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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 structurally novel Ca sup 2+ channel blocker Ro 40-5967, which binds to the ( sup 3 H) desmethoxyverapamil receptor, is devoid of the negative inotropic effects of verapamil in normal and failing rat hearts

    SciTech Connect

    Clozel, J.P.; Veniant, M.; Osterrieder, W. )

    1990-06-01

    Ro 40-5967 is a structurally novel Ca{sup 2+} channel blocker that binds to the verapamil-type receptor of cardiac membranes but that has been shown in isolated guinea-pig hearts to be about ten times less potent a negative inotropic agent than verapamil. The goals of the present study were to confirm these findings in vitro in isolated perfused rat hearts as well as in vivo in conscious rats and to compare Ro 40-5967 to verapamil. The effects of Ro 40-5967 and verapamil were tested not only in normal rats, but also in rats with heart failure induced by chronic myocardial infarction. In isolated Langendorff hearts (without heart failure), no decrease of contractility was observed with Ro 40-5967 up to complete AV block. In contrast, verapamil decreased contractility with an IC50 of 100 nM. In isolated, electrically stimulated rat papillary muscles, the IC50 values for the decrease of contractile force were 15,000 and 440 nM for Ro 40-5967 and verapamil, respectively. In vivo, Ro 40-5967 did not decrease left ventricular contractility (as assessed by changes of dP/dt max +) in rats without and with heart failure. In contrast, verapamil was markedly negative inotropic in both conditions.

  3. Potassium changes the relationship between receptor occupancy and the inotropic effect of cardiac glycosides in guinea-pig myocardium.

    PubMed Central

    Bachmaier, A.; Ebner, F.; Reiter, M.

    1985-01-01

    K+ (2.4-15.6 mmol l-1) antagonized the positive inotropic effect of dihydro-ouabain. The concentration-effect curves became steeper with the shift to higher concentrations of the glycoside. At 1.2 mmol l-1 Ca2+, an increase in K+ from 2.4 to 12 mmol l-1 required tenfold higher concentrations of dihydro-ouabain to produce equal inotropic effects. This factor was reduced to four at 3.2 mmol l-1 Ca2+. The same change in K+ concentration, at 1.2 mmol l-1 Ca2+, diminished the inotropic effect of ouabain on rested-state contractions by a factor of six. The positive inotropic effect of Ca2+ was also antagonized by K+ (1.2-12 mmol l-1). Reduction of Na+ from 140 to 70 mmol l-1 abolished the antagonistic action of K+ (1.2-8.0 mmol l-1). Moreover the inotropic effect of Ca2+ was enhanced. Reduction of Na+, from 140 to 70 mmol l-1, antagonized the positive inotropic effect of dihydro-ouabain more at low (2.4 mmol l-1) than at high (8.0 mmol l-1) K+. Accordingly, the extent of the dihydro-ouabain-K+ antagonism was reduced. When the K+ concentration was increased from 2.4 to 12 mmol l-1, [3H]-ouabain binding was reduced by a factor of three. This is less than the reduction in the inotropic effectiveness of ouabain or dihydro-ouabain. Reduction of stimulation frequency from 1 to 0.1215 Hz did not significantly alter the antagonistic effect of K+. Diminution of Vmax of the action potential was observed only at K+ concentrations greater than 5.9 mmol l-1, whereas the resting membrane potential was continuously depolarized over the entire range of K+ concentrations. The results support the view that the reduction in receptor affinity cannot be the sole cause of the antagonism between the glycoside and K+. Impairment of passive Na+ influx during diastole, due to the K+-dependent depolarization of the resting membrane potential, contributed to about one half of the glycoside-K+ antagonism. PMID:4041678

  4. Muscarinic receptors mediate negative and positive inotropic effects in mammalian ventricular myocardium: differentiation by agonists.

    PubMed Central

    Korth, M.; Kühlkamp, V.

    1987-01-01

    The concentration-dependence of the negative and positive inotropic effect of choline esters and of oxotremorine was studied in isometrically contracting papillary muscles of the guinea-pig. The preparations were obtained from reserpine-pretreated animals and were electrically driven at a frequency of 0.2 Hz. In the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine (IBMX, 100 mumol l-1), choline esters and oxotremorine produced concentration-dependent negative inotropic effects. Oxotremorine exhibited the highest negative inotropic potency (with a half-maximal effective concentration, EC50, of 20 nmol l-1) followed by carbachol (139 nmol l-1), methacholine (490 nmol l-1), acetylcholine in the presence of 10 mumol l-1 physostigmine (1.36 mumol l-1) and bethanechol (10 mumol l-1). Atropine was a competitive antagonist of the negative inotropic effects. Carbachol and oxotremorine decreased Vmax, overshoot and duration of slow Ca2+-dependent action potentials which had been elicited in the presence of 100 mumol l-1 IBMX. Choline esters produced a concentration-dependent positive inotropic effect. With an EC50 of 32 mumol l-1, carbachol was the most potent compound, followed by methacholine (35 mumol l-1), acetylcholine in the presence of 10 mumol l-1 physostigmine (46 mumol l-1) and bethanechol (142 mumol l-1). Compared to carbachol and methacholine which increased force by 100% of control, the increase induced by acetylcholine and bethanechol was only 64 and 58%, respectively. Atropine shifted the concentration-effect curves of all choline esters to higher concentrations. Choline esters caused intracellular Na+ activity to increase in the quiescent papillary muscle. This effect was reversed by atropine. Oxotremorine produced a small concentration-dependent positive inotropic effect (about 30% of the maximal effect of carbachol) which was resistant to atropine. Oxotremorine was a potent inhibitor of the positive inotropic effect of choline esters

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

    PubMed Central

    Bellemann, P.; Scholz, H.

    1974-01-01

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

  6. Functional studies on alpha 1-adrenoceptor subtypes mediating inotropic effects in rat right ventricle.

    PubMed Central

    Michel, M. C.; Hanft, G.; Gross, G.

    1994-01-01

    1. We have studied the alpha 1-adrenoceptor subtypes mediating inotropic effects of adrenaline in rat right ventricle and the Ca2+ sources used to elicit these effects. alpha 1A-Adrenoceptor-mediated contractile effects in rat vas deferens were studied for comparison in some cases. 2. Treatment with chloroethylclonidine did not affect the maximal beta-adrenoceptor-mediated inotropic effects in rat right ventricle or the maximal alpha 1A-adrenoceptor-mediated contractile effects in rat vas deferens; it did not alter the potency of isoprenaline in the ventricle and reduced the potency of the alpha-adrenoceptor antagonists in vas deferens only slightly. Treatment of right ventricular strips with CdCl2 markedly reduced resting tension and enhanced maximal inotropic effects of isoprenaline but did not affect its potency. 3. Inactivation of cardiac alpha 1B-adrenoceptors by treatment with chloroethylclonidine slightly enhanced the maximal inotropic effects of the full agonist, adrenaline and of several partial agonists. 4. Schild analysis of inhibition experiments with the alpha 1A-adrenoceptor-selective antagonists, 5-methyl-urapidil and (+/-)-tamsulosin, demonstrated that adrenaline causes its inotropic effects mainly via the alpha 1B-adrenoceptor subtype. Schild analysis of 5-methyl-urapidil inhibition experiments in chloroethylclonidine-treated ventricles indicated that only alpha 1A-adrenoceptors mediate the inotropic effects of adrenaline following inactivation of the alpha 1B-adrenoceptors. 5. In control ventricles the organic Ca2+ entry blocker, nitrendipine and treatment with the inorganic Ca2+ entry blocker, CdCl2 did not reduce inotropic effects of adrenaline whereas ryanodine treatment inhibited them. In contrast, nitrendipine and CdCl2 treatment had major inhibitory effects in chloroethylclonidine-treated but lacked inhibitory effects in phenoxybenzamine-treated ventricular strips. 6. We conclude that inotropic effects of adrenaline in rat heart are mediated

  7. Positive inotropic effects of Tityus cambridgei and T. serrulatus scorpion venoms on skeletal muscle.

    PubMed

    Borja-Oliveira, C R; Pertinhez, T A; Rodrigues-Simioni, L; Spisni, A

    2009-04-01

    Toxins that block voltage-dependent K+ channels and those that modify Na+ channel gating exhibit positive inotropic effect on skeletal muscle. We compared the effect of the venom of Tityus cambridgei (Tc) and Tityus serrulatus (Ts) scorpions on mouse diaphragm force, in vitro. In indirect and direct (using D-tubocurarine 7.3 microM) stimulation, Tc, 10microg/mL, increased the contractile force, an effect prevented by tetrodotoxin (TTX) while Ts, 0.5 microg/mL, potentiated only indirectly stimulated diaphragm, thus indicating its activity is mainly mediated through acetylcholine release from nerve terminal. This effect is prevented by TTX and attenuated by the K+ channel opener cromakalim. In conclusion, our data show that while the positive inotropic effect of both venoms appears associated to the activity of Na+ and K+ channels, only Tc venom acts also directly on skeletal muscle. This finding call for further studies on Tc venom to identify the toxin responsible for its direct inotropic activity as it may have clinical applications. PMID:18926933

  8. Inotropic and lusitropic effects of calcitonin gene-related peptide in the heart

    PubMed Central

    Al-Rubaiee, Mustafa; Gangula, Pandu R.; Millis, Richard M.; Walker, Robin K.; Umoh, Nsini A.; Cousins, Valerie M.; Jeffress, Miara A.

    2013-01-01

    Previous studies have demonstrated positive-inotropic effects of calcitonin gene-related peptide (CGRP), but the mechanisms remain unclear. Therefore, two experiments were performed to determine the physiological correlates of the positive-inotropic effects of CGRP. Treatments designed to antagonize the effects of physiologically active CGRP1–37 included posttreatment with CGRP8–37 and pretreatment with LY-294002 (LY, an inhibitor of phosphatidylinositol 3-kinase), 17β-estradiol (E), and progesterone (P) were also used to modulate the effects of CGRP1–37. Experiment 1 was in vitro studies on sarcomeres and cells of isolated adult rat cardiac myocytes. CGRP1–37, alone and in combination with E and P, decreased sarcomere shortening velocities and increased shortening percentages, effects that were antagonized by CGRP8–37, but not by LY. CGRP1–37 increased resting intracellular calcium ion concentrations and Ca2+ influxes, effects that were also antagonized by both CGRP8–37 and LY. Experiment 2 was in vivo studies on left ventricular pressure-volume (PV) loops. CGRP1–37 increased end-systolic pressure, ejection fraction, and velocities of contraction and relaxation while decreasing stroke volume, cardiac output, stroke work, PV area, and compliance. After partial occlusion of the vena cava, CGRP1–37 increased the slope of the end-systolic PV relationship. CGRP8–37 and LY attenuated most of the CGRP-induced changes. These findings suggest that CGRP-induced positive-inotropic effects may be increased by treatments with estradiol and progesterone and inhibited by LY. The physiological correlates of CGRP-induced positive inotropy observed in rat sarcomeres, cells, and intact hearts are likely to reveal novel mechanisms of heart failure in humans. PMID:23585136

  9. Negative inotropic and hypotensive effects of the superoxide dismutase mimetic tempol in pigs.

    PubMed

    Kristensen, Mads Nyboe; Frederiksen, Christian Alcaraz; Sivén, Eleonora; Hyldebrandt, Janus Adler; Juhl-Olsen, Peter; Sloth, Erik; Simonsen, Ulf; Buus, Niels Henrik

    2014-05-15

    Through interference with free radicals, the nitroxide tempol potentially increases bioavailability of nitric oxide (NO) and along with modulation of potassium channels reduces blood pressure (BP). We studied whether tempol in pigs lowers BP by mechanisms sensitive to inhibition of NO synthase or large conductance calcium-activated potassium channels (BKCa). The cardiovascular effects of intravenous tempol (25-50mg/kg) were examined in anesthetized pigs with myocardial function being evaluated by echocardiography. While saline-treated animals remained hemodynamically stable, tempol induced fast, dose-dependent and transient reductions in BP lasting 5-10 min with a simultaneous impairment of left ventricular contraction. Pretreatment with the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 4 mg/kg) or a blocker of BKCa (tetraethylammonium (TEA), 100mg/h) increased baseline BP but also enhanced BP reductions to tempol. Isolated myocardial trabeculae subjected to an identical protocol also demonstrated dose-related reductions in contractility to tempol. This effect was not affected by l-NAME, but attenuated by TEA. In isolated mesenteric resistance arteries contracted with noradrenaline, tempol caused small postjunctional l-NAME sensitive relaxations, while neurogenic contractions were inhibited by tempol by TEA-sensitive mechanisms and mechanisms insensitive to TEA and l-NAME. In conclusion intravenous tempol induces fast transient reductions in BP associated with simultaneous reductions in myocardial contraction. Tempol exerts direct negative inotropic effects which are partly sensitive to BKCa-blockade but independent of NOS inhibition. In addition tempol has direct vasodilatory effects despite NOS and potassium channel blockade. The negative inotropic and hypotensive effects raise concerns using tempol, or structurally similar drugs, for intravenous use. PMID:24632458

  10. Effects of commonly used inotropes on myocardial function and oxygen consumption under constant ventricular loading conditions.

    PubMed

    DeWitt, Elizabeth S; Black, Katherine J; Thiagarajan, Ravi R; DiNardo, James A; Colan, Steven D; McGowan, Francis X; Kheir, John N

    2016-07-01

    Inotropic medications are routinely used to increase cardiac output and arterial blood pressure during critical illness. However, few comparative data exist between these medications, particularly independent of their effects on venous capacitance and systemic vascular resistance. We hypothesized that an isolated working heart model that maintained constant left atrial pressure and aortic blood pressure could identify load-independent differences between inotropic medications. In an isolated heart preparation, the aorta and left atrium of Sprague Dawley rats were cannulated and placed in working mode with fixed left atrial and aortic pressure. Hearts were then exposed to common doses of a catecholamine (dopamine, epinephrine, norepinephrine, or dobutamine), milrinone, or triiodothyronine (n = 10 per dose per combination). Cardiac output, contractility (dP/dtmax), diastolic performance (dP/dtmin and tau), stroke work, heart rate, and myocardial oxygen consumption were compared during each 10-min infusion to an immediately preceding baseline. Of the catecholamines, dobutamine increased cardiac output, contractility, and diastolic performance more than clinically equivalent doses of norepinephrine (second most potent), dopamine, or epinephrine (P < 0.001). The use of triiodothyronine and milrinone was not associated with significant changes in cardiac output, contractility or diastolic function, either alone or added to a baseline catecholamine infusion. Myocardial oxygen consumption was closely related to dP/dtmax (r(2) = 0.72), dP/dtmin (r(2) = 0.70), and stroke work (r(2) = 0.53). In uninjured, isolated working rodent hearts under constant ventricular loading conditions, dobutamine increased contractility and cardiac output more than clinically equivalent doses of norepinephrine, dopamine, and epinephrine; milrinone and triiodothyronine did not have significant effects on contractility. PMID:27150829

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

    PubMed Central

    Rousseau, M. F.; Vincent, M. F.; Cheron, P.; Van Den Berghe, G.; Charlier, A. A.; Pouleur, H.

    1985-01-01

    1 The effects of intravenous nicardipine (2.5 mg) on the left ventricular (LV) inotropic state, LV metabolism, and coronary haemodynamics were analysed in 22 patients with angina pectoris. 2 Measurements were made at fixed heart rate (atrial pacing), under basal state, and during a cold pressor test. 3 After nicardipine, coronary blood flow and oxygen content in the coronary sinus increased significantly. 4 The indices of inotropic state increased slightly, and the rate of isovolumic LV pressure fall improved. 5 Myocardial oxygen consumption was unchanged despite the significant reduction in pressure-rate product, but LV lactate uptake increased, particularly during the cold pressor test. 6 When nicardipine was administered after propranolol, the indices of inotropic state were unaffected. 7 The lack of direct effect of nicardipine on LV inotropic state was further confirmed by intracoronary injection of 0.1 and 0.2 mg in a separate group of 10 patients. 8 It is concluded that the nicardipine-induced coronary dilatation seems to improve perfusion and aerobic metabolism in areas with chronic ischaemia, resulting in reduced lactate production and augmented oxygen consumption. PMID:2862900

  13. Treprostinil potentiates the positive inotropic effect of catecholamines in adult rat ventricular cardiomyocytes

    PubMed Central

    Fontana, M; Olschewski, H; Olschewski, A; Schlüter, K-D

    2007-01-01

    Background and purpose: Prostanoids have been shown to improve exercise tolerance, hemodynamics and quality of life in patients with pulmonary arterial hypertension (PAH). We investigated whether treprostinil exerts direct contractile effects on cardiomyocytes that may explain partly the beneficial effects of these drugs. Experimental approach: Ventricular cardiomyocytes from adult rats were paced at a constant frequency of 0.5 to 2.0 Hz and cell shortening was monitored via a cell edge detection system. Twitch amplitudes, expressed as percent cell shortening of the diastolic cell length, and maximal contraction velocity, relaxation velocity, time to peak of contraction and time to reach 50% of relaxation were analyzed. Key results: Treprostinil (0.15 – 15 ng ml−1) slightly increased contractile dynamics of cardiomyocytes at clinically relevant concentrations. However, the drug significantly improved cell shortening of cardiomyocytes in the presence of isoprenaline, a β-adrenoceptor agonist. Treprostinil exerted this effect at all beating frequencies under investigation. Treprostinil mimicked this potentiating effect in a Langendorff preparation as well. The potentiating effect of treprostinil on isoprenaline-dependent cell shortening was no longer seen after phosphodiesterase inhibition. Long-term cultivation of cardiomyocytes with treprostinil did not modify load free cell shortening of these cells, but reduces the duration of contraction. Conclusions and implications: We conclude that the clinically used prostanoid treprostinil potentiates the positive inotropic effects of catecholamines in adult ventricular cardiomyocytes. This newly described effect may contribute to the beneficial clinical effects of prostanoids in patients with PAH. PMID:17533419

  14. [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

  15. 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

  16. Comparison of the intrinsic vasorelaxant and inotropic effects of the antiarrhythmic agents vernakalant and flecainide in human isolated vascular and cardiac tissues.

    PubMed

    Lynch, Joseph J; Regan, Christopher P; Beatch, Gregory N; Gleim, Gilbert W; Morabito, Christopher J

    2013-03-01

    This study explored the intrinsic vasorelaxant and inotropic effects of the mixed potassium and sodium channel blocker atrial antiarrhythmic vernakalant and the class IC antiarrhythmic agent flecainide in human isolated subcutaneous resistance artery and in ventricular trabecular muscle preparations. At test concentrations encompassing free plasma concentrations associated with clinical efficacy for conversion of atrial fibrillation, vernakalant (1-10 μM) displayed no significant direct effects on human resistance artery tone or ventricular contractility. In contrast, tested at equimolar concentrations, flecainide significantly reduced peak isometric contractile force (10 μM) and maximal rates of force development and decline (3 and 10 μM) in the human ventricular muscle preparation while displaying no significant effect on human resistance artery tone. The lack of effects of vernakalant on human resistance artery tone and ventricular muscle contractile function suggests that direct vasorelaxant and inotropic effects do not underlie the rare hypotensive events observed clinically with vernakalant, raising the possibility that secondary (eg, reflex) effects may mediate these events. The demonstration of negative inotropic effects with flecainide in the human ventricular muscle preparations in the absence of an effect on resistance artery tone suggests that the hemodynamic effects of flecainide observed clinically result primarily from direct negative inotropic effects. PMID:23188129

  17. Predictivity of in vitro non-clinical cardiac contractility assays for inotropic effects in humans--A literature search.

    PubMed

    Wallis, Rob; Gharanei, Mayel; Maddock, Helen

    2015-01-01

    Adverse drug effects on the cardiovascular system are a major cause of compound attrition throughout compound discovery and development. There are many ways by which drugs can affect the cardiovascular system, including effects on the electrocardiogram, vascular resistance, heart rate and the force of contraction of the heart (inotropy). Compounds that increase the force of contraction of the heart can be harmful in patients with ischemic heart disease, whilst negative inotropes can induce symptoms of heart failure. There is a range of non-clinical in vitro and in vivo assays used to detect inotropic effects of drugs. We have conducted a literature review of the in vitro assays and compared the findings from these with known effects on cardiac contractility in man. There was a wide variety of assays used, ranging from perfuse whole hearts to isolated regions of the heart (papillary muscle, ventricle and atria), which were removed from a number of species (cat, guinea pig, rabbit and rat). We conducted two analyses. The first was investigating the concordance of the findings from the in vitro assays at any concentration with those observed in man (an assessment of hazard identification) and the second was the concordance of the in vitro findings at concentrations tested up to 10-fold higher than those tested in the clinic. We found that when used as a hazard identification tool, the available assays had good sensitivity (88%), although the specificity was not so good (60%), but when used as a risk management tool the sensitivity was considerably reduced (sensitivity 58-70% and specificity 60%). These data would suggest that the available in vitro assays can be used as hazard identification tools for adverse drug effects on cardiac contractility, but there is a need for new assays to better predict the exposures in man that may cause a change in cardiac contractility and therefore better predict the likely therapeutic index of compounds prior to nomination of

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

    PubMed

    Gonano, Luis Alberto; Petroff, Martín Vila

    2014-12-01

    Cardiotonic glycosides or digitalis are positive inotropes used in clinical practice for the treatment of heart failure, which also exist as endogenous ligands of the Na(+)/K(+) ATPase. An increase in the intracellular Ca2+ content mediates their positive inotropic effect, but has also been proposed as a trigger of life-threatening arrhythmias. Although the mechanisms involved in the positive inotropic effect of these compounds have been extensively studied, those underlying their arrhythmogenic action remain ill defined. Recent evidence has placed posttranslational modifications of the ryanodine receptor (RyR2), leading to arrhythmogenic Ca2+ release, in the centre of the storm. In this review we will examine, in depth, the mechanisms that generate the arrhythmogenic substrate, focussing on the role played by the RyR2 and how its CaMKII-dependent regulation may shift the balance from an inotropic to an arrhythmogenic Ca2+ release. Finally, we will provide evidence suggesting that stabilising RyR2 function could result in a potential new strategy to prevent cardiotonic glycoside-induced arrhythmias that could lead to a safer and more extensive use of these compounds. PMID:25201479

  19. Inotropic and lusitropic effects of ghrelin and their modulation by the endocardial endothelium, NO, prostaglandins, GHS-R1a and KCa channels.

    PubMed

    Soares, João-Bruno; Rocha-Sousa, Amândio; Castro-Chaves, Paulo; Henriques-Coelho, Tiago; Leite-Moreira, Adelino F

    2006-07-01

    Contractile effects of ghrelin (10(-9) to 10(-6) M) were tested in rat papillary muscles of normal (n = 50) and hypertrophic (n = 16) right ventricles (RV). RV hypertrophy was induced by pulmonary hypertension using monocrotaline. In normal muscles, ghrelin was added either alone (n = 9) or after pre-treatment with indomethacin (cycloxygenase inhibitor, 10(-5) M; n = 10), L-nitro-L-arginin (NO synthase inhibitor, 10(-4) M; n = 9), D-Lys(3)-GHRP-6 (GHS-R1a antagonist; 10(-4) M; n = 8) or apamin+charybdotoxin (KCa channels blockers; 10(-6) M, n =7 ), as well as after damaging the endocardial endothelium (n = 7). In hypertrophic muscles, ghrelin was added either alone (n = 9) or after pre-treatment with apamin+charybdotoxin (10(-6 M, n=7). Ghrelin concentration-dependently decreased active tension (AT) and maximal velocity of tension rise (negative inotropic effect), as well as, maximal velocity of tension decay (negative lusitropic effect) and time to AT (onset of relaxation). These effects were maximal at 10(-6) M, similar in normal and hypertrophic muscles and were significantly altered only by apamin+charybdotoxin, indomethacin and L-nitro-L-arginin. Apamin+charybdotoxin attenuated the negative inotropic effect, while indomethacin and L-nitro-L-arginin, respectively, blunted and exacerbated the premature onset of relaxation. In conclusion, ghrelin induces negative inotropic and lusitropic effects and an earlier onset of relaxation in normal and hypertrophic myocardium, which are independent of GHS-R1a, since they were not affected by D-Lys(3)-GHRP-6. The negative inotropic effect is partly mediated by KCa channels, while the earlier onset of relaxation is modulated by prostaglandins and NO. PMID:16417945

  20. PDE3, but not PDE4, reduces β1- and β2-adrenoceptor-mediated inotropic and lusitropic effects in failing ventricle from metoprolol-treated patients

    PubMed Central

    Molenaar, Peter; Christ, Torsten; Hussain, Rizwan I; Engel, Andreas; Berk, Emanuel; Gillette, Katherine T; Chen, Lu; Galindo-Tovar, Alejandro; Krobert, Kurt A; Ravens, Ursula; Levy, Finn Olav; Kaumann, Alberto J

    2013-01-01

    Background and Purpose PDE3 and/or PDE4 control ventricular effects of catecholamines in several species but their relative effects in failing human ventricle are unknown. We investigated whether the PDE3-selective inhibitor cilostamide (0.3–1 μM) or PDE4 inhibitor rolipram (1–10 μM) modified the positive inotropic and lusitropic effects of catecholamines in human failing myocardium. Experimental Approach Right and left ventricular trabeculae from freshly explanted hearts of 5 non-β-blocker-treated and 15 metoprolol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through β1 adrenoceptors (β2 adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through β2 adrenoceptors (β1 adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of PDE inhibitors. Catecholamine potencies were estimated from –logEC50s. Key Results Cilostamide did not significantly potentiate the inotropic effects of the catecholamines in non-β-blocker-treated patients. Cilostamide caused greater potentiation (P = 0.037) of the positive inotropic effects of (-)-adrenaline (0.78 ± 0.12 log units) than (-)-noradrenaline (0.47 ± 0.12 log units) in metoprolol-treated patients. Lusitropic effects of the catecholamines were also potentiated by cilostamide. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline on right and left ventricular trabeculae from metoprolol-treated patients. Conclusions and Implications Metoprolol induces a control by PDE3 of ventricular effects mediated through both β1 and β2 adrenoceptors, thereby further reducing sympathetic cardiostimulation in patients with terminal heart failure. Concurrent therapy with a PDE3 blocker and metoprolol could conceivably facilitate cardiostimulation evoked by adrenaline through β2 adrenoceptors. PDE4 does not appear to reduce inotropic and lusitropic effects of

  1. 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

  2. Augmentation of the Inotropic Effects of Dopamine Following Linezolid Administration in an Infant.

    PubMed

    Lovett, Marlina E; Estrada, Maria; Sargel, Cheryl; Tobias, Joseph D

    2016-08-01

    Although generally safe and effective, one of the unique properties of linezolid is its weak inhibitory effect on monoamine oxidase. As such, it may interact with other medications that act through the adrenergic or serotonergic systems, including selective-serotonin reuptake inhibitors and vasoactive agents. We present a 3-month-old infant who was being treated with dopamine to maintain mean arterial pressure during mechanical ventilation following viral-induced respiratory failure. Hypertension and tachycardia developed during the administration of linezolid on two separate occasions. The physiology of catecholamine metabolism is reviewed including the role of the monoamine oxidase system. The potential interaction between linezolid and vasoactive agents such as dopamine is discussed. PMID:26864168

  3. (−)-Timolol is a more potent antagonist of the positive inotropic effects of (−)-adrenaline than of those of (−)-noradrenaline in human atrium

    PubMed Central

    WANG, T; KAUMANN, A J; BROWN, M J

    1996-01-01

    The affinity of (−)-timolol for β1- and β2-adrenoceptors was determined on isolated atrial preparations from patients undergoing open heart surgery. The times for onset and offset of antagonism of the positive inotropic effects of (−)-adrenaline and (−)-noradrenaline by (−)-timolol were measured. The antagonism of the positive inotropic effects of (−)-adrenaline and (−)-noradrenaline by (−)-timolol (0.1–100 nm) was simple competitive in human atrium tissue. The slope of Schild-plots was not significantly different from 1.0 [0.93±0.09 for (−)-adrenaline, 0.97±0.09 for (−)-noradrenaline]. The inotropic effects of (−)-adrenaline were antagonized significantly more by each concentration of (−)-timolol than those of (−)-noradrenaline. KB-values (-log m) were 10.10±0.09 against (−)-adrenaline and 9.43±0.07 against (−)-noradrenaline (P<0.001). Blocking kinetics of (−)-timolol for the β-adrenoceptor were relatively slow. Half-times for the onset of blockade by 10 times KB of (−)-timolol were approximately 30 min for both (−)-adrenaline and (−)-noradrenaline; offset times were similar. It is concluded that (−)-timolol has a higher affinity for the β2-adrenoceptor than for the β1-adrenoceptor in human atrium. This property may be beneficial clinically in protecting against the β2-adrenoceptor hypersensitivity induced by cardiac β1-adrenoceptor blockade, but also explain why severe asthma can occur after administration of very low intra-ocular doses of the drug. PMID:8864321

  4. Positive inotropic effect of the inhibition of cyclic GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2) on guinea pig left atria in eu- and hyperthyroidism.

    PubMed

    Gesztelyi, R; Zsuga, J; Hajdú, P; Szabó, J Zs; Cseppento, A; Szentmiklósi, A J

    2003-12-01

    The significance of PDE2 on the atrial inotropy was studied in eu- and hyperthyroidism. The contractile force was measured and negative inotropic capacity of N6-cyclopentyladenosine (CPA) was determined on left atria isolated from 8-day thyroxine- or solvent-treated guinea pigs, in the presence or absence of EHNA (adenosine deaminase and PDE2 inhibitor) or NBTI (nucleoside transporter inhibitor). EHNA was administered to inhibit PDE2, while NBTI was used to model the accumulation of endogenous adenosine. The reduction of the contractile force caused by EHNA was smaller in the thyroxine-treated atria than in the solvent-treated samples. Contrary, NBTI induced a decrease in the contractile force without significant difference between the two groups. In addition, EHNA enhanced the efficiency of CPA in thyroxine-treated atria and did not affect it in solvent-treated samples, while the response to CPA was decreased by NBTI in all atria, especially in hyperthyroidism. On the basis of greater retention of the contractile force and sustained/enhanced responsiveness to CPA in the presence of EHNA we conclude that PDE2's inhibition has a significant positive inotropic effect in guinea pig atria and this effect is proven to be augmented in hyperthyroidism. PMID:15113122

  5. High concentrations of drug in target tissues following local controlled release are utilized for both drug distribution and biologic effect: an example with epicardial inotropic drug delivery.

    PubMed

    Maslov, Mikhail Y; Edelman, Elazer R; Wei, Abraham E; Pezone, Matthew J; Lovich, Mark A

    2013-10-28

    Local drug delivery preferentially loads target tissues with a concentration gradient from the surface or point of release that tapers down to more distant sites. Drug that diffuses down this gradient must be in unbound form, but such drug can only elicit a biologic effect through receptor interactions. Drug excess loads tissues, increasing gradients and driving penetration, but with limited added biological response. We examined the hypothesis that local application reduces dramatically systemic circulating drug levels but leads to significantly higher tissue drug concentration than might be needed with systemic infusion in a rat model of local epicardial inotropic therapy. Epinephrine was infused systemically or released locally to the anterior wall of the heart using a novel polymeric platform that provides steady, sustained release over a range of precise doses. Epinephrine tissue concentration, upregulation of cAMP, and global left ventricular response were measured at equivalent doses and at doses equally effective in raising indices of contractility. The contractile stimulation by epinephrine was linked to drug tissue levels and commensurate cAMP upregulation for IV systemic infusion, but not with local epicardial delivery. Though cAMP was a powerful predictor of contractility with local application, tissue epinephrine levels were high and variable--only a small fraction of the deposited epinephrine was utilized in second messenger signaling and biologic effect. The remainder of deposited drug was likely used in diffusive transport and distribution. Systemic side effects were far more profound with IV infusion which, though it increased contractility, also induced tachycardia and loss of systemic vascular resistance, which were not seen with local application. Local epicardial inotropic delivery illustrates then a paradigm of how target tissues differentially handle and utilize drug compared to systemic infusion. PMID:23872515

  6. The Inotropic Effect of the Active Metabolite of Levosimendan, OR-1896, Is Mediated through Inhibition of PDE3 in Rat Ventricular Myocardium

    PubMed Central

    Ørstavik, Øivind; Manfra, Ornella; Andressen, Kjetil Wessel; Andersen, Geir Øystein; Skomedal, Tor; Osnes, Jan-Bjørn; Levy, Finn Olav; Krobert, Kurt Allen

    2015-01-01

    Aims We recently published that the positive inotropic response (PIR) to levosimendan can be fully accounted for by phosphodiesterase (PDE) inhibition in both failing human heart and normal rat heart. To determine if the PIR of the active metabolite OR-1896, an important mediator of the long-term clinical effects of levosimendan, also results from PDE3 inhibition, we compared the effects of OR-1896, a representative Ca2+ sensitizer EMD57033 (EMD), levosimendan and other PDE inhibitors. Methods Contractile force was measured in rat ventricular strips. PDE assay was conducted on rat ventricular homogenate. cAMP was measured using RII_epac FRET-based sensors. Results OR-1896 evoked a maximum PIR of 33±10% above basal at 1 μM. This response was amplified in the presence of the PDE4 inhibitor rolipram (89±14%) and absent in the presence of the PDE3 inhibitors cilostamide (0.5±5.3%) or milrinone (3.2±4.4%). The PIR was accompanied by a lusitropic response, and both were reversed by muscarinic receptor stimulation with carbachol and absent in the presence of β-AR blockade with timolol. OR-1896 inhibited PDE activity and increased cAMP levels at concentrations giving PIRs. OR-1896 did not sensitize the concentration-response relationship to extracellular Ca2+. Levosimendan, OR-1896 and EMD all increased the sensitivity to β-AR stimulation. The combination of either EMD and levosimendan or EMD and OR-1896 further sensitized the response, indicating at least two different mechanisms responsible for the sensitization. Only EMD sensitized the α1-AR response. Conclusion The observed PIR to OR-1896 in rat ventricular strips is mediated through PDE3 inhibition, enhancing cAMP-mediated effects. These results further reinforce our previous finding that Ca2+ sensitization does not play a significant role in the inotropic (and lusitropic) effect of levosimendan, nor of its main metabolite OR-1896. PMID:25738589

  7. Pharmacological investigation on asclepin--a new cardenolide from Asclepias curassavica. Part II. Comparative studies on the inotropic and toxic effects of asclepin, g-strophantin, digoxin and digitoxin).

    PubMed

    Patnaik, G K; Köhler, E

    1978-01-01

    The cardiac effects of asclepin, a new glycoside from the plant Asclepias curassavica, were studies in vitro (isolated atrium and heart of guineapig) and in vivo (anaesthetized cat) and were compared with g-strophanthin, digoxin, digitoxin, or digitoxigenin, resp. Asclepin showed a marked positive inotropic effect as evidenced by the increase in the force of contraction, measured by (dp/dt)max and (formula: see text). It was found to be more active than the other glycosides. PMID:380581

  8. An overview of inotropic agents.

    PubMed

    Vroom, Margreeth B

    2006-09-01

    The use of inotropic agents has been surrounded by many controversies. Recent guidelines for the treatment of patients with chronic and acute heart failure have elucidated some of the issues, but many remain. As a result, a substantial variability in the use of agents between institutions and caregivers remains, which mainly results from the lack of uniform data in the literature. Prospective randomized trials with a long-term follow-up and sufficient power are clearly needed, and a number of trials are currently in progress. PMID:16959760

  9. cAMP- and Ca2+/calmodulin-dependent protein kinases mediate inotropic, lusitropic and arrhythmogenic effects of urocortin 2 in mouse ventricular myocytes

    PubMed Central

    Yang, Li-Zhen; Kockskämper, Jens; Khan, Shelina; Suarez, Jorge; Walther, Stefanie; Doleschal, Bernhard; Unterer, Gregor; Khafaga, Mounir; Mächler, Heinrich; Heinzel, Frank R; Dillmann, Wolfgang H; Pieske, Burkert; Spiess, Joachim

    2011-01-01

    BACKGROUND AND PURPOSE Urocortin 2 is beneficial in heart failure, but the underlying cellular mechanisms are not completely understood. Here we have characterized the functional effects of urocortin 2 on mouse cardiomyocytes and elucidated the underlying signalling pathways and mechanisms. EXPERIMENTAL APPROACH Mouse ventricular myocytes were field-stimulated at 0.5 Hz at room temperature. Fractional shortening and [Ca2+]i transients were measured by an edge detection and epifluorescence system respectively. Western blots were carried out on myocyte extracts with antibodies against total phospholamban (PLN) and PLN phosphorylated at serine-16. KEY RESULTS Urocortin 2 elicited time- and concentration-dependent positive inotropic and lusitropic effects (EC50: 19 nM) that were abolished by antisauvagine-30 (10 nM, n = 6), a specific antagonist of corticotrophin releasing factor (CRF) CRF2 receptors. Urocortin 2 (100 nM) increased the amplitude and decreased the time constant of decay of the underlying [Ca2+]i transients. Urocortin 2 also increased PLN phosphorylation at serine-16. H89 (2 µM) or KT5720 (1 µM), two inhibitors of protein kinase A (PKA), as well as KN93 (1 µM), an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), suppressed the urocortin 2 effects on shortening and [Ca2+]i transients. In addition, urocortin 2 also elicited arrhythmogenic events consisting of extra cell shortenings and extra [Ca2+]i increases in diastole. Urocortin 2-induced arrhythmogenic events were significantly reduced in cells pretreated with KT5720 or KN93. CONCLUSIONS AND IMPLICATIONS Urocortin 2 enhanced contractility in mouse ventricular myocytes via activation of CRF2 receptors in a cAMP/PKA- and Ca2+/CaMKII-dependent manner. This enhancement was accompanied by Ca2+-dependent arrhythmogenic effects mediated by PKA and CaMKII. PMID:20942811

  10. Flunarizine but not theophylline modulates inotropic responses of the isolated rat heart to diazepam.

    PubMed

    Leeuwin, R S; Zeegers, A; Van Wilgenburg, H

    1996-11-14

    Diazepam (2 x 10(-5)-6 x 10(-4) M) induced a concentration-dependent positive inotropic effect on the perfused rat heart which was preceded by a transient concentration-dependent negative inotropic response. The influence of the Ca(2+)-entry blocking drug, flunarizine, and the adenosine receptor blocking drug, theophylline on these inotropic responses was studied. Flunarizine in concentrations of 10(-9)-10(-6) M antagonized the positive inotropic response to diazepam significantly; the negative inotropic response was reduced as well. At the lower concentrations of diazepam the negative inotropic response was completely abolished in the presence of flunarizine. The actions of the Ca(2+)-entry blocker were related to the concentrations used. Theophylline in concentrations up to 5 x 10(-5) M did not interfere with either inotropic response to diazepam. The results suggest that Ca2+ currents in the myocardium are involved with the response of the isolated heart to diazepam. It is concluded that the finding that the negative inotropic effect of diazepam was almost abolished by flunarizine suggests that the site of this response most be associated with Ca(2+)-current mechanisms. PMID:8960878

  11. [Vasoactive and inotropic drugs in acute heart failure].

    PubMed

    Ruiz-Laiglesia, Fernando José; Camafort-Babkowski, Miguel

    2014-03-01

    Vasoactive and inotropic drugs provide effective symptomatic and hemodynamic relief in the short term but can increase mortality in the long-term. Consequently, their use should be restricted to the indications described in clinical practice guidelines. The present article reviews the main drugs and the available evidence on their use. PMID:24930084

  12. 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

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

    PubMed Central

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

    1989-01-01

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

  14. Acute heart failure: inotropic agents and their clinical uses.

    PubMed

    Endoh, Masao; Hori, Masatsugu

    2006-11-01

    Inotropic agents are indispensable for the improvement of cardiac contractile dysfunction in acute or decompensated heart failure. Clinically available agents, including sympathomimetic amines (dopamine, dobutamine, noradrenaline) and selective phosphodiesterase-3 inhibitors (amrinone, milrinone, olprinone and enoximone) act via cAMP/protein kinase A (PKA)-mediated facilitation of intracellular Ca2+ mobilisation. Phosphodiesterase-3 inhibitors also have a vasodilatory action, which plays a role in improving haemodynamic parameters in certain patients, and are termed inodilators. The available inotropic agents suffer from risks of Ca2+ overload leading to arrhythmias, myocardial cell injury and ultimately, cell death. In addition, they are energetically disadvantageous because of an increase in activation energy and cellular metabolism. Furthermore, they lose their effectiveness under pathophysiological conditions, such as acidosis, stunned myocardium and heart failure. Pimobendan and levosimendan (that act by a combination of an increase in Ca2+ sensitivity and phosphodiesterase-3 inhibition) appear to be more beneficial among existing agents. Novel Ca2+ sensitisers that are under basic research warrant clinical trials to replace available inotropic agents. PMID:17059376

  15. Positive inotropic action of saponins on isolated atrial and papillary muscles from the guinea-pig.

    PubMed Central

    Enomoto, Y.; Ito, K.; Kawagoe, Y.; Morio, Y.; Yamasaki, Y.

    1986-01-01

    The effects of several saponins of animal and plant origin on the contractile activity of atrial and papillary muscles of the guinea-pig were tested. In a concentration of 1 X 10(-5)M, holothurin-A (HL-A), holothurin-B, echinoside-A, echinoside-B and sakuraso-saponin (Saku) exhibited positive inotropic and chronotropic actions whereas desacyl-jego-saponin and ginsenoside-Rd did not. Saponins having a positive inotropic action caused haemolysis of rabbit erythrocytes whereas those without inotropic action did not cause haemolysis. The positive inotropic action of saponins was not affected by practolol, chlorpheniramine, cimetidine and indomethacin. Verapamil (10(-6)M) inhibited the inotropic actions due to HL-A and isoprenaline (10(-8)M) to the same extent but had a small effect on those due to ouabain (10(-7)M). In high K+ (30 mM K+) medium where the action potential and the contraction were depressed, HL-A, Saku and isoprenaline restored the action potential and the contraction of the 'slow response' type whereas ouabain failed to do so. In normal medium HL-A and Saku reduced the resting membrane potential by 15-20 mV. These results suggest that modification of the Ca channel is involved in the positive inotropic action of saponins. PMID:3708218

  16. Understanding Oxadiazolothiazinone Biological Properties: Negative Inotropic Activity versus Cytochrome P450-Mediated Metabolism.

    PubMed

    Carosati, Emanuele; Cosimelli, Barbara; Ioan, Pierfranco; Severi, Elda; Katneni, Kasiram; Chiu, Francis C K; Saponara, Simona; Fusi, Fabio; Frosini, Maria; Matucci, Rosanna; Micucci, Matteo; Chiarini, Alberto; Spinelli, Domenico; Budriesi, Roberta

    2016-04-14

    We present a series of oxadiazolothiazinones, selective inotropic agents on isolated cardiac tissues, devoid of chronotropy and vasorelaxant activity. Functional and binding data for the precursor of the series (compound 1) let us hypothesize LTCC blocking activity and the existence of a recognition site specific for this scaffold. We synthesized and tested 22 new derivatives: introducing a para-methoxyphenyl at C-8 led to compound 12 (EC50 = 0.022 μM), twice as potent as its para-bromo analogue (1). For 10 analogues, we extended the characterization of the biological properties by including the assessment of metabolic stability in human liver microsomes and cytochrome P450 inhibition potential. We observed that the methoxy group led to active compounds with low metabolic stability and high CYP inhibition, whereas the protective effect of bromine resulted in enhanced metabolic stability and reduced CYP inhibition. Thus, we identified two para-bromo benzothiazino-analogues as candidates for further studies. PMID:26962886

  17. Hospital Patterns of Use of Positive Inotropic Agents in Patients with Heart Failure

    PubMed Central

    Partovian, Chohreh; Gleim, Scott R.; Mody, Purav S.; Li, Shu-Xia; Wang, Haiyan; Strait, Kelly M.; Allen, Larry A.; Lagu, Tara C.; Normand, Sharon-Lise T.; Krumholz, Harlan M.

    2013-01-01

    Objective We sought to determine hospital variation in the use of positive inotropic agents in patients with heart failure. Background Clinical guidelines recommend targeted use of positive inotropic agents in highly selected patients, but data are limited and the recommendations are not specific. Methods We analyzed data from 376 hospitals including 189,948 hospitalizations for heart failure during 2009–10. We used hierarchical logistic regression models to estimate hospital-level risk-standardized rates of inotrope use and risk-standardized in-hospital mortality rates. Results The risk-standardized rates of inotrope use ranged across hospitals from 0.9% to 44.6% (median: 6.3%, inter-quartile range: 4.3% to 9.2%). We identified various hospital patterns based on the type of agents: dobutamine-predominant (29% of hospitals), dopamine-predominant (25%), milrinone-predominant (1%), mixed dobutamine/dopamine pattern (32%), and mixed pattern including all 3 agents (13%). When studying the factors associated with inter-hospital variation, the best model performance was with the HGLM models that adjusted for patient case mix and an individual hospital effect (ROCs from 0.77 to 0.88). The intra-class correlation coefficients of the HGLMs (0.113 for any inotrope) indicated that a noteworthy proportion of the observed variation was related to an “individual institutional effect.” Hospital rates or patterns of use were not associated with differences in length of stay or risk-standardized mortality rates. Conclusions We found marked differences in the use of inotropic agents for heart failure patients among a diverse group of hospitals. This variability, occurring in the context of little clinical evidence, indicates an urgent need to define the appropriate use of these medications. PMID:22981548

  18. Elevated Serum Creatine Phosphokinase is Associated with Mortality and Inotropic Requirement in Critically Injured Adults

    PubMed Central

    Sowards, Kendell J.; Mukherjee, Kaushik; Norris, Patrick R.; Shintani, Ayumi; Ware, Lorraine B.; Roberts, L. Jackson; May, Addison K.

    2016-01-01

    Background Hemeproteins such as free myoglobin can undergo autoxidation and catalyze lipid peroxidation, increasing oxidative stress. Creatine phosphokinase (CPK) elevation is a marker for free myoglobin after myocyte damage. Since oxidative injury is a key mechanism of injury-related organ dysfunction, we hypothesized that serum CPK levels correlate with mortality and need for and duration of inotropic support, i.e. shock, among critically injured patients. Methods We conducted a retrospective review of 17,847 patients admitted to a single Trauma Intensive Care Unit over 9 years. 2,583 patients with serum CPK levels were included in the analysis. Patient data were collected continuously into an electronic ICU repository. Univariate analysis was accomplished using Spearman correlation and the Mann Whitney U test. Propensity score adjustment models accounting for potential confounders were used to assess the independent effect of CPK level on mortality, need for inotropic support, and duration of inotropic support. Results Median CPK was significantly higher in patients who died (916 [IQR 332, 2472] vs. 711 [253, 1971], p= 0.004) and in those who required inotropic medications (950 [353, 2525] vs. 469 [188, 1220], p< 0.001). After adjusting for propensity score and potential confounders the odds of mortality increased by 1.10 (95% CI 1.02– 1.19, p= 0.020) and the odds of inotropic medication use increased by 1.30 (95% CI 1.22–1.38, p <0.001) per natural log unit increase in CPK. There was a significant association between CPK level and duration of inotropic support (Spearman’s rho .237, p< 0.001) that remained significant in a propensity score adjusted model. Conclusion In critically injured patients, elevated serum CPK level is independently associated with mortality, need for inotropic medication, and duration of inotropic support. This study is the first to evaluate the relationship of CPK level and mortality in addition to surrogate measures of shock in a

  19. Pharmacotherapy update on the use of vasopressors and inotropes in the intensive care unit.

    PubMed

    Jentzer, Jacob C; Coons, James C; Link, Christopher B; Schmidhofer, Mark

    2015-05-01

    This paper summarizes the pharmacologic properties of vasoactive medications used in the treatment of shock, including the inotropes and vasopressors. The clinical application of these therapies is discussed and recent studies describing their use and associated outcomes are also reported. Comprehension of hemodynamic principles and adrenergic and non-adrenergic receptor mechanisms are salient to the appropriate therapeutic utility of vasoactive medications for shock. Vasoactive medications can be classified based on their direct effects on vascular tone (vasoconstriction or vasodilation) and on the heart (presence or absence of positive inotropic effects). This classification highlights key similarities and differences with respect to pharmacology and hemodynamic effects. Vasopressors include pure vasoconstrictors (phenylephrine and vasopressin) and inoconstrictors (dopamine, norepinephrine, and epinephrine). Each of these medications acts as vasopressors to increase mean arterial pressure by augmenting vascular tone. Inotropes include inodilators (dobutamine and milrinone) and the aforementioned inoconstrictors. These medications act as inotropes by enhancing cardiac output through enhanced contractility. The inodilators also reduce afterload from systemic vasodilation. The relative hemodynamic effect of each agent varies depending on the dose administered, but is particularly apparent with dopamine. Recent large-scale clinical trials have evaluated vasopressors and determined that norepinephrine may be preferred as a first-line therapy for a broad range of shock states, most notably septic shock. Consequently, careful selection of vasoactive medications based on desired pharmacologic effects that are matched to the patient's underlying pathophysiology of shock may optimize hemodynamics while reducing the potential for adverse effects. PMID:25432872

  20. Vasopressor and Inotropic Management Of Patients With Septic Shock

    PubMed Central

    Pollard, Sacha; Edwin, Stephanie B.; Alaniz, Cesar

    2015-01-01

    Numerous studies have evaluated the role of vasopressors and inotropes in the management of septic shock. This review assesses available evidence for the use of specific vasopressors in the management of septic shock. Use of adjunctive vasopressor therapy is also evaluated, examining the potential value of individual agents. Lastly, inotropic agents are evaluated for use in patients with myocardial dysfunction. PMID:26185405

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

    PubMed Central

    Tariq, Sohaib; Aronow, Wilbert S.

    2015-01-01

    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. PMID:26690127

  2. Inotropes do not increase mortality in advanced heart failure

    PubMed Central

    Guglin, Maya; Kaufman, Marc

    2014-01-01

    Inotrope use is one of the most controversial topics in the management of heart failure. While the heart failure community utilizes them and recognizes the state of inotrope dependency, retrospective analyses and registry data have overwhelmingly suggested high mortality, which is logically to be expected given the advanced disease states of those requiring their use. Currently, there is a relative paucity of randomized control trials due to the ethical dilemma of creating control groups by withholding inotropes from patients who require them. Nonetheless, results of such trials have been mixed. Many were also performed with agents no longer in use, on patients without an indication for inotropes, or at a time before automatic cardio-defibrillators were recommended for primary prevention. Thus, their results may not be generalizable to current clinical practice. In this review, we discuss current indications for inotrope use, specifically dobutamine and milrinone, depicting their mechanisms of action, delineating their patterns of use in clinical practice, defining the state of inotrope dependency, and ultimately examining the literature to ascertain whether evidence is sufficient to support the current view that these agents increase mortality in patients with heart failure. Our conclusion is that the evidence is insufficient to link inotropes and increased mortality in low output heart failure. PMID:24899821

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

    SciTech Connect

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

    1986-03-05

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

  4. Positive inotropic effect of purified green tea catechin derivative in guinea pig hearts: the measurements of cellular Ca2+ and nitric oxide release.

    PubMed

    Hotta, Yoshihiro; Huang, Lei; Muto, Tatsuya; Yajima, Michio; Miyazeki, Kunihiro; Ishikawa, Naohisa; Fukuzawa, Yoshitaka; Wakida, Yasushi; Tushima, Hiromi; Ando, Hiroaki; Nonogaki, Tunemasa

    2006-12-15

    Each individual and pure catechin isolated from green tea was investigated as to its myocardial or blood pressure effects. The nitric oxide (NO) electrode and fluorometry were used to monitor changes in the NO and Ca(2+) contents of the heart, together with simultaneous recordings of the left ventricular developed pressure. The low dose of (-)-epigallocatechin-3-gallate (EGCg: 10(-6), 10(-5 )M) increased the left ventricular developed pressure with elevation of the transient fura-2 Ca(2+) signal (T(Ca)), but the high dose of EGCg (10(-4 )M) produced a maximum left ventricular developed pressure with decreases in the basal level of T(Ca) in a manner similar to the administration of the Ca-sensitizer pimobendan. However, the level of the transient NO signal (T(NO)) increased dose-dependently without any increases in the width of T(NO). In the isolated right atria, the contractile force of (-)-gallocatechin-3-gallate (GCg) at 10(-8)-10(-4 )M produced the highest pD(2) value, 6.7, in catechins (EGCg: 5.2, pimobendan: 5.1), but did not affect the heart rate. GCg, an artifact due to the epimerization of EGCg during the heating procedure, showed the most prolonged hypotensive effect in rabbits among the catechins. Each catechin (GCg or EGCg), like the NO donor, may have a therapeutic use as an NO-mediated vasorelaxant and may have an additional protective action in myocardial ischemia-reperfusion induced injury. PMID:17059816

  5. Positive inotropes in heart failure: a review article

    PubMed Central

    Amin, Ahmad; Maleki, Majid

    2012-01-01

    Increasing myocardial contractility has long been considered a big help for patients with systolic heart failure, conferring an augmented haemodynamic profile in terms of higher cardiac output, lower cardiac filling pressure and better organ perfusion. Though concerns have been raised over the safety issues regarding the clinical trials of different inotropes in hearts with systolic dysfunction, they still stand as a main therapeutic strategy in many centres dealing with such patients. They must be used as short in duration, low in dose and stopped as early as possible. Evidence-based guidelines have provided clinicians with valuable data for better applying inotropes in heart failure patients. In this paper, the authors address clinical trials with different agents used for increasing cardiac contractility in heart failure patients. Furthermore, the authors focus on recent guidelines on making the most out of inotropes in heart failure patients.

  6. 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

  7. 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

  8. Pharmacological characterization of the activity of endogenous inotropic factor from porcine left ventricle.

    PubMed

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

    1993-01-01

    We report some of the unique pharmacological properties of a semipurified endogenous inotropic factor (EIF) present in the extract of the porcine left ventricle. EIF produced the following effects: (a) increase in isometric contractile force developed by electrically driven canine right ventricular trabecula, reaching a maximum with 60-100 microliters/ml concentration; (b) inhibition of Na-pump activity in canine portal vein; (c) no digitalis-like cardiac toxicity, e.g., increased diastolic tension or spontaneous diastolic mechanical oscillatory activity, despite inhibition of the sodium pump; (d) a small increase in sarcoplasmic reticular Ca release from the heart but a large increase in transsarcolemmal Ca influx as seen in biphasic contractions, an action similar to that produced by digitalis-like substances; and (e) prolongation of the action potential duration and refractory period of the canine isolated trabeculae. This latter action may confer a unique antiarrhythmic property to EIF. PMID:7508042

  9. 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

  10. Inotropes and Inodilators for Acute Heart Failure: Sarcomere Active Drugs in Focus

    PubMed Central

    Nagy, László; Papp, Zoltán

    2014-01-01

    Abstract: Acute heart failure (AHF) emerges as a major and growing epidemiological concern with high morbidity and mortality rates. Current therapies in patients with acute heart failure rely on different strategies. Patients with hypotension, hypoperfusion, or shock require inotropic support, whereas diuretics and vasodilators are recommended in patients with systemic or pulmonary congestion. Traditionally inotropic agents, referred to as Ca2+ mobilizers load the cardiomyocyte with Ca2+ and thereby increase oxygen consumption and risk for arrhythmias. These limitations of traditional inotropes may be avoided by sarcomere targeted agents. Direct activation of the cardiac sarcomere may be achieved by either sensitizing the cardiac myofilaments to Ca2+ or activating directly the cardiac myosin. In this review, we focus on sarcomere targeted inotropic agents, emphasizing their mechanisms of action and overview the most relevant clinical considerations. PMID:24785346

  11. Effects of contamination on radioligand binding parameters.

    PubMed

    Lazareno, S; Birdsall, N J

    2000-02-01

    Radioligand binding studies are used to provide quantitative estimates of parameters such as the receptor density of a tissue and the affinity values of labelled and unlabelled ligands. The presence of an unlabelled competing contaminant, which might be present because of actual contamination, inadequate radioligand purification or the breakdown of the radioligand to an active species, has surprising effects on these estimates: the apparent affinity of the radioligand is increased but the Ki values of unlabelled ligands are unaffected. The most striking and sensitive effects are on radioligand association kinetics, which become independent of radioligand concentration at high radioligand concentrations. PMID:10664609

  12. Efficacy and limitations of oral inotropic agents for the treatment of chronic heart failure.

    PubMed

    Murai, Koji; Seino, Yoshihiko; Kimata, Nakahisa; Inami, Toru; Murakami, Daisuke; Abe, Junko; Yodogawa, Kenji; Maruyama, Mitsunori; Takano, Masamichi; Ohba, Takayoshi; Ibuki, Chikao; Mizuno, Kyoichi

    2013-01-01

    The heart failure guideline in Japan has stated the necessity of investigating the role of oral inotropic agents in patients with chronic heart failure (CHF), which are clinically available only in Japan. A total of 1,846 consecutive patients with heart failure (mean: 69.5 years old, 1,279 males) treated at our institute from November 2009 to August 2010 were investigated retrospectively. Thirty-one patients (1.84%) who had taken oral inotropic agents (pimobendan 27, docarpamine 6, and denopamine 4) were extracted for this study, and the efficacy and limitations of the treatments were analyzed. Following the oral inotropic treatment, the NYHA functional class (P = 0.017), cardiothoracic ratio (P = 0.002) and B-type natriuretic peptide levels (P = 0.011) were significantly improved, and the number of emergency room (ER) visits (P < 0.001) and hospitalizations (P < 0.001) were significantly reduced. The nonsurviving patients (n = 7/31, 22.6%) were significantly older (P = 0.02) and tended to have a larger cardiothoracic ratio (P = 0.084) compared with the survivors. An absence of concomitant beta-blocker therapy was significantly associated with a worse prognosis (oneyear mortality 2/21 versus 5/10, log rank, P = 0.011). Oral inotropic agents brought about improvements in the clinical parameters of CHF and a reduction in ER visits and hospitalizations. However, concomitant beta-blocker therapy should be considered for patients receiving oral inotropic treatment. PMID:23676366

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

    PubMed Central

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

    1989-01-01

    1. Isolated left atria from reserpine-pretreated guinea-pigs, electrically driven (3 Hz) in the presence of atropine (1 microM), phentolamine (0.3 microM) and propranolol (1 microM), responded to a train of stimuli (10 Hz for 2.5s) with a delayed neurogenic positive inotropic response which was insensitive to hexamethonium (10 microM) but abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to calcitonin gene-related peptide (CGRP). 2. In these experimental conditions, gamma-aminobutyric acid (GABA) produced a concentration-related (10 microM-1 mM) positive inotropic response similar to that produced by electrical field stimulation. The effect of GABA was competitively antagonized by bicuculline methiodide (10 microM), a GABAA receptor antagonist. 3. The selective GABAA receptor agonists, muscimol and homotaurine mimicked the positive inotropic effect of GABA while baclofen, the selective GABAB receptor agonist, did not. 4. The action of GABA (1 mM) was abolished by either tetrodotoxin (1 microM), omega-conotoxin (0.1 microM), in vitro capsaicin desensitization or desensitization to CGRP, while it was unaffected by hexamethonium. In contrast, the inotropic response to CGRP was unaffected by tetrodotoxin, omega-conotoxin, bicuculline methiodide, hexamethonium or in vitro capsaicin desensitization, but was abolished by CGRP desensitization. 5. In the spontaneously beating guinea-pig right atrium, GABA (1 microM) produced a small and transient positive chronotropic effect that was no longer observed after in vitro desensitization with capsaicin (1 microM). 6. In the guinea-pig isolated perfused heart from reserpine-pretreated animals (with atropine, phentolamine and propranolol in the perfusion medium), GABA (1 microM) produced a transient tachycardia and a small increase in coronary flow.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2541852

  14. 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

  15. Involvement of cyclooxygenase-2 in carbachol-induced positive inotropic response in mouse isolated left atrium.

    PubMed

    Hara, Yukio; Ike, Asako; Tanida, Riyo; Okada, Muneyoshi; Yamawaki, Hideyuki

    2009-12-01

    The mouse heart is expected to have characteristic contractile properties. However, basic information on the function of the mouse heart has not been accumulated sufficiently. In this study, the involvement of cyclooxygenase (COX)-2 in carbachol (CCh)-induced inotropic response was investigated in mouse isolated left atrium. Influences of CCh and their mechanisms of action on developed tension elicited by electrical stimulation were examined pharmacologically. The presence of COX-2 in atrium was examined by Western blotting and immunohistochemical analysis. CCh (3 microM for 15 min) produced a biphasic inotropic response: a transient decrease in contractile force followed by a late increase. Atropine suppressed the biphasic inotropic response to CCh. A muscarinic M(3) receptor antagonist, 4-diphenyl-acetoxy-N-methlpiperidine, inhibited the late positive inotropic action. Blockade of prostaglandin (PG) E(2) or F(2alpha) receptor by 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH6809) or 9alpha, 15R-dihydroxy-11beta-fluoro-15-(2,3-dihydro-1H-inden-2-yl)-16,17,18,19,20-pentanor-prosta 5Z, 13E-dien-1-oic acid (AL8810), respectively, significantly suppressed the positive inotropic response to CCh. A nonselective COX inhibitor, indomethacin, and a selective COX-2 inhibitor, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398) inhibited the positive response. A COX-1 inhibitor, valeroyl salicylate, did not affect the positive response. The positive response was almost completely abolished in the endocardial endothelium-deprived atria. Existence of COX-2 in endocardial endothelium was confirmed by Western blotting and immunohistochemical analysis. The present study indicated that the CCh-induced positive inotropic response was mediated by PGs, possibly PGE(2) and PGF(2alpha), released in part from endocardial endothelium. Furthermore, for the first time, we demonstrated that the production of PGs depended in part on COX-2 in endocardial endothelium through the

  16. 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

  17. Stabilization of Kv1.5 channel protein by the inotropic agent olprinone.

    PubMed

    Endo, Ryo; Kurata, Yasutaka; Notsu, Tomomi; Li, Peili; Morikawa, Kumi; Kondo, Takehito; Ogura, Kazuyoshi; Miake, Junichiro; Yoshida, Akio; Shirayoshi, Yasuaki; Ninomiya, Haruaki; Higaki, Katsumi; Kuwabara, Masanari; Yamamoto, Kazuhiro; Inagaki, Yoshimi; Hisatome, Ichiro

    2015-10-15

    Olprinone is an inotropic agent that inhibits phosphodiesterase (PDE) III and causes vasodilation. Olprinone has been shown to be less proarrhythmic and possibly affect expression of functional Kv1.5 channels that confer the ultra-rapid delayed-rectifier K+ channel current (IKur) responsible for action potential repolarization. To reveal involvement of Kv1.5 channels in the less arrhythmic effect of olprinone, we examined effects of the agent on the stability of Kv1.5 channel proteins expressed in COS7 cells. Olprinone at 30-1000 nM increased the protein level of Kv1.5 channels in a concentration-dependent manner. Chase experiments showed that olprinone delayed degradation of Kv1.5 channels. Olprinone increased the immunofluorescent signal of Kv1.5 channels in the endoplasmic reticulum (ER) and Golgi apparatus as well as on the cell surface. Kv1.5-mediated membrane currents, measured as 4-aminopyridine-sensitive currents, were increased by olprinone without changes in their activation kinetics. A protein transporter inhibitor, colchicine, abolished the olprinone-induced increase of Kv.1.5-mediated currents. The action of olprinone was inhibited by 4-aminopyridine, and was not mimicked by the application of 8-Bromo-cAMP. Taken together, we conclude that olprinone stabilizes Kv1.5 proteins at the ER through an action as a chemical chaperone, and thereby increases the density of Kv1.5 channels on the cell membrane. The enhancement of Kv1.5 currents could underlie less arrhythmogenicity of olprinone. PMID:26368666

  18. Parenteral inotropic therapy in the home: an update for home care and hospice.

    PubMed

    Lyons, Margaret G; Carey, Lawrence

    2013-04-01

    This article provides an evidence-based overview of heart failure (HF), including its pathogenesis, staging, assessment, prognosis, and treatment with intravenous inotropic medications in the home. Inotropic infusions in the home setting require advanced care planning, symptom management, and knowledge about ambulatory pumps and devices. These medications can be safely used throughout the continuum of care as pediatric/adult bridges from transplant to hospice care. Nurses who recognize advanced HF symptomatology and use prognostic/risk-stratification models will be better prepared to facilitate this advanced care planning, thus supporting optimal treatment outcomes. The ultimate goal of care for heart failure therapy in the home is to integrate pharmacotherapeutic knowledge of treatment advances with comfort measures and to provide them to patients concurrently and in a seamless process. PMID:23549250

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

    PubMed

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

    2016-06-01

    Intravenous inotropic therapy may be necessary to achieve short-term survival in end-stage heart failure patients with cardiogenic shock or extreme low output and severe organ hypoperfusion. However, mid- or long-term intravenous inotropic therapy is associated with an increased mortality in advanced stage D heart failure patients using β-adrenoceptor agonists (dobutamine) or PDE-3-inhibitors (milrinone). Intermittent levosimendan may evolve as a reasonable therapeutic option. Randomized trials or other meaningful scientific evidence addressing the optimal treatment of exclusively the most threatened subgroup of hospitalized patients with persistent severe organ hypoperfusion are missing, but urgently needed. Despite a lack of other beneficial pharmacological options, the use of long-term intravenous inotropic therapy as a treatment for refractory heart failure or as an obligatory criterion for high urgency (HU) listing of heart transplant candidates with a median waiting time of 66 days in Germany is not based on scientific evidence. In addition, it might create a disincentive to achieve the HU status as well as keeping it, thereby potentially exposing the patient to an unnecessary additional risk. Upcoming new allocation algorithms may possibly help to improve the inadequate present situation. There is need for both, a better definition and a better treatment of high risk terminal heart failure requiring high urgent transplant listing. PMID:26879807

  20. Effects of heparin on insulin binding and biological activity

    SciTech Connect

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

    1987-02-01

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

  1. 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

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

    PubMed

    Tahri-Jouti, M A; Chaby, R

    1991-07-01

    The respective roles of serum lipoproteins, and of the complement component C3, in the binding of endotoxin (LPS) to macrophages were analyzed by an in vitro assay using [3H]LPS. The addition of an anti-C3 serum in the medium induced an apparent abolishment of the specific binding of LPS to mouse macrophages, but this effect appeared to be due to an actual increase of nonspecific binding. Isolated complexes of LPS with lipoproteins of high density (HDL3) and of very high density (VHDL) did not bind to macrophages. Furthermore, addition of HDL3 and VHDL in the incubation medium inhibited the specific binding of LPS to macrophages. These results suggest that C3 reduces nonspecific interactions between LPS and macrophages whereas associations between LPS and HDL3 or VHDL inhibit specific LPS-macrophage interactions. PMID:1937584

  3. 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

  4. 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

  5. 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

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

    NASA Astrophysics Data System (ADS)

    Trynda-Lemiesz, Lilianna; Wiglusz, Katarzyna

    2011-05-01

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

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

    SciTech Connect

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

    1991-01-01

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

  8. Effects of exercise on insulin binding to human muscle.

    PubMed

    Bonen, A; Tan, M H; Clune, P; Kirby, R L

    1985-04-01

    A procedure was developed to measure insulin binding to human skeletal muscle obtained via the percutaneous muscle biopsy technique. With this method the effects of exercise on insulin binding were investigated. Subjects (n = 9) exercised for 60 min on a bicycle ergometer at intensities ranging from 20-86% maximum O2 consumption (VO2max). Blood samples were obtained before, during, and after exercise and analyzed for glucose and insulin. Muscle samples (250 mg) for the vastus lateralis were obtained 30 min before exercise, at the end of exercise, and 60 min after exercise. Two subjects rested during the experimental period. There was no linear relationship between exercise intensities and the changes in insulin binding to human muscle. At rest (n = 2) and at exercise intensities below 60% VO2max (n = 5) no change in insulin binding occurred (P greater than 0.05). However, when exercise occurred at greater than or equal to 69% VO2max (n = 4), a pronounced decrement in insulin binding (30-50%) was observed (P less than 0.05). This persisted for 60 min after exercise. These results indicate that insulin binding in human muscle is not altered by 60 min of exercise at less than or equal to 60% VO2max but that a marked decrement occurs when exercise is greater than or equal to 69% VO2max. PMID:3885753

  9. 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.

  10. Discriminatory effects in the optical binding of chiral nanoparticles

    NASA Astrophysics Data System (ADS)

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

    2015-08-01

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

  11. 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

  12. Adenosine deaminase inhibition enhances the inotropic response mediated by A1 adenosine receptor in hyperthyroid guinea pig atrium.

    PubMed

    Kemeny-Beke, Adam; Jakab, Anita; Zsuga, Judit; Vecsernyes, Miklos; Karsai, Denes; Pasztor, Fanni; Grenczer, Maria; Szentmiklosi, Andras Jozsef; Berta, Andras; Gesztelyi, Rudolf

    2007-08-01

    The aim of the present study was to test the hypothesis that inhibition of adenosine deaminase (ADA) enhances the efficiency of signal-transduction of myocardial A1 adenosine receptors in hyperthyroidism. The inotropic response to N6-cyclopentyladenosine (CPA), a selective A1 adenosine receptor agonist resistant to ADA, was investigated in the absence or presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an ADA and cGMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE2) inhibitor, or of pentostatin (2'-deoxycoformycin; DCF), an exclusive ADA inhibitor, in left atria isolated from eu- or hyperthyroid guinea pigs. Both ADA inhibitors enhanced the effect of CPA only in hyperthyroid atria. EHNA significantly increased the Emax (mean+/-S.E.M.) from 83.8+/-1.2% to 93.4+/-1.2%, while DCF significantly decreased the logEC50 from -7.5+/-0.07 to -7.83+/-0.07 in hyperthyroid samples. Conversely, EHNA also diminished the logEC50 (from -7.5+/-0.07 to -7.65+/-0.07) and DCF also raised the Emax (from 83.8+/-1.2% to 85.7+/-2%) in hyperthyroidism, but these changes were not significant. In conclusion, ADA inhibition moderately but significantly enhanced the efficiency of A(1) adenosine receptor signaling pathway in the hyperthyroid guinea pig atrium. This suggests that elevated intracellular adenosine level caused by ADA inhibition may improve the suppressed responsiveness to A1 adenosine receptor agonists associated with the hyperthyroid state. Alternatively or in addition, the role of decreased concentration of adenosine degradation products cannot be excluded. Furthermore, in the case of EHNA, inhibition of PDE2 also appears to contribute to the enhanced A1 adenosine receptor signaling in the hyperthyroid guinea pig atrium. PMID:17574432

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

    PubMed Central

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

    1988-01-01

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

  14. 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. PMID:26581834

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

    SciTech Connect

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

    1990-01-01

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

  16. Effective Binding of Methane Using a Weak Hydrogen Bond.

    PubMed

    Henley, Alice; Bound, Michelle; Besley, Elena

    2016-05-26

    The weak hydrogen bond is an important type of noncovalent interaction, which has been shown to contribute to stability and conformation of proteins and large biochemical membranes, stereoselectivity, crystal packing, and effective gas storage in porous materials. In this work, we systematically explore the interaction of methane with a series of functionalized organic molecules specifically selected to exhibit a weak hydrogen bond with methane molecules. To enhance the strength of hydrogen bond interactions, the functional groups include electron-enriched sites to allow sufficient polarization of the C-H bond of methane. The binding between nine functionalized benzene molecules and methane has been studied using the second order Møller-Plesset perturbation theory to reveal that benzenesulfonic acid (C6H5-SO3H) and phenylphosphonic acid (C6H5-PO3H2) have the greatest potential for efficient methane capture through hydrogen bonding interactions. Both acids exhibit efficient binding potential with up to three methane molecules. For additional insight, the atomic charge distribution associated with each binding site is presented. PMID:27148999

  17. Less Invasive and Inotrope-Reduction Approach to Automated Closed-Loop Control of Hemodynamics in Decompensated Heart Failure.

    PubMed

    Uemura, Kazunori; Kawada, Toru; Zheng, Can; Sugimachi, Masaru

    2016-08-01

    We have been developing an automated cardiovascular drug infusion system for simultaneous control of arterial pressure (AP), cardiac output (CO), and left atrial pressure (PLA) in decompensated heart failure (HF). In our prototype system, CO and PLA were measured invasively through thoracotomy. Furthermore, the control logic inevitably required use of inotropes to improve hemodynamics, which was not in line with clinical HF guidelines. The goal of this study was to solve these problems and develop a clinically feasible system. We integrated to the system minimally invasive monitors of CO and pulmonary capillary wedge pressure (PCWP, surrogates for PLA) that we developed recently. We also redesigned the control logic to reduce the use of inotrope. We applied the newly developed system to nine dogs with decompensated HF. Once activated, our system started to control the infusion of vasodilator and diuretics in all the animals. Inotrope was not infused in three animals, and infused at minimal doses in six animals that were intolerant of vasodilator infusion alone. Within 50 min, our system controlled AP, CO, and PCWP to their respective targets accurately. Pulmonary artery catheterization confirmed optimization of hemodynamics (AP, from 98 ± 4 to 74 ± 11 mmHg; CO, from 2.2 ± 0.5 to 2.9 ± 0.3 L·min(-1)·m(-2); PCWP, from 27.0 ± 6.6 to 13.8 ± 3.0 mmHg). In a minimally invasive setting while reducing the use of inotrope, our system succeeded in automatically optimizing the overall hemodynamics in canine models of HF. The present results pave the way for clinical application of our automated drug infusion system. PMID:26571509

  18. Successful use of levosimendan as a primary inotrope in pediatric cardiac surgery: An observational study in 110 patients

    PubMed Central

    Joshi, Reena Khantwal; Aggarwal, Neeraj; Aggarwal, Mridul; Pandey, Rakesh; Dinand, Veronique; Joshi, Raja

    2016-01-01

    Context: Levosimendan is a new generation inotrope with calcium sensitizing properties and proven benefits in adults. Aims: This study investigates the use of levosimendan as a first line inotrope in congenital heart surgery. Settings and Design: Prospective, observational study in a tertiary care center. Materials and Methods: One hundred and ten patients undergoing congenital cardiac surgery received levosimendan at a loading dose of 12 mcg/kg during rewarming on cardiopulmonary bypass followed by continuous infusion of 0.1 mcg/kg/min for 48 h. Hemodynamic parameters were recorded at the time of admission to Intensive Care Unit, and at 3 h, 6 h, 12 h, 24 h, and 48 h thereafter. Statistical Analysis: Categorical variables were compared using Chi-square test. Non-normally distributed quantitative variables were compared between groups using Kruskal-Wallis test. Results: At discharge from operating room (OR), 36 (32.7%) patients required levosimendan alone to maintain optimum cardiac output, 59 (53.6%) patients required the addition of low-dose adrenaline (<0.1 mcg/kg/min) and 15 (13.6%) patients required either increment in adrenaline to high-dose (≥0.1 mcg/kg/min) or starting another inotrope/vasoactive agent. Overall, there were five mortalities. Hypotension leading to discontinuation of levosimendan was not found in any patient. Arrhythmias were observed in three patients. Fifty-four patients were extubated in the OR. Conclusions: Levosimendan-based inotropic regime offers optimized cardiac output with a well-controlled heart rate and a low incidence of arrhythmias in patients undergoing all categories of congenital heart surgeries. PMID:27011685

  19. 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

  20. Effect of ECM Stiffness on Integrin-Ligand Binding Strength

    NASA Astrophysics Data System (ADS)

    Thomas, Gawain; Wen, Qi

    2014-03-01

    Many studies have shown that cells respond to the stiffness of their extracellular matrix (ECM). However, the mechanism of this stiffness sensing is not fully understood. We believe that cells probe stiffness by applying intracellular force to the ECM via the integrin-mediated adhesions. The linkage of integrins to the cytoskeleton has been modeled as a slip clutch, which has been shown to affect focal adhesion formation and hence force transmission in a stiffness dependent manner. In contrast, the bonds between integrins and ECM have been characterized as ``catch bonds.'' It is unclear how ECM viscoelasticity affects these catch bonds. We report, for the first time, the effects of ECM stiffness on the binding strength of integrins to ECM ligands by measuring the rupture force of individual integrin-ligand bonds of cells on collagen-coated polyacrylamide gels. Results show that the integrin-collagen bonds of 3T3 fibroblasts are nearly four times stronger on a stiff (30 kPa) gel than on a soft (3 kPa) gel. The stronger integrin bonds on stiffer substrates can promote focal adhesion formation. This suggests that the substrate stiffness regulates the cell-ECM adhesions not only by affecting the cytoskeleton-integrin links but also by modulating the binding of integrins to the ECM.

  1. Investigating binding particles distribution effects on polymer translocation through nanopore

    NASA Astrophysics Data System (ADS)

    Haji Abdolvahab, Rouhollah

    2016-03-01

    Chaperone driven polymer translocation is an important model for biopolymer's translocation in vivo. Binding proteins spatial distribution is a significant factor in calculating the translocation time of the polymer in this type of translocation. Here using a dynamical Monte Carlo simulation we compare the results of the usual uniform distribution with the exponential distribution of different rates for a stiff polymer. Our simulation results show that just by changing the chaperones spatial distribution the translocation time of the biopolymer will change by as large as an order. It can change the translocation regime of the polymer completely from a diffusive to a ballistic one. Although generally increasing the exponential rate and the background concentration will increase the translocation velocity, it is not always true and one should consider both the sequence and the background concentration. We show that the results depend on the sequence and changing the distribution rates for increasing the translocation velocity will change the whole Probability Density Function (PDF) of the polymer translocation time accordance to its sequence. The translocation time sequence dependency will change in the extreme cases e.g. in the high exponential rate. Investigating the binding protein size, λ, also shows the importance of the so called parking lot effect in distribution dependency of the translocation velocity. Although there is not any important dependency for λ = 1, translocation time depends clearly on the chaperone spatial distribution for the case of λ ≥ 2.

  2. Effect of aeration of sediment on cadmium binding

    SciTech Connect

    Zhuang, Y.; Allen, H.E.; Fu, G. . Dept. of Civil Engineering)

    1994-05-01

    Acid-volatile sulfide (AVS) has been shown to be the dominant phase reacting with metals in anoxic sediments. The AVS in sediment decreases upon resuspension due to storm and dredging, and in winter when the rate of aeration processes exceeds that of the formation of sulfide. The authors conducted a series of lab aeration experiments in batch reactors to investigate the effects of aeration of sediment on the sulfide content of sediment and on the partitioning of cadmium, a model toxic metal, to the sediment. Aeration of sediment results in rapid decrease of the AVS. The authors studied the sediment characteristics for aeration periods of approximately a month. During this time, the concentrations of dissolved metals increased by 200 to 400% or more, relative to the concentrations present at the beginning of the test. The concentration of metal associated with AVS and with pyrite decreased. During the aeration, there are increases in the concentrations of hydrous iron and manganese oxides, and these materials become increasingly more important in the binding of cadmium. Following the aeration, > 50% of the cadmium was associated with the extractable iron and manganese components of the sediment. Overall, the binding capacity of the sediments for cadmium decreased after aeration.

  3. Pharmacological effects and binding studies of new methylxanthine thioderivatives.

    PubMed

    Ragazzi, E; Froldi, G; Santi Soncin, E; Borea, P A; Fassina, G

    1989-01-01

    The effects of two methylxanthine derivatives, 6-thiocaffeine (TC) and 6-thiotheophylline (TT), were investigated in different in vitro and in vivo conditions. On guinea-pig isolated trachea, both TC and TT showed a relaxant effect (EC50 50 microM and 60 microM, respectively), more potent than theophylline (300 microM). In guinea-pig isolated atria TC (30-50 microM) was able to antagonize R-PIA (a stable agonist on adenosine receptors) negative effect in not a clearly competitive way. Higher concentration (100 microM) began to reverse that inhibitory effect. In vitro Ki of TC and TT for A1 and A2 adenosine receptors was intermediate in comparison to caffeine and theophylline. On the contrary, the two thioderivatives showed a higher affinity for [3H]-nitrendipine binding sites, in comparison to the original methylxanthines. All these data suggest a complex mechanism of action, probably at the level of adenosine extracellular receptors and L-type Ca2+ channels. In vivo experiments in mice provided evidence for a lack of CNS stimulant effects, but a loss of motor coordination was observed. Both thioderivatives showed a reduced acute toxicity. These data can be useful for the development of drugs for the therapy of asthma with reduced side effects. PMID:2560547

  4. Microwave effect on camphor binding to rat olfactory epithelium

    SciTech Connect

    Philippova, T.M.; Novoselov, V.I.; Bystrova, M.F.; Alekseev, S.I.

    1988-01-01

    Microwave radiation decreased specific camphor binding to a membrane fraction of rat epithelium but not to a Triton X-100 extract of this fraction. Inhibition of the ligand binding did not depend on the modulation frequency of the microwave field in the region 1-100 Hz and was not a linear function of specific absorption rate (SAR). The decreased ligand binding was due to a shedding or release of the specific camphor-binding protein from the membrane into solution. It is highly probable that several other membrane proteins may be shed into solution during microwave exposure.

  5. Dual Effects of Adp and Adenylylimidodiphosphate on Cftr Channel Kinetics Show Binding to Two Different Nucleotide Binding Sites

    PubMed Central

    Weinreich, Frank; Riordan, John R.; Nagel, Georg

    1999-01-01

    The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A. PMID:10398692

  6. Dual effects of ADP and adenylylimidodiphosphate on CFTR channel kinetics show binding to two different nucleotide binding sites.

    PubMed

    Weinreich, F; Riordan, J R; Nagel, G

    1999-07-01

    The CFTR chloride channel is regulated by phosphorylation by protein kinases, especially PKA, and by nucleotides interacting with the two nucleotide binding domains, NBD-A and NBD-B. Giant excised inside-out membrane patches from Xenopus oocytes expressing human epithelial cystic fibrosis transmembrane conductance regulator (CFTR) were tested for their chloride conductance in response to the application of PKA and nucleotides. Rapid changes in the concentration of ATP, its nonhydrolyzable analogue adenylylimidodiphosphate (AMP-PNP), its photolabile derivative ATP-P3-[1-(2-nitrophenyl)ethyl]ester, or ADP led to changes in chloride conductance with characteristic time constants, which reflected interaction of CFTR with these nucleotides. The conductance changes of strongly phosphorylated channels were slower than those of partially phosphorylated CFTR. AMP-PNP decelerated relaxations of conductance increase and decay, whereas ATP-P3-[1-(2-nitrophenyl)ethyl]ester only decelerated the conductance increase upon ATP addition. ADP decelerated the conductance increase upon ATP addition and accelerated the conductance decay upon ATP withdrawal. The results present the first direct evidence that AMP-PNP binds to two sites on the CFTR. The effects of ADP also suggest two different binding sites because of the two different modes of inhibition observed: it competes with ATP for binding (to NBD-A) on the closed channel, but it also binds to channels opened by ATP, which might either reflect binding to NBD-A (i.e., product inhibition in the hydrolysis cycle) or allosteric binding to NBD-B, which accelerates the hydrolysis cycle at NBD-A. PMID:10398692

  7. 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

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

    PubMed Central

    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

  9. 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.

  10. 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

  11. 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.

  12. Myocardial energy production and consumption remain balanced during positive inotropic stimulation when coronary flow is restricted to basal rates in rabbit heart.

    PubMed Central

    Marshall, R C; Nash, W W; Bersohn, M M; Wong, G A

    1987-01-01

    The effect on myocardial energy balance of increasing oxygen demand without altering basal myocardial perfusion rate was assessed in isolated, isovolumic, retrograde blood perfused rabbit hearts. Myocardial energy requirements were increased with paired stimulation. The capacity of rapid paired stimulation to increase mechanical energy consumption was demonstrated in the presence of increased perfusion with the rate X pressure product and oxygen consumption increasing 86 and 148%, respectively, compared with control values. In contrast, rapid paired stimulation under constant, basal flow conditions did not alter the rate X pressure product, while oxygen extraction and consumption increased only 40% relative to control. Myocardial ATP, creatine-phosphate, and lactate content were identical under control and constant flow-paired stimulation conditions. The results of this study indicate that no detectable energy imbalance was produced by rapid paired stimulation with flow held constant at basal rates. These results suggest that the myocardium does not increase mechanical energy expenditure in response to inotropic or rate stimulation in the presence of restricted flow reserve and are inconsistent with the concept of "demand-induced" or "relative" myocardial ischemia. PMID:3654976

  13. Effect of total binding capacity of thyroxine binding globulin on the free thyroxine index

    SciTech Connect

    Cuaron, A.

    1986-06-01

    In search of a definite source of misleading free thyroxine index (FT/sub 4/I), the relationship between in vitro thyroid testing results and thyroxine-binding globulin (TBG) capacities were reexamined in sera from a population with a relatively high prevalence of serum TBG alterations. Sera from 21 subjects with different total thyroxine-binding globulin capacities (TTBG), were loaded with graded amounts of thyroxine (T/sub 4/) and assayed for T/sub 4/, T/sub 3/ uptake (T/sub 3/U), TTBG, and free T/sub 4/ concentration (FT/sub 4/I). Serum T/sub 4/, T/sub 3/U, and the calculated FT/sub 4/ index (FT/sub 4/I) were able to separate efficiently the samples according to their FT/sub 4/, but their respective normal ranges varied with TTBG. Interpretation of the results of the in vitro tests, in the light of TTBG, greatly improved their operating characteristics in the study of 141 patients with a high prevalence of TBG alterations. The misleading FT/sub 4/I is not the outcome of reduced intrinsic sensitivities of the in vitro tests, but a consequence of a shift of their normal ranges caused by a change of TTBG. By estimating TTBG from the values of T/sub 4/ and T/sub 3/U, this problem is easily solved without adding cost.

  14. 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. PMID:25132465

  15. 40 CFR 72.32 - Permit application shield and binding effect of permit application.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 16 2010-07-01 2010-07-01 false Permit application shield and binding effect of permit application. 72.32 Section 72.32 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) PERMITS REGULATION Acid Rain Permit Applications § 72.32 Permit application shield and binding effect...

  16. 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.

  17. Effects of the Hydroxyl Group on Phenyl Based Ligand/ERRγ Protein Binding

    PubMed Central

    2015-01-01

    Bisphenol-A (4,4′-dihydroxy-2,2-diphenylpropane, BPA, or BPA-A) and its derivatives, when exposed to humans, may affect functions of multiple organs by specific binding to the human estrogen-related receptor γ (ERRγ). We carried out atomistic molecular dynamics (MD) simulations of three ligand compounds including BPA-A, 4-α-cumylphenol (BPA-C), and 2,2-diphenylpropane (BPA-D) binding to the ligand binding domain (LBD) of a human ERRγ to study the structures and energies associated with the binding. We used the implicit Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) method to estimate the free energies of binding for the phenyl based compound/ERRγ systems. The addition of hydroxyl groups to the aromatic ring had only a minor effect on binding structures and a significant effect on ligand/protein binding energy in an aqueous solution. Free binding energies of BPA-D to the ERRγ were found to be considerably less than those of BPA-A and BPA-C to the ERRγ. These results are well correlated with those from experiments where no binding affinities were determined in the BPA-D/ERRγ complex. No conformational change was observed for the helix 12 (H-12) of ERRγ upon binding of these compounds preserving an active transcriptional conformation state. PMID:25098505

  18. 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

  19. Parameterization of an effective potential for protein–ligand binding from host–guest affinity data

    PubMed Central

    Wickstrom, Lauren; Deng, Nanjie; He, Peng; Mentes, Ahmet; Nguyen, Crystal; Gilson, Michael K.; Kurtzman, Tom; Gallicchio, Emilio; Levy, Ronald M.

    2015-01-01

    Force field accuracy is still one of the “stalemates” in biomolecular modeling. Model systems with high quality experimental data are valuable instruments for the validation and improvement of effective potentials. With respect to protein–ligand binding, organic host–guest complexes have long served as models for both experimental and computational studies because of the abundance of binding affinity data available for such systems. Binding affinity data collected for cyclodextrin (CD) inclusion complexes, a popular model for molecular recognition, is potentially a more reliable resource for tuning energy parameters than hydration free energy measurements. Convergence of binding free energy calculations on CD host–guest systems can also be obtained rapidly, thus offering the opportunity to assess the robustness of these parameters. In this work, we demonstrate how implicit solvent parameters can be developed using binding affinity experimental data and the binding energy distribution analysis method (BEDAM) and validated using the Grid Inhomogeneous Solvation Theory analysis. These new solvation parameters were used to study protein–ligand binding in two drug targets against the HIV-1 virus and improved the agreement between the calculated and the experimental binding affinities. This work illustrates how benchmark sets of high quality experimental binding affinity data and physics-based binding free energy models can be used to evaluate and optimize force fields for protein–ligand systems. PMID:26256816

  20. In vitro effects of cytosolic inhibitor and opiates on the binding of [3H]oestradiol to nuclear type II binding sites of rat uterus and hypothalamus.

    PubMed

    Garai, J; Vértes, M; Kovács, S

    1989-03-01

    The effect of cytosolic ultrafiltrates prepared from intact rat uteri, brain hemispheres and hypothalami and of some opiate analogues on oestradiol binding to nuclear type II sites in rat uterus and hypothalamus was studied. Opiate binding in nuclear fraction of rat uteri was also evaluated. Both uterine and hypothalamic low affinity nuclear oestradiol binding was inhibited by filtrate from uteri, while only hypothalamic nuclear binding was decreased in presence of hypothalamic filtrate. Filtrate from brain was ineffective on nuclear oestradiol binding of the studied tissues. Concentration dependent inhibition of uterine nuclear oestradiol binding could be demonstrated by some opiate analogues in vitro. Specific low affinity nuclear binding of opiate antagonist naloxone and agonist dihydromorphine was observed in rat uteri which could be inhibited by uterine filtrate and oestradiol but not by hypothalamic filtrate or other steroids. Present findings support the probable intracellular interplay of opiates and oestradiol action and suggest that cytosolic inhibitor factor might be involved. PMID:2704239

  1. 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.

  2. 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

  3. Effect of a high intensity laser beam on impurity binding energy in a nanocone

    NASA Astrophysics Data System (ADS)

    Paredes, H.; Beltrán Ríos, C. L.; Gutíerrez, W.

    2016-02-01

    This paper presents theoretical results of a study that analyzed the effect of a high- frequency laser in the ground state binding energy of a hydrogenic donnor impurity. For these results, the trigonometric sweep method and framework of the effective mass approximation is applied. The results showed that the binding energy changes depending on the laser intensity and the impurity position across of the nanocone axis. The results agree with previous results obtained in similar systems.

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

    SciTech Connect

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

    1990-02-26

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

  5. Effect of carbon nanofibre structure on the binding of antibodies

    NASA Astrophysics Data System (ADS)

    Naguib, Nevin N.; Mueller, Yvonne M.; Bojczuk, Paul M.; Pía Rossi, María; Katsikis, Peter D.; Gogotsi, Yury

    2005-04-01

    Potential biomedical applications for carbon nanofibres include, but are not limited to, biosensors and drug delivery vehicles. For such applications, it is essential to know how carbon nanotubes interact with antibodies and proteins. We report on the successful adsorption of monoclonal CD3 antibodies on two types of carbon nanofibre produced by the same method and having the same average size and shape, but differing in surface structure and chemistry due to dissimilar post-treatments. Binding of proteins to nanofibres is enhanced by poly (L-lysine) (PLL) and improves with increasing disorder and hydrophilicity of the nanofibres' surface. Oxidized and disordered surfaces of pyrolytically stripped nanofibres show improved wetting and attachment of PLL and proteins compared to hydrophobic and well-ordered surfaces of heat-treated nanofibres. These results show that the surface of carbon nanofibres can be tailored for their use in biomedical applications.

  6. Quantum Physics Inspired Optical Effects in Tight-Binding Lattices

    NASA Astrophysics Data System (ADS)

    Thompson, Clinton; Vemuri, Gautam

    We theoretically investigated the propagation of light inside an array of single-mode evanescently coupled waveguides that can be described by the tight-binding Hamiltonian. We show that directed photonic transport can be achieved with phase-displaced inputs. In addition, the form of a parity-symmetric waveguide-dependent coupling constant can tune the dynamics of the photon's wavepacket. Lastly, we examine the statistical aspects of the output light for different input fields when disorder is present in the waveguide array. We find that the light will undergo Anderson localization independent of the type of field and that the intensity fluctuations of the output light will increase with disorder at the initial waveguide.

  7. 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.

  8. Effects of ligand binding upon flexibility of proteins.

    PubMed

    Erman, Burak

    2015-05-01

    Binding of a ligand on a protein changes the flexibility of certain parts of the protein, which directly affects its function. These changes are not the same at each point, some parts become more flexible and some others become stiffer. Here, an equation is derived that gives the stiffness map for proteins. The model is based on correlations of fluctuations of pairs of points in proteins, which may be evaluated at different levels of refinement, ranging from all atom molecular dynamics to general elastic network models, including the simplest case of isotropic Gaussian Network Model. The latter is used, as an example, to evaluate the changes of stiffness upon dimerization of ACK1. PMID:25737428

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

    SciTech Connect

    Sweeney, C.; Cutroneo, K.R.

    1987-05-01

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

  10. Quantitative effects of antihydrophobic agents on binding constants and solubilities in water.

    PubMed Central

    Breslow, R; Halfon, S

    1992-01-01

    The effects of urea and of guanidinium chloride on binding constants in water for 6-(4-tert-butylanilino)-naphthalene-2-sulfonate and of bis(p-tert-butylphenyl) phosphate binding to beta-cyclodextrin and to N,N'-bis(6-beta-cyclo-dextrinyl)imidazolium ion have been determined. Their effects on the water solubility of p-tert-butylbenzyl alcohol and p-methylbenzyl alcohol have also been examined. Quantitative correlations show that the effects of these additives, which diminish hydrophobic effects, are similar for release of a tert-butylphenyl group from a cyclodextrin cavity into water or for solubilizing such a group from a second phase. The effects of these agents on the binding constants for double-ended substrates binding to the bis(cyclodextrin) host are much larger than for a simple substrate binding to monomeric cyclodextrin, consistent with additivity of free-energy perturbations. Ethanol also decreases binding in these systems, and increases solubilities, but the quantitative correlations are less straightforward. Images PMID:1495980

  11. 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

  12. 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.

  13. 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

  14. Identification of tail binding effect of kinesin-1 using an elastic network model.

    PubMed

    Kim, Jae In; Chang, Hyun Joon; Na, Sungsoo

    2015-10-01

    Kinesin is a motor protein that delivers cargo inside a cell. Kinesin has many different families, but they perform basically same function and have same motions. The walking motion of kinesin enables the cargo delivery inside the cell. Autoinhibition of kinesin is important because it explains how function of kinesin inside a cell is stopped. Former researches showed that tail binding is related to autoinhibition of kinesin. In this work, we performed normal mode analysis with elastic network model using different conformation of kinesin to determine the effect of tail binding by considering four models such as functional form, autoinhibited form, autoinhibited form without tail, and autoinhibited form with carbon structure. Our calculation of the thermal fluctuation and cross-correlation shows the change of tail-binding region in structural motion. Also strain energy of kinesin showed that elimination of tail binding effect leads the structure to have energetically similar behavior with the functional form. PMID:25676575

  15. Cellular retinol-binding protein and retinoic acid-binding protein in rat testes: effect of retinol depletion.

    PubMed

    Ong, D E; Tsai, C H; Chytil, F

    1976-02-01

    Testes of rats contain two cellular binding proteins of interest in vitamin A metabolism. One protein binds retinoic acid with high specificity; the other binds retinol with high specificity. When the cellular retinol-binding protein was partially purified from rat testes, it exhibited fluorescence excitation and emission spectra similar to that of all-trans-retinol in hexane. Exposure of this preparation to UV light destroyed this fluorescence but spectra identical to the original were obtained after addition of retinol. Hexane extracts of the binding protein had fluorescence spectra identical to all-trans-retinol, suggesting that this compound is bound to the protein in vivo. Extracts of testes from retinol depleted rats were submitted to gel filtration but failed to show a retinol-like fluorescence at the elution position of retinol binding protein. This fluorescence was observed in the preparations from pair fed control animals. However, after addition of all-trans-retinol to the extracts from the depleted rats, fluorescence at that elution position was observed. This indicates that in testes of retinol depleted rats the cellular retinol binding protein is present but without bound retinol, in contrast to the non-depleted rats where 30-43% of the binding protein had bound retinol. The amounts of cellular retinol binding protein and retinoic acid binding protein in testes, as determined by sucrose gradient centrifugation, were found to be similar for retinol depleted and pair fed control rats. PMID:942996

  16. Aluminium competitive effect on rare earth elements binding to humic acid

    NASA Astrophysics Data System (ADS)

    Marsac, Rémi; Davranche, Mélanie; Gruau, Gérard; Dia, Aline; Bouhnik-Le Coz, Martine

    2012-07-01

    Competitive mechanisms between rare earth elements (REE) and aluminium for humic acid (HA) binding were investigated by combining laboratory experiments and modeling to evaluate the effect of Al on REE-HA complexation. Results indicates that Al3+ competes more efficiently with heavy REE (HREE) than with light REE (LREE) in acidic (pH = 3) and low REE/HA concentration ratio conditions providing evidence for the Al high affinity for the few HA multidentate sites. Under higher pH - 5 to 6 - and high REE/HA conditions, Al is more competitive for LREE suggesting that Al is bound to HA carboxylic rather than phenolic sites. PHREEQC/Model VI Al-HA binding parameters were optimized to simulate precisely both Al binding to HA and Al competitive effect on REE binding to HA. REE-HA binding pattern is satisfactorily simulated for the whole experimental conditions by the ΔLK1A optimization (i.e. ΔLK1A controls the distribution width of log K around log KMA). The present study provides fundamental knowledge on Al binding mechanisms to HA. Aluminium competitive effect on other cations binding to HA depends clearly on its affinity for carboxylic, phenolic or chelate ligands, which is pH dependent. Under circumneutral pH such as in natural waters, Al should lead to LREE-depleted patterns since Al is expected to be bound to weak HA carboxylic groups. As deduced from the behavior of Al species, other potential competitor cations are expected to have their own competitive effect on REE-HA binding. Therefore, in order to reliably understand and model REE-HA patterns in natural waters, a precise knowledge of the exact behavior of the different REE competitor cations is required. Finally, this study highlights the ability of the REE to be used as a “speciation probe” to precisely describe cation interactions with HA as here evidenced for Al.

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

    PubMed

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

    1991-09-01

    Binding of the synthetic glucocorticoid dexamethasone to the rat liver cytosol glucocorticoid receptor was inhibited by physiological concentrations of nonesterified fatty acids as a function of increasing dose, degree of unsaturation, and chain length of the fatty acid. Polyunsaturated fatty acids were the most potent inhibitors. Scatchard analysis and Line-weaver-Burk plots of the binding data revealed that both the association constants and number of binding sites decreased and that polyunsaturated fatty acids inhibition was of a mixed non-competitive type. The dissociation rate constant of [3H]dexamethasone from glucocorticoid receptors was increased by up to 10 times in the presence of docosahexaenoic acid, whereas a competitive inhibitor like the glucocorticoid antagonist RU 38486 had no effect. Moreover, sucrose density gradient analysis showed that docosahexaenoic acid inhibited the binding of [3H] dexamethasone to both the 8.8S and 4S forms. The results strongly suggest that unsaturated fatty acids are interacting at a site on the receptor different from the hormone binding site and the heat shock protein and that by binding to a second site unsaturated fatty acids greatly change the conformation of the hormone binding site to reduce its affinity for the hormone, either partially or completely depending on the concentration and the class of the fatty acid. PMID:1874175

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

    ERIC Educational Resources Information Center

    Rummel, Lynda; And Others

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

  19. Phosphorylation in protein-protein binding: effect on stability and function

    PubMed Central

    Nishi, Hafumi; Hashimoto, Kosuke; Panchenko, Anna R.

    2011-01-01

    Summary Post-translational modifications offer a dynamic way to regulate protein activity, subcellular localization and stability. Here we estimate the effect of phosphorylation on protein binding and function for different types of complexes from human proteome. We find that phosphorylation sites have a tendency to be located on binding interfaces in heterooligomeric and weak transient homooligomeric complexes. The analysis of molecular mechanisms of phosphorylation shows that phosphorylation may modulate the strength of interactions directly on interfaces and binding hotspots have a tendency to be phosphorylated in heterooligomers. Although majority of phosphosites do not show significant estimated stability differences upon attaching the phosphate groups, for about one third of all complexes it causes relatively large changes in binding energy. We discuss the cases where phosphorylation mediates the complex formation and regulates the function. We show that phosphorylation sites are not only more likely to be evolutionary conserved than surface residues but even more so than other interfacial residues. PMID:22153503

  20. Effect of Thiol-Binding Reagents on the Metabolism of Thiosulfate and Tetrathionate by Thiobacillus neapolitanus

    PubMed Central

    Trudinger, P. A.

    1965-01-01

    Trudinger, P. A. (Division of Plant Industry, Canberra, Australia). Effect of thiol-binding reagents on the metabolism of thiosulfate and tetrathionate by Thiobacillus neapolitanus. J. Bacteriol. 89:617–625. 1965.—Iodoacetamide, N-ethyl maleimide (NEM), p-chloromercuribenzoate (CMB), Mercurochrome, and HgCl2 inhibited the oxidation of thiosulfate to sulfate by Thiobacillus neapolitanus; tetrathionate accumulated under these conditions. High concentrations of the thiol-binding reagents lowered the rate of oxidation of thiosulfate to tetrathionate; inhibition by CMB was reversed by high concentrations of thiosulfate. Relatively low concentrations of the thiol-binding reagents completely inhibited the oxidation and anaerobic metabolism of tetrathionate. Similar reagents had no effect on a soluble thiosulfate-oxidizing enzyme. Inhibition by thiol-binding reagents was overcome by washing the bacteria with Na2S or thioethanol after their exposure to the inhibitors. Under some conditions, the addition of thiosulfate or tetrathionate to bacterial suspensions before the addition of the thiol-binding reagents prevented the inhibition of thiosulfate and tetrathionate metabolism by these reagents. Thiosulfate catalyzed a rapid chemical breakdown of NEM and reacted with iodoacetamide. A complex between thiosulfate and mercuribenzoate was demonstrated. Three types of thiol group appear to be associated with the metabolism of thiosulfate and tetrathionate; one of these types may be located at the bacterial cell membrane. The results are consistent with the hypothesis that thiols (or disulfide groups) are binding sites for the substrates. PMID:14273636

  1. NADP(+) binding effects tryptophan accessibility, folding and stability of recombinant B. malayi G6PD.

    PubMed

    Verma, Anita; Chandra, Sharat; Suthar, Manish Kumar; Doharey, Pawan Kumar; Siddiqi, Mohammad Imran; Saxena, Jitendra Kumar

    2016-04-01

    Brugia malayi Glucose 6-phosphate dehydrogenase apoenzyme (BmG6PD) was expressed and purified by affinity chromatography to study the differences in kinetic properties of enzyme and the effect of the cofactor NADP(+) binding on enzyme stability. The presence of cofactor NADP(+) influenced the tertiary structure of enzyme due to significant differences in the tryptophan microenvironment. However, NADP(+) binding have no effect on secondary structure of the enzyme. Quenching with acrylamide indicated that two or more tryptophan residues became accessible upon cofactor binding. Unfolding and cross linking study of BmG6PD showed that NADP(+) stabilized the protein in presence of high concentration of urea/GdmCl. A homology model of BmG6PD constructed using human G6PD (PDB id: 2BH9) as a template indicated 34% α-helix, 19% β-sheet and 47% random coil conformations in the predicted model of the enzyme. In the predicted model binding of NADP(+) to BmG6PD was less tight with the structural sites (-10.96kJ/mol binding score) as compared with the coenzyme site (-15.47kJ/mol binding score). PMID:26763177

  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. PMID:25330347

  3. Milk matrix effects on antibody binding analyzed by enzyme-linked immunosorbent assay and biolayer interferometry.

    PubMed

    Brandon, David L; Adams, Lisa M

    2015-04-01

    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 used ligands immobilized covalently or via biotin-streptavidin linkage, and the results were compared to those obtained by enzyme-linked immunosorbent assay (ELISA). In sandwich ELISA, the binding of ricin to ASF was dramatically decreased when galactose was present during the analyte or detection antibody binding step. Low concentrations of milk (1%, v/v) produced a similar reduction in ricin binding to ASF but not to a high-affinity monoclonal antibody (mAb), increasing the dissociation rate of ASF-ricin complexes up to 100-fold. The effect of milk on the binding of ricin to ASF was ascribable to dialyzable factors, and milk sugar can account for these effects. The use of high-affinity mAbs in ELISA effectively limits the milk matrix effect on ricin analysis. PMID:25822824

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

    PubMed

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

    2007-06-01

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

  6. Positive inotropic activity of 5-amino-6-cyano-1,3-dimethyl-1,2,3,4-tetrahydropyrido[2,3-d]pyrim idine-2,4-dione in cardiac muscle from guinea-pig and man. Part 6: Compounds with positive inotropic activity.

    PubMed

    Heber, D; Heers, C; Ravens, U

    1993-07-01

    In screening experiments, several 5-aminopyrido[2,3-d]-pyrimidine derivatives 1-14 were found to possess a positive inotropic action in guinea-pig left atria. The size of the effect varied between 10 and 60% of the maximum response to isoprenaline (3 x 10(-7) mol/l). Of these compounds, only 7 and 14 increased force of contraction also in papillary muscles. The latter effect was not accompanied by any changes in the shapes of the transmembrane action potentials and was reversible after addition of carbachol indicating that an increase in intracellular levels of cAMP might be involved. In Langendorff-perfused hearts of the guinea-pig 7 (10(-5) mol/l) increased force of contraction and spontaneous beating frequency like isoprenaline, but unlike isoprenaline, reduced perfusion pressure. Like 3-isobutyl-1-methylxanthine (IBMX) and milrinone, 7 also increased force of contraction of isolated right atrial trabeculae obtained from man during cardiac surgery. The influence of 7 on phosphodiesterase (PDE) activity was investigated in partially purified isoenzymes from guinea-pig ventricles. Compound 7 inhibited preferably PDE III with an IC50 value of 15.2 +/- 4.5 mumol/l. More than tenfold higher concentrations were needed to inhibit PDE II. The IC50 value was 198 +/- 91 mumol/l. PDE I and IV were inhibited by 7 only by a minor extent. At a drug concentration of 1 mmol/l PDE activity was reduced to 83 +/- 30 and 55 +/- 8% of control value, respectively.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7692456

  7. Quantitative and qualitative effects of N10-methylfolate on high-affinity folate binding in human leukocytes.

    PubMed

    Holm, J; Hansen, S I; Lyngbye, J

    1984-01-01

    N10-methylfolate acted as a potent competitive inhibitor of high-affinity [3H] folate binding in human leukocytes, while methotrexate had no effect. Furthermore, folate binding changed into a non-cooperative type in the presence of N10-methylfolate. Hence, in qualitative and quantitative respects, the substrate specificity characteristics of leukocyte folate binding resemble those of other high-affinity folate binding systems. PMID:6500843

  8. [The effect of cocaine on binding of tricyclic antidepressives in the synaptic plasma membranes in the brain].

    PubMed

    Bures, P; Krulík, R; Fisar, Z; Fuksová, K

    1993-10-01

    Effect of cocaine on binding of 3H-imipramine, 3H-desmethylimipramine, 3H-didesmethylimipramine and 3H-amitriptyline to brain synaptic plasma membranes (SPM) was studied. Binding of methylated tricyclic antidepressants was more affected. Cocaine inhibits 3H-imipramine binding at concentrations higher than 10(-5) mol/l. Binding stimulated by phosphatidylserine was affected more significantly. PMID:8269521

  9. 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).

  10. Effect of dipole polarizability on positron binding by strongly polar molecules

    NASA Astrophysics Data System (ADS)

    Gribakin, G. F.; Swann, A. R.

    2015-11-01

    A model for positron binding to polar molecules is considered by combining the dipole potential outside the molecule with a strongly repulsive core of a given radius. Using existing experimental data on binding energies leads to unphysically small core radii for all of the molecules studied. This suggests that electron-positron correlations neglected in the simple model play a large role in determining the binding energy. We account for these by including the polarization potential via perturbation theory and non-perturbatively. The perturbative model makes reliable predictions of binding energies for a range of polar organic molecules and hydrogen cyanide. The model also agrees with the linear dependence of the binding energies on the polarizability inferred from the experimental data (Danielson et al 2009 J. Phys. B: At. Mol. Opt. Phys. 42 235203). The effective core radii, however, remain unphysically small for most molecules. Treating molecular polarization non-perturbatively leads to physically meaningful core radii for all of the molecules studied and enables even more accurate predictions of binding energies to be made for nearly all of the molecules considered.

  11. Effect of oxidative DNA damage in promoter elements on transcription factor binding.

    PubMed Central

    Ghosh, R; Mitchell, D L

    1999-01-01

    Reactive oxygen species produced by endogenous metabolic activity and exposure to a multitude of exogenous agents impact cells in a variety of ways. The DNA base damage 8-oxodeoxyguanosine (8-oxodG) is a prominent indicator of oxidative stress and has been well-characterized as a premutagenic lesion in mammalian cells and putative initiator of the carcinogenic process. Commensurate with the recent interest in epigenetic pathways of cancer causation we investigated how 8-oxodG alters the interaction between cis elements located on gene promoters and sequence-specific DNA binding proteins associated with these promoters. Consensus binding sequences for the transcription factors AP-1, NF-kappaB and Sp1 were modified site-specifically at guanine residues and electrophoretic mobility shift assays were performed to assess DNA-protein interactions. Our results indicate that whereas a single 8-oxodG was sufficient to inhibit transcription factor binding to AP-1 and Sp1 sequences it had no effect on binding to NF-kappaB, regardless of its position. We conclude from these data that minor alterations in base composition at a crucial position within some, but not all, promoter elements have the ability to disrupt transcription factor binding. The lack of inhibition by damaged NF-kappaB sequences suggests that DNA-protein contact sites may not be as determinative for stable p50 binding to this promoter as other, as yet undefined, structural parameters. PMID:10454620

  12. 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

  13. Lead and calcium binding to fulvic acids: Salt effect and competition

    SciTech Connect

    Pinheiro, J.P.; Mota, A.M.; Benedetti, M.F.

    1999-10-01

    Knowledge of the speciation of Pb in natural aquatic systems is important if the authors want to understand the bioavailability and mobility of Pb in polluted and natural environments. The results given in this paper were obtained under conditions as close as possible to natural conditions. These new data show that Pb strongly binds to fulvic acids. The authors also show that the competitive effect of Pb on Ca binding to the same fulvic acid is smaller than the salt effect on Ca binding to fulvic acids as pH varies from 4 to 8. All the data were analyzed with the NICCA-Donnan model developed to describe metal ion binding to natural organic matter. The model predictions of competitive and salt effects are excellent. Comparison of their results with previously published data suggests that metal ion binding strength is similar for fulvic acids from different origins. Thus, all data sets could be interpreted within the framework of a unified modeling approach.

  14. 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

  15. 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. PMID:26254986

  16. 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

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

    SciTech Connect

    Asano, T.; Ogasawara, N.

    1986-03-01

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

  18. Differential effect of glucocorticoid receptor antagonists on glucocorticoid receptor nuclear translocation and DNA binding

    PubMed Central

    Spiga, Francesca; Knight, David M; Droste, Susanne K; Conway-Campbell, Becky; Kershaw, Yvonne; MacSweeney, Cliona P; Thomson, Fiona J; Craighead, Mark; Peeters, Bernard WMM; Lightman, Stafford L

    2016-01-01

    The effects of RU486 and S-P, a more selective glucocorticoid receptor antagonist from Schering-Plough, were investigated on glucocorticoid receptor nuclear translocation and DNA binding. In the in vitro study, AtT20 cells were treated with vehicle or with RU486, S-P or corticosterone (3–300 nM) or co-treated with vehicle or glucocorticoid receptor antagonists (3–300 nM) and 30 nM corticosterone. Both glucocorticoid receptor antagonists induced glucocorticoid receptor nuclear translocation but only RU486 induced DNA binding. RU486 potentiated the effect of corticosterone on glucocorticoid receptor nuclear translocation and DNA binding, S-P inhibited corticosterone-induced glucocorticoid receptor nuclear translocation, but not glucocorticoid receptor-DNA binding. In the in vivo study, adrenalectomized rats were treated with vehicle, RU486 (20 mg/kg) and S-P (50 mg/kg) alone or in combination with corticosterone (3 mg/kg). RU486 induced glucocorticoid receptor nuclear translocation in the pituitary, hippocampus and prefrontal cortex and glucocorticoid receptor-DNA binding in the hippocampus, whereas no effect of S-P on glucocorticoid receptor nuclear translocation or DNA binding was observed in any of the areas analysed. These findings reveal differential effects of RU486 and S-P on areas involved in regulation of hypothalamic–pituitary–adrenal axis activity in vivo and they are important in light of the potential use of this class of compounds in the treatment of disorders associated with hyperactivity of the hypothalamic–pituitary–adrenal axis. PMID:20093322

  19. The effect of diffusion on the binding of membrane-bound receptors to coated pits.

    PubMed Central

    Keizer, J; Ramirez, J; Peacock-López, E

    1985-01-01

    We have formulated a kinetic model for the primary steps that occur at the cell membrane during receptor-mediated endocytosis. This model includes the diffusion of receptor molecules, the binding of receptors to coated pits, the loss of coated pits by invagination, and random reinsertion of receptors and coated pits. Using the mechanistic statistical theory of nonequilibrium thermodynamics, we employ this mechanism to calculate the two-dimensional radial distribution of receptors around coated pits at steady state. From this we obtain an equation that describes the effect of receptor diffusion on the rate of binding to coated pits. Our equation does not assume that ligand binding is instantaneous and can be used to assess the effect of diffusion on the binding rate. Using experimental data for low density lipoprotein receptors on fibroblast cells, we conclude that the effect of diffusion on the binding of these receptors to coated pits is no more than 84% diffusion controlled. This corresponds to a dissociation rate constant for receptors on coated pits (k-) that is much less than the rate constant for invagination of the pits (lambda = 3.3 X 10(-3)/s) and a correlation length for the radial distribution function of six times the radius of a coated pit. Although the existing experimental data are compatible with any value of k-, we obtain a lower bound for the value of the binding constant (k+) of 2.3 X 10(-2)(micron)2/s. Comparison of the predicted radial distributions with experiment should provide a clear indication of the effect of diffusion on k+. PMID:2858230

  20. 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

  1. 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

  2. H274Y's Effect on Oseltamivir Resistance: What Happens Before the Drug Enters the Binding Site.

    PubMed

    Yusuf, Muhammad; Mohamed, Nornisah; Mohamad, Suriyati; Janezic, Dusanka; Damodaran, K V; Wahab, Habibah A

    2016-01-25

    Increased reports of oseltamivir (OTV)-resistant strains of the influenza virus, such as the H274Y mutation on its neuraminidase (NA), have created some cause for concern. Many studies have been conducted in the attempt to uncover the mechanism of OTV resistance in H274Y NA. However, most of the reported studies on H274Y focused only on the drug-bound system, so the direct effects of the mutation on NA itself prior to drug binding still remain unclear. Therefore, molecular dynamics simulations of NA in apo form, followed by principal component analysis and interaction energy calculations, were performed to investigate the structural changes of the NA binding site as a result of the H274Y mutation. It was observed that the disruption of the NA binding site due to the H274Y mutation was initiated by the repulsive effect of Y274 on the 250-loop, which in turn altered the hydrogen-bonding network around residue 274. The rotated W295 side chain caused the upward movement of the 340-loop. Consequently, sliding box docking results suggested that the binding pathway of OTV was compromised because of the disruption of this binding site. This study also highlighted the importance of the functional group at C6 of the sialic acid mimicry. It is hoped that these results will improve the understanding of OTV resistance and shed some light on the design of a novel anti-influenza drug. PMID:26703840

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

    PubMed

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

    2014-06-01

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

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

    PubMed Central

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

    2014-01-01

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

  5. Optimizing the affinity and specificity of ligand binding with the inclusion of solvation effect.

    PubMed

    Yan, Zhiqiang; Wang, Jin

    2015-09-01

    Solvation effect is an important factor for protein-ligand binding in aqueous water. Previous scoring function of protein-ligand interactions rarely incorporates the solvation model into the quantification of protein-ligand interactions, mainly due to the immense computational cost, especially in the structure-based virtual screening, and nontransferable application of independently optimized atomic solvation parameters. In order to overcome these barriers, we effectively combine knowledge-based atom-pair potentials and the atomic solvation energy of charge-independent implicit solvent model in the optimization of binding affinity and specificity. The resulting scoring functions with optimized atomic solvation parameters is named as specificity and affinity with solvation effect (SPA-SE). The performance of SPA-SE is evaluated and compared to 20 other scoring functions, as well as SPA. The comparative results show that SPA-SE outperforms all other scoring functions in binding affinity prediction and "native" pose identification. Our optimization validates that solvation effect is an important regulator to the stability and specificity of protein-ligand binding. The development strategy of SPA-SE sets an example for other scoring function to account for the solvation effect in biomolecular recognitions. PMID:26111900

  6. 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.

  7. 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

  8. 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...

  9. In situ analysis of cisplatin binding to DNA: the effects of physiological ionic conditions.

    PubMed

    Park, Jin-Sung; Kim, Sook Ho; Lee, Nam-Kyung; Lee, Kyoung J; Hong, Seok-Cheol

    2012-03-01

    Platinum-based anti-cancer drugs form a major family of cancer chemotherapeutic agents. Cisplatin, the first member of the family, remains a potent anti-cancer drug and exhibits its clinical effect by inducing local DNA kinks and subsequently interfering with DNA metabolism. Although its mechanism is reasonably well understood, effects of intracellular ions on cisplatin activity are left to be elucidated because cisplatin binding to DNA, thus its drug efficacy, is modified by various ions. One such issue is the effect of carbonate ions: cisplatin binding to DNA is suppressed under physiological carbonate conditions. Here, we examined the role of common cellular ions (carbonate and chloride) by measuring cisplatin binding in relevant physiological buffers via a DNA micromanipulation technique. Using two orthogonal single-molecule methods, we succeeded in detecting hidden monofunctional adducts (kink-free, presumably clinically inactive form) and clearly showed that the major effect of carbonates was to form such adducts and to prevent them from converting to bifunctional adducts (kinked, clinically active). The chloride-rich environment also led to the formation of monofunctional adducts. Our approach is widely applicable to the study of the transient behaviours of various drugs and proteins that bind to DNA in different modes depending on various physical and chemical factors such as tension, torsion, ligands, and ions. PMID:22286168

  10. Effect of Cropping Pattern and Crop Residue on Herbicide Binding to Soil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The binding of herbicides to soil is dependent on many factors, including soil texture, organic matter, and pH. In 2007 we were conducting an experiment to determine the effect of cropping patterns on atrazine efficacy and fate, and found that there was a significant relationship between cropping p...

  11. Effects of Fasting on IGF-Binding Proteins, Glucose, and Cortisol in Channel Catfish (Ictalurus punctatus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of fasting on IGF-binding proteins, glucose, and cortisol in channel catfish were examined. Fed fish (controls) were compared to 14-, 30-, and 45-day fasted fish and 45-day fasted fish refed for 15 additional days. Body length and body weight changes, condition factor(CF), hepatosomati...

  12. 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. PMID:22350963

  13. Survey of phosphorylation near drug binding sites in the Protein Data Bank (PDB) and their effects

    PubMed Central

    Smith, Kyle P.; Gifford, Kathleen M.; Waitzman, Joshua S.; Rice, Sarah E.

    2014-01-01

    While it is currently estimated that 40–50% of eukaryotic proteins are phosphorylated, little is known about the frequency and local effects of phosphorylation near pharmaceutical inhibitor binding sites. In this study, we investigated how frequently phosphorylation may affect the binding of drug inhibitors to target proteins. We examined the 453 non-redundant structures of soluble mammalian drug target proteins bound to inhibitors currently available in the Protein Data Bank (PDB). We cross-referenced these structures with phosphorylation data available from the PhosphoSitePlus database. 322/453 (71%) of drug targets have evidence of phosphorylation that has been validated by multiple methods or labs. For 132/453 (29%) of those, the phosphorylation site is within 12Å of the small molecule-binding site, where it would likely alter small molecule binding affinity. We propose a framework for distinguishing between drug-phosphorylation site interactions that are likely to alter the efficacy of drugs vs. those that are not. In addition we highlight examples of well-established drug targets, such as estrogen receptor alpha, for which phosphorylation may affect drug affinity and clinical efficacy. Our data suggest that phosphorylation may affect drug binding and efficacy for a significant fraction of drug target proteins. PMID:24833420

  14. Survey of phosphorylation near drug binding sites in the Protein Data Bank (PDB) and their effects.

    PubMed

    Smith, Kyle P; Gifford, Kathleen M; Waitzman, Joshua S; Rice, Sarah E

    2015-01-01

    While it is currently estimated that 40 to 50% of eukaryotic proteins are phosphorylated, little is known about the frequency and local effects of phosphorylation near pharmaceutical inhibitor binding sites. In this study, we investigated how frequently phosphorylation may affect the binding of drug inhibitors to target proteins. We examined the 453 non-redundant structures of soluble mammalian drug target proteins bound to inhibitors currently available in the Protein Data Bank (PDB). We cross-referenced these structures with phosphorylation data available from the PhosphoSitePlus database. Three hundred twenty-two of 453 (71%) of drug targets have evidence of phosphorylation that has been validated by multiple methods or labs. For 132 of 453 (29%) of those, the phosphorylation site is within 12 Å of the small molecule-binding site, where it would likely alter small molecule binding affinity. We propose a framework for distinguishing between drug-phosphorylation site interactions that are likely to alter the efficacy of drugs versus those that are not. In addition we highlight examples of well-established drug targets, such as estrogen receptor alpha, for which phosphorylation may affect drug affinity and clinical efficacy. Our data suggest that phosphorylation may affect drug binding and efficacy for a significant fraction of drug target proteins. PMID:24833420

  15. 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.

  16. 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.

  17. Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist

    PubMed Central

    Latypov, Ramil F.; Liu, Dingjiang; Gunasekaran, Kannan; Harvey, Timothy S.; Razinkov, Vladimir I.; Raibekas, Andrei A.

    2008-01-01

    Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using high-resolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANS–protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25°C and 37°C. Overall, the affinity of ANS was lower at 37°C compared to 25°C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solvent-exposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation. PMID:18305195

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  19. Effects of co-operative ligand binding on protein amide NH hydrogen exchange.

    PubMed

    Polshakov, Vladimir I; Birdsall, Berry; Feeney, James

    2006-03-01

    Amide protection factors have been determined from NMR measurements of hydrogen/deuterium amide NH exchange rates measured on assigned signals from Lactobacillus casei apo-DHFR and its binary and ternary complexes with trimethoprim (TMP), folinic acid and coenzymes (NADPH/NADP(+)). The substantial sizes of the residue-specific DeltaH and TDeltaS values for the opening/closing events in NH exchange for most of the measurable residues in apo-DHFR indicate that sub-global or global rather than local exchange mechanisms are usually involved. The amide groups of residues in helices and sheets are those most protected in apo-DHFR and its complexes, and the protection factors are generally related to the tightness of ligand binding. The effects of ligand binding that lead to changes in amide protection are not localised to specific binding sites but are spread throughout the structure via a network of intramolecular interactions. Although the increase in protein stability in the DHFR.TMP.NADPH complex involves increased ordering in the protein structure (requiring TDeltaS energy) this is recovered, to a large extent, by the stronger binding (enthalpic DeltaH) interactions made possible by the reduced motion in the protein. The ligand-induced protection effects in the ternary complexes DHFR.TMP.NADPH (large positive binding co-operativity) and DHFR.folinic acid.NADPH (large negative binding co-operativity) mirror the co-operative effects seen in the ligand binding. For the DHFR.TMP.NADPH complex, the ligand-induced protection factors result in DeltaDeltaG(o) values for many residues being larger than the DeltaDeltaG(o) values in the corresponding binary complexes. In contrast, for DHFR.folinic acid.NADPH, the DeltaDeltaG(o) values are generally smaller than many of those in the corresponding binary complexes. The results indicate that changes in protein conformational flexibility on formation of the ligand complex play an important role in determining the co-operativity in

  20. CFTR gating II: Effects of nucleotide binding on the stability of open states.

    PubMed

    Bompadre, Silvia G; Cho, Jeong Han; Wang, Xiaohui; Zou, Xiaoqin; Sohma, Yoshiro; Li, Min; Hwang, Tzyh-Chang

    2005-04-01

    Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADP. To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of DeltaR/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. DeltaR/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of approximately 100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Single-channel kinetic analysis for DeltaR/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation. PMID:15767296

  1. (/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.

  2. 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

  3. 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.

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

    PubMed

    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

  5. 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

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

    PubMed

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

    2014-06-01

    In this study, we investigated the neural correlates of age-related differences in the binding of verbal and spatial information utilizing event-related working memory tasks. Twenty-one right handed younger adults and twenty-one right handed older adults performed two versions of a dual task of verbal and spatial working memory. In the unbound dual task version letters and locations were presented simultaneously in separate locations, while in the bound dual task version each letter was paired with a specific location. In order to identify binding-specific differences, mixed-effects ANOVAs were run with the interaction of age and task as the effect of interest. Although older adults performed worse in the bound task than younger adults, there was no significant interaction between task and age on working memory performance. However, interactions of age and task were observed in brain activity analyses. Older adults did not display the greater unbound than bound task activity that younger adults did at the encoding phase in bilateral inferior parietal lobule, right putamen, and globus pallidus as well as at the maintenance phase in the cerebellum. We conclude that the binding of letters and locations in working memory is not as efficient in older adults as it is in younger adults, possibly due to the decline of cognitive control processes that are specific to working memory binding. PMID:24631396

  7. Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

    PubMed

    Wojtas, Magdalena; Hołubowicz, Rafał; Poznar, Monika; Maciejewska, Marta; Ożyhar, Andrzej; Dobryszycki, Piotr

    2015-10-27

    Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 μM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 μM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins. PMID:26445027

  8. 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

  9. 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

  10. 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.

  11. Effect of corticosteroid binding proteins on the steroidogenic activity of bovine adrenocortical cell suspensions.

    PubMed

    Basset, M; Rostaing-Metz, B; Chambaz, E M

    1982-07-01

    The possible role of steroid binding proteins in the hormonal secretion process of a steroidogenic tissue was examined using bovine adrenocortical cell suspensions, either under basal conditions or in the presence of half-maximally active concentration (1 x 10(-9) M) of synthetic adrenocorticotropic hormone (ACTH). Three types of plasma cortisol binding proteins were used, namely bovine serum albumine (BSA), purified transcortin (CBG) and purified anticortisol immunoglobulins (IgG). When added to the incubation medium, CBG (at 1 x 10(-10) to 2 x 10(-9) M cortisol binding sites) and anticortisol IgG (at 4.8 x 10(-10) to 3 x 10(-9) M cortisol binding sites) did not influence either the basal nor the ACTH-stimulated net cortisol production of the cell preparations. Whereas crystallized and delipidated BSA showed also no effect, crude commercial BSA preparation (Cohn fraction V) exhibited an ACTH-like cofactor effect which resulted in a marked increase in the net cortisol production by stimulated cells. These observations might be explained by the presence in crude BSA of lipoprotein-cholesterol complexes, possibly acting as an extracellular source of cholesterol available for corticosteroidogenesis. It may be concluded that specific high affinity cortisol binding systems present outside adrenocortical steroidogenic cells do not influence their secretory activity under short term in vitro condition. In addition, it can be stressed that use of ill defined protein preparations (e.g. crude BSA) may lead to artifactual observations in the study of the differentiated functions of isolated steroidogenic cells. PMID:6287106

  12. Binding Selectivity of Abaloparatide for PTH-Type-1-Receptor Conformations and Effects on Downstream Signaling.

    PubMed

    Hattersley, Gary; Dean, Thomas; Corbin, Braden A; Bahar, Hila; Gardella, Thomas J

    2016-01-01

    The PTH receptor type 1 (PTHR1) mediates the actions of two endogenous polypeptide ligands, PTH and PTHrP, and thereby plays key roles in bone biology. Based on its capacity to stimulate bone formation, the peptide fragment PTH (1-34) is currently in use as therapy for osteoporosis. Abaloparatide (ABL) is a novel synthetic analog of human PTHrP (1-34) that holds promise as a new osteoporosis therapy, as studies in animals suggest that it can stimulate bone formation with less of the accompanying bone resorption and hypercalcemic effects that can occur with PTH (1-34). Recent studies in vitro suggest that certain PTH or PTHrP ligand analogs can distinguish between two high-affinity PTHR1 conformations, R(0) and RG, and that efficient binding to R(0) results in prolonged signaling responses in cells and prolonged calcemic responses in animals, whereas selective binding to RG results in more transient responses. As intermittent PTH ligand action is known to favor the bone-formation response, whereas continuous ligand action favors the net bone-resorption/calcemic response, we hypothesized that ABL binds more selectively to the RG vs the R(0) PTHR1 conformation than does PTH (1-34), and thus induces more transient signaling responses in cells. We show that ABL indeed binds with greater selectivity to the RG conformation than does PTH (1-34), and as a result of this RG bias, ABL mediates more transient cAMP responses in PTHR1-expressing cells. The findings provide a plausible mechanism (ie, transient signaling via RG-selective binding) that can help account for the favorable anabolic effects that ABL has on bone. PMID:26562265

  13. 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.

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

    PubMed

    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

  15. 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

  16. 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.

  17. Control of glycolytic enzyme binding: effect of changing enzyme substrate concentrations on in vivo enzyme distributions.

    PubMed

    Brooks, S P; Storey, K B

    1993-05-12

    The effect of changing concentrations of glycolytic intermediates on the binding of phosphofructokinase, aldolase and pyruvate kinase to cellular particulate matter was investigated. Concentrations of glycolytic intermediates were altered by adding 2 mM iodoacetic acid (IAA) to an incubation medium containing tissues isolated from the channelled whelk Busycon canaliculatum. Iodoacetic acid inhibited glyceraldehyde 3-phosphate dehydrogenase activity causing a 100-400 fold increase in the concentration of fructose 1,6-bisphosphate as well as 3-20 fold increases in glucose 6-phosphate, fructose 6-phosphate, and dihydroxyacetone phosphate levels depending on the experimental protocol. Cellular pH values were not statistically different in the presence of IAA. Measurement of enzyme binding to particulate matter showed that the binding of phosphofructokinase, aldolase and pyruvate kinase was unaffected by iodoacetic acid under any experimental condition. These results show that changes in the tissue concentrations of enzyme substrates and products do not regulate enzyme binding to particulate matter in the cell. PMID:8350861

  18. Modulation of the chloroplast ATPase by tight ADP binding. Effect of uncouplers and ATP.

    PubMed

    Bar-Zvi, D; Shavit, N

    1982-12-01

    Inactivation of the membrane-bound ATPase by tight ADP binding was studied under nonenergized conditions. The energy state of the system was controlled either by omitting MgCl2, preventing ATP hydrolysis, or by addition of an uncoupler which dissipates the delta mu H+. In the absence of Mg2+, ATP prevents the inactivation of the enzyme by ADP, in a competitive manner. This effect of ATP resembles that of GDP with Mg2+ present. In the presence of nigericin, Mg2+, and ATP, inactivation occurs after a 10-15-sec interval, during which the enzyme is able to hydrolyze ATP at a relatively rapid rate. The degree of inactivation is proportional to the level of bound ADP detected. This behavior is different from that of the coupled ATPase (no uncoupler added), where inactivation is attained only upon exhaustion of the ATP by its hydrolysis, despite the finding that ADP binds tightly to the active ATPase at all stages of the reaction. Higher levels of tightly bound ADP were detected in the presence of an uncoupler. We suggest that the interval during which the enzyme becomes inactive is that required for the enzyme to generate and bind ADP, and to change from the active to the inactive conformation. These results support the mechanism suggested previously for the modulation of the ATPase by tight nucleotide binding. PMID:6219104

  19. Partial MHC class II constructs inhibit MIF/CD74 binding and downstream effects.

    PubMed

    Benedek, Gil; Meza-Romero, Roberto; Andrew, Shayne; Leng, Lin; Burrows, Gregory G; Bourdette, Dennis; Offner, Halina; Bucala, Richard; Vandenbark, Arthur A

    2013-05-01

    MIF and its receptor, CD74, are pivotal regulators of the immune system. Here, we demonstrate for the first time that partial MHC class II constructs comprised of linked β1α1 domains with covalently attached antigenic peptides (also referred to as recombinant T-cell receptor ligands - RTLs) can inhibit MIF activity by not only blocking the binding of rhMIF to immunopurified CD74, but also downregulating CD74 cell-surface expression. This bifunctional inhibition of MIF/CD74 interactions blocked downstream MIF effects, including enhanced secretion of proinflammatory cytokines, anti-apoptotic activity, and inhibition of random migration that all contribute to the reversal of clinical and histological signs of EAE. Moreover, we demonstrate that enhanced CD74 cell-surface expression on monocytes in mice with EAE and subjects with multiple sclerosis can be downregulated by humanized RTLs, resulting in reduced MIF binding to the cells. Thus, binding of partial MHC complexes to CD74 blocks both the accessibility and availability of CD74 for MIF binding and downstream inflammatory activity. PMID:23576302

  20. Partial MHC class II constructs inhibit MIF/CD74 binding and downstream effects

    PubMed Central

    Benedek, Gil; Meza-Romero, Roberto; Andrew, Shayne; Leng, Lin; Burrows, Gregory G.; Bourdette, Dennis; Offner, Halina; Bucala, Richard; Vandenbark, Arthur A.

    2013-01-01

    Macrophage migration inhibitory factor (MIF) and its receptor, CD74, are pivotal regulators of the immune system. Here we demonstrate for the first time that partial MHC class II constructs comprised of linked β1α1 domains with covalently attached antigenic peptides (also referred to as recombinant T-cell receptor ligands - RTLs) can inhibit MIF activity by not only blocking the binding of rhMIF to immunopurified CD74, but also down-regulating CD74 cell-surface expression. This bi-functional inhibition of MIF/CD74 interactions blocked downstream MIF effects, including enhanced secretion of proinflammatory cytokines, anti-apoptotic activity and inhibition of random migration that all contribute to the reversal of clinical and histological signs of experimental autoimmune encephalomyelitis (EAE). Moreover, we demonstrate that enhanced CD74 cell surface expression on monocytes in mice with EAE and subjects with multiple sclerosis (MS) can be down-regulated by humanized RTLs, resulting in reduced MIF binding to the cells. Thus, binding of partial MHC complexes to CD74 blocks both the accessibility and availability of CD74 for MIF binding and downstream inflammatory activity. PMID:23576302

  1. Comparison of Binding Properties and Early Biological Effects of Elicitins in Tobacco Cells1

    PubMed Central

    Bourque, Stéphane; Ponchet, Michel; Binet, Marie-Noëlle; Ricci, Pierre; Pugin, Alain; Lebrun-Garcia, Angela

    1998-01-01

    Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca2+ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca2+ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin. PMID:9847105

  2. 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.

  3. Protein binding of chlorpromazine in vivo and in vitro: effect of chlorpromazine metabolite on chlorpromazine protein binding in rat.

    PubMed

    Sato, S; Koshiro, A

    1995-04-01

    The serum protein binding curve of chlorpromazine (CPZ) on the Scatchard plot in vitro was a two-phase downward curve. However, after i.v. administration of CPZ the curve was altered to an upward curve. To clarify the reasons for these in vivo changes, the influence of chlorpromazine S-oxide (CPZSO), chlorpromazine N-oxide (CPZNO), desmethylchlorpromazine (nor1-CPZ), chlorpromazine sulfone (sul-CPZ) and 7-hydroxychlorpromazine (7-OH-CPZ) on CPZ protein binding were studied in vitro. The results indicated that the characteristics of the CPZ protein binding are altered by the combination of CPZSO or CPZNO or by either of them. Since it was very difficult to explain the relationship between serum total and free concentrations of CPZ in vivo using mass-balance equations like Hill's equation or a competitive inhibition equation on the multiple binding sites for drug, the correlation between the ratio ot total concentration of CPZ metabolites and CPZ (CPZSO/CPZ or CPZNO/CPZ) and the free fraction of CPZ was examined using the in vitro and in vivo data. The correlation between the ratio of CPZSO/CPZ and the free fraction of CPZ was good in both the in vivo and the in vitro studies. There was no statistically significant difference between the population regression coefficient of the two studies. The values of the slope and the intercept became almost the same as those obtained using the in vivo studies when combined with CPZNO.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7655433

  4. Voluntary action and tactile sensory feedback in the intentional binding effect.

    PubMed

    Zhao, Ke; Hu, Li; Qu, Fangbing; Cui, Qian; Piao, Qiuhong; Xu, Hui; Li, Yanyan; Wang, Liang; Fu, Xiaolan

    2016-08-01

    The intentional binding effect refers to a subjective compression over a temporal interval between the start point initialized by a voluntary action and the endpoint signaled by an external sensory (visual or audio) feedback. The present study aimed to explore the influence of tactile sensory feedback on this binding effect by comparing voluntary key-press actions with voluntary key-release actions. In experiment 1, each participant was instructed to report the perceived interval (in ms) between an action and the subsequent visual sensory feedback. In this task, either the action (key-press or key-release) was voluntarily performed by the participant or a kinematically identical movement was passively applied to the left index finger of the participant. In experiment 2, we explored whether the difference in the perception of time was affected by the direction of action. In experiment 3, we developed an apparatus in which two parallel laser beams were generated by a laser emission unit and detected by a laser receiver unit; this allowed the movement of the left index finger to be detected without it touching a keyboard (i.e., without any tactile sensory feedback). Convergent results from all of the experiments showed that the temporal binding effect was only observed when the action was both voluntary and involved physical contact with the key, suggesting that the combination of intention and tactile sensory feedback, as a form of top-down processing, likely distracted attention from temporal events and caused the different binding effects. PMID:27038203

  5. Probing nucleotide-binding effects on backbone dynamics and folding of the nucleotide-binding domain of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase.

    PubMed Central

    Abu-Abed, Mona; Millet, Oscar; MacLennan, David H; Ikura, Mitsuhiko

    2004-01-01

    In muscle cells, SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) plays a key role in restoring cytoplasmic Ca2+ levels to resting concentrations after transient surges caused by excitation-coupling cycles. The mechanism by which Ca2+ is translocated to the lumen of the ER (endoplasmic reticulum) involves major conformational rearrangements among the three cytoplasmic domains: actuator (A), nucleotide-binding (N) and phosphorylation (P) domains; and within the transmembrane Ca2+-binding domain of SERCA. CD, fluorescence spectroscopy and NMR spectroscopy were used in the present study to probe the conformation and stability of the isolated N domain of SERCA (SERCA-N), in the presence and absence of AMP-PNP (adenosine 5'-[beta,gamma-imido]triphosphate). CD and tryptophan fluorescence spectroscopy results established that the effects of nucleotide binding were not readily manifested on the global fold and structural stability of SERCA-N. 15N-backbone-relaxation experiments revealed site-specific changes in backbone dynamics that converge on the central beta-sheet domain. Nucleotide binding produced diverse effects on dynamics, with enhanced mobility observed for Ile369, Cys420, Arg467, Asp568, Phe593 and Gly598, whereas rigidifying effects were found for Ser383, Leu419, Thr484 and Thr532. These results demonstrate that the overall fold and backbone motional properties of SERCA-N remained essentially the same in the presence of AMP-PNP, yet revealing evidence for internal counter-balancing effects on backbone dynamics upon binding the nucleotide, which propagate through the central beta-sheet. PMID:14987197

  6. [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

  7. 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.

  8. Effects of class I heparin binding growth factor and fibronectin on platelet adhesion and aggregation

    SciTech Connect

    Greisler, H.P.; Klosak, J.J.; Steinam, S.J.; Lam, T.M.; Burgess, W.H.; Kim, D.U. )

    1990-05-01

    Fibronectin and heparin binding growth factor-type 1 have been affixed to vascular graft surfaces to enhance the attachment and the proliferation of transplanted endothelial cells, respectively. The current study examines the effect of fibronectin and heparin binding growth factor-type 1 on platelet adhesion and activation in vivo and on platelet aggregation in vitro. Expanded polytetrafluoroethylene prostheses (5 cm x 4 mm internal diameter) were treated either with fibronectin (n = 9), fibronectin/heparin/heparin binding growth factor-type 1/heparin (n = 12), or neither (n = 13) and were interposed into canine aortoiliac systems bilaterally. Autogenous radiolabeled (Indium 111 oxine, 650 microCi) platelets were injected intravenously before reestablishment of circulation. Perfusion was maintained for 30 minutes, and prostheses were removed with segments of native aorta and distal iliac arteries bilaterally. Specimens were examined for thrombus-free surface area, by gamma well counting for adherent radiolabeled platelets, and by light microscopy and transmission and scanning electron microscopic techniques. Results showed that both the fibronectin and fibronectin/heparin/heparin binding growth factor-type 1/heparin pretreated prostheses contained significantly greater numbers of platelets and adherent radioactivity than did control graft segments when normalized to their ipsilateral iliac arteries. Fibronectin/heparin/heparin binding growth factor-type 1/heparin pretreated prostheses contained 27 +/- 16 times more radioactivity per square millimeter than ipsilateral iliac arteries, fibronectin pretreated prostheses had 13 +/- 8 times more radioactivity per square millimeter than ipsilateral iliac arteries, and untreated expanded polytetrafluoroethylene had 4 +/- 3 times more radioactivity per square millimeter than ipsilateral iliac arteries.

  9. Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

    PubMed Central

    De Filippis, Vincenzo; De Dea, Elisa; Lucatello, Filippo; Frasson, Roberta

    2005-01-01

    In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by ∼2 kcal/mol (1 cal≡4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the ‘fast’ form. Finally, we have used hirudin fragment 1–47 as a molecular probe of the conformation of thrombin recognition sites in the fast and ‘slow’ form. From the effects of amino acid substitutions on the affinity of fragment 1–47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation. PMID:15971999

  10. Three dimensional simulation on binding efficiency of immunoassay for a biosensor with applying electrothermal effect

    NASA Astrophysics Data System (ADS)

    Huang, Kuan-Rong; Chang, Jeng-Shian

    2013-11-01

    In this work, we perform three dimensional finite element simulations on the binding reaction kinetics of the commonly used analyte-ligand protein pairs, namely, C-reactive protein (CRP) and anti-CRP, in a reaction chamber (microchannel) of a biosensor. For the diffusion limited binding biomolecular pairs, due to the slower transport speed of the analyte and the faster reaction rate of analyte-ligand complex, diffusion boundary layers often develop on the reaction surface. To enhance the performance of a biosensor by accelerating the transport speed, a non-uniform AC electric field is applied to induce the electrothermal force to stir the flow field. The swirling flow in the fluid can accelerate the transport of the analyte to and from the reaction surface and hence enhance the association and dissociation of analyte-ligand complex. Four types of biosensors with different arrangements of the geometric locations of the electrode pair and the reaction surface are designed to study the effects of varying geometric configuration on the binding efficiency. The simulation results show that the performance of a biosensor can be better improved by placing the electrodes and the reaction surface on the same side of the microchannel against the opposite side. For the best case studied in this work, the maximum initial slope of the binding curve can be raised up to 6.94 times (with respect to the field-free value) in the association phase, under applying AC field of 15 Vrms and operating frequency of 100 kHz. Another important result with applying electrothermal effect is that it is feasible to use the slower sample flow in the microchannel to save a lot of sample consumption without sacrificing the performance of a biosensor. Several control factors not studied in our previous works such as the thermal boundary condition and the effect of electrical conductivity are also discussed.

  11. 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...

  12. 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…

  13. Molecular crowding effect on dynamics of DNA-binding proteins search for their targets

    NASA Astrophysics Data System (ADS)

    Liu, Lin; Luo, Kaifu

    2014-12-01

    DNA-binding proteins locate and bind their target sequences positioned on DNA in crowded environments, but the molecular crowding effect on this search process is not clear. Using analytical techniques and Langevin dynamics simulations in two dimensions (2D), we find that the essential physics for facilitated diffusion in 2D search and 3D search is the same. We observe that the average search times have minima at the same optimal nonspecific binding energy for the cases with and without the crowding particle. Moreover, the molecular crowding increases the search time by increasing the average search rounds and the one-dimensional (1D) sliding time of a round, but almost not changing the average 2D diffusion time of a round. In addition, the fraction of 1D sliding time out of the total search time increases with increasing the concentration of crowders. For 2D diffusion, the molecular crowding decreases the jumping length and narrows its distribution due to the cage effect from crowders. These results shed light on the role of facilitated diffusion in DNA targeting kinetics in living cells.

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

    PubMed Central

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

    2014-01-01

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

  15. Actin-binding proteins coronin-1a and IBA-1 are effective microglial markers for immunohistochemistry.

    PubMed

    Ahmed, Zeshan; Shaw, Gerry; Sharma, Ved P; Yang, Cui; McGowan, Eileen; Dickson, Dennis W

    2007-07-01

    This study identifies the actin-binding protein, coronin-1a, as a novel and effective immunohistochemical marker for microglia in both cell cultures and in formaldehyde-fixed, paraffin-embedded tissue. Antibodies to coronin-1a effectively immunostained microglia in human, monkey, horse, rat, and mouse tissues, even in tissues stored for long periods of time. The identity of coronin-1a-immunoreactive cells as microglia was confirmed using double immunolabeling with cell type-specific markers as well as by morphological features and the distribution of immunoreactive cells. These properties are shared by another actin-binding protein, IBA-1. Unlike IBA-1, coronin-1a immunoreactivity was also detected in lymphocytes and certain other hematopoietic cells. The results indicate that both coronin-1a and IBA-1 are robust markers for microglia that can be used in routinely processed tissue of humans and animals. Because both coronin-1a and IBA-1 are actin-binding proteins that play a role in rearrangement of the membrane cytoskeleton, it suggests that these proteins are critical to dynamic properties of microglia. PMID:17341475

  16. 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.

  17. 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

  18. A study into the effects of protein binding on nucleotide conformation.

    PubMed Central

    Moodie, S L; Thornton, J M

    1993-01-01

    In this study, we examine the effects of binding to protein upon nucleotide conformation, by the comparison of X-ray crystal structures of free and protein-bound nucleotides. A dataset of structurally non-homologous protein-nucleotide complexes was derived from the Brookhaven Protein Data Bank by a novel protocol of dual sequential and structural alignments, and a dataset of native nucleotide structures was obtained from the Cambridge Structural Database. The nucleotide torsion angles and sugar puckers, which describe nucleotide conformation, were analysed in both datasets and compared. Differences between them are described and discussed. Overall, the nucleotides were found to bind in low energy conformations, not significantly different from their 'free' conformations except that they adopted an extended conformation in preference to the 'closed' structure predominantly observed by free nucleotide. The archetypal conformation of a protein-bound nucleotide is derived from these observations. PMID:8464727

  19. 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

  20. An investigation of large inhibitors binding to phosphoglycerate kinase and their effect on anion activation.

    PubMed

    Joao, H C; Williams, R J; Littlechild, J A; Nagasuma, R; Watson, H C

    1992-05-01

    This study extends, to a series of larger anions, our earlier investigation of the interaction of the trypanocidal drug suramin and other small negatively charged molecules with yeast phosphoglycerate kinase. 1H-NMR structural studies of phosphoglycerate kinase in the presence of varying concentrations of these large molecules (designed to mimic, at one end, the anionic charge distribution in the substrate 3-phosphoglycerate, while possibly being able to interact across the cleft of the enzyme) including inositol 1,4,5-triphosphate, 4-amino-6-trichloroethenyl-1,3- benzenedisulphonamide, gallic acid and sulphasalazine are described. The anion activation and/or inhibition of the enzyme by these molecules are also reported. Evidence that binding to the general anion site in the 'basic patch' region of the protein may be responsible for either the activating or inhibiting effects, while binding at the hydrophobic (catalytic) site leads to inhibition only is presented. A reaction scheme which explains these observations is given. PMID:1349525

  1. 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.

  2. 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

  3. The Effect of Hydraulic Loading Rate and Influent Source on the Binding Capacity of Phosphorus Filters

    PubMed Central

    Herrmann, Inga; Jourak, Amir; Hedström, Annelie; Lundström, T. Staffan; Viklander, Maria

    2013-01-01

    Sorption by active filter media can be a convenient option for phosphorus (P) removal and recovery from wastewater for on-site treatment systems. There is a need for a robust laboratory method for the investigation of filter materials to enable a reliable estimation of their longevity. The objectives of this study were to (1) investigate and (2) quantify the effect of hydraulic loading rate and influent source (secondary wastewater and synthetic phosphate solution) on P binding capacity determined in laboratory column tests and (3) to study how much time is needed for the P to react with the filter material (reaction time). To study the effects of these factors, a 22 factorial experiment with 11 filter columns was performed. The reaction time was studied in a batch experiment. Both factors significantly (α = 0.05) affected the P binding capacity negatively, but the interaction of the two factors was not significant. Increasing the loading rate from 100 to 1200 L m−2 d−1 decreased P binding capacity from 1.152 to 0.070 g kg−1 for wastewater filters and from 1.382 to 0.300 g kg−1 for phosphate solution filters. At a loading rate of 100 L m−2 d−1, the average P binding capacity of wastewater filters was 1.152 g kg−1 as opposed to 1.382 g kg−1 for phosphate solution filters. Therefore, influent source or hydraulic loading rate should be carefully controlled in the laboratory. When phosphate solution and wastewater were used, the reaction times for the filters to remove P were determined to be 5 and 15 minutes, respectively, suggesting that a short residence time is required. However, breakthrough in this study occurred unexpectedly quickly, implying that more time is needed for the P that has reacted to be physically retained in the filter. PMID:23936313

  4. Radioligand binding studies of alpha 1-adrenoceptor subtypes in rat heart.

    PubMed Central

    Michel, M. C.; Hanft, G.; Gross, G.

    1994-01-01

    1. In order to characterize the alpha 1-adrenoceptor subtypes mediating positive inotropic effects of adrenaline (in the presence of propranolol) in rat right ventricular strips and the Ca2+ sources used to elicit them, we have used radioligand binding to identify the alpha 1-adrenoceptor subtypes present in rat heart and the alpha 1-adrenoceptor affinity and subtype-selectivity of various pharmacological tools. 2. Amitryptiline, mianserin, trimipramine, oxaprotiline, clonidine, chloroethylclonidine, phenoxybenzamine, BE 2254 and 8-OH-DPAT competed for [3H]-prazosin binding in rat heart, vas deferens, liver, spleen, cerebral cortex and hippocampus but none of them displayed detectable alpha 1-adrenoceptor subtype-selectivity; nitrendipine did not compete for [3H]-prazosin binding in concentrations up to 5 mumol 1(-1). 3. The alpha 1 A-adrenoceptor-selective, 5-methyl-urapidil, (+)-niguldipine, and to a lesser extent (-)-niguldipine competed for [3H]-prazosin binding in rat heart, vas deferens, cerebral cortex and hippocampus with shallow and biphasic curves; analysis of these curves demonstrated that rat heart contains alpha 1A-and alpha 1B-adrenoceptors in a 20:80 ratio. 4. Treatment of rat right ventricular strips with 100 mumol l-1 chloroethylclonidine for 30 min at 30 degrees C followed by 60 min washout reduced the number of alpha 1-adrenoceptors, as assessed by [3H]-prazosin saturation experiments, by 74%. Treatment with 100 mumol l(-1) CdCl2 did not affect number or affinity of cardiac alpha 1-adrenoceptors and combined treatment with chlorethylclonidine and CdCl2 reduced alpha 1-adrenoceptor number by 90%.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7911718

  5. Calcium binding to calmodulin mutants having domain-specific effects on the regulation of ion channels.

    PubMed

    VanScyoc, Wendy S; Newman, Rhonda A; Sorensen, Brenda R; Shea, Madeline A

    2006-12-01

    Calmodulin (CaM) is an essential, eukaryotic protein comprised of two highly homologous domains (N and C). CaM binds four calcium ions cooperatively, regulating a wide array of target proteins. A genetic screen of Paramecia by Kung [Kung, C. et al. (1992) Cell Calcium 13, 413-425] demonstrated that the domains of CaM have separable physiological roles: "under-reactive" mutations affecting calcium-dependent sodium currents mapped to the N-domain, while "over-reactive" mutations affecting calcium-dependent potassium currents localized to the C-domain of CaM. To determine whether and how these mutations affected intrinsic calcium-binding properties of CaM domains, phenylalanine fluorescence was used to monitor calcium binding to sites I and II (N-domain) and tyrosine fluorescence was used to monitor sites III and IV (C-domain). To explore interdomain interactions, binding properties of each full-length mutant were compared to those of its corresponding domain fragments. The calcium-binding properties of six under-reactive mutants (V35I/D50N, G40E, G40E/D50N, D50G, E54K, and G59S) and one over-reactive mutant (M145V) were indistinguishable from those of wild-type CaM, despite their deleterious physiological effects on ion-channel regulation. Four over-reactive mutants (D95G, S101F, E104K, and H135R) significantly decreased the calcium affinity of the C-domain. Of these, one (E104K) also increased the calcium affinity of the N-domain, demonstrating that the magnitude and direction of wild-type interdomain coupling had been perturbed. This suggests that, while some of these mutations alter calcium-binding directly, others probably alter CaM-channel association or calcium-triggered conformational change in the context of a ternary complex with the affected ion channel. PMID:17128970

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

    PubMed

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

    2016-07-01

    Large amount of data of experimental and theoretical studies have shown that ethidium bromide (EtBr) and methylene blue (MB) may bind to nucleic acids via three modes: intercalation between two adjacent base pairs, insertion into the plane between neighboring bases in the same strand (semi-intercalation), and outside binding with negatively charged backbone phosphate groups. The aim of the given research is to examine the behavior of these two ligands at both separate and joint DNA binding. The obtained experimental data show that the effect of simultaneous binding of EtBr and MB on double-stranded DNA has a non-additive effect of separate binding. The analyses of the melting thermodynamic parameters of DNA complexes with two bound ligands suggest competitive mechanism of interaction. PMID:26239502

  7. 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.

  8. The effect of macromolecular crowding, ionic strength and calcium binding on calmodulin dynamics

    NASA Astrophysics Data System (ADS)

    Wang, Qian; Liang, Kao-Chen; Waxham, Neal; Cheung, Margaret

    2011-03-01

    The flexibility in the structure of calmodulin (CaM) allows its binding to over 300 target proteins in the cell. To investigate the structure-function relationship of CaM in response to the changing intracellular environment, we use a combined method of computer simulation and experiments based on circular dichroism (CD). The conformation, helicity and EF hand orientation of CaM are analyzed computationally to address the effect of macromolecular crowding, ionic strength and calcium binding in the experiments. We applied a unique solution of charges computed from QM/MM to accurately represent the charge distribution in the transition from apo-CaM to holo-CaM. Computationally, we found that a high level of macromolecular crowding, in addition to calcium binding and ionic strength, can impact the conformation, helicity and the EF hand orientation of CaM. Our result may provide unique insight into understanding the promiscuous behavior of calmodulin in target selection inside cells. This work is supported by National Science Foundation, Molecular & Cellular Biosciences (MCB0919974).

  9. 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.

  10. The Effect of Nonspecific Binding of Lambda Repressor on DNA Looping Dynamics

    PubMed Central

    Manzo, Carlo; Zurla, Chiara; Dunlap, David D.; Finzi, Laura

    2012-01-01

    The λ repressor (CI) protein-induced DNA loop maintains stable lysogeny, yet allows efficient switching to lysis. Herein, the kinetics of loop formation and breakdown has been characterized at various concentrations of protein using tethered particle microscopy and a novel, to our knowledge, method of analysis. Our results show that a broad distribution of rate constants and complex kinetics underlie loop formation and breakdown. In addition, comparison of the kinetics of looping in wild-type DNA and DNA with mutated o3 operators showed that these sites may trigger nucleation of nonspecific binding at the closure of the loop. The average activation energy calculated from the rate constant distribution is consistent with a model in which nonspecific binding of CI between the operators shortens their effective separation, thereby lowering the energy barrier for loop formation and broadening the rate constant distribution for looping. Similarly, nonspecific binding affects the kinetics of loop breakdown by increasing the number of loop-securing protein interactions, and broadens the rate constant distribution for this reaction. Therefore, simultaneous increase of the rate constant for loop formation and reduction of that for loop breakdown stabilizes lysogeny. Given these simultaneous changes, the frequency of transitions between the looped and the unlooped state remains nearly constant. Although the loop becomes more stable thermodynamically with increasing CI concentration, it still opens periodically, conferring sensitivity to environmental changes, which may require switching to lytic conditions. PMID:23083719

  11. 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.

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

    SciTech Connect

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

    1986-08-01

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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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.

  18. Effect of 60Co-gamma radiation on the binding properties in furs

    NASA Astrophysics Data System (ADS)

    Raina, R. K.

    New Zealand white rabbit pelts were pickled by the usual procedure and were tanned with basic aluminium sulphate, basic chromium sulphate and their combinations. Tanned furs were irradiated with 60Co-gamma radiations in the dose range of 5.0-114.0 kGy. The effect of radiation on the binding properties of various added substances like mineral tannins, fats, moisture and shrinkage temperature has been assessed by their comparison with the control samples. The results of these investigations show that radiation on furs causes detannage, increases the moisture and bound fat content and decreases the shrinkage temperature of the furs.

  19. 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

  20. Honey bee odorant-binding protein 14: effects on thermal stability upon odorant binding revealed by FT-IR spectroscopy and CD measurements.

    PubMed

    Schwaighofer, Andreas; Kotlowski, Caroline; Araman, Can; Chu, Nam; Mastrogiacomo, Rosa; Becker, Christian; Pelosi, Paolo; Knoll, Wolfgang; Larisika, Melanie; Nowak, Christoph

    2014-03-01

    In the present work, we study the effect of odorant binding on the thermal stability of honey bee (Apis mellifera L.) odorant-binding protein 14. Thermal denaturation of the protein in the absence and presence of different odorant molecules was monitored by Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). FT-IR spectra show characteristic bands for intermolecular aggregation through the formation of intermolecular β-sheets during the heating process. Transition temperatures in the FT-IR spectra were evaluated using moving-window 2D correlation maps and confirmed by CD measurements. The obtained results reveal an increase of the denaturation temperature of the protein when bound to an odorant molecule. We could also discriminate between high- and low-affinity odorants by determining transition temperatures, as demonstrated independently by the two applied methodologies. The increased thermal stability in the presence of ligands is attributed to a stabilizing effect of non-covalent interactions between odorant-binding protein 14 and the odorant molecule. PMID:24362824

  1. Binding and unbinding the auditory and visual streams in the McGurk effect.

    PubMed

    Nahorna, Olha; Berthommier, Frédéric; Schwartz, Jean-Luc

    2012-08-01

    Subjects presented with coherent auditory and visual streams generally fuse them into a single percept. This results in enhanced intelligibility in noise, or in visual modification of the auditory percept in the McGurk effect. It is classically considered that processing is done independently in the auditory and visual systems before interaction occurs at a certain representational stage, resulting in an integrated percept. However, some behavioral and neurophysiological data suggest the existence of a two-stage process. A first stage would involve binding together the appropriate pieces of audio and video information before fusion per se in a second stage. Then it should be possible to design experiments leading to unbinding. It is shown here that if a given McGurk stimulus is preceded by an incoherent audiovisual context, the amount of McGurk effect is largely reduced. Various kinds of incoherent contexts (acoustic syllables dubbed on video sentences or phonetic or temporal modifications of the acoustic content of a regular sequence of audiovisual syllables) can significantly reduce the McGurk effect even when they are short (less than 4 s). The data are interpreted in the framework of a two-stage "binding and fusion" model for audiovisual speech perception. PMID:22894226

  2. 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

  3. In vitro anti-cancer effects of the actin-binding natural compound rhizopodin.

    PubMed

    Zhang, S; Menche, D; Zahler, S; Vollmar, A M; Liebl, J; Förster, F

    2015-09-01

    Several natural compound interfere with microtubules or the actin cytoskeleton. Compounds interfering with the microtubules like Vinca-alkaloids or taxanes, are extensively used for cancer therapy. In contrast, knowledge about pharmacological properties of actin binding drugs is poor and drugs interfering with actin are far from clinical use. Rhizopodin is a natural compound that strongly affects the actin cytoskeleton at nanomolar concentrations. Initial work revealed interesting anti-bacterial and cytotoxic effects, but the cellular effects and pharmacological properties of rhizopodin have not been characterized. We hypothesized that rhizopodin might exert anti-cancer activity. Therefore, the aim of this study was to characterize the cellular and pharmacological effects of rhizopodin in cancer. Effects of rhizopodin demonstrated prominent effects on the actin cytoskeleton as shown in the actin-pyrene assay and by immunostaining of cancer cells. To investigate cellular effects of rhizopodin, we analyzed cell proliferation, cell death induction by propidium iodide exclusion and western blot, as well as migration by impedance measurement using the xCELLligence device in MDA-MB-231 breast cancer and T24 bladder cancer cell lines. Rhizopodin inhibited proliferation and induced cell death of MDA-MB-231 and T24 cells at nanomolar concentrations. PARP cleavage by rhizopodin suggests caspase-dependent cell death induction. Importantly, rhizopodin potently inhibited MDA-MB-231 and T24 cancer cell migration at subtoxic doses where no actin aggregation was observed, indicating a specific underlying signaling of rhizopodin. In summary, our study elucidates rhizopodin as actin-binding natural compound that exerts potent anti-cancer effects. Therefore, our work provides the basis for further in depth characterization of rhizopodin as an antitumoral agent. PMID:26492647

  4. PROSPECTIVE VALIDATION OF THE VASOACTIVE-INOTROPIC SCORE AND CORRELATION TO SHORT TERM OUTCOMES IN NEONATES AND INFANTS AFTER CARDIOTHORACIC SURGERY

    PubMed Central

    Davidson, Jesse; Tong, Suhong; Hancock, Hayley; Hauck, Amanda; da Cruz, Eduardo; Kaufman, Jon

    2016-01-01

    Purpose Prospective validation of vasoactive-inotropic score (VIS) and inotrope score (IS) in infants after cardiovascular surgery Methods Prospective observational study of 70 infants (≤90 days of age) undergoing cardiothoracic surgery. VIS and IS were assessed at 24 (VIS24, IS24), 48 (VIS48, IS48), and 72 (VIS72, IS72) hours after surgery. Maximum VIS and IS scores in the first 48 hours were also calculated (VIS48max and IS48max). The primary outcome was length of intubation. Additional outcomes included length of intensive care (ICU) stay and hospitalization, cardiac arrest, mortality, time to negative fluid balance, peak lactate, and change in creatinine. Results Based on Receiver Operating Characteristic (ROC) analysis, area under the curve (AUC) was highest for VIS48 to identify prolonged intubation time. AUC for the primary outcome was higher for VIS than IS at all time points assessed. On multivariate analysis VIS48 was independently associated with prolonged intubation (OR 22.3, p=0.002), prolonged ICU stay (OR 8.1, p=0.017), and prolonged hospitalization (OR 11.3, p=0.011). VIS48max, IS48max, and IS48 were also associated with prolonged intubation, but not prolonged ICU or hospital stay. None of the scores were associated with time to negative fluid balance, peak lactate, or change in creatinine. Conclusion In neonates and infants, a higher VIS at 48 hours after cardiothoracic surgery is strongly associated with increased length of ventilation, and prolonged ICU and total hospital stay. At all time points assessed, VIS is more predictive of poor short term outcome than IS. VIS may be useful as an independent predictor of outcomes. PMID:22527067

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

    PubMed

    Zafra Ruano, Ana; Cilia, Elisa; Couceiro, José R; Ruiz Sanz, Javier; Schymkowitz, Joost; Rousseau, Frederic; Luque, Irene; Lenaerts, Tom

    2016-05-01

    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

  6. 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

  7. 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.

  8. Binding modes of DL-2-haloacid dehalogenase revealed by crystallography, modeling and isotope effects studies.

    PubMed

    Siwek, Agata; Omi, Rie; Hirotsu, Ken; Jitsumori, Keiji; Esaki, Nobuyoshi; Kurihara, Tatsuo; Paneth, Piotr

    2013-12-01

    Several pathways of biotic dechlorination can be found in enzymes, each characterized by different chlorine isotopic fractionation, which can thus serve as a signature of a particular mechanism. Unlike other dehalogenases, DL-2-haloacid dehalogenase, DL-DEX, converts both enantiomers of the substrate. Chlorine isotope effects for this enzyme are larger than in the case of other dehalogenases. Recently, the 3D structure of this enzyme became available and enabled us to model these isotope effects and seek their origin. We show that the elevated values of the chlorine kinetic isotope effects originate in part in the processes of binding and migration within the enzyme active site that precede the dehalogenation step. PMID:24071515

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

    SciTech Connect

    Hansen, D.C.; McCafferty, E.

    1996-01-01

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

  10. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  12. 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.

  13. Effects of ion binding on the backbone dynamics of calbindin D9k determined by 15N NMR relaxation.

    PubMed

    Akke, M; Skelton, N J; Kördel, J; Palmer, A G; Chazin, W J

    1993-09-21

    The backbone dynamics of apo- and (Cd2+)1-calbindin D9k have been characterized by 15N nuclear magnetic resonance spectroscopy. Spin-lattice and spin-spin relaxation rate constants and steady-state [1H]-15N nuclear Overhauser effects were measured at a magnetic field strength of 11.74 T by two-dimensional, proton-detected heteronuclear NMR experiments using 15N-enriched samples. The relaxation parameters were analyzed using a model-free formalism that characterizes the dynamics of the N-H bond vectors in terms of generalized order parameters and effective correlation times. The data for the apo and (Cd2+)1 states were compared to those for the (Ca2+)2 state [Kördel, J., Skelton, N. J., Akke, M., Palmer, A. G., & Chazin, W. J. (1992) Biochemistry 31, 4856-4866] to ascertain the effects on ion ligation on the backbone dynamics of calbindin D9k. The two binding loops respond differently to ligation by metal ions: high-frequency (10(9)-10(12) s-1) fluctuations of the N-terminal ion-binding loop are not affected by ion binding, whereas residues G57, D58, G59, and E60 in the C-terminal ion-binding loop have significantly lower order parameters in the apo state than in the metal-bound states. The dynamical responses of the four helices to binding of ions are much smaller than that for the C-terminal binding loop, with the strongest effect on helix III, which is located between the linker loop and binding site II. Significant fluctuations on slower time scales also were detected in the unoccupied N-terminal ion-binding loop of the apo and (Cd2+)1 states; the apparent rates were greater for the (Cd2+)1 state. These results on the dynamical response to ion binding in calbindin D9k provide insights into the molecular details of the binding process and qualitative evidence for entropic contributions to the cooperative phenomenon of calcium binding for the pathway in which the ion binds first in the C-terminal site. PMID:8373781

  14. Effect of binding of lanthanide ions on the bacteriorhodopsin hexagonal structure: An x-ray study

    SciTech Connect

    Griffiths, J.A.; El-Sayed, M.A.; Capel, M.

    1996-07-18

    The effect of the binding of trivalent lanthanide metal cations (Eu{sup 3+}, Ho{sup 3+}, and Dy{sup 3+}) on the hexagonal structure of bacteriorhodopsin (bR) is investigated at different pH using x-Ray diffraction to examine films made by slow evaporation of the corresponding regenerated bR. It is observed that the lanthanide-regenerated bR (at a ratio of 2:1 metal ion to bR) does not form a 2D structure isomorphous to that of native bR or Ca{sup 2+}-regenerated samples at low sample pH. The native bR hexagonal structure is recovered by titration of the sample with sodium hydroxide. The pH at which the hexagonal structure is recovered depends on the charge density of the lanthanide ion used for the regeneration. The higher the charge density of the ion, the higher pH at which an isomorphous lattice is formed. A model is proposed in which at normal or low pH a complex bidentate and monodentate type binding (which disrupts the lattice hexagonal structure) exists between a lanthanide ion, the O{sup -} of PO{sub 2}{sup -} groups, and/or the amino acid residues. At high pH, complexation with OH{sup -} takes place, which converts this binding to a simple monodentate type complex that leads to the recovery of the lattice structure. An equation is derived for the pH at which this conversion takes place. 48 refs., 4 figs.

  15. 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

  16. 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

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

    PubMed

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

    2016-05-14

    Semiconducting polymers are soft materials with many conformational degrees of freedom. The limited understanding of how conformational disorder affects their optoelectronic properties is a key source of difficulties that limits their widespread usage in electronic devices. We develop a coarse-grained approach based on the tight binding approximation to model the electronic degrees of freedom of polythiophene chains, taking into account conformational degrees of freedom. Particularly important is dihedral disorder, which disrupts extended electronic states. Our tight binding model is parameterized using density functional theory (DFT) calculations of the one-dimensional band structures for chains with imposed periodic variations in dihedral angles. The model predicts valence and conduction bands for these chain conformations that compare well to DFT results. As an initial application of our model, we compute the optical absorption spectrum of poly(3-hexylthiophene) chains in solution. We observe a broadening of the absorption edge resulting from dihedral disorder, just shy of the experimental broadening. We conclude that the effects of molecular disorder on the optoelectronic properties of conjugated polymer single chains can be mostly accounted for by torsional disorder alone. PMID:27087455

  18. 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

  19. 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.

  20. Measurement in vivo of dopamine receptor density II: Effect of d-amphetamine on spiroperidol binding

    SciTech Connect

    Friedman, A.M.; De Jesus, O.T.; Woolverton, W.; Dinerstein, R.J.

    1984-01-01

    In the authors continuing studies to measure dopamine (DA) receptors in vivo using the DA antagonist bromospiroperidol (BrSP) and positron emission tomography (PET). The authors have examined the effect of d-amphetamine (d-AMP) on BrSP distribution in primate brain. Using the University of Chicago PETT VI scanner, /sup 76/Br-BrSP was found to localize in the caudate and putamen of anesthetized rhesus monkeys. The maximum level of this drug in these regions was reached at 100 minutes post-injection and remained constant for the next 200 minutes. Levels in the cerebellum, on the other hand, decline steadily after an hour post-injection. This is consistent with the presence of high level of DA receptors in the basal ganglia and low levels in the cerebellum. Preliminary studies showed that the administration of d-AMP (0.5 mg/kg i.v.) resulted in a small but statistically significant decrease in caudate /sup 76/Br-BrSP levels. Since d-AMP is known to release DA in the caudate, these findings are consistent with the competition of released DA for BrSP binding at caudate DA binding sites.

  1. 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.

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

    PubMed

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

    2014-07-21

    Fullerene and its derivatives with different surface chemistry have great potential in biomedical applications. Accordingly, it is important to delineate the impact of these carbon-based nanoparticles on protein structure, dynamics, and subsequently function. Here, we focused on the effect of hydroxylation - a common strategy for solubilizing and functionalizing fullerene - on protein-nanoparticle interactions using a model protein, ubiquitin. We applied a set of complementary computational modeling methods, including docking and molecular dynamics simulations with both explicit and implicit solvent, to illustrate the impact of hydroxylated fullerenes on the structure and dynamics of ubiquitin. We found that all derivatives bound to the model protein. Specifically, the more hydrophilic nanoparticles with a higher number of hydroxyl groups bound to the surface of the protein via hydrogen bonds, which stabilized the protein without inducing large conformational changes in the protein structure. In contrast, fullerene derivatives with a smaller number of hydroxyl groups buried their hydrophobic surface inside the protein, thereby causing protein denaturation. Overall, our results revealed a distinct role of surface chemistry on nanoparticle-protein binding and binding-induced protein misfolding. PMID:24934397

  3. 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. PMID:24874236

  4. Effect of Ghrelin on Hepatic IGF-Binding Protein-1 Production

    PubMed Central

    Lewitt, Moira S.

    2013-01-01

    Ghrelin plays key roles in energy homeostasis by central and peripheral actions that include effects on insulin signalling pathways in liver. Insulin is an important inhibitor of production by hepatocytes of insulin-like growth factor-binding protein-1 (IGFBP-1) which has an endocrine role to inhibit IGF availability. The effects of ghrelin, insulin, an AMPK activator, and an AMPK inhibitor on IGFBP-1 secretion were studied in H4-II-E rat liver cells. Ghrelin (100 nM) blocked the inhibitory effect of a maximally effective concentration of insulin (10 ng/mL) on IGFBP-1 secretion during a 5 h incubation period (P < 0.001) in the absence and presence of an AMPK inhibitor. Ghrelin, alone, had no effect on IGFBP-1 production, but enhanced secretion independently of insulin under conditions of AMPK activation (P < 0.001). In conclusion, IGFBP-1 is identified as a novel target of ghrelin action in liver that may contribute to its metabolic effects in obesity. PMID:24555152

  5. 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

  6. Effect of drug-binding-induced deformation on the vibrational spectrum of a DNA.daunomycin complex

    NASA Astrophysics Data System (ADS)

    Chen, Y. Z.; Szabó, A.; Schroeter, D. F.; Powell, J. W.; Lee, S. A.; Prohofsky, E. W.

    1997-06-01

    Vibrational frequencies of a DNA.daunomycin complex and those of a free DNA helix and an isolated daunomycin are calculated and compared with the infrared spectrum of similar systems at frequencies above 600 cm-1. Our study indicates that the binding induces a considerable change in the vibrational spectrum of both DNA and the binding drug. The frequency shifts appear to be closely related to the conformational deformation in the complex caused by drug binding. Significant frequency shift is found in the normal modes in the DNA.drug complex that are primarily vibrations localized to the sugar-phosphate backbone of the binding site. Sizable frequency change is also found in the modes associated with base atoms involved in the drug binding and in the modes in regions of the binding daunomycin that are deformed by the binding. In contrast the frequency of the modes in the region with no significant deformation is relatively unchanged. The modification of the DNA dynamical force field by the nonbonded interactions between DNA and the drug is found to have little effect on the modes in DNA above 600 cm-1. The modification to the daunomycin dynamical force field appears to be sizable since the frequency of several daunomycin modes is changed by several cm-1. The close relationship between structure and spectrum revealed in this work is of potential application in the identification of sites and types of deformation of a biomolecule from Raman and infrared spectra.

  7. Improving the scoring of protein-ligand binding affinity by including the effects of structural water and electronic polarization.

    PubMed

    Liu, Jinfeng; He, Xiao; Zhang, John Z H

    2013-06-24

    Docking programs that use scoring functions to estimate binding affinities of small molecules to biological targets are widely applied in drug design and drug screening with partial success. But accurate and efficient scoring functions for protein-ligand binding affinity still present a grand challenge to computational chemists. In this study, the polarized protein-specific charge model (PPC) is incorporated into the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method to rescore the binding poses of some protein-ligand complexes, for which docking programs, such as Autodock, could not predict their binding modes correctly. Different sampling techniques (single minimized conformation and multiple molecular dynamics (MD) snapshots) are used to test the performance of MM/PBSA combined with the PPC model. Our results show the availability and effectiveness of this approach in correctly ranking the binding poses. More importantly, the bridging water molecules are found to play an important role in correctly determining the protein-ligand binding modes. Explicitly including these bridging water molecules in MM/PBSA calculations improves the prediction accuracy significantly. Our study sheds light on the importance of both bridging water molecules and the electronic polarization in the development of more reliable scoring functions for predicting molecular docking and protein-ligand binding affinity. PMID:23651068

  8. 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

  9. 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.

  10. 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