytochrome P450. However, there are no prior theoretical reports to help understand their formation mechanisms and identify key information governing the binding mode, formation feasibility, and stability/reactivity. A systematic quantum chemical study was performed to investigate the effects of carbene substituent, porphyrin substituent, and axial ligand on IPC formation pathways. Results not only are consistent with available experimental data but also provide a number of unprecedented insights into electronic, steric, and H-bonding effects of various structural factors on IPC formation mechanisms. These results shall facilitate research on IPC and related systems for sustainable chemical catalysis and biocatalysis. PMID:26067900

  7. Effects of Supplemental Calcium or Calcium-binding Agents on Staphylococcal Bacteriophage Proliferation in Skim Milk1

    PubMed Central

    Das, N. K.; Marshall, R. T.

    1967-01-01

    Additions of 0.0005 N calcium borogluconate to Trypticase Soy Broth (TSB) produced an increase in phage titer about 1 million-fold, whereas its addition to skim milk resulted in about a 100-fold decrease in the maximal titer. Supplemental calcium had a stimulatory influence on bacterial growth in TSB but not in skim milk. Studies were made of the effect of binding of calcium of skim milk on the proliferation of staphylococcal bacteriophage. Sequestering the calcium with 2% phosphate mixture inactivated the phages without affecting the bacterial growth. However, chelation of calcium by 0.012% ethylenediaminetetraacetic acid produced an inhibitory effect on both the phages and the bacteria. PMID:16349770

  8. In Silico and In Vitro Investigation of the Piperine's Male Contraceptive Effect: Docking and Molecular Dynamics Simulation Studies in Androgen-Binding Protein and Androgen Receptor.

    PubMed

    Chinta, Gopichand; Ramya Chandar Charles, Mariasoosai; Klopčič, Ivana; Sollner Dolenc, Marija; Periyasamy, Latha; Selvaraj Coumar, Mohane

    2015-07-01

    Understanding the molecular mechanism of action of traditional medicines is an important step towards developing marketable drugs from them. Piperine, an active constituent present in the Piper species, is used extensively in Ayurvedic medicines (practiced on the Indian subcontinent). Among others, piperine is known to possess a male contraceptive effect; however, the molecular mechanism of action for this effect is not very clear. In this regard, detailed docking and molecular dynamics simulation studies of piperine with the androgen-binding protein and androgen receptors were carried out. Androgen receptors control male sexual behavior and fertility, while the androgen-binding protein binds testosterone and maintains its concentration at optimal levels to stimulate spermatogenesis in the testis. It was found that piperine docks to the androgen-binding protein, similar to dihydrotestosterone, and to androgen receptors, similar to cyproterone acetate (antagonist). Also, the piperine-androgen-binding protein and piperine-androgen receptors interactions were found to be stable throughout 30 ns of molecular dynamics simulation. Further, two independent simulations for 10 ns each also confirmed the stability of these interactions. Detailed analysis of the piperine-androgen-binding protein interactions shows that piperine interacts with Ser42 of the androgen-binding protein and could block the binding with its natural ligands dihydrotestosterone/testosterone. Moreover, piperine interacts with Thr577 of the androgen receptors in a manner similar to the antagonist cyproterone acetate. Based on the in silico results, piperine was tested in the MDA-kb2 cell line using the luciferase reporter gene assay and was found to antagonize the effect of dihydrotestosterone at nanomolar concentrations. Further detailed biochemical experiments could help to develop piperine as an effective male contraceptive agent in the future.

  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. Recombinant Hamster Oviductin Is Biologically Active and Exerts Positive Effects on Sperm Functions and Sperm-Oocyte Binding

    PubMed Central

    Yang, Xiaojing; Zhao, Yuewen; Yang, Xiaolong; Kan, Frederick W. K.

    2015-01-01

    Studies carried out in several mammalian species suggest that oviductin, also known as oviduct-specific glycoprotein or OVGP1, plays a key role in sperm capacitation, fertilization, and development of early embryos. In the present study, we used recombinant DNA technology to produce, for the first time, recombinant hamster OVGP1 (rHamOVGP1) in human embryonic kidney 293 (HEK293) cells. rHamOVGP1 secreted in the culture medium was purified by affinity chromatography. The resulting protein migrated as a poly-dispersed band of 160-350 kDa on SDS-PAGE corresponding to the molecular mass of the native HamOVGP1. Subsequent mass spectrometric analysis of the purified rHamOVGP1 confirmed its identity as HamOVGP1. Immunocytochemistry demonstrated binding of rHamOVGP1 to the mid-piece and head of hamster sperm and to the zona pellucida (ZP) of ovarian oocytes. In vitro functional experiments showed that addition of rHamOVGP1 in the capacitation medium further enhanced tyrosine phosphorylation of two sperm proteins of approximately 75 kDa and 83 kDa in a time-dependent manner. After 3 hours of incubation in the presence of rHamOVGP1, a significant increase in acrosome reaction was measured. Pretreatment of either sperm or oocyte with 20 μg/ml of rHamOVGP1 prior to sperm-egg binding assay significantly increased the number of sperm bound to the ZP. Addition of rHamOVGP1 in the medium during sperm-egg binding with either oocyte or sperm pretreated with rHamOVGP1 also saw an increase in the number of sperm bound to ZP. In all experimental conditions, the effect of rHamOVGP1 on sperm-oocyte binding was negated by the addition of monoclonal anti-HamOVGP1 antibody. The successful production and purification of a biologically active rHamOVGP1 will allow further exploration of the function of this glycoprotein in reproductive function. PMID:25849110

  11. Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.

    PubMed Central

    Reynaldo, L. P.; Villafranca, J. J.; Horrocks, W. D.

    1996-01-01

    Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation

  12. 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

  13. 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

  14. Effect of Zn2+ binding and enzyme active site on the transition state for RNA 2'-O-transphosphorylation interpreted through kinetic isotope effects.

    PubMed

    Chen, Haoyuan; Piccirilli, Joseph A; Harris, Michael E; York, Darrin M

    2015-11-01

    Divalent metal ions, due to their ability to stabilize high concentrations of negative charge, are important for RNA folding and catalysis. Detailed models derived from the structures and kinetics of enzymes and from computational simulations have been developed. However, in most cases the specific catalytic modes involving metal ions and their mechanistic roles and effects on transition state structures remain controversial. Valuable information about the nature of the transition state is provided by measurement of kinetic isotope effects (KIEs). However, KIEs reflect changes in all bond vibrational modes that differ between the ground state and transition state. QM calculations are therefore essential for developing structural models of the transition state and evaluating mechanistic alternatives. Herein, we present computational models for Zn2+ binding to RNA 2'O-transphosphorylation reaction models that aid in the interpretation of KIE experiments. Different Zn2+ binding modes produce distinct KIE signatures, and one binding mode involving two zinc ions is in close agreement with KIEs measured for non-enzymatic catalysis by Zn2+ aquo ions alone. Interestingly, the KIE signatures in this specific model are also very close to those in RNase A catalysis. These results allow a quantitative connection to be made between experimental KIE measurements and transition state structure and bonding, and provide insight into RNA 2'O-ransphosphorylation reactions catalyzed by metal ions and enzymes. This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment.

  15. Sensitive electrical detection of human prion proteins using field effect transistor biosensor with dual-ligand binding amplification.

    PubMed

    Wustoni, Shofarul; Hideshima, Sho; Kuroiwa, Shigeki; Nakanishi, Takuya; Hashimoto, Masahiro; Mori, Yasuro; Osaka, Tetsuya

    2015-05-15

    Simple and accurate detection of prion proteins in biological samples is of utmost importance in recent years. In this study, we developed a label-free electrical detection-based field effect transistor (FET) biosensor using thiamine as a probe molecule for a non-invasive and specific test of human prion protein detection. We found that thiamine-immobilized FETs can be used to observe the prion protein oligomer, and might be a significant test for the early diagnosis of prion-related diseases. The thiamine-immobilized FET was also demonstrated for the detection of prion proteins in blood serum without any complex pre-treatments. Furthermore, we designed a dual-ligand binding approach by the addition of metal ions as a second ligand to bind with the adsorbed prion protein on the thiamine-immobilized surface. When the prion attached to metal ions, the additional positive charge was induced on the gate surface of the FET. This approach was capable of amplifying the magnitude of the FET response and of enhancing the sensitivity of the FET biosensor. Detection of prion proteins has achieved the required concentration for clinical diagnosis in blood serum, which is less than 2 nM. In summary, this FET biosensor was successfully applied to prion detection and proved useful as a simple, fast, sensitive and low-cost method towards a mass-scale and routine blood screening-based test.

  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. 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

  18. Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding.

    PubMed

    Panda, Satya Prakash; Polusani, Srikanth R; Kellogg, Dean L; Venkatakrishnan, Priya; Roman, Madeline G; Demeler, Borries; Masters, Bettie Sue S; Roman, Linda J

    2013-05-01

    Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca(2+)/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ~3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20% to 60%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding. PMID:23507581

  19. Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding

    PubMed Central

    Panda, Satya Prakash; Polusani, Srikanth R.; Kellogg, Dean L.; Venkatakrishnan, Priya; Roman, Madeline G.; Demeler, Borries; Masters, Bettie Sue S.; Roman, Linda J.

    2013-01-01

    Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca2+/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ∼3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20% to 60%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding. PMID:23507581

  20. 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.

  1. 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.

  2. Inhibitory effects of hesperetin derivatives on guinea pig phosphodiesterases and their ratios between high- and low-affinity rolipram binding.

    PubMed

    Hsu, Hsin-Te; Wang, Wen-Hung; Han, Cheng-Ying; Chen, Chun-Nan; Chen, Chi-Ming; Ko, Wun-Chang

    2013-07-01

    The phosphodiesterase (PDE)4 molecule exists as two distinct conformers, PDE4H and PDE4L , which have high and low affinities, respectively, for the selective PDE4 inhibitor, rolipram. The inhibition of PDE4H and PDE4L is associated with adverse responses, such as nausea, vomiting, and gastric hypersecretion, and with anti-inflammatory and bronchodilator effects, respectively. We determined the therapeutic (PDE4H/PDE4L) ratios of hesperetin-7-O-methylether, hesperetin-5,7,3'-O-trimethylether (HTME), hesperetin-7-O-acetate, hesperetin-7,3'-O-diacetate, hesperetin-5,7,3'-O-triacetate (HTA), hesperetin-5,7,3'-O-tripropionate, hesperetin-5,7,3'-O-tributyrate, hesperetin-5,7,3'-O-triisobutyrate, and hesperetin-5,7,3'-O-tripivatate, and compared these ratios to those of hesperetin, hesperetin-7,3'-O-dimethylether, hesperidin, and hesperidin-3'-O-methylether to identify derivatives with therapeutic ratios and to characterize the structure-activity relationships among these compounds. The activities of PDE isozymes 1 through 5 were measured using a two-step procedure using [(3)H]adenosine 3',5'-cyclic monophosphate or [(3)H]guanosine 3',5'-cyclic monophosphate as substrates. The inhibitory concentration (IC50) for 50% of PDE4 inhibition and effective concentration (EC50) for replacing 50% of [(3)H]rolipram binding on high-affinity rolipram-binding sites was taken as the PDE4L and PDE4H value, respectively. The HTME and the HTA dually inhibited PDE3 and PDE4, and displayed PDE4H/PDE4L ratios of 18.3 and 20.8, respectively, suggesting that they may be candidate drugs for treating asthma and chronic obstructive pulmonary disease (COPD) because the combined inhibition of PDE3 and PDE4 has synergistically anti-inflammatory and bronchodilator effects in COPD patients.

  3. Oxidation and nitrosylation of cysteines proximal to the intermediate filament (IF)-binding site of plectin: effects on structure and vimentin binding and involvement in IF collapse.

    PubMed

    Spurny, Radovan; Abdoulrahman, Kamaran; Janda, Lubomir; Rünzler, Dominik; Köhler, Gottfried; Castañón, Maria J; Wiche, Gerhard

    2007-03-16

    As an intermediate filament (IF)-based cytolinker protein, plectin plays a key role in the maintenance of cellular cytoarchitecture and serves at the same time as a scaffolding platform for signaling cascades. Consisting of six structural repeats (R1-6) and harboring binding sites for different IF proteins and proteins involved in signaling, the plectin C-terminal domain is of strategic functional importance. Depending on the species, it contains at least 13 cysteines, 4 of which reside in the R5 domain. To investigate the structural and biological functions of R5 cysteines, we used cysteine-to-serine mutagenesis and spectroscopic, biochemical, and functional analyses. Urea-induced unfolding experiments indicated that wild-type R5 in the oxidized, disulfide bond-mediated conformation was more stable than its cysteine-free mutant derivative. The binding affinity of R5 for vimentin was significantly higher, however, when the protein was in the reduced, more relaxed conformation. Of the four R5 cysteines, one (Cys4) was particularly reactive as reflected by its ability to form disulfide bridges with R5 Cys1 and to serve as a target for nitrosylation in vitro. Using immortalized endothelial cell cultures from mice, we show that endogenous plectin is nitrosylated in vivo, and we found that NO donor-induced IF collapse proceeds dramatically faster in plectin-deficient compared with wild-type cells. Our data suggest an antagonistic role of plectin in nitrosylation (oxidative stress)-mediated alterations of IF cytoarchitecture and a possible role of R5 Cys4 as a regulatory switch. PMID:17224453

  4. Influence of stimulation of myocardial alpha- as well as beta-adrenoceptors on the effect of digoxin in isolated electrically driven rabbit papillary muscles.

    PubMed

    Knorr, A; Wagner, J

    1979-12-01

    On isolated electrically driven rabbit papillary muscle the cardiac glycoside digoxin was infused at driving rates of 0.5, 1.0 and 2.0 Hz. Two effective concentrations of digoxin were determined: 1. that inducing the maximal inotropic effect (maximal inotropic concentration) and 2, that causing cardiac arrest (toxic concentration). The influence of the alpha-sympathomimetic drug phenylephrine and for comparison that of the beta-sympathomimetic drug isoprenaline on either concentration of digoxin was investigated. 1. Stimulation of alpha-adrenoceptors by phenylephrine at a rate of 0.5 Hz significantly decreased the maximal inotropic as well as the toxic concentration of digoxin by about 36%; this decrease was maximal under the influence of the EC25 of phenylephrine and could be blocked by phentolamine. Phenylephrine did not alter the maximal inotropic effect of digoxin. At a stimulus rate of 1.0 Hz the EC75 of phenylephrine still diminished significantly the effective concentrations of digoxin whereas under these conditions the EC25 was ineffective. At 2.0 Hz stimulation of myocardial alpha-adrenoceptors had no effect anymore on either the maximal inotropic or the toxic concentration of digoxin. 2. In contrast, stimulation of beta-adrenoceptors by isoprenaline at a driving rate of 2.0 Hz resulted in a pronounced decrease of maximal inotropic and toxic concentration of digoxin while the maximal positive inotropic effect exerted by digoxin was found to be not altered by isoprenaline. The decrease of the effective concentration of digoxin caused by isoprenaline was abolished by pindolol. At a driving rate of 1.0 Hz this effect was slightly attenuated but was completely absent at 0.5 Hz. 3. From these results it can be concluded that stimulation of either adrenoceptor, alpha- and beta-, increases the effectiveness of the cardiac glycoside digoxin, i.e. diminishes the maximal inotropic as well as its toxic concentration. While stimulation of alpha-adrenoceptors is

  5. 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…