Steady-state [Ca2+]i-force relationship in intact twitching cardiac muscle: direct evidence for modulation by isoproterenol and EMD 53998.

PubMed Central

Dobrunz, L E; Backx, P H; Yue, D T

1995-01-01

We have developed a novel method for measuring steady-state force-[Ca2+]i relations in isolated, membrane-intact rat trabeculae that are microinjected with Fura-2 salt. Twitches are markedly slowed after inhibition of phasic Ca2+ release and uptake from the sarcoplasmic reticulum by addition of cyclopiazonic acid and ryanodine. During relaxation of slowed twitches, force and [Ca2+]i trace a common trajectory in plots of force versus [Ca2+]i, despite very different histories of contraction. The common trajectory thereby provides a high resolution determination of the steady-state relation between force and [Ca2+]i. Using this method, we show that 1 microM isoproterenol, a beta-adrenergic agonist, causes a rightward shift (Hill function K1/2 increased from 0.39 +/- 0.07 microM to 0.82 +/- 0.23 microM, p < 0.02, n = 6) and a decreased slope (nH decreased from 5.4 +/- 1.1 to 4.0 +/- 1.4, p < 0.02) of the steady-state force-[Ca2+]i curve, with no change in maximal force (Fmax = 99.2 +/- 2.2% of control). In contrast, 2 microM EMD 53998, a racemic thiadiazinone derivative, causes a leftward shift (K1/2 decreased from 0.42 +/- 0.02 microM to 0.30 +/- 0.06 microM, p < 0.02, n = 4) with no change in slope of the steady-state force-[Ca2+]i curve, accompanied by a modest increase in maximal force (Fmax = 107.1 +/- 4.6% of control, p < 0.02). To gain mechanistic insight into these modulatory events, we developed a simple model of cooperative thin filament activation that predicts steady-state force-[Ca2+]i relationships. Model analysis suggests that isoproterenol decreases cooperativity arising from nearest-neighbor interactions between regulatory units on the thin filament, without change in the equilibrium constant for Ca2+ binding. In contrast, the effects of EMD 53998 are consistent with an increase in the affinity of strong-binding cross-bridges, without change in either the affinity of troponin C for Ca2+ or cooperative interactions. PMID:7669896

On the interaction of luminol with human serum albumin: Nature and thermodynamics of ligand binding

NASA Astrophysics Data System (ADS)

Moyon, N. Shaemningwar; Mitra, Sivaprasad

2010-09-01

The mechanism and thermodynamic parameters for the binding of luminol (LH 2) with human serum albumin was explored by steady state and picosecond time-resolved fluorescence spectroscopy. It was shown that out of two possible LH 2 conformers present is solution, only one is accessible for binding with HSA. The thermodynamic parameters like enthalpy (Δ H) and entropy (Δ S) change corresponding to the ligand binding process were also estimated by performing the experiment at different temperatures. The ligand replacement experiment with bilirubin confirms that LH 2 binds into the sub-domain IIA of the protein.

Quantization of bovine serum albumin by fluorescence enhancement effects and corresponding binding of macrocyclic host-protein assembly.

PubMed

Bardhan, Munmun; Misra, Tapas; Ganguly, Tapan

2012-01-05

The present paper reports the investigations on the spectroscopic behavior of the binary complexes of the dye aurintricarboxylic acid (ATA) with protein bovine serum albumin (BSA) and 18-crown 6 (CW) (ATA·BSA, ATA·CW) and the ternary complex ATA·CW·BSA by using UV-vis steady state and time resolved fluorescence spectroscopy. The primary aim of the work is to determine the protein (BSA) quantization by fluorescence enhancement method and investigate the 'enhancer' activity of crown ether (CW) on it to increase the resolution. Steady state and time resolved fluorescence measurements demonstrated how fluorescence intensity of ATA could be used for the determination of the protein BSA in aqueous solution. The binding of dye (probe/fluorescent medicinal molecule) with protein and the denaturing effect in the polar environment of acetonitrile of the dye protein complex act as drug binding as well as drug release activity. Apart from its basic research point of view, the present study also possesses significant importance and applications in the field of medicinal chemistry. Copyright © 2011 Elsevier B.V. All rights reserved.

Deriving an explicit hepatic clearance equation accounting for plasma protein binding and hepatocellular uptake.

PubMed

Yoon, Miyoung; Clewell, Harvey J; Andersen, Melvin E

2013-02-01

High throughput in vitro biochemical and cell-based assays have the promise to provide more mechanism-based assessments of the adverse effects of large numbers of chemicals. One of the most challenging hurdles for interpreting in vitro toxicity findings is the need for reverse dosimetry tools that estimate the exposures that will give concentrations in vivo similar to the active concentrations in vitro. Recent experience using IVIVE approaches to estimate in vivo pharmacokinetics (Wetmore et al., 2012) identified the need to develop a hepatic clearance equation that explicitly accounted for a broader set of protein binding and membrane transport processes and did not depend on a well-mixed description of the liver compartment. Here we derive an explicit steady-state hepatic clearance equation that includes these factors. In addition to the derivation, we provide simple computer code to calculate steady-state extraction for any combination of blood flow, membrane transport processes and plasma protein-chemical binding rates. This expanded equation provides a tool to estimate hepatic clearance for a more diverse array of compounds. Copyright © 2012 Elsevier Ltd. All rights reserved.

Elucidation of the factors affecting the oxidative activity of Acremonium sp. HI-25 ascorbate oxidase by an electrochemical approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murata, Kenichi; Nakamura, Nobuhumi; Ohno, Hiroyuki

Steady-state kinetics of Acremonium sp. HI-25 ascorbate oxidase toward p-hydroquinone derivatives have been examined by using an electrochemical analysis based on the theory of steady-state bioelectrocatalysis. The electrochemical technique has enabled one to examine the influence of electronic and chemical properties of substrates on the activity. It was proven that the oxidative activity of ascorbate oxidase was dominated by the highly selective substrate-binding affinity based on electrostatic interaction beyond the one-electron redox potential difference between ascorbate oxidase's type 1 copper site and substrate.

Steady-state pharmacokinetics of (R)- and (S)-methadone in methadone maintenance patients

PubMed Central

Foster, David J R; Somogyi, Andrew A; Dyer, Kyle R; White, Jason M; Bochner, Felix

2000-01-01

Aims To investigate the steady-state pharmacokinetics of (R)- and (S)-methadone in a methadone maintenance population. Methods Eighteen patients recruited from a public methadone maintenance program underwent an interdosing interval pharmacokinetic study. Plasma and urine samples were collected and analysed for methadone and its major metabolite (EDDP) using stereoselective h.p.l.c. Methadone plasma protein binding was examined using ultrafiltration, and plasma α1-acid glycoprotein concentrations were quantified by radial immunoassay. Results (R)-methadone had a significantly (P < 0.05) greater unbound fraction (mean 173%) and total renal clearance (182%) compared with (S)-methadone, while maximum measured plasma concentrations (83%) and apparent partial clearance of methadone to EDDP (76%) were significantly (P < 0.001) lower. When protein binding was considered (R)-methadone plasma clearance of the unbound fraction (59%) and apparent partial intrinsic clearance to EDDP (44%) were significantly (P < 0.01) lower than for (S)-methadone, while AUCτSS, (167%) was significantly (P < 0.001) greater. There were no significant (P > 0.2) differences between the methadone enantiomers for AUCτSS, steady-state plasma clearance, trough plasma concentrations and unbound renal clearance. Patients excreted significantly (P < 0.0001) more (R)-methadone and (S)-EDDP than the corresponding enantiomers. Considerable interindividual variability was observed for the pharmacokinetic parameters, with coefficients of variation of up to 70%. Conclusions Steady-state pharmacokinetics of unbound methadone are stereoselective, and there is large interindividual variability consistent with CYP3A4 mediated metabolism to the major metabolite EDDP; the variability did not obscure a significant dose-plasma concentration relationship. Stereoselective differences in the pharmacokinetics of methadone may have important implications for pharmacokinetic-pharmacodynamic modelling but is unlikely to be important for therapeutic drug monitoring of methadone, in the setting of opioid dependence. PMID:11069437

Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance*

PubMed Central

Schermerhorn, Kelly M.; Gardner, Andrew F.

2015-01-01

Family D DNA polymerases (polDs) have been implicated as the major replicative polymerase in archaea, excluding the Crenarchaeota branch, and bear little sequence homology to other DNA polymerase families. Here we report a detailed kinetic analysis of nucleotide incorporation and exonuclease activity for a Family D DNA polymerase from Thermococcus sp. 9°N. Pre-steady-state single-turnover nucleotide incorporation assays were performed to obtain the kinetic parameters, kpol and Kd, for correct nucleotide incorporation, incorrect nucleotide incorporation, and ribonucleotide incorporation by exonuclease-deficient polD. Correct nucleotide incorporation kinetics revealed a relatively slow maximal rate of polymerization (kpol ∼2.5 s−1) and especially tight nucleotide binding (Kd(dNTP) ∼1.7 μm), compared with DNA polymerases from Families A, B, C, X, and Y. Furthermore, pre-steady-state nucleotide incorporation assays revealed that polD prevents the incorporation of incorrect nucleotides and ribonucleotides primarily through reduced nucleotide binding affinity. Pre-steady-state single-turnover assays on wild-type 9°N polD were used to examine 3′-5′ exonuclease hydrolysis activity in the presence of Mg2+ and Mn2+. Interestingly, substituting Mn2+ for Mg2+ accelerated hydrolysis rates >40-fold (kexo ≥110 s−1 versus ≥2.5 s−1). Preference for Mn2+ over Mg2+ in exonuclease hydrolysis activity is a property unique to the polD family. The kinetic assays performed in this work provide critical insight into the mechanisms that polD employs to accurately and efficiently replicate the archaeal genome. Furthermore, despite the unique properties of polD, this work suggests that a conserved polymerase kinetic pathway is present in all known DNA polymerase families. PMID:26160179

Ferrocene-Boronic Acid-Fructose Binding Based on Dual-Plate Generator-Collector Voltammetry and Square-Wave Voltammetry.

PubMed

Li, Meng; Xu, Su-Ying; Gross, Andrew J; Hammond, Jules L; Estrela, Pedro; Weber, James; Lacina, Karel; James, Tony D; Marken, Frank

2015-06-10

The interaction of ferrocene-boronic acid with fructose is investigated in aqueous 0.1 m phosphate buffer at pH 7, 8 and 9. Two voltammetric methods, based on 1) a dual-plate generator-collector micro-trench electrode (steady state) and 2) a square-wave voltammetry (transient) method, are applied and compared in terms of mechanistic resolution. A combination of experimental data is employed to obtain new insights into the binding rates and the cumulative binding constants for both the reduced ferrocene-boronic acid (pH dependent and weakly binding) and for the oxidised ferrocene-boronic acid (pH independent and strongly binding).

Interaction of Merocyanine 540 with serum albumins: photophysical and binding studies.

PubMed

Banerjee, Mousumi; Pal, Uttam; Subudhhi, Arijita; Chakrabarti, Abhijit; Basu, Samita

2012-03-01

Photophysical studies on binding interactions of a negatively charged anti-tumor photosensitizer, Merocyanine 540 (MC 540), with serum proteins, bovine serum albumin (BSA) and human serum albumin (HSA), have been performed using absorption and steady-state as well as time-resolved fluorescence techniques. Formation of ground state complex has been confirmed from the detailed studies of absorption spectra of MC 540 in presence of SAs producing isosbestic points. Binding between the proteins and MC 540, which perturbs the existing equilibrium between the fluorescent monomer and its non-fluorescent dimer, induces a remarkable enhancement in fluorescence anisotropy and intensity of MC 540 along with a red shift of its maximum. The binding stoichiometry of MC 540 and SAs are more than 1.0 which depicts that two types of complexes, i.e., 1:1 and 2:1 are formed with addition of varied concentration of protein. Both the steady-state and time-resolved fluorescence results show that in 2:1 complex one of the MC 540 molecules is exposed towards aqueous environment with a greater extent when bound with HSA compared to BSA due to the structural flexibility of that protein. Thermodynamic analyses using van't Hoff plot indicate that the binding between MC 540 and individual SA is an entropy-driven phenomenon. The probable hydrophobic binding site has been located by denaturation of proteins, micropolarity measurement and Förster resonance energy transfer and that is further supported by molecular docking studies. Changes in circular dichroism spectra of BSA in presence of MC 540 depict secondary structural changes of the protein. The induced-CD shows that BSA due to its rigid structure generates chirality in MC 540 much more efficiently compared to HSA. Copyright © 2011 Elsevier B.V. All rights reserved.

Plasma sex steroid binding in Chiroptera.

PubMed

Kwiecinski, G G; Damassa, D A; Gustafson, A W; Armao, M E

1987-04-01

Plasma steroid binding was examined in samples obtained from seven species of bats representing four different families. A specific sex steroid-binding protein (SBP) was identified by steady-state polyacrylamide gel electrophoresis in representatives of two families, the phyllostomids and the vespertilionids. In these species, as in primates, SBP not only exhibited high affinity for the androgens testosterone and dihydrotestosterone (DHT), but also for estradiol. A specific SBP was not identified in the tropical American vampire bat or in the two species of pteropodids examined. In all species examined, except for the vampire bat, a specific corticosteroid-binding globulin (CBG) was also identified. In addition to binding glucocorticoids, CBG in these species appeared to bind androgens as well.

Effect of humoral immunity on HIV-1 dynamics with virus-to-target and infected-to-target infections

NASA Astrophysics Data System (ADS)

Elaiw, A. M.; Raezah, A. A.; Alofi, A. S.

2016-08-01

We consider an HIV-1 dynamics model by incorporating (i) two routes of infection via, respectively, binding of a virus to a receptor on the surface of a target cell to start genetic reactions (virus-to-target infection), and the direct transmission from infected cells to uninfected cells through the concept of virological synapse in vivo (infected-to-target infection); (ii) two types of distributed-time delays to describe the time between the virus or infected cell contacts an uninfected CD4+ T cell and the emission of new active viruses; (iii) humoral immune response, where the HIV-1 particles are attacked by the antibodies that are produced from the B lymphocytes. The existence and stability of all steady states are completely established by two bifurcation parameters, R 0 (the basic reproduction number) and R 1 (the viral reproduction number at the chronic-infection steady state without humoral immune response). By constructing Lyapunov functionals and using LaSalle's invariance principle, we have proven that, if R 0 ≤ 1 , then the infection-free steady state is globally asymptotically stable, if R 1 ≤ 1 < R 0 , then the chronic-infection steady state without humoral immune response is globally asymptotically stable, and if R 1 > 1 , then the chronic-infection steady state with humoral immune response is globally asymptotically stable. We have performed numerical simulations to confirm our theoretical results.

Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis.

PubMed

Zakharova, Maria Yu; Kuznetsova, Alexandra A; Kaliberda, Elena N; Dronina, Maria A; Kolesnikov, Alexander V; Kozyr, Arina V; Smirnov, Ivan V; Rumsh, Lev D; Fedorova, Olga S; Knorre, Dmitry G; Gabibov, Alexander G; Kuznetsov, Nikita A

2017-11-01

Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage "minimal" kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of "elementary" kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Kinetics of nitrogenase of Klebsiella pneumoniae. Heterotropic interactions between magnesium-adenosine 5'-diphosphate and magnesium-adenosine 5'-triphosphate.

PubMed Central

Thorneley, R N; Cornish-Bowden, A

1977-01-01

The effects of MgADP and MgATP on the kinetics of a pre-steady-state electron-transfer reaction and on the steady-state kinetics of H2 evulution for nitrogenase proteins of K. pneumoniae were studied. MgADP was a competitive inhibitor of MgATP in the MgATP-induced electron transfer from the Fe-protein to the Mo-Fe-protein. A dissociation constant K'i = 20 micron was determined for MgADP. The release of MgADP or a coupled conformation change in the Fe-protein of K.pneumoniae occurred with a rate comparable with that of electron transfer, k approximately 2 X 10(2)S-1. Neither homotropic nor heterotropic interactions involving MgATP and MgADP were observed for this reaction. Steady-state kinetic data for H2 evolution exhibited heterotropic effects between MgADP and MgATP. The data have been fitted to symmetry and sequential-type models involving conformation changes in two identical subunits. The data suggest that the enzyme can bind up to molecules of either MgATP or MgADP, but is unable to bind both nucleotides simultaneously. The control of H2 evolution by the MgATP/MgADP ratio is not at the level of electron transfer between the Fe- and Mo-Fe-proteins. Images Fig. 2. PMID:336036

Processive movement of single kinesins on crowded microtubules visualized using quantum dots

PubMed Central

Seitz, Arne; Surrey, Thomas

2006-01-01

Kinesin-1 is a processive molecular motor transporting cargo along microtubules. Inside cells, several motors and microtubule-associated proteins compete for binding to microtubules. Therefore, the question arises how processive movement of kinesin-1 is affected by crowding on the microtubule. Here we use total internal reflection fluorescence microscopy to image in vitro the runs of single quantum dot-labelled kinesins on crowded microtubules under steady-state conditions and to measure the degree of crowding on a microtubule at steady-state. We find that the runs of kinesins are little affected by high kinesin densities on a microtubule. However, the presence of high densities of a mutant kinesin that is not able to step efficiently reduces the average speed of wild-type kinesin, while hardly changing its processivity. This indicates that kinesin waits in a strongly bound state on the microtubule when encountering an obstacle until the obstacle unbinds and frees the binding site for kinesin's next step. A simple kinetic model can explain quantitatively the behaviour of kinesin under both crowding conditions. PMID:16407972

Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

NASA Astrophysics Data System (ADS)

Just Sørensen, Thomas; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

2013-06-01

Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

Fluorescence measurements of activity associated with a molecularly imprinted polymer imprinted to dipicolinic acid

NASA Astrophysics Data System (ADS)

Anderson, John; Pestov, Dmitry; Fischer, Robert L.; Webb, Stanley; Tepper, Gary C.

2004-03-01

Steady state and lifetime fluorescence measurements were acquired to measure the binding activity associated with molecularly imprinted polymer (MIP) microparticles imprinted to dipicolinic acid. Dipicolinic acid is a unique compound associated with the sporulation phase of spore-forming bacteria (e.g., genus Bacillus and Clostridium). Vinylic monomers were polymerized in a dimethylformamide solution containing the dipicolinic acid as a template. The resulting MIP was then pulverized and size selected into small microscale particles. Samplers were adapted incorporating the MIP particles within a dialyzer (500 MW). Tests were run on replicate samples of biologically active cultures representing both stationary phase and sporulation post fermentation products in standard media. The permeability of the membrane permitted diffusion of lighter molecular weight constituents from media effluents to enter the dialyzer chamber and contact the MIP. Extractions of the media were measured using steady state and lifetime fluorescence. Results showed dramatic steady state fluorescence changes as a function of excitation, emission and intensity and an estimated lifetime of 5.8 ns.

Ultrasensitivity and sharp threshold theorems for multisite systems

NASA Astrophysics Data System (ADS)

Dougoud, M.; Mazza, C.; Vinckenbosch, L.

2017-02-01

This work studies the ultrasensitivity of multisite binding processes where ligand molecules can bind to several binding sites. It considers more particularly recent models involving complex chemical reactions in allosteric phosphorylation processes and for transcription factors and nucleosomes competing for binding on DNA. New statistics-based formulas for the Hill coefficient and the effective Hill coefficient are provided and necessary conditions for a system to be ultrasensitive are exhibited. It is first shown that the ultrasensitivity of binding processes can be approached using sharp-threshold theorems which have been developed in applied probability theory and statistical mechanics for studying sharp threshold phenomena in reliability theory, random graph theory and percolation theory. Special classes of binding process are then introduced and are described as density dependent birth and death process. New precise large deviation results for the steady state distribution of the process are obtained, which permits to show that switch-like ultrasensitive responses are strongly related to the multi-modality of the steady state distribution. Ultrasensitivity occurs if and only if the entropy of the dynamical system has more than one global minimum for some critical ligand concentration. In this case, the Hill coefficient is proportional to the number of binding sites, and the system is highly ultrasensitive. The classical effective Hill coefficient I is extended to a new cooperativity index I q , for which we recommend the computation of a broad range of values of q instead of just the standard one I  =  I 0.9 corresponding to the 10%-90% variation in the dose-response. It is shown that this single choice can sometimes mislead the conclusion by not detecting ultrasensitivity. This new approach allows a better understanding of multisite ultrasensitive systems and provides new tools for the design of such systems.

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA

PubMed Central

Cristóvão, Michele; Sisamakis, Evangelos; Hingorani, Manju M.; Marx, Andreas D.; Jung, Caroline P.; Rothwell, Paul J.; Seidel, Claus A. M.; Friedhoff, Peter

2012-01-01

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS–mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand. PMID:22367846

Characterization of Protein Tyrosine Phosphatase 1B Inhibition by Chlorogenic Acid and Cichoric Acid.

PubMed

Lipchock, James M; Hendrickson, Heidi P; Douglas, Bonnie B; Bird, Kelly E; Ginther, Patrick S; Rivalta, Ivan; Ten, Nicholas S; Batista, Victor S; Loria, J Patrick

2017-01-10

Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of the insulin and leptin signaling pathways and is an active target for the design of inhibitors for the treatment of type II diabetes and obesity. Recently, cichoric acid (CHA) and chlorogenic acid (CGA) were predicted by docking methods to be allosteric inhibitors that bind distal to the active site. However, using a combination of steady-state inhibition kinetics, solution nuclear magnetic resonance experiments, and molecular dynamics simulations, we show that CHA is a competitive inhibitor that binds in the active site of PTP1B. CGA, while a noncompetitive inhibitor, binds in the second aryl phosphate binding site, rather than the predicted benzfuran binding pocket. The molecular dynamics simulations of the apo enzyme and cysteine-phosphoryl intermediate states with and without bound CGA suggest CGA binding inhibits PTP1B by altering hydrogen bonding patterns at the active site. This study provides a mechanistic understanding of the allosteric inhibition of PTP1B.

Lacosamide Inhibition of Nav1.7 Voltage-Gated Sodium Channels: Slow Binding to Fast-Inactivated States

PubMed Central

Jo, Sooyeon

2017-01-01

Lacosamide is an antiseizure agent that targets voltage-dependent sodium channels. Previous experiments have suggested that lacosamide is unusual in binding selectively to the slow-inactivated state of sodium channels, in contrast to drugs like carbamazepine and phenytoin, which bind tightly to fast-inactivated states. Using heterologously expressed human Nav1.7 sodium channels, we examined the state-dependent effects of lacosamide. Lacosamide induced a reversible shift in the voltage dependence of fast inactivation studied with 100-millisecond prepulses, suggesting binding to fast-inactivated states. Using steady holding potentials, lacosamide block was very weak at −120 mV (3% inhibition by 100 µM lacosamide) but greatly enhanced at −80 mV (43% inhibition by 100 µM lacosamide), where there is partial fast inactivation but little or no slow inactivation. During long depolarizations, lacosamide slowly (over seconds) put channels into states that recovered availability slowly (hundreds of milliseconds) at −120 mV. This resembles enhancement of slow inactivation, but the effect was much more pronounced at −40 mV, where fast inactivation is complete, but slow inactivation is not, than at 0 mV, where slow inactivation is maximal, more consistent with slow binding to fast-inactivated states than selective binding to slow-inactivated states. Furthermore, inhibition by lacosamide was greatly reduced by pretreatment with 300 µM lidocaine or 300 µM carbamazepine, suggesting that lacosamide, lidocaine, and carbamazepine all bind to the same site. The results suggest that lacosamide binds to fast-inactivated states in a manner similar to other antiseizure agents but with slower kinetics of binding and unbinding. PMID:28119481

Lacosamide Inhibition of Nav1.7 Voltage-Gated Sodium Channels: Slow Binding to Fast-Inactivated States.

PubMed

Jo, Sooyeon; Bean, Bruce P

2017-04-01

Lacosamide is an antiseizure agent that targets voltage-dependent sodium channels. Previous experiments have suggested that lacosamide is unusual in binding selectively to the slow-inactivated state of sodium channels, in contrast to drugs like carbamazepine and phenytoin, which bind tightly to fast-inactivated states. Using heterologously expressed human Nav1.7 sodium channels, we examined the state-dependent effects of lacosamide. Lacosamide induced a reversible shift in the voltage dependence of fast inactivation studied with 100-millisecond prepulses, suggesting binding to fast-inactivated states. Using steady holding potentials, lacosamide block was very weak at -120 mV (3% inhibition by 100 µ M lacosamide) but greatly enhanced at -80 mV (43% inhibition by 100 µ M lacosamide), where there is partial fast inactivation but little or no slow inactivation. During long depolarizations, lacosamide slowly (over seconds) put channels into states that recovered availability slowly (hundreds of milliseconds) at -120 mV. This resembles enhancement of slow inactivation, but the effect was much more pronounced at -40 mV, where fast inactivation is complete, but slow inactivation is not, than at 0 mV, where slow inactivation is maximal, more consistent with slow binding to fast-inactivated states than selective binding to slow-inactivated states. Furthermore, inhibition by lacosamide was greatly reduced by pretreatment with 300 µ M lidocaine or 300 µ M carbamazepine, suggesting that lacosamide, lidocaine, and carbamazepine all bind to the same site. The results suggest that lacosamide binds to fast-inactivated states in a manner similar to other antiseizure agents but with slower kinetics of binding and unbinding. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Slow-binding inhibition of sialidase from influenza virus.

PubMed

Pegg, M S; von Itzstein, M

1994-04-01

Sialidase from influenza virus A (Tokyo/3/67, N2) is inhibited in slow-binding fashion by 2,3-didehydro-2,4-dideoxy-4-guanidino-N-acetyl-D-neuraminic acid. The Ki observed for the tightly-bound form at steady-state is 3 x 10(-11) M. Slow-binding, which is a consequence of the guanidinyl moiety of the inhibitor, is observed only for influenza virus A sialidase and not for influenza virus B or any other viral, bacterial, or mammalian sialidase investigated. The different results obtained for sialidases from influenza virus A and B, whose active sites are conserved, point to the involvement of the expulsion of a structural water molecule in the slow-binding mechanism.

Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169

PubMed Central

Min, Weihong; Li, Huiying; Li, Hongmei; Liu, Chunlei; Liu, Jingsheng

2015-01-01

Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni2+. Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter Vmax of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production. PMID:26633359

Cooperativity and pseudo-cooperativity in the glutathione S-transferase from Plasmodium falciparum.

PubMed

Liebau, Eva; De Maria, Francesca; Burmeister, Cora; Perbandt, Markus; Turella, Paola; Antonini, Giovanni; Federici, Giorgio; Giansanti, Francesco; Stella, Lorenzo; Lo Bello, Mario; Caccuri, Anna Maria; Ricci, Giorgio

2005-07-15

Binding and catalytic properties of glutathione S-transferase from Plasmodium falciparum (PfGST) have been studied by means of fluorescence, steady state and pre-steady state kinetic experiments, and docking simulations. This enzyme displays a peculiar reversible low-high affinity transition, never observed in other GSTs, which involves the G-site and shifts the apparent K(D) for glutathione (GSH) from 200 to 0.18 mM. The transition toward the high affinity conformation is triggered by the simultaneous binding of two GSH molecules to the dimeric enzyme, and it is manifested as an uncorrected homotropic behavior, termed "pseudo-cooperativity." The high affinity enzyme is able to activate GSH, lowering its pK(a) value from 9.0 to 7.0, a behavior similar to that found in all known GSTs. Using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, this enzyme reveals a potential optimized mechanism for the GSH conjugation but a low catalytic efficiency mainly due to a very low affinity for this co-substrate. Conversely, PfGST efficiently binds one molecule of hemin/monomer. The binding is highly cooperative (n(H) = 1.8) and occurs only when GSH is bound to the enzyme. The thiolate of GSH plays a crucial role in the intersubunit communication because no cooperativity is observed when S-methylglutathione replaces GSH. Docking simulations suggest that hemin binds to a pocket leaning into both the G-site and the H-site. The iron is coordinated by the amidic nitrogen of Asn-115, and the two carboxylate groups are in electrostatic interaction with the epsilon-amino group of Lys-15. Kinetic and structural data suggest that PfGST evolved by optimizing its binding property with the parasitotoxic hemin rather than its catalytic efficiency toward toxic electrophilic compounds.

Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site.

PubMed

Tang, Lixia; Torres Pazmiño, Daniel E; Fraaije, Marco W; de Jong, René M; Dijkstra, Bauke W; Janssen, Dick B

2005-05-03

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, resulting in the formation of the corresponding epoxide, a halide ion, and a proton. Halide release is rate-limiting during the catalytic cycle of the conversion of (R)-p-nitro-2-bromo-1-phenylethanol by the enzyme. The recent elucidation of the X-ray structure of HheC showed that hydrogen bonds between the OH group of Tyr187 and between the Odelta1 atom of Asn176 and Nepsilon1 atom of Trp249 could play a role in stabilizing the conformation of the halide-binding site. The possibility that these hydrogen bonds are important for halide binding and release was studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F, which has lost both hydrogen bonds, has a higher catalytic activity (k(cat)) with two of the three tested substrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced k(cat) value with these two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalytic efficiency (k(cat)/K(m)) was obtained, which is mainly due to an increase of the K(m) value of the enzyme. Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicating that halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescence experiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for the wild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that the disruption of two hydrogen bonds around the halide-binding site increases the rate of halide release and can enhance the overall catalytic activity of HheC.

Binding and relaxation behavior of Coumarin-153 in lecithin-taurocholate mixed micelles: A time resolved fluorescence spectroscopic study

NASA Astrophysics Data System (ADS)

Chakrabarty, Debdeep; Chakraborty, Anjan; Seth, Debabrata; Hazra, Partha; Sarkar, Nilmoni

2005-09-01

The microenvironment of the bile salt-lecithin mixed aggregates has been investigated using steady state and picosecond time resolved fluorescence spectroscopy. The steady state spectra show that the polarity of the bile salt is higher compared to lecithin vesicles or the mixed aggregates. We have observed slow solvent relaxation in bile salt micelles and lecithin vesicles. The solvation time is gradually slowed down due to gradual addition of the bile salt in lecithin vesicles. Addition of bile salt leads to the tighter head group packing in lecithin. Thus, mobility of the water molecules becomes slower and consequently the solvation time is also retarded. We have observed bimodal slow rotational relaxation time in all these systems.

Phase-plane analysis of the totally asymmetric simple exclusion process with binding kinetics and switching between antiparallel lanes

PubMed Central

Kuan, Hui-Shun; Betterton, Meredith D.

2016-01-01

Motor protein motion on biopolymers can be described by models related to the totally asymmetric simple exclusion process (TASEP). Inspired by experiments on the motion of kinesin-4 motors on antiparallel microtubule overlaps, we analyze a model incorporating the TASEP on two antiparallel lanes with binding kinetics and lane switching. We determine the steady-state motor density profiles using phase-plane analysis of the steady-state mean field equations and kinetic Monte Carlo simulations. We focus on the density-density phase plane, where we find an analytic solution to the mean field model. By studying the phase-space flows, we determine the model’s fixed points and their changes with parameters. Phases previously identified for the single-lane model occur for low switching rate between lanes. We predict a multiple coexistence phase due to additional fixed points that appear as the switching rate increases: switching moves motors from the higher-density to the lower-density lane, causing local jamming and creating multiple domain walls. We determine the phase diagram of the model for both symmetric and general boundary conditions. PMID:27627345

Error-prone bypass of O6-methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1.

PubMed

Gu, Shiling; Xiong, Jingyuan; Shi, Ying; You, Jia; Zou, Zhenyu; Liu, Xiaoying; Zhang, Huidong

2017-09-01

O 6 -Methylguanine (O 6 -MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O 6 -MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O 6 -MeG by gp90 exo - . O 6 -MeG partially inhibited full-length extension by gp90 exo - . O 6 -MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo - extends beyond T:O 6 -MeG 2-fold more efficiently than C:O 6 -MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O 6 -MeG show fast burst phases. The pre-steady-state incorporation efficiency (k pol /K d,dNTP ) is decreased in the order of dCTP:G>dTTP:O 6 -MeG>dCTP:O 6 -MeG. The presence of O 6 -MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg 2+ . Misincorporation of dTTP opposite O 6 -MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O 6 -MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O 6 -MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional Hemispheric Asymmetries of Global/Local Processing Mirrored by the Steady-State Visual Evoked Potential

ERIC Educational Resources Information Center

Martens, Ulla; Hubner, Ronald

2013-01-01

While hemispheric differences in global/local processing have been reported by various studies, it is still under dispute at which processing stage they occur. Primarily, it was assumed that these asymmetries originate from an early perceptual stage. Instead, the content-level binding theory (Hubner & Volberg, 2005) suggests that the hemispheres…

Interaction of ligands with the opiate receptors of brain membranes: Regulation by ions and nucleotides

PubMed Central

Blume, Arthur J.

1978-01-01

This study shows that nucleotides, as well as ions, regulate the opiate receptors of brain. GMP-P(NH)P and Na+ reduce the amount of steady-state specific [3H]dihydromorphine binding and increase the rate of dissociation of the ligand from the opiate receptor. In contrast, Mn2+ decreases the rate of ligand dissociation and antagonizes the ability of Na+ to increase dissociation. The effects of GMP-P(NH)P on steady-state binding and dissociation are not reversed by washing. Only GTP, GDP, ITP, and IMP-P(NH)P, in addition to GMP-P(NH)P, increase the rate of dihydromorphine dissociation. The site of nucleotide action appears to have high affinity: <1 μM GMP-P(NH)P produces half-maximal increases in ligand dissociation. GMP-P(NH)P- and Na+-directed increases in dissociation have also been found for the opiate agonists [3H]etorphine, [3H]Leu-enkephalin, and [3H]Met-enkephalin and the opiate antagonist [3H]naltrexone. Mn2+-directed decreases in dissociation have been found for the agonist [3H]-etorphine and the antagonist [3H]naltrexone. Although the plasma membrane receptors for a number of other neuro-transmitters and hormones are also regulated by guanine nucleotides, the opiate receptors appear unique because only they show nucleotide regulation of both agonist and antagonist binding. PMID:205867

Characterization of Streptococcus pneumoniae thymidylate kinase: steady-state kinetics of the forward reaction and isothermal titration calorimetry.

PubMed Central

Petit, Chantal M; Koretke, Kristin K

2002-01-01

Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis. The tmk gene from the bacterial pathogen Streptococcus pneumoniae was identified. The gene, encoding a 212-amino-acid polypeptide (23352 Da), was cloned and overexpressed in Escherichia coli with an N-terminal hexahistidine tag. The enzyme was purified to homogeneity, and characterized in the forward reaction. The pH profile of TMK indicates that its activity is optimal at pH 8.5. The substrate specificity of the enzyme was examined; it was found that not only ATP, but also dATP and to a lesser extent CTP, could act as phosphate donors, and dTMP and dUMP could serve as phosphate acceptors. Furthermore, AZT-MP (3'-azido-3'-deoxythymidine 5'-monophosphate) was shown not to be a substrate for S. pneumoniae TMK. Steady-state kinetics and inhibition studies with adenosine 5'-[beta-thio]diphosphate and dTDP in addition to isothermal titration calorimetry were performed. The data showed that binding follows an ordered pathway, in which ATP binds first with a K(m) of 235 +/- 46 microM and a K(d) of 116 +/- 3 microM, and dTMP binds secondly with a K(m) of 66 +/- 12 microM and a K(d) of 53 +/- 2 microM. PMID:11964185

Long-Chain Perfluoroalkyl acids (PFAAs) Affect the Bioconcentration and Tissue Distribution of Short-Chain PFAAs in Zebrafish (Danio rerio).

PubMed

Wen, Wu; Xia, Xinghui; Hu, Diexuan; Zhou, Dong; Wang, Haotian; Zhai, Yawei; Lin, Hui

2017-11-07

Short- and long-chain perfluoroalkyl acids (PFAAs), ubiquitously coexisting in the environment, can be accumulated in organisms by binding with proteins and their binding affinities generally increase with their chain length. Therefore, we hypothesized that long-chain PFAAs will affect the bioconcentration of short-chain PFAAs in organisms. To testify this hypothesis, the bioconcentration and tissue distribution of five short-chain PFAAs (linear C-F = 3-6) were investigated in zebrafish in the absence and presence of six long-chain PFAAs (linear C-F = 7-11). The results showed that the concentrations of the short-chain PFAAs in zebrafish tissues increased with exposure time until steady states reached in the absence of long-chain PFAAs. However, in the presence of long-chain PFAAs, these short-chain PFAAs in tissues increased until peak values reached and then decreased until steady states, and the uptake and elimination rate constants of short-chain PFAAs declined in all tissues and their BCF ss decreased by 24-89%. The inhibitive effect of long-chain PFAAs may be attributed to their competition for transporters and binding sites of proteins in zebrafish with short-chain PFAAs. These results suggest that the effect of long-chain PFAAs on the bioconcentration of short-chain PFAAs should be taken into account in assessing the ecological and environmental effects of short-chain PFAAs.

The effect of tenidap sodium on the disposition and plasma protein binding of phenytoin in healthy male volunteers

PubMed Central

Blum, R. A.; Schentag, J. J.; Gardner, M. J.; Wilner, K. D.

1995-01-01

1 The effects of tenidap sodium 120 mg day-1 at steady state and placebo on the plasma protein binding and pharmacokinetics of phenytoin were compared in this randomised, double-blind, placebo-controlled, parallel-group study, involving 12 healthy young men, conducted over 34 days. 2 Single oral doses of phenytoin 200 mg were given on days 1-3 and 29-31, and intravenous phenytoin, 250 mg infused over 20 min, was given on days 4 and 32. Tenidap (120 mg day-1), or matching placebo, was administered as single oral daily doses from days 8 to 34 inclusive. 3 The plasma protein binding of phenytoin was determined immediately before oral phenytoin administration on days 1 and 29. Pharmacokinetic parameters were estimated from the serum phenytoin concentration-time curves derived on days 4 and 32 following the phenytoin infusions. The differences between the pre- and post-treatment mean percentage of unbound plasma phenytoin and mean pharmacokinetic parameters were compared between treatment groups. 4 Tenidap sodium 120 mg day-1, at steady state, increased the percentage of unbound phenytoin in plasma by approximately 25%, but did not significantly affect AUC(0,48h) or Cmax. 5 Since tenidap increases the percentage of unbound phenytoin in plasma, when monitoring phenytoin plasma concentrations free concentrations of phenytoin should be considered. 6 Tenidap was well tolerated throughout the study. PMID:7547092

Probing the binding interaction of a phenazinium dye with serum transport proteins: a combined fluorometric and circular dichroism study.

PubMed

Bose, Debosreeta; Sarkar, Deboleena; Chattopadhyay, Nitin

2010-01-01

In the present investigation, an attempt has been made to study the interaction of phenosafranin (PSF), a cationic phenazinium dye with the transport proteins, bovine serum albumin (BSA) and human serum albumin (HSA), employing steady-state and time-resolved fluorometric and circular dichroism (CD) techniques. The photophysical properties of the dye are altered on binding with the serum proteins. An explicit study with respect to the modification of the fluorescence and fluorescence anisotropy upon binding, effect of denaturant, fluorescence lifetime and CD measurements reveal that the dye binds to both BSA and HSA with almost the same affinity. Far-UV CD spectra indicate a decrease in the percentage of alpha-helicity only for BSA upon binding with the probe. Near-UV CD responses indicate an alteration in the tertiary structure of both the transport proteins because of binding.

The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex.

PubMed

Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

2013-07-01

A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy=2,2-bipyridine, Hbcpip=2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5±1.4)×10(5) M(-1) in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived. Copyright © 2013 Elsevier B.V. All rights reserved.

Design and operation of a continuous integrated monoclonal antibody production process.

PubMed

Steinebach, Fabian; Ulmer, Nicole; Wolf, Moritz; Decker, Lara; Schneider, Veronika; Wälchli, Ruben; Karst, Daniel; Souquet, Jonathan; Morbidelli, Massimo

2017-09-01

The realization of an end-to-end integrated continuous lab-scale process for monoclonal antibody manufacturing is described. For this, a continuous cultivation with filter-based cell-retention, a continuous two column capture process, a virus inactivation step, a semi-continuous polishing step (twin-column MCSGP), and a batch-wise flow-through polishing step were integrated and operated together. In each unit, the implementation of internal recycle loops allows to improve the performance: (a) in the bioreactor, to simultaneously increase the cell density and volumetric productivity, (b) in the capture process, to achieve improved capacity utilization at high productivity and yield, and (c) in the MCSGP process, to overcome the purity-yield trade-off of classical batch-wise bind-elute polishing steps. Furthermore, the design principles, which allow the direct connection of these steps, some at steady state and some at cyclic steady state, as well as straight-through processing, are discussed. The setup was operated for the continuous production of a commercial monoclonal antibody, resulting in stable operation and uniform product quality over the 17 cycles of the end-to-end integration. The steady-state operation was fully characterized by analyzing at the outlet of each unit at steady state the product titer as well as the process (HCP, DNA, leached Protein A) and product (aggregates, fragments) related impurities. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1303-1313, 2017. © 2017 American Institute of Chemical Engineers.

Effect of bioaugmentation and biostimulation on sulfate-reducing column startup captured by functional gene profiling.

PubMed

Pereyra, Luciana P; Hiibel, Sage R; Perrault, Elizabeth M; Reardon, Kenneth F; Pruden, Amy

2012-10-01

Sulfate-reducing permeable reactive zones (SR-PRZs) depend upon a complex microbial community to utilize a lignocellulosic substrate and produce sulfides, which remediate mine drainage by binding heavy metals. To gain insight into the impact of the microbial community composition on the startup time and pseudo-steady-state performance, functional genes corresponding to cellulose-degrading (CD), fermentative, sulfate-reducing, and methanogenic microorganisms were characterized in columns simulating SR-PRZs using quantitative polymerase chain reaction (qPCR) and denaturing gradient gel electrophoresis (DGGE). Duplicate columns were bioaugmented with sulfate-reducing or CD bacteria or biostimulated with ethanol or carboxymethyl cellulose and compared with baseline dairy manure inoculum and uninoculated controls. Sulfate removal began after ~ 15 days for all columns and pseudo-steady state was achieved by Day 30. Despite similar performance, DGGE profiles of 16S rRNA gene and functional genes at pseudo-steady state were distinct among the column treatments, suggesting the potential to control ultimate microbial community composition via bioaugmentation and biostimulation. qPCR revealed enrichment of functional genes in all columns between the initial and pseudo-steady-state time points. This is the first functional gene-based study of CD, fermentative and sulfate-reducing bacteria and methanogenic archaea in a lignocellulose-based environment and provides new qualitative and quantitative insight into startup of a complex microbial system. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Designing Flavoprotein-GFP Fusion Probes for Analyte-Specific Ratiometric Fluorescence Imaging.

PubMed

Hudson, Devin A; Caplan, Jeffrey L; Thorpe, Colin

2018-02-20

The development of genetically encoded fluorescent probes for analyte-specific imaging has revolutionized our understanding of intracellular processes. Current classes of intracellular probes depend on the selection of binding domains that either undergo conformational changes on analyte binding or can be linked to thiol redox chemistry. Here we have designed novel probes by fusing a flavoenzyme, whose fluorescence is quenched on reduction by the analyte of interest, with a GFP domain to allow for rapid and specific ratiometric sensing. Two flavoproteins, Escherichia coli thioredoxin reductase and Saccharomyces cerevisiae lipoamide dehydrogenase, were successfully developed into thioredoxin and NAD + /NADH specific probes, respectively, and their performance was evaluated in vitro and in vivo. A flow cell format, which allowed dynamic measurements, was utilized in both bacterial and mammalian systems. In E. coli the first reported intracellular steady-state of the cytoplasmic thioredoxin pool was measured. In HEK293T mammalian cells, the steady-state cytosolic ratio of NAD + /NADH induced by glucose was determined. These genetically encoded fluorescent constructs represent a modular approach to intracellular probe design that should extend the range of metabolites that can be quantitated in live cells.

Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP*

PubMed Central

Narayan, Vikram; Pion, Emmanuelle; Landré, Vivien; Müller, Petr; Ball, Kathryn L.

2011-01-01

Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20–40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP (C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1·CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 (residues 106–140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or “docking” of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase. PMID:20947504

Investigations on the interactions of aurintricarboxylic acid with bovine serum albumin: Steady state/time resolved spectroscopic and docking studies.

PubMed

Bardhan, Munmun; Chowdhury, Joydeep; Ganguly, Tapan

2011-01-10

In this paper, the nature of the interactions between bovine serum albumin (BSA) and aurintricarboxylic acid (ATA) has been investigated by measuring steady state and time-resolved fluorescence, circular dichroism (CD), FT-IR and fluorescence anisotropy in protein environment under physiological conditions. From the analysis of the steady state and time-resolved fluorescence quenching of BSA in aqueous solution in presence of ATA it has been inferred that the nature of the quenching originates from the combined effect of static and dynamic modes. From the determination of the thermodynamic parameters obtained from temperature-dependent changes in K(b) (binding constant) it was apparent that the combined effect of hydrophobic association and electrostatic attraction is responsible for the interaction of ATA with BSA. The effect of ATA on the conformation of BSA has been examined by analyzing CD spectrum. Though the observed results demonstrate some conformational changes in BSA in presence of ATA but the secondary structure of BSA, predominantly of α-helix, is found to retain its identity. Molecular docking of ATA with BSA also indicates that ATA docks through hydrophobic interaction. Copyright © 2010 Elsevier B.V. All rights reserved.

Interaction of Human Hemoglobin with Methotrexate

NASA Astrophysics Data System (ADS)

Zaharia, M.; Gradinaru, R.

2015-05-01

This study focuses on the interaction between methotrexate and human hemoglobin using steady-state ultraviolet-visible and fluorescence quenching methods. Fluorescence quenching was found to be valuable in assessing drug binding to hemoglobin. The quenching of methotrexate is slightly smaller than the quenching observed with related analogs (dihydrofolate and tetrahydrofolate). The quenching studies were performed at four different temperatures and various pH values. The number of binding sites for tryptophan is ~1. Parameter-dependent assays revealed that electrostatic forces play an essential role in the methotrexate-hemoglobin interaction. Furthermore, the complex was easily eluted using gel filtration chromatography.

The Role and Specificity of the Catalytic and Regulatory Cation-binding Sites of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae*

PubMed Central

Juárez, Oscar; Shea, Michael E.; Makhatadze, George I.; Barquera, Blanca

2011-01-01

The Na+-translocating NADH:quinone oxidoreductase is the entry site for electrons into the respiratory chain and the main sodium pump in Vibrio cholerae and many other pathogenic bacteria. In this work, we have employed steady-state and transient kinetics, together with equilibrium binding measurements to define the number of cation-binding sites and characterize their roles in the enzyme. Our results show that sodium and lithium ions stimulate enzyme activity, and that Na+-NQR enables pumping of Li+, as well as Na+ across the membrane. We also confirm that the enzyme is not able to translocate other monovalent cations, such as potassium or rubidium. Although potassium is not used as a substrate, Na+-NQR contains a regulatory site for this ion, which acts as a nonessential activator, increasing the activity and affinity for sodium. Rubidium can bind to the same site as potassium, but instead of being activated, enzyme turnover is inhibited. Activity measurements in the presence of both sodium and lithium indicate that the enzyme contains at least two functional sodium-binding sites. We also show that the binding sites are not exclusively responsible for ion selectivity, and other steps downstream in the mechanism also play a role. Finally, equilibrium-binding measurements with 22Na+ show that, in both its oxidized and reduced states, Na+-NQR binds three sodium ions, and that the affinity for sodium is the same for both of these states. PMID:21652714

Multispectroscopic and Theoretical Exploration of the Comparative Binding Aspects of Bioflavonoid Fisetin with Triple- and Double-Helical Forms of RNA.

PubMed

Bhuiya, Sutanwi; Haque, Lucy; Goswami, Rapti; Das, Suman

2017-12-14

The interactions of RNA triplex (U.A*U) and duplex (A.U) with naturally occurring flavonoid fisetin (FTN) have been examined at pH 7.0 using various spectroscopic, viscometric, and theoretical studies. Experimental observations showed that the ligand binds with both double- and triple-helical forms of RNA, although the binding affinity is greater for the triplex structure (5.94 × 10 6 M -1 ) compared to that for the duplex counterpart (1.0 × 10 5 M -1 ). Thermal melting experiments revealed that the Hoogsteen base-paired third strand of triplex was stabilized to a greater extent (∼14 °C) compared with the Watson-Crick base-paired second strand (∼4 °C) in the presence of FTN. From fluorimetric study, we observed that U.A*U and A.U primarily bind to the photoproduced tautomer of FTN in the excited state. Steady-state and time-resolved anisotropy measurements illustrate considerable modulations of the spectroscopic properties of the tautomeric FTN within the RNA environment. Viscometric, fluorescence quenching, and thermal melting studies all together support the mode of binding to be intercalation. Theoretical study explains the experimental absorption and emission (dual fluorescence) behavior of FTN along with the excited-state intramolecular proton transfer process.

Mutational definition of binding requirements of an hnRNP-like protein in Arabidopsis using fluorescence correlation spectroscopy.

PubMed

Leder, Verena; Lummer, Martina; Tegeler, Kathrin; Humpert, Fabian; Lewinski, Martin; Schüttpelz, Mark; Staiger, Dorothee

2014-10-10

Arabidopsis thaliana glycine-rich RNA binding protein 7 (AtGRP7) is part of a negative feedback loop through which it regulates alternative splicing and steady-state abundance of its pre-mRNA. Here we use fluorescence correlation spectroscopy to investigate the requirements for AtGRP7 binding to its intron using fluorescently-labelled synthetic oligonucleotides. By systematically introducing point mutations we identify three nucleotides that lead to an increased Kd value when mutated and thus are critical for AtGRP7 binding. Simultaneous mutation of all three residues abrogates binding. The paralogue AtGRP8 binds to an overlapping motif but with a different sequence preference, in line with overlapping but not identical functions of this protein pair. Truncation of the glycine-rich domain reduces the binding affinity of AtGRP7, showing for the first time that the glycine-rich stretch of a plant hnRNP-like protein contributes to binding. Mutation of the conserved R(49) that is crucial for AtGRP7 function in pathogen defence and splicing abolishes binding. Copyright © 2014 Elsevier Inc. All rights reserved.

Loss of RUNX1/AML1 arginine-methylation impairs in peripheral T cell homeostasis

PubMed Central

Mizutani, Shinsuke; Yoshida, Tatsushi; Zhao, Xinyang; Nimer, Stephen D.; Taniwaki, Masafumi; Okuda, Tsukasa

2016-01-01

Summary RUNX1 (previously termed AML1) is a frequent target of human leukaemia-associated gene aberrations, and it encodes the DNA-binding subunit of the Core-Binding Factor transcription factor complex. RUNX1 expression is essential for the initiation of definitive haematopoiesis, for steady-state thrombopoiesis, and for normal lymphocytes development. Recent studies revealed that protein arginine methyltransferase 1 (PRMT1), which accounts for the majority of the type I PRMT activity in cells, methylates two arginine residues in RUNX1 (R206 and R210), and these modifications inhibit corepressor-binding to RUNX1 thereby enhancing its transcriptional activity. In order to elucidate the biological significance of these methylations, we established novel knock-in mouse lines with non-methylable, double arginine-to-lysine (RTAMR-to-KTAMK) mutations in RUNX1. Homozygous Runx1KTAMK/KTAMK mice are born alive and appear normal during adulthood. However, Runx1KTAMK/KTAMK mice showed a reduction in CD3+ T lymphoid cells and a decrease in CD4+ T cells in peripheral lymphoid organs, in comparison to their wild-type littermates, leading to a reduction in the CD4+ to CD8+ T-cell ratio. These findings suggest that arginine-methylation of RUNX1 in the RTAMR-motif is dispensable for the development of definitive haematopoiesis and for steady-state platelet production, however this modification affects the role of RUNX1 in the maintenance of the peripheral CD4+ T-cell population. PMID:26010396

A new MIF4G domain-containing protein, CTIF, directs nuclear cap-binding protein CBP80/20-dependent translation

PubMed Central

Kim, Kyoung Mi; Cho, Hana; Choi, Kobong; Kim, Jaedong; Kim, Bong-Woo; Ko, Young-Gyu; Jang, Sung Key; Kim, Yoon Ki

2009-01-01

During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF. PMID:19648179

Functional hemispheric asymmetries of global/local processing mirrored by the steady-state visual evoked potential.

PubMed

Martens, Ulla; Hübner, Ronald

2013-03-01

While hemispheric differences in global/local processing have been reported by various studies, it is still under dispute at which processing stage they occur. Primarily, it was assumed that these asymmetries originate from an early perceptual stage. Instead, the content-level binding theory (Hübner & Volberg, 2005) suggests that the hemispheres differ at a later stage at which the stimulus information is bound to its respective level. The present study tested this assumption by means of steady-state evoked potentials (SSVEPs). In particular, we presented hierarchical letters flickering at 12Hz while participants categorised the letters at a pre- cued level (global or local). The information at the two levels could be congruent or incongruent with respect to the required response. Since content-binding is only necessary if there is a response conflict, asymmetric hemispheric processing should be observed only for incongruent stimuli. Indeed, our results show that the cue and congruent stimuli elicited equal SSVEP global/local effects in both hemispheres. In contrast, incongruent stimuli elicited lower SSVEP amplitudes for a local than for a global target level at left posterior electrodes, whereas a reversed pattern was seen at right hemispheric electrodes. These findings provide further evidence for a level-specific hemispheric advantage with respect to content-level binding. Moreover, the fact that the SSVEP is sensitive to these processes offers the possibility to separately track global and local processing by presenting both level contents with different frequencies. Copyright © 2012 Elsevier Inc. All rights reserved.

A detailed spectroscopic study on the interaction of Rhodamine 6G with human hemoglobin.

PubMed

Mandal, Paulami; Bardhan, Munmun; Ganguly, Tapan

2010-05-03

UV-vis, time-resolved fluorescence and circular dichroism spectroscopic investigations have been made to reveal the nature of the interactions between xanthene dye Rhodamine 6G and the well known protein hemoglobin. From the analysis of the steady-state and time-resolved fluorescence quenching of Rhodamine 6G in aqueous solutions in presence of hemoglobin, it is revealed that the quenching is static in nature. The primary binding pattern between Rhodamine and hemoglobin has been interpreted as combined effect of hydrophobic association and electrostatic interaction. The binding constants, number of binding sites and thermodynamic parameters at various pH of the environment have been computed. The binding average distance between the energy donor Rhodamine and acceptor hemoglobin has been determined from the Forster's theory. Copyright 2010 Elsevier B.V. All rights reserved.

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization.

PubMed

Perdih, Andrej; Hrast, Martina; Pureber, Kaja; Barreteau, Hélène; Grdadolnik, Simona Golič; Kocjan, Darko; Gobec, Stanislav; Solmajer, Tom; Wolber, Gerhard

2015-06-01

Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization

NASA Astrophysics Data System (ADS)

Perdih, Andrej; Hrast, Martina; Pureber, Kaja; Barreteau, Hélène; Grdadolnik, Simona Golič; Kocjan, Darko; Gobec, Stanislav; Solmajer, Tom; Wolber, Gerhard

2015-06-01

Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/ d-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/ d-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8- 11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

Transport direction determines the kinetics of substrate transport by the glutamate transporter EAAC1

PubMed Central

Zhang, Zhou; Tao, Zhen; Gameiro, Armanda; Barcelona, Stephanie; Braams, Simona; Rauen, Thomas; Grewer, Christof

2007-01-01

Glutamate transport by the excitatory amino acid carrier EAAC1 is known to be reversible. Thus, glutamate can either be taken up into cells, or it can be released from cells through reverse transport, depending on the electrochemical gradient of the co- and countertransported ions. However, it is unknown how fast and by which reverse transport mechanism glutamate can be released from cells. Here, we determined the steady- and pre-steady-state kinetics of reverse glutamate transport with submillisecond time resolution. First, our results suggest that glutamate and Na+ dissociate from their cytoplasmic binding sites sequentially, with glutamate dissociating first, followed by the three cotransported Na+ ions. Second, the kinetics of glutamate transport depend strongly on transport direction, with reverse transport being faster but less voltage-dependent than forward transport. Third, electrogenicity is distributed over several reverse transport steps, including intracellular Na+ binding, reverse translocation, and reverse relocation of the K+-bound EAAC1. We propose a kinetic model, which is based on a “first-in-first-out” mechanism, suggesting that glutamate association, with its extracellular binding site as well as dissociation from its intracellular binding site, precedes association and dissociation of at least one Na+ ion. Our model can be used to predict rates of glutamate release from neurons under physiological and pathophysiological conditions. PMID:17991780

Remediation of RDX- and HMX-contaminated groundwater using organic mulch permeable reactive barriers.

PubMed

Ahmad, Farrukh; Schnitker, Stephen P; Newell, Charles J

2007-02-20

Organic mulch is a complex organic material that is typically populated with its own consortium of microorganisms. The organisms in mulch breakdown complex organics to soluble carbon, which can then be used by these and other microorganisms as an electron donor for treating RDX and HMX via reductive pathways. A bench-scale treatability study with organic mulch was conducted for the treatment of RDX- and HMX-contaminated groundwater obtained from a plume at the Pueblo Chemical Depot (PCD) in Pueblo, Colorado. The site-specific cleanup criteria of 0.55 ppb RDX and 602 ppb HMX were used as the logical goals of the study. Column flow-through tests were run to steady-state at the average site seepage velocity, using a 70%:30% (vol.:vol.) mulch:pea gravel packing to approach the formation's permeability. Significant results included: (1) Complete removal of 90 ppb influent RDX and 8 ppb influent HMX in steady-state mulch column effluent; (2) pseudo-first-order steady-state kinetic rate constant, k, of 0.20 to 0.27 h(-1) based on RDX data, using triplicate parallel column runs; (3) accumulation of reduced RDX intermediates in the steady-state column effluent at less than 2% of the influent RDX mass; (4) no binding of RDX to the column fill material; and (5) no leaching of RDX, HMX or reduction intermediates from the column fill material. The results of the bench-scale study will be used to design and implement a pilot-scale organic mulch/pea gravel permeable reactive barrier (PRB) at the site.

The p53/p21(WAF/CIP) pathway mediates oxidative stress and senescence in dyskeratosis congenita cells with telomerase insufficiency.

PubMed

Westin, Erik R; Aykin-Burns, Nukhet; Buckingham, Erin M; Spitz, Douglas R; Goldman, Frederick D; Klingelhutz, Aloysius J

2011-03-15

Telomere attrition is a natural process that occurs due to inadequate telomere maintenance. Once at a critically short threshold, telomeres signal growth arrest, leading to senescence. Telomeres can be elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Mutations in genes for telomere binding proteins or components of telomerase give rise to the premature aging disorder dyskeratosis congenita (DC), which is characterized by extremely short telomeres and an aging phenotype. The current study demonstrates that DC cells signal a DNA damage response through p53 and its downstream mediator, p21(WAF/CIP), which is accompanied by an elevation in steady-state levels of superoxide and percent glutathione disulfide, both indicators of oxidative stress. Poor proliferation of DC cells can be partially overcome by reducing O(2) tension from 21% to 4%. Further, restoring telomerase activity or inhibiting p53 or p21(WAF/CIP) significantly mitigated growth inhibition as well as caused a significant decrease in steady-state levels of superoxide. Our results support a model in which telomerase insufficiency in DC leads to p21(WAF/CIP) signaling, via p53, to cause increased steady-state levels of superoxide, metabolic oxidative stress, and senescence.

Dynamical tides in highly eccentric binaries: chaos, dissipation, and quasi-steady state

NASA Astrophysics Data System (ADS)

Vick, Michelle; Lai, Dong

2018-05-01

Highly eccentric binary systems appear in many astrophysical contexts, ranging from tidal capture in dense star clusters, precursors of stellar disruption by massive black holes, to high-eccentricity migration of giant planets. In a highly eccentric binary, the tidal potential of one body can excite oscillatory modes in the other during a pericentre passage, resulting in energy exchange between the modes and the binary orbit. These modes exhibit one of three behaviours over multiple passages: low-amplitude oscillations, large-amplitude oscillations corresponding to a resonance between the orbital frequency and the mode frequency, and chaotic growth, with the mode energy reaching a level comparable to the orbital binding energy. We study these phenomena with an iterative map that includes mode dissipation, fully exploring how the mode evolution depends on the orbital and mode properties of the system. The dissipation of mode energy drives the system towards a quasi-steady state, with gradual orbital decay punctuated by resonances. We quantify the quasi-steady state and the long-term evolution of the system. A newly captured star around a black hole can experience significant orbital decay and heating due to the chaotic growth of the mode amplitude and dissipation. A giant planet pushed into a high-eccentricity orbit may experience a similar effect and become a hot or warm Jupiter.

New insights into the organization of plasma membrane and its role in signal transduction.

PubMed

Suzuki, Kenichi G N

2015-01-01

Plasma membranes have heterogeneous structures for efficient signal transduction, required to perform cell functions. Recent evidence indicates that the heterogeneous structures are produced by (1) compartmentalization by actin-based membrane skeleton, (2) raft domains, (3) receptor-receptor interactions, and (4) the binding of receptors to cytoskeletal proteins. This chapter provides an overview of recent studies on diffusion, clustering, raft association, actin binding, and signal transduction of membrane receptors, especially glycosylphosphatidylinositol (GPI)-anchored receptors. Studies on diffusion of GPI-anchored receptors suggest that rafts may be small and/or short-lived in plasma membranes. In steady state conditions, GPI-anchored receptors form transient homodimers, which may represent the "standby state" for the stable homodimers and oligomers upon ligation. Furthermore, It is proposed that upon ligation, the binding of GPI-anchored receptor clusters to cytoskeletal actin filaments produces a platform for downstream signaling, and that the pulse-like signaling easily maintains the stability of the overall signaling activity. Copyright © 2015 Elsevier Inc. All rights reserved.

To reveal the nature of interactions of human hemoglobin with gold nanoparticles having two different morphologies (sphere and star-shaped) by using various spectroscopic techniques.

PubMed

Chakraborty, Madhurima; Paul, Somnath; Mitra, Ishani; Bardhan, Munmun; Bose, Mridul; Saha, Abhijit; Ganguly, Tapan

2018-01-01

The nature of interactions between heme protein human hemoglobin (HHb) and gold nanoparticles of two different morphologies that is GNP (spherical) and GNS (star-shaped) have been investigated by using UV-vis absorption, steady state fluorescence, synchronous fluorescence, resonance light scattering (RLS), time resolved fluorescence, FT-IR, and circular dichroism (CD) techniques under physiological condition of pH ~7 at ambient and different temperatures. Analysis of the steady state fluorescence quenching of HHb in aqueous solution in the presence of GNP and GNS suggests that the nature of the quenching is of static type. The static nature of the quenching is also confirmed from time resolved data. The static type of quenching also indicates the possibility of formation of ground state complex for both HHb-GNP and HHb-GNS systems. From the measurements of Stern-Volmer (SV) constants K SV and binding constants, K A and number of binding sites it appears that HHb forms stronger binding with GNP relative to GNS. Analysis of the thermodynamic parameters indicates that the formation of HHb-GNP and HHb-GNS complexes are spontaneous molecular interaction processes (∆G<0). In both cases hydrogen bonding and van der Waals interactions play a dominant role (∆H<0, ∆S<0). Synchronous fluorescence spectroscopy further reveals that the ground state complex formations of HHb-GNP and HHb-GNS preferably occur by binding with the amino acid tyrosine through hydrogen bonding interactions. Moreover the α-helicity contents of the proteins as obtained from the circular dichroism (CD) spectra appears to be marginally reduced by increasing concentrations of GNP and GNS and the α-helical structures of HHb retain its identity as native secondary structure in spite of complex formations with GNP or GNS. These findings demonstrate the efficiency of biomedical applications of GNP and GNS nanoparticles as well as in elucidating their mechanisms of action as drugs or drug delivery systems in human. Copyright © 2017 Elsevier B.V. All rights reserved.

Poly(A)-binding proteins and mRNA localization: who rules the roost?

PubMed

Gray, Nicola K; Hrabálková, Lenka; Scanlon, Jessica P; Smith, Richard W P

2015-12-01

RNA-binding proteins are often multifunctional, interact with a variety of protein partners and display complex localizations within cells. Mammalian cytoplasmic poly(A)-binding proteins (PABPs) are multifunctional RNA-binding proteins that regulate multiple aspects of mRNA translation and stability. Although predominantly diffusely cytoplasmic at steady state, they shuttle through the nucleus and can be localized to a variety of cytoplasmic foci, including those associated with mRNA storage and localized translation. Intriguingly, PABP sub-cellular distribution can alter dramatically in response to cellular stress or viral infection, becoming predominantly nuclear and/or being enriched in induced cytoplasmic foci. However, relatively little is known about the mechanisms that govern this distribution/relocalization and in many cases PABP functions within specific sites remain unclear. Here we discuss the emerging evidence with respect to these questions in mammals. © 2015 Authors; published by Portland Press Limited.

Analysis of the binding interaction in uric acid - Human hemoglobin system by spectroscopic techniques

NASA Astrophysics Data System (ADS)

Makarska-Bialokoz, Magdalena

2017-05-01

The binding interaction between human hemoglobin and uric acid has been studied for the first time, by UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence techniques. Characteristic effects observed for human hemoglobin intrinsic fluorescence during interaction with uric acid at neutral pH point at the formation of stacking non-covalent and non-fluorescent complexes. All the calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants, as well as Förster resonance energy transfer parameters confirm the existence of static quenching. The results of synchronous fluorescence measurements indicate that the fluorescence quenching of human hemoglobin originates both from Trp and Tyr residues and that the addition of uric acid could significantly hinder the physiological functions of human hemoglobin.

Characterization of insulin-like growth factor I receptor on human erythrocytes.

PubMed

Hizuka, N; Takano, K; Tanaka, I; Honda, N; Tsushima, T; Shizume, K

1985-12-01

[125I]Insulin-like growth factor I (IGF-I) specifically bound to erythrocytes; the binding was saturable, and time and temperature dependent. Steady state binding was reached at 16 h at 4 C, and specific binding averaged 14.3 +/- 0.7% (+/- SEM) at a concentration of 3.6 X 10(9) cells/ml in seven normal subjects. [125I]IGF-I binding to the cells was displaced by unlabeled IGF-I in a dose-dependent manner. Scatchard analysis indicated a linear plot, and Ka and number of binding sites/cell were 1.43 +/- 0.07 X 10(9) M-1 and 20.7 +/- 2.2, respectively. Compared to IGF-I, the relative potencies of multiplication-stimulating activity and insulin for displacing [125I]IGF-I binding were 20% and 1%, respectively. [125I]IGF-I binding to erythrocytes from patients with acromegaly was lower than binding to cells from pituitary dwarfs. An inverse correlation between plasma IGF-I level and the number of IGF-I-binding sites per cell was found (r = -0.75; P less than 0.005). These results demonstrate that [125I]IGF-I binding to erythrocytes can be used for clinical measurement of the IGF-I receptor.

Structural Dynamics as a Contributor to Error-prone Replication by an RNA-dependent RNA Polymerase*

PubMed Central

Moustafa, Ibrahim M.; Korboukh, Victoria K.; Arnold, Jamie J.; Smidansky, Eric D.; Marcotte, Laura L.; Gohara, David W.; Yang, Xiaorong; Sánchez-Farrán, María Antonieta; Filman, David; Maranas, Janna K.; Boehr, David D.; Hogle, James M.; Colina, Coray M.; Cameron, Craig E.

2014-01-01

RNA viruses encoding high- or low-fidelity RNA-dependent RNA polymerases (RdRp) are attenuated. The ability to predict residues of the RdRp required for faithful incorporation of nucleotides represents an essential step in any pipeline intended to exploit perturbed fidelity as the basis for rational design of vaccine candidates. We used x-ray crystallography, molecular dynamics simulations, NMR spectroscopy, and pre-steady-state kinetics to compare a mutator (H273R) RdRp from poliovirus to the wild-type (WT) enzyme. We show that the nucleotide-binding site toggles between the nucleotide binding-occluded and nucleotide binding-competent states. The conformational dynamics between these states were enhanced by binding to primed template RNA. For the WT, the occluded conformation was favored; for H273R, the competent conformation was favored. The resonance for Met-187 in our NMR spectra reported on the ability of the enzyme to check the correctness of the bound nucleotide. Kinetic experiments were consistent with the conformational dynamics contributing to the established pre-incorporation conformational change and fidelity checkpoint. For H273R, residues comprising the active site spent more time in the catalytically competent conformation and were more positively correlated than the WT. We propose that by linking the equilibrium between the binding-occluded and binding-competent conformations of the nucleotide-binding pocket and other active-site dynamics to the correctness of the bound nucleotide, faithful nucleotide incorporation is achieved. These studies underscore the need to apply multiple biophysical and biochemical approaches to the elucidation of the physical basis for polymerase fidelity. PMID:25378410

Thermodynamic and kinetic analyses of curcumin and bovine serum albumin binding.

PubMed

Hudson, Eliara Acipreste; de Paula, Hauster Maximiler Campos; Ferreira, Guilherme Max Dias; Ferreira, Gabriel Max Dias; Hespanhol, Maria do Carmo; da Silva, Luis Henrique Mendes; Pires, Ana Clarissa Dos S

2018-03-01

Bovine serum albumin (BSA)/curcumin binding and dye photodegradation stability were evaluated. BSA/curcumin complex showed 1:1 stoichiometry, but the thermodynamic binding parameters depended on the technique used and BSA conformation. The binding constant was of the order of 10 5 L·mol -1 by fluorescence and microcalorimetric, and 10 3 and 10 4 L·mol -1 by surface plasmon resonance (steady-state equilibrium and kinetic experiments, respectively). For native BSA/curcumin, fluorescence indicated an enthalpic and entropic driven process based on the standard enthalpy change (ΔH ○ F =-8.67kJ·mol -1 ), while microcalorimetry showed an entropic driven binding process (ΔH ○ cal =29.11kJ·mol -1 ). For the unfolded BSA/curcumin complex, it was found thatp ΔH ○ F =-16.12kJ·mol -1 and ΔH ○ cal =-42.63kJ·mol -1 . BSA (mainly native) increased the curcumin photodegradation stability. This work proved the importance of using different techniques to characterize the protein-ligand binding. Copyright © 2017 Elsevier Ltd. All rights reserved.

Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

PubMed

Thrower, J S; Blalock, R; Klinman, J P

2001-08-14

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

Mechanisms by which herpes simplex virus DNA polymerase limits translesion synthesis through abasic sites.

PubMed

Zhu, Yali; Song, Liping; Stroud, Jason; Parris, Deborah S

2008-01-01

Results suggest a high probability that abasic (AP) sites occur at least once per herpes simplex virus type 1 (HSV-1) genome. The parameters that control the ability of HSV-1 DNA polymerase (pol) to engage in AP translesion synthesis (TLS) were examined because AP lesions could influence the completion and fidelity of viral DNA synthesis. Pre-steady-state kinetic experiments demonstrated that wildtype (WT) and exonuclease-deficient (exo-) pol could incorporate opposite an AP lesion, but full TLS required absence of exo function. Virtually all of the WT pol was bound at the exo site to AP-containing primer-templates (P/Ts) at equilibrium, and the pre-steady-state rate of excision by WT pol was higher on AP-containing than on matched DNA. However, several factors influencing polymerization work synergistically with exo activity to prevent HSV-1 pol from engaging in TLS. Although the pre-steady-state catalytic rate constant for insertion of dATP opposite a T or AP site was similar, ground-state-binding affinity of dATP for insertion opposite an AP site was reduced 3-9-fold. Single-turnover running-start experiments demonstrated a reduced proportion of P/Ts extended to the AP site compared to the preceding site during processive synthesis by WT or exo- pol. Only the exo- pol engaged in TLS, though inefficiently and without burst kinetics, suggesting a much slower rate-limiting step for extension beyond the AP site.

Ligand binding to telomeric G-quadruplex DNA investigated by funnel-metadynamics simulations

PubMed Central

Moraca, Federica; Amato, Jussara; Ortuso, Francesco; Artese, Anna; Novellino, Ettore; Alcaro, Stefano; Parrinello, Michele; Limongelli, Vittorio

2017-01-01

G-quadruplexes (G4s) are higher-order DNA structures typically present at promoter regions of genes and telomeres. Here, the G4 formation decreases the replicative DNA at each cell cycle, finally leading to apoptosis. The ability to control this mitotic clock, particularly in cancer cells, is fascinating and passes through a rational understanding of the ligand/G4 interaction. We demonstrate that an accurate description of the ligand/G4 binding mechanism is possible using an innovative free-energy method called funnel-metadynamics (FM), which we have recently developed to investigate ligand/protein interaction. Using FM simulations, we have elucidated the binding mechanism of the anticancer alkaloid berberine to the human telomeric G4 (d[AG3(T2AG3)3]), computing also the binding free-energy landscape. Two ligand binding modes have been identified as the lowest energy states. Furthermore, we have found prebinding sites, which are preparatory to reach the final binding mode. In our simulations, the ions and the water molecules have been explicitly represented and the energetic contribution of the solvent during ligand binding evaluated. Our theoretical results provide an accurate estimate of the absolute ligand/DNA binding free energy (ΔGb0 = −10.3 ± 0.5 kcal/mol) that we validated through steady-state fluorescence binding assays. The good agreement between the theoretical and experimental value demonstrates that FM is a most powerful method to investigate ligand/DNA interaction and can be a useful tool for the rational design also of G4 ligands. PMID:28232513

Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate.

PubMed Central

Nimmo, H G

1986-01-01

The inhibition of Escherichia coli isocitrate dehydrogenase by glyoxylate and oxaloacetate was examined. The shapes of the progress curves in the presence of the inhibitors depended on the order of addition of the assay components. When isocitrate dehydrogenase or NADP+ was added last, the rate slowly decreased until a new, inhibited, steady state was obtained. When isocitrate was added last, the initial rate was almost zero, but the rate increased slowly until the same steady-state value was obtained. Glyoxylate and oxaloacetate gave competitive inhibition against isocitrate and uncompetitive inhibition against NADP+. Product-inhibition studies showed that isocitrate dehydrogenase obeys a compulsory-order mechanism, with coenzyme binding first. Glyoxylate and oxaloacetate bind to and dissociate from isocitrate dehydrogenase slowly. These observations can account for the shapes of the progress curves observed in the presence of the inhibitors. Condensation of glyoxylate and oxaloacetate produced an extremely potent inhibitor of isocitrate dehydrogenase. Analysis of the reaction by h.p.l.c. showed that this correlated with the formation of oxalomalate. This compound decomposed spontaneously in assay mixtures, giving 4-hydroxy-2-oxoglutarate, which was a much less potent inhibitor of the enzyme. Oxalomalate inhibited isocitrate dehydrogenase competitively with respect to isocitrate and was a very poor substrate for the enzyme. The data suggest that the inhibition of isocitrate dehydrogenase by glyoxylate and oxaloacetate is not physiologically significant. PMID:3521584

Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells*

PubMed Central

Chan, Chun-Yuan; Dominguez, Dennis; Parra, Karlett J.

2016-01-01

Yeast 6-phosphofructo-1-kinase (PFK-1) has two subunits, Pfk1p and Pfk2p. Deletion of Pfk2p alters glucose-dependent V-ATPase reassembly and vacuolar acidification (Chan, C. Y., and Parra, K. J. (2014) Yeast phosphofructokinase-1 subunit Pfk2p is necessary for pH homeostasis and glucose-dependent vacuolar ATPase reassembly. J. Biol. Chem. 289, 19448–19457). This study capitalized on the mechanisms suppressing vacuolar H+-ATPase (V-ATPase) in pfk2Δ to gain new knowledge of the mechanisms underlying glucose-dependent V-ATPase regulation. Because V-ATPase is fully assembled in pfk2Δ, and glycolysis partially suppressed at steady state, we manipulated glycolysis and assessed its direct involvement on V-ATPase function. At steady state, the ratio of proton transport to ATP hydrolysis increased 24% after increasing the glucose concentration from 2% to 4% to enhance the glycolysis flow in pfk2Δ. Tighter coupling restored vacuolar pH when glucose was abundant and glycolysis operated below capacity. After readdition of glucose to glucose-deprived cells, glucose-dependent V1Vo reassembly was proportional to the glycolysis flow. Readdition of 2% glucose to pfk2Δ cells, which restored 62% of ethanol concentration, led to equivalent 60% V1Vo reassembly levels. Steady-state level of assembly (100% reassembly) was reached at 4% glucose when glycolysis reached a threshold in pfk2Δ (≥40% the wild-type flow). At 4% glucose, the level of Pfk1p co-immunoprecipitated with V-ATPase decreased 58% in pfk2Δ, suggesting that Pfk1p binding to V-ATPase may be inhibitory in the mutant. We concluded that V-ATPase activity at steady state and V-ATPase reassembly after readdition of glucose to glucose-deprived cells are controlled by the glycolysis flow. We propose a new mechanism by which glucose regulates V-ATPase catalytic activity that occurs at steady state without changing V1Vo assembly. PMID:27226568

Interaction of the iron(II) cage complexes with proteins: protein fluorescence quenching study.

PubMed

Losytskyy, Mykhaylo Y; Kovalska, Vladyslava B; Varzatskii, Oleg A; Sergeev, Alexander M; Yarmoluk, Sergiy M; Voloshin, Yan Z

2013-09-01

Interaction of the iron(II) mono- and bis-clathrochelates with bovine serum albumin (BSA), β-lactoglobulin, lysozyme and insulin was studied by the steady-state and time-resolved fluorescent spectroscopies. These cage complexes do not make significant impact on fluorescent properties of β-lactoglobulin, lysozyme and insulin. At the same time, the monoclathrochelates strongly quench a fluorescence intensity of BSA and substantially decrease its excited state lifetime due to their binding to this protein. This occurs due to the excitation energy transfer from a tryptophan residue to a cage molecule or/and to the change of the tryptophan nearest environment caused by either clathrochelate binding or an alteration of the BSA conformation. The effect of the iron(II) bis-clathrochelate on BSA fluorescence is much weaker as compared to its monomacrobicyclic analogs as a result of an increase in its size.

Prospect of Bioflavonoid Fisetin as a Quadruplex DNA Ligand: A Biophysical Approach

PubMed Central

Sengupta, Bidisha; Pahari, Biswapathik; Blackmon, Laura; Sengupta, Pradeep K.

2013-01-01

Quadruplex (G4) forming sequences in telomeric DNA and c-myc promoter regions of human DNA are associated with tumorogenesis. Ligands that can facilitate or stabilize the formation and increase the stabilization of G4 can prevent tumor cell proliferation and have been regarded as potential anti-cancer drugs. In the present study, steady state and time-resolved fluorescence measurements provide important structural and dynamical insights into the free and bound states of the therapeutically potent plant flavonoid fisetin (3,3′,4′,7-tetrahydroxyflavone) in a G4 DNA matrix. The excited state intra-molecular proton transfer (ESPT) of fisetin plays an important role in observing and understanding the binding of fisetin with the G4 DNA. Differential absorption spectra, thermal melting, and circular dichroism spectroscopic studies provide evidences for the formation of G4 DNA and size exclusion chromatography (SEC) proves the binding and 1∶1 stoichiometry of fisetin in the DNA matrix. Comparative analysis of binding in the presence of EtBr proves that fisetin favors binding at the face of the G-quartet, mostly along the diagonal loop. Time resolved fluorescence anisotropy decay analysis indicates the increase in the restrictions in motion from the free to bound fisetin. We have also investigated the fingerprints of the binding of fisetin in the antiparallel quadruplex using Raman spectroscopy. Preliminary results indicate fisetin to be a prospective candidate as a G4 ligand. PMID:23785423

The discovery of slowness: low-capacity transport and slow anion channel gating by the glutamate transporter EAAT5.

PubMed

Gameiro, Armanda; Braams, Simona; Rauen, Thomas; Grewer, Christof

2011-06-08

Excitatory amino acid transporters (EAATs) control the glutamate concentration in the synaptic cleft by glial and neuronal glutamate uptake. Uphill glutamate transport is achieved by the co-/countertransport of Na(+) and other ions down their concentration gradients. Glutamate transporters also display an anion conductance that is activated by the binding of Na(+) and glutamate but is not thermodynamically coupled to the transport process. Of the five known glutamate transporter subtypes, the retina-specific subtype EAAT5 has the largest conductance relative to glutamate uptake activity. Our results suggest that EAAT5 behaves as a slow-gated anion channel with little glutamate transport activity. At steady state, EAAT5 was activated by glutamate, with a K(m)= 61 ± 11 μM. Binding of Na(+) to the empty transporter is associated with a K(m) = 229 ± 37 mM, and binding to the glutamate-bound form is associated with a K(m) = 76 ± 40 mM. Using laser-pulse photolysis of caged glutamate, we determined the pre-steady-state kinetics of the glutamate-induced anion current of EAAT5. This was characterized by two exponential components with time constants of 30 ± 1 ms and 200 ± 15 ms, which is an order of magnitude slower than those observed in other glutamate transporters. A voltage-jump analysis of the anion currents indicates that the slow activation behavior is caused by two slow, rate-limiting steps in the transport cycle, Na(+) binding to the empty transporter, and translocation of the fully loaded transporter. We propose a kinetic transport scheme that includes these two slow steps and can account for the experimentally observed data. Overall, our results suggest that EAAT5 may not act as a classical high-capacity glutamate transporter in the retina; rather, it may function as a slow-gated glutamate receptor and/or glutamate buffering system. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Comparative study of flavins binding with human serum albumin: a fluorometric, thermodynamic, and molecular dynamics approach.

PubMed

Sengupta, Abhigyan; Sasikala, Wilbee D; Mukherjee, Arnab; Hazra, Partha

2012-06-04

Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derivatives of riboflavin (RF), a water-soluble vitamin, more commonly known as vitamin B(2). Flavins have attracted special attention in the last few years because of the recent discovery of a large number of flavoproteins. In this work, these flavins are used as extrinsic fluorescence markers for probing the microheterogeneous environment of a well-known transport protein, human serum albumin (HSA). Steady-state and time-resolved fluorescence experiments confirm that both FMN and FAD bind to the Sudlow's site-1 (SS1) binding pocket of HSA, where Trp214 resides. In the case of RF, a fraction of RF molecules binds at the SS1, whereas the major fraction of RF molecules remains unbound or surface bound to the protein. Moreover, flavin(s)-HSA interactions are monitored with the help of isothermal titration calorimetry, which provides free energy, enthalpy, and entropy changes of binding along with the binding constants. The molecular picture of binding interaction between flavins and HSA is well explored by docking and molecular dynamics studies. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Broadening the cofactor specificity of a thermostable alcohol dehydrogenase using rational protein design introduces novel kinetic transient behavior.

PubMed

Campbell, Elliot; Wheeldon, Ian R; Banta, Scott

2010-12-01

Cofactor specificity in the aldo-keto reductase (AKR) superfamily has been well studied, and several groups have reported the rational alteration of cofactor specificity in these enzymes. Although most efforts have focused on mesostable AKRs, several putative AKRs have recently been identified from hyperthermophiles. The few that have been characterized exhibit a strong preference for NAD(H) as a cofactor, in contrast to the NADP(H) preference of the mesophilic AKRs. Using the design rules elucidated from mesostable AKRs, we introduced two site-directed mutations in the cofactor binding pocket to investigate cofactor specificity in a thermostable AKR, AdhD, which is an alcohol dehydrogenase from Pyrococcus furiosus. The resulting double mutant exhibited significantly improved activity and broadened cofactor specificity as compared to the wild-type. Results of previous pre-steady-state kinetic experiments suggest that the high affinity of the mesostable AKRs for NADP(H) stems from a conformational change upon cofactor binding which is mediated by interactions between a canonical arginine and the 2'-phosphate of the cofactor. Pre-steady-state kinetics with AdhD and the new mutants show a rich conformational behavior that is independent of the canonical arginine or the 2'-phosphate. Additionally, experiments with the highly active double mutant using NADPH as a cofactor demonstrate an unprecedented transient behavior where the binding mechanism appears to be dependent on cofactor concentration. These results suggest that the structural features involved in cofactor specificity in the AKRs are conserved within the superfamily, but the dynamic interactions of the enzyme with cofactors are unexpectedly complex. © 2010 Wiley Periodicals, Inc.

Numerical simulation of particle transport and deposition in the pulmonary vasculature.

PubMed

Sohrabi, Salman; Zheng, Junda; Finol, Ender A; Liu, Yaling

2014-12-01

To quantify the transport and adhesion of drug particles in a complex vascular environment, computational fluid particle dynamics (CFPD) simulations of blood flow and drug particulate were conducted in three different geometries representing the human lung vasculature for steady and pulsatile flow conditions. A fully developed flow profile was assumed as the inlet velocity, and a lumped mathematical model was used for the calculation of the outlet pressure boundary condition. A receptor-ligand model was used to simulate the particle binding probability. The results indicate that bigger particles have lower deposition fraction due to less chance of successful binding. Realistic unsteady flow significantly accelerates the binding activity over a wide range of particle sizes and also improves the particle deposition fraction in bifurcation regions when comparing with steady flow condition. Furthermore, surface imperfections and geometrical complexity coupled with the pulsatility effect can enhance fluid mixing and accordingly particle binding efficiency. The particle binding density at bifurcation regions increases with generation order and drug carriers are washed away faster in steady flow. Thus, when studying drug delivery mechanism in vitro and in vivo, it is important to take into account blood flow pulsatility in realistic geometry. Moreover, tissues close to bifurcations are more susceptible to deterioration due to higher uptake.

Critical insight into the interaction of naringenin with human haemoglobin: A combined spectroscopic and computational modeling approaches

NASA Astrophysics Data System (ADS)

Maity, Subhajit; Chakraborty, Sandipan; Chakraborti, Abhay Sankar

2017-02-01

The present study demonstrates critical insight into the binding of a bioactive flavanone naringenin with normal human haemoglobin (NHb). Both spectrophotometric and spectrofluorimetric studies reveal that naringenin interacts with NHb. The binding affinity constant and number of binding sites appear to be approximately (1.5 ± 0.2) × 104 M-1 and 1, respectively. Static quenching seems to be an important factor in binding process, as evident from steady-state and time-resolved fluorescence spectroscopic studies. Far UV circular dichroism spectroscopy depicts that binding of naringenin to NHb causes no change in the secondary structure of the protein, which is also evident from Fourier transform infrared spectroscopic study. Free energy change (ΔG0) for naringenin-NHb interaction, determined by spectroscopic and isothermal calorimetric method, appears to be -5.67 kcal/mol and -6.90 kcal/mol, respectively, and is close to the docking energy -6.84 kcal/mol. Molecular docking suggests that naringenin binds near the cavity of the tetrameric heme protein, forming hydrogen bonds with surrounding amino acid residues. The binding site is away from the heme moieties, implicating naringenin binding does not affect the oxygen binding capacity of NHb, which makes the protein a suitable carrier of the flavonoid.

Monoclonal antibody binding to the macrophage-specific receptor sialoadhesin alters the phagocytic properties of human and mouse macrophages.

PubMed

De Schryver, Marjorie; Cappoen, Davie; Elewaut, Dirk; Nauwynck, Hans J; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

2017-02-01

Sialoadhesin (Sn) is a surface receptor expressed on macrophages in steady state conditions, but during inflammation, Sn can be upregulated both on macrophages and on circulating monocytes. It was shown for different species that Sn becomes internalized after binding with monoclonal antibodies. These features suggest that Sn is a potential target for immunotherapies. In this study, human and mouse macrophages were treated with anti-Sn monoclonal antibodies or F(ab') 2 fragments and the effect of their binding to Sn on phagocytosis was analyzed. Binding of antibodies to Sn resulted in delayed and reduced phagocytosis of fluorescent beads. No effect was observed on Fc-mediated phagocytosis or phagocytosis of bacteria by human macrophages. In contrast, an enhanced phagocytosis of bacteria by mouse macrophages was detected. These results showed that stimulation of Sn could have different effects on macrophage phagocytosis, depending both on the type of phagocytosis and cellular background. Copyright © 2016 Elsevier Inc. All rights reserved.

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

PubMed Central

Ryden, T A; de Mars, M; Beemon, K

1993-01-01

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive. Images PMID:8386280

Kinetic and Thermodynamic Characterization of Dihydrotestosterone-Induced Conformational Perturbations in Androgen Receptor Ligand-Binding Domain

PubMed Central

Jasuja, Ravi; Ulloor, Jagadish; Yengo, Christopher M.; Choong, Karen; Istomin, Andrei Y.; Livesay, Dennis R.; Jacobs, Donald J.; Swerdloff, Ronald S.; Mikšovská, Jaroslava; Larsen, Randy W.; Bhasin, Shalender

2009-01-01

Ligand-induced conformational perturbations in androgen receptor (AR) are important in coactivator recruitment and transactivation. However, molecular rearrangements in AR ligand-binding domain (AR-LBD) associated with agonist binding and their kinetic and thermodynamic parameters are poorly understood. We used steady-state second-derivative absorption and emission spectroscopy, pressure and temperature perturbations, and 4,4′-bis-anilinonaphthalene 8-sulfonate (bis-ANS) partitioning to determine the kinetics and thermodynamics of the conformational changes in AR-LBD after dihydrotestosterone (DHT) binding. In presence of DHT, the second-derivative absorption spectrum showed a red shift and a change in peak-to-peak distance. Emission intensity increased upon DHT binding, and center of spectral mass was blue shifted, denoting conformational changes resulting in more hydrophobic environment for tyrosines and tryptophans within a more compact DHT-bound receptor. In pressure perturbation calorimetry, DHT-induced energetic stabilization increased the Gibbs free energy of unfolding to 8.4 ± 1.3 kcal/mol from 3.5 ± 1.6 kcal/mol. Bis-ANS partitioning studies revealed that upon DHT binding, AR-LBD underwent biphasic rearrangement with a high activation energy (13.4 kcal/mol). An initial, molten globule-like burst phase (k ∼30 sec−1) with greater solvent accessibility was followed by rearrangement (k ∼0.01 sec−1), leading to a more compact conformation than apo-AR-LBD. Molecular simulations demonstrated unique sensitivity of tyrosine and tryptophan residues during pressure unfolding with rearrangement of residues in the coactivator recruitment surfaces distant from the ligand-binding pocket. In conclusion, DHT binding leads to energetic stabilization of AR-LBD domain and substantial rearrangement of residues distant from the ligand-binding pocket. DHT binding to AR-LBD involves biphasic receptor rearrangement including population of a molten globule-like intermediate state. PMID:19443608

Role of recoverin in rod photoreceptor light adaptation.

PubMed

Morshedian, Ala; Woodruff, Michael L; Fain, Gordon L

2018-04-15

Recoverin is a small molecular-weight, calcium-binding protein in rod outer segments that can modulate the rate of rhodopsin phosphorylation. We describe two additional and perhaps more important functions during photoreceptor light adaptation. Recoverin influences the rate of change of adaptation. In wild-type rods, sensitivity and response integration time adapt with similar time constants of 150-200 ms. In Rv-/- rods lacking recoverin, sensitivity declines faster and integration time is already shorter and not significantly altered. During steady light exposure, rod circulating current slowly increases during a time course of tens of seconds, gradually extending the operating range of the rod. In Rv-/- rods, this mechanism is deleted, steady-state currents are already larger and rods saturate at brighter intensities. We propose that recoverin modulates spontaneous and light-activated phophodiesterase-6, the phototransduction effector enzyme, to increase sensitivity in dim light but improve responsiveness to change in brighter illumination. Recoverin is a small molecular-weight, calcium-binding protein in rod outer segments that binds to G-protein receptor kinase 1 and can alter the rate of rhodopsin phosphorylation. A change in phosphorylation should change the lifetime of light-activated rhodopsin and the gain of phototransduction, but deletion of recoverin has little effect on the sensitivity of rods either in the dark or in dim-to-moderate background light. We describe two additional functions perhaps of greater physiological significance. (i) When the ambient intensity increases, sensitivity and integration time decrease in wild-type (WT) rods with similar time constants of 150-200 ms. Recoverin is part of the mechanism controlling this process because, in Rv-/- rods lacking recoverin, sensitivity declines more rapidly and integration time is already shorter and not further altered. (ii) During steady light exposure, WT rod circulating current slowly increases during a time course of tens of seconds, gradually extending the operating range of the rod. In Rv-/- rods, this mechanism is also deleted, steady-state currents are already larger and rods saturate at brighter intensities. We argue that neither (i) nor (ii) can be caused by modulation of rhodopsin phosphorylation but may instead be produced by direct modulation of phophodiesterase-6 (PDE6), the phototransduction effector enzyme. We propose that recoverin in dark-adapted rods keeps the integration time long and the spontaneous PDE6 rate relatively high to improve sensitivity. In background light, the integration time is decreased to facilitate detection of change and motion and the spontaneous PDE6 rate decreases to augment the rod working range. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

Modulated photophysics of a cationic DNA-staining dye inside protein bovine serum albumin: Study of binding interaction and structural changes of protein

NASA Astrophysics Data System (ADS)

Samanta, Anuva; Jana, Sankar; Ray, Debarati; Guchhait, Nikhil

2014-03-01

The binding affinity of cationic DNA-staining dye, propidium iodide, with transport protein, bovine serum albumin, has been explored using UV-vis absorption, fluorescence, and circular dichroism spectroscopy. Steady state and time resolved fluorescence studies authenticate that fluorescence quenching of bovine serum albumin by propidium iodide is due to bovine serum albumin-propidium iodide complex formation. Thermodynamic parameters obtained from temperature dependent spectral studies cast light on binding interaction between the probe and protein. Site marker competitive binding has been encountered using phenylbutazone and flufenamic acid for site I and site II, respectively. Energy transfer efficiency and distance between bovine serum albumin and propidium iodide have been determined using Förster mechanism. Structural stabilization or destabilization of protein by propidium iodide has been investigated by urea denaturation study. The circular dichroism study as well as FT-IR measurement demonstrates some configurational changes of the protein in presence of the dye. Docking studies support the experimental data thereby reinforcing the binding site of the probe to the subdomain IIA of bovine serum albumin.

Dynamic contact network between ribosomal subunits enables rapid large-scale rotation during spontaneous translocation

PubMed Central

Bock, Lars V.; Blau, Christian; Vaiana, Andrea C.; Grubmüller, Helmut

2015-01-01

During ribosomal translation, the two ribosomal subunits remain associated through intersubunit bridges, despite rapid large-scale intersubunit rotation. The absence of large barriers hindering rotation is a prerequisite for rapid rotation. Here, we investigate how such a flat free-energy landscape is achieved, in particular considering the large shifts the bridges undergo at the periphery. The dynamics and energetics of the intersubunit contact network are studied using molecular dynamics simulations of the prokaryotic ribosome in intermediate states of spontaneous translocation. Based on observed occupancies of intersubunit contacts, residues were grouped into clusters. In addition to the central contact clusters, peripheral clusters were found to maintain strong steady interactions by changing contacts in the course of rotation. The peripheral B1 bridges are stabilized by a changing contact pattern of charged residues that adapts to the rotational state. In contrast, steady strong interactions of the B4 bridge are ensured by the flexible helix H34 following the movement of protein S15. The tRNAs which span the subunits contribute to the intersubunit binding enthalpy to an almost constant degree, despite their different positions in the ribosome. These mechanisms keep the intersubunit interaction strong and steady during rotation, thereby preventing dissociation and enabling rapid rotation. PMID:26109353

Obstructing Androgen Receptor Activation in Prostate Cancer Cells Through Post-translational Modification by NEDD8

DTIC Science & Technology

2012-11-01

FACS flow cytometer analysis . In addition, we will measure the steady state protein level of p53, p21, p27, and pRb. In the Jab1 silencing cell...affected by DHT treatment, and the endogenous AR level was not affected by Jab1 silencing. Interestingly, Western blot analysis of immunoprecipitated AR...Avantaggiati, and R. G. Pestell . 2003. Acetylation of androgen receptor enhances coactivator binding and promotes prostate cancer cell growth. Mol

Mimicking Nonequilibrium Steady States with Time-Periodic Driving

DTIC Science & Technology

2016-08-29

nonequilibrium steady states, and vice versa, within the theoretical framework of discrete-state stochastic thermodynamics . Nonequilibrium steady states...equilibrium [2], spontaneous relaxation towards equilibrium [3], nonequilibrium steady states generated by fixed thermodynamic forces [4], and stochastic pumps...paradigm, a system driven by fixed thermodynamic forces—such as temperature gradients or chemical potential differences— reaches a steady state in

Mimicking Nonequilibrium Steady States with Time-Periodic Driving (Open Source)

DTIC Science & Technology

2016-05-18

nonequilibrium steady states, and vice versa, within the theoretical framework of discrete-state stochastic thermodynamics . Nonequilibrium steady states...equilibrium [2], spontaneous relaxation towards equilibrium [3], nonequilibrium steady states generated by fixed thermodynamic forces [4], and stochastic pumps...paradigm, a system driven by fixed thermodynamic forces—such as temperature gradients or chemical potential differences— reaches a steady state in

Influences of urea, pH and metal ions on the interaction between cepharanthine and lysozyme by steady state fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Yumin; Li, Daojin; Xu, Chen

2015-03-01

The study on the binding mode of drug with protein is important to understand the pharmacokinetics and toxicity of the drug as well as the relationship of structure and function of the protein. In the study, the interaction between cepharanthine and lysozyme (Lys) in aqueous solution was first investigated by fluorescence spectroscopic techniques at pH 7.4. The obtained quenching rate constant and binding constant indicated the static quenching mechanism and medium binding force. The effect of cepharanthine on the conformation of Lys was analyzed using synchronous fluorescence and three-dimensional (3D) fluorescence. In addition, the effect of urea on the interaction of cepharanthine with Lys was studied and the binding capacity of cepharanthine to the denatured Lys deceases dramatically, as compared with that of cepharanthine to native Lys. Moreover, influence of pH on the interaction of cepharanthine with Lys was investigated. As compared with that at pH 7.4, the binding abilities of the drug to Lys under other pH conditions (pH 9.0, 5.5, 3.5, and 1.9) deceased. Furthermore, the effect of metal ions on the binding constant of cepharanthine with Lys was investigated.

Model-Based Therapeutic Correction of Hypothalamic-Pituitary-Adrenal Axis Dysfunction

PubMed Central

Ben-Zvi, Amos; Vernon, Suzanne D.; Broderick, Gordon

2009-01-01

The hypothalamic-pituitary-adrenal (HPA) axis is a major system maintaining body homeostasis by regulating the neuroendocrine and sympathetic nervous systems as well modulating immune function. Recent work has shown that the complex dynamics of this system accommodate several stable steady states, one of which corresponds to the hypocortisol state observed in patients with chronic fatigue syndrome (CFS). At present these dynamics are not formally considered in the development of treatment strategies. Here we use model-based predictive control (MPC) methodology to estimate robust treatment courses for displacing the HPA axis from an abnormal hypocortisol steady state back to a healthy cortisol level. This approach was applied to a recent model of HPA axis dynamics incorporating glucocorticoid receptor kinetics. A candidate treatment that displays robust properties in the face of significant biological variability and measurement uncertainty requires that cortisol be further suppressed for a short period until adrenocorticotropic hormone levels exceed 30% of baseline. Treatment may then be discontinued, and the HPA axis will naturally progress to a stable attractor defined by normal hormone levels. Suppression of biologically available cortisol may be achieved through the use of binding proteins such as CBG and certain metabolizing enzymes, thus offering possible avenues for deployment in a clinical setting. Treatment strategies can therefore be designed that maximally exploit system dynamics to provide a robust response to treatment and ensure a positive outcome over a wide range of conditions. Perhaps most importantly, a treatment course involving further reduction in cortisol, even transient, is quite counterintuitive and challenges the conventional strategy of supplementing cortisol levels, an approach based on steady-state reasoning. PMID:19165314

A second dihydroorotate dehydrogenase (Type A) of the human pathogen Enterococcus faecalis: expression, purification, and steady-state kinetic mechanism.

PubMed

Marcinkeviciene, J; Jiang, W; Locke, G; Kopcho, L M; Rogers, M J; Copeland, R A

2000-05-01

We report the identification, expression, and characterization of a second Dihydroorotate dehydrogenase (DHODase A) from the human pathogen Enterococcus faecalis. The enzyme consists of a polypeptide chain of 322 amino acids that shares 68% identity with the cognate type A enzyme from the bacterium Lactococcus lactis. E. faecalis DHODase A catalyzed the oxidation of l-dihydroorotate while reducing a number of substrates, including fumarate, coenzyme Q(0), and menadione. The steady-state kinetic mechanism has been determined with menadione as an oxidizing substrate at pH 7.5. Initial velocity and product inhibition data suggest that the enzyme follows a two-site nonclassical ping-pong kinetic mechanism. The absorbance of the active site FMN cofactor is quenched in a concentration-dependent manner by titration with orotate and barbituric acid, two competitive inhibitors with respect to dihydroorotate. In contrast, titration of the enzyme with menadione had no effect on FMN absorbance, consistent with nonoverlapping binding sites for dihyroorotate and menadione, as suggested from the kinetic mechanism. The reductive half-reaction has been shown to be only partially rate limiting, and an attempt to evaluate the slow step in the overall reaction has been made by simulating orotate production under steady-state conditions. Our data indicate that the oxidative half-reaction is a rate-limiting segment, while orotate, most likely, retains significant affinity for the reduced enzyme, as suggested by the product inhibition pattern. Copyright 2000 Academic Press.

Structure dependent selective efficacy of pyridine and pyrrole based Cu(II) Schiff base complexes towards in vitro cytotoxicity, apoptosis and DNA-bases binding in ground and excited state.

PubMed

Koley Seth, Banabithi; Saha, Arpita; Haldar, Srijan; Chakraborty, Partha Pratim; Saha, Partha; Basu, Samita

2016-09-01

This work highlights a systematic and comparative study of the structure-dependent influence of a series of biologically active Cu(II) Schiff base complexes (CSCs) on their in vitro cytotoxicity, apoptosis and binding with polymeric DNA-bases in ground and photo-excited states. The structure-activity relationship of the closely resembled CSCs towards in vitro cytotoxicity and apoptosis against cervical cancerous HeLa and normal human diploid WI-38 cell lines has been investigated by MTT assay and FACS techniques respectively. The steady-state and time-resolved spectroscopic studies have also been carried out to explore the selective binding affinities of the potential complexes towards different polymeric nucleic acid bases (poly d(A), poly d(T), poly d(G), poly d(C), Poly d(G)-Poly d(C)), which enlighten the knowledge regarding their ability in controlling the structure and medium dependent interactions in 'ground' and 'excited' states. The pyridine containing water soluble complexes (CuL(1) and CuL(3)) are much more cytotoxic than the corresponding pyrrole counterparts (CuL(2) and CuL(4)). Moreover the acidic hydrogens in CuL(1) increase its cytotoxicity much more than methyl substitution as in CuL(3). The results of MTT assay and double staining FACS experiments indicate selective inhibition of cell growth (cell viability 39% (HeLa) versus 85% (WI-38)) and occurrence of apoptosis rather than necrosis. The ground state binding of CuL(1) with polymeric DNA bases, especially with guanine rich DNA (Kb=6.41±0.122×10(5)), that enhances its cytotoxic activity, is further confirmed from its binding isotherms. On the other hand the pyrrole substituted CuL(4) complex exhibits the structure and medium dependent selective electron-transfer in triplet state as observed in laser flash photolysis studies followed by magnetic field (MF) effect. Copyright © 2016 Elsevier B.V. All rights reserved.

Revealing kinetics and state-dependent binding properties of IKur-targeting drugs that maximize atrial fibrillation selectivity

NASA Astrophysics Data System (ADS)

Ellinwood, Nicholas; Dobrev, Dobromir; Morotti, Stefano; Grandi, Eleonora

2017-09-01

The KV1.5 potassium channel, which underlies the ultra-rapid delayed-rectifier current (IKur) and is predominantly expressed in atria vs. ventricles, has emerged as a promising target to treat atrial fibrillation (AF). However, while numerous KV1.5-selective compounds have been screened, characterized, and tested in various animal models of AF, evidence of antiarrhythmic efficacy in humans is still lacking. Moreover, current guidelines for pre-clinical assessment of candidate drugs heavily rely on steady-state concentration-response curves or IC50 values, which can overlook adverse cardiotoxic effects. We sought to investigate the effects of kinetics and state-dependent binding of IKur-targeting drugs on atrial electrophysiology in silico and reveal the ideal properties of IKur blockers that maximize anti-AF efficacy and minimize pro-arrhythmic risk. To this aim, we developed a new Markov model of IKur that describes KV1.5 gating based on experimental voltage-clamp data in atrial myocytes from patient right-atrial samples in normal sinus rhythm. We extended the IKur formulation to account for state-specificity and kinetics of KV1.5-drug interactions and incorporated it into our human atrial cell model. We simulated 1- and 3-Hz pacing protocols in drug-free conditions and with a [drug] equal to the IC50 value. The effects of binding and unbinding kinetics were determined by examining permutations of the forward (kon) and reverse (koff) binding rates to the closed, open, and inactivated states of the KV1.5 channel. We identified a subset of ideal drugs exhibiting anti-AF electrophysiological parameter changes at fast pacing rates (effective refractory period prolongation), while having little effect on normal sinus rhythm (limited action potential prolongation). Our results highlight that accurately accounting for channel interactions with drugs, including kinetics and state-dependent binding, is critical for developing safer and more effective pharmacological anti-AF options.

Modeling of the blood rheology in steady-state shear flows

DOE Office of Scientific and Technical Information (OSTI.GOV)

Apostolidis, Alex J.; Beris, Antony N., E-mail: beris@udel.edu

We undertake here a systematic study of the rheology of blood in steady-state shear flows. As blood is a complex fluid, the first question that we try to answer is whether, even in steady-state shear flows, we can model it as a rheologically simple fluid, i.e., we can describe its behavior through a constitutive model that involves only local kinematic quantities. Having answered that question positively, we then probe as to which non-Newtonian model best fits available shear stress vs shear-rate literature data. We show that under physiological conditions blood is typically viscoplastic, i.e., it exhibits a yield stress thatmore » acts as a minimum threshold for flow. We further show that the Casson model emerges naturally as the best approximation, at least for low and moderate shear-rates. We then develop systematically a parametric dependence of the rheological parameters entering the Casson model on key physiological quantities, such as the red blood cell volume fraction (hematocrit). For the yield stress, we base our description on its critical, percolation-originated nature. Thus, we first determine onset conditions, i.e., the critical threshold value that the hematocrit has to have in order for yield stress to appear. It is shown that this is a function of the concentration of a key red blood cell binding protein, fibrinogen. Then, we establish a parametric dependence as a function of the fibrinogen and the square of the difference of the hematocrit from its critical onset value. Similarly, we provide an expression for the Casson viscosity, in terms of the hematocrit and the temperature. A successful validation of the proposed formula is performed against additional experimental literature data. The proposed expression is anticipated to be useful not only for steady-state blood flow modeling but also as providing the starting point for transient shear, or more general flow modeling.« less

Pseudo Steady-State Free Precession for MR-Fingerprinting.

PubMed

Assländer, Jakob; Glaser, Steffen J; Hennig, Jürgen

2017-03-01

This article discusses the signal behavior in the case the flip angle in steady-state free precession sequences is continuously varied as suggested for MR-fingerprinting sequences. Flip angle variations prevent the establishment of a steady state and introduce instabilities regarding to magnetic field inhomogeneities and intravoxel dephasing. We show how a pseudo steady state can be achieved, which restores the spin echo nature of steady-state free precession. Based on geometrical considerations, relationships between the flip angle, repetition and echo time are derived that suffice to the establishment of a pseudo steady state. The theory is tested with Bloch simulations as well as phantom and in vivo experiments. A typical steady-state free precession passband can be restored with the proposed conditions. The stability of the pseudo steady state is demonstrated by comparing the evolution of the signal of a single isochromat to one resulting from a spin ensemble. As confirmed by experiments, magnetization in a pseudo steady state can be described with fewer degrees of freedom compared to the original fingerprinting and the pseudo steady state results in more reliable parameter maps. The proposed conditions restore the spin-echo-like signal behavior typical for steady-state free precession in fingerprinting sequences, making this approach more robust to B 0 variations. Magn Reson Med 77:1151-1161, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Mechanism for multiplicity of steady states with distinct cell concentration in continuous culture of mammalian cells.

PubMed

Yongky, Andrew; Lee, Jongchan; Le, Tung; Mulukutla, Bhanu Chandra; Daoutidis, Prodromos; Hu, Wei-Shou

2015-07-01

Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi-scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells. © 2015 Wiley Periodicals, Inc.

Probing the behavior of bovine serum albumin upon binding to atenolol: insights from spectroscopic and molecular docking approaches.

PubMed

Jiang, Tuo-Ying; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi; Shi, Jie-Hua

2018-04-01

Molecular interaction of atenolol, a selective β 1 receptor antagonist with the major carrier protein, bovine serum albumin (BSA), was investigated under imitated physiological conditions (pH 7.4) by means of fluorescence spectroscopy, UV absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and molecular modeling studies. The steady-state fluorescence spectra manifested that static type, due to formation of the atenolol-BSA complex, was the dominant mechanism for fluorescence quenching. The characteristic information about the binding interaction of atenolol with BSA in terms of binding constant (K b ) were determined by the UV-vis absorption titration, and were found to be in the order of 10 3  M -1 at different temperatures, indicating the existence of a weak binding in this system. Thermodynamic analysis revealed that the binding process was primarily mediated by van der Waals force and hydrogen bonds due to the negative sign for enthalpy change (ΔH 0 ), entropy change (ΔS 0 ). The molecular docking results elucidated that atenolol preferred binding on the site II of BSA according to the findings observed in competitive binding experiments. Moreover, via alterations in synchronous fluorescence, three-dimensional fluorescence and FT-IR spectral properties, it was concluded that atenolol could arouse slight configurational and micro-environmental changes of BSA.

Probing the Allosteric Modulator Binding Site of GluR2 with Thiazide Derivatives

PubMed Central

Ptak, Christopher P.; Ahmed, Ahmed H.; Oswald, Robert E.

2009-01-01

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission and are therapeutic targets for cognitive enhancement and treatment of schizophrenia. The binding domains of these tetrameric receptors consist of two dimers, and the dissociation of the dimer interface of the ligand-binding domain leads to desensitization in the continued presence of agonist. Positive allosteric modulators act by strengthening the dimer interface and reducing desensitization, thereby increasing steady-state activation. Removing the desensitized state for simplified analysis of receptor activation is commonly achieved using cyclothiazide (CTZ), the most potent modulator of the benzothiadiazide class, with the flip form of the AMPA receptor subtype. IDRA-21, the first benzothiadiazide to have an effect in behavioral tests, is an important lead compound in clinical trials for cognitive enhancement as it can cross the blood-brain barrier. Intermediate structures between CTZ and IDRA-21 show reduced potency suggesting that these two compounds have different contact points associated with binding. To understand how benzothiadiazides bind to the pocket bridging the dimer interface, we generated a series of crystal structures of the GluR2 ligand-binding domain complexed with benzothiadiazide derivatives (IDRA-21, hydroflumethiazide, hydrochlorothiazide, chlorothiazide, trichlormethiazide, and althiazide) for comparison with an existing structure for cyclothiazide. The structures detail how changes in the substituents in the 3- and 7-positions of the hydrobenzothiadiazide ring shift the orientation of the drug in the binding site and, in some cases, change the stoichiometry of binding. All derivatives maintain a hydrogen bond with the Ser754 hydroxyl, affirming the partial selectivity of the benzothiadiazides for the flip form of AMPA receptors. PMID:19673491

Excited-State Proton Transfer on the Surface of a Therapeutic Protein, Protamine.

PubMed

Awasthi, Ankur A; Singh, Prabhat K

2017-11-16

Proton transfer reactions on biosurfaces play an important role in a myriad of biological processes. Herein, the excited-state proton transfer reaction of 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) has been investigated in the presence of an important therapeutic protein, Protamine (PrS), using ground-state absorption, steady-state, and detailed time-resolved emission measurements. HPTS forms a 1:1 complex with Protamine with a high association constant of 2.6 × 10 4 M -1 . The binding of HPTS with Protamine leads to a significant modulation in the ground-state prototropic equilibrium causing a downward shift of 1.1 unit in the acidity constant (pK a ). In contrast to a large number of reports of slow proton transfer of HPTS on biosurfaces, interestingly, HPTS registers a faster proton transfer event in the presence of Protamine as compared to that of even the bulk aqueous buffer medium. Furthermore, the dimensionality of the proton diffusion process is also significantly reduced on the surface of Protamine that is in contrast to the behavior of HPTS in the bulk aqueous buffer medium, where the proton diffusion process is three-dimensional. The effect of ionic strength on the binding of HPTS toward PrS suggests a predominant role of electrostatic interaction between anionic HPTS and cationic Protamine, which is further supported by molecular docking simulations which predict that the most preferable binding site for HPTS on the surface of Protamine is surrounded by multiple cationic arginine residues.

Minimal physical requirements for crystal growth self-poisoning

DOE PAGES

Whitelam, Stephen; Dahal, Yuba Raj; Schmit, Jeremy D.

2016-02-10

Self-poisoning is a kinetic trap that can impair or prevent crystal growth in a wide variety of physical settings. In this paper, we use dynamic mean-field theory and computer simulation to argue that poisoning is ubiquitous because its emergence requires only the notion that a molecule can bind in two (or more) ways to a crystal; that those ways are not energetically equivalent; and that the associated binding events occur with sufficiently unequal probability. If these conditions are met then the steady-state growth rate is in general a non-monotonic function of the thermodynamic driving force for crystal growth, which ismore » the characteristic of poisoning. Finally, our results also indicate that relatively small changes of system parameters could be used to induce recovery from poisoning.« less

Salmonella Enterica Serovar Typhimurium BipA Exhibits Two Distinct Ribosome Binding Modes

DOE Office of Scientific and Technical Information (OSTI.GOV)

deLivron, M.; Robinson, V

BipA is a highly conserved prokaryotic GTPase that functions to influence numerous cellular processes in bacteria. In Escherichia coli and Salmonella enterica serovar Typhimurium, BipA has been implicated in controlling bacterial motility, modulating attachment and effacement processes, and upregulating the expression of virulence genes and is also responsible for avoidance of host defense mechanisms. In addition, BipA is thought to be involved in bacterial stress responses, such as those associated with virulence, temperature, and symbiosis. Thus, BipA is necessary for securing bacterial survival and successful invasion of the host. Steady-state kinetic analysis and pelleting assays were used to assess themore » GTPase and ribosome-binding properties of S. enterica BipA. Under normal bacterial growth, BipA associates with the ribosome in the GTP-bound state. However, using sucrose density gradients, we demonstrate that the association of BipA and the ribosome is altered under stress conditions in bacteria similar to those experienced during virulence. The data show that this differential binding is brought about by the presence of ppGpp, an alarmone that signals the onset of stress-related events in bacteria.« less

Paxillin associates with poly(A)-binding protein 1 at the dense endoplasmic reticulum and the leading edge of migrating cells.

PubMed

Woods, Alison J; Roberts, Marnie S; Choudhary, Jyoti; Barry, Simon T; Mazaki, Yuichi; Sabe, Hisataka; Morley, Simon J; Critchley, David R; Norman, Jim C

2002-02-22

Using mass spectrometry we have identified proteins which co-immunoprecipitate with paxillin, an adaptor protein implicated in the integrin-mediated signaling pathways of cell motility. A major component of paxillin immunoprecipitates was poly(A)-binding protein 1, a 70-kDa mRNA-binding protein. Poly(A)-binding protein 1 associated with both the alpha and beta isoforms of paxillin, and this was unaffected by RNase treatment consistent with a protein-protein interaction. The NH(2)-terminal region of paxillin (residues 54-313) associated directly with poly(A)-binding protein 1 in cell lysates, and with His-poly(A)-binding protein 1 immobilized in microtiter wells. Binding was specific, saturable and of high affinity (K(d) of approximately 10 nm). Cell fractionation studies showed that at steady state, the bulk of paxillin and poly(A)-binding protein 1 was present in the "dense" polyribosome-associated endoplasmic reticulum. However, inhibition of nuclear export with leptomycin B caused paxillin and poly(A)-binding protein 1 to accumulate in the nucleus, indicating that they shuttle between the nuclear and cytoplasmic compartments. When cells migrate, poly(A)-binding protein 1 colocalized with paxillin-beta at the tips of lamellipodia. Our results suggest a new mechanism whereby a paxillin x poly(A)-binding protein 1 complex facilitates transport of mRNA from the nucleus to sites of protein synthesis at the endoplasmic reticulum and the leading lamella during cell migration.

Electroosmotic flow in capillary channels filled with nonconstant viscosity electrolytes: exact solution of the Navier-Stokes equation.

PubMed

Otevrel, Marek; Klepárník, Karel

2002-10-01

The partial differential equation describing unsteady velocity profile of electroosmotic flow (EOF) in a cylindrical capillary filled with a nonconstant viscosity electrolyte was derived. Analytical solution, based on the general Navier-Stokes equation, was found for constant viscosity electrolytes using the separation of variables (Fourier method). For the case of a nonconstant viscosity electrolyte, the steady-state velocity profile was calculated assuming that the viscosity decreases exponentially in the direction from the wall to the capillary center. Since the respective equations with nonconstant viscosity term are not solvable in general, the method of continuous binding conditions was used to solve this problem. In this method, an arbitrary viscosity profile can be modeled. The theoretical conclusions show that the relaxation times at which an EOF approaches the steady state are too short to have an impact on a separation process in any real systems. A viscous layer at the wall affects EOF significantly, if it is thicker than the Debye length of the electric double layer. The presented description of the EOF dynamics is applicable to any microfluidic systems.

Fluorescence life-time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles.

PubMed

Schwartz, Shmulik; Fixler, Dror; Popovtzer, Rachela; Shefi, Orit

2015-11-01

Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications. Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY-GNP (Middle) enable the differentiation between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

c-Myb is required for progenitor cell homeostasis in colonic crypts

PubMed Central

Malaterre, Jordane; Carpinelli, Marina; Ernst, Matthias; Alexander, Warren; Cooke, Michael; Sutton, Susan; Dworkin, Sebastian; Heath, Joan K.; Frampton, Jon; McArthur, Grant; Clevers, Hans; Hilton, Douglas; Mantamadiotis, Theo; Ramsay, Robert G.

2007-01-01

The colonic crypt is the functional unit of the colon mucosa with a central role in ion and water reabsorption. Under steady-state conditions, the distal colonic crypt harbors a single stem cell at its base that gives rise to highly proliferative progenitor cells that differentiate into columnar, goblet, and endocrine cells. The role of c-Myb in crypt homeostasis has not been elucidated. Here we have studied three genetically distinct hypomorphic c-myb mutant mouse strains, all of which show reduced colonic crypt size. The mutations target the key domains of the transcription factor: the DNA binding, transactivation, and negative regulatory domains. In vivo proliferation and cell cycle marker studies suggest that these mice have a progenitor cell proliferation defect mediated in part by reduced Cyclin E1 expression. To independently assess the extent to which c-myb is required for colonic crypt homeostasis we also generated a novel tissue-specific mouse model to allow the deletion of c-myb in adult colon, and using these mice we show that c-Myb is required for crypt integrity, normal differentiation, and steady-state proliferation. PMID:17360438

Evidences of trapping in tungsten and implications for plasma-facing components

NASA Astrophysics Data System (ADS)

Longhurst, G. R.; Anderl, R. A.; Holland, D. F.

Trapping effects that include significant delays in permeation saturation, abrupt changes in permeation rate associated with temperature changes, and larger than expected inventories of hydrogen isotopes in the material, were seen in implantation-driven permeation experiments using 25- and 50-micron thick tungsten foils at temperatures of 638 to 825 K. Computer models that simulate permeation transients reproduce the steady-state permeation and reemission behavior of these experiments with expected values of material parameters. However, the transient time characteristics were not successfully simulated without the assumption of traps of substantial trap energy and concentration. An analytical model based on the assumptions of thermodynamic equilibrium between trapped hydrogen atoms and a comparatively low mobile atom concentration successfully accounts for the observed behavior. Using steady-state and transient permeation data from experiments at different temperatures, the effective trap binding energy may be inferred. We analyze a tungsten coated divertor plate design representative of those proposed for ITER and ARIES and consider the implications for tritium permeation and retention if the same trapping we observed was present in that tungsten. Inventory increases of several orders of magnitude may result.

HPV 5 and 8 E6 Expression Reduces ATM Protein Levels and Attenuates LINE-1 Retrotransposition

PubMed Central

Wallace, Nicholas A.; Gasior, Stephen L.; Faber, Zachary J.; Howie, Heather L.; Deininger, Prescott L.; Galloway, Denise A.

2013-01-01

The expression of the E6 protein from certain members of the HPV genus β (β HPV 5 and 8 E6) can disrupt p53 signaling by diminishing the steady state levels of two p53 modifying enzymes, ATR and p300. Here, we show that β-HPV 5 and 8 E6 are also capable of reducing the steady state levels of another p53 modifying enzyme, ATM, and as a result restrict LINE-1 retrotransposition. Furthermore, we show that the reduction of both ATM and LINE-1 retrotransposition is dependent upon the ability of β-HPV 8 E6 to bind and degrade p300. We use inhibitors and dominant negative mutants to confirm that ATM is needed for efficient LINE-1 retrotransposition. Furthermore, neither sensitivity to LINE-1 expression nor LINE-1 induced DSB formation is altered in an ATM deficient background. Together, these data illustrate the broad impact some β-HPVs have on DNA damage signaling by promoting p300 degradation. PMID:23706308

Quantitative analysis of small molecule-nucleic acid interactions with a biosensor surface and surface plasmon resonance detection.

PubMed

Liu, Yang; Wilson, W David

2010-01-01

Surface plasmon resonance (SPR) technology with biosensor surfaces has become a widely-used tool for the study of nucleic acid interactions without any labeling requirements. The method provides simultaneous kinetic and equilibrium characterization of the interactions of biomolecules as well as small molecule-biopolymer binding. SPR monitors molecular interactions in real time and provides significant advantages over optical or calorimetic methods for systems with strong binding coupled to small spectroscopic signals and/or reaction heats. A detailed and practical guide for nucleic acid interaction analysis using SPR-biosensor methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples, as well as extensive information on experimental design, quantitative and qualitative data analysis and presentation. A specific example of the interaction of a minor-groove-binding agent with DNA is evaluated by both kinetic and steady-state SPR methods to illustrate the technique. Since the molecules that bind cooperatively to specific DNA sequences are attractive for many applications, a cooperative small molecule-DNA interaction is also presented.

Quasi-steady state conditions in heterogeneous aquifers during pumping tests

NASA Astrophysics Data System (ADS)

Zha, Yuanyuan; Yeh, Tian-Chyi J.; Shi, Liangsheng; Huang, Shao-Yang; Wang, Wenke; Wen, Jet-Chau

2017-08-01

Classical Thiem's well hydraulic theory, other aquifer test analyses, and flow modeling efforts often assume the existence of ;quasi-steady; state conditions. That is, while drawdowns due to pumping continue to grow, the hydraulic gradient in the vicinity of the pumping well does not change significantly. These conditions have built upon two-dimensional and equivalent homogeneous conceptual models, but few field data have been available to affirm the existence of these conditions. Moreover, effects of heterogeneity and three-dimensional flow on this quasi-steady state concept have not been thoroughly investigated and discussed before. In this study, we first present a quantitative definition of quasi-steady state (or steady-shape conditions) and steady state conditions based on the analytical solution of two- or three-dimensional flow induced by pumping in unbounded, homogeneous aquifers. Afterward, we use a stochastic analysis to investigate the influence of heterogeneity on the quasi-steady state concept in heterogeneous aquifers. The results of the analysis indicate that the time to reach an approximate quasi-steady state in a heterogeneous aquifer could be quite different from that estimated based on a homogeneous model. We find that heterogeneity of aquifer properties, especially hydraulic conductivity, impedes the development of the quasi-steady state condition before the flow reaching steady state. Finally, 280 drawdown-time data from the hydraulic tomographic survey conducted at a field site corroborate our finding that the quasi-steady state condition likely would not take place in heterogeneous aquifers unless pumping tests last a long period. Research significance (1) Approximate quasi-steady and steady state conditions are defined for two- or three-dimensional flow induced by pumping in unbounded, equivalent homogeneous aquifers. (2) Analysis demonstrates effects of boundary condition, well screen interval, and heterogeneity of parameters on the existence of the quasi-steady, and validity of approximate quasi-steady concept. (3) Temporal evaluation of information content about heterogeneity in head observations are analyzed in heterogeneous aquifer. (4) 280 observed drawdown-time data corroborate the stochastic analysis that quasi-steady is difficult to reach in highly heterogeneous aquifers.

Estimating systemic exposure to levonorgestrel from an oral contraceptive.

PubMed

Basaraba, Cale N; Westhoff, Carolyn L; Pike, Malcolm C; Nandakumar, Renu; Cremers, Serge

2017-04-01

The gold standard for measuring oral contraceptive (OC) pharmacokinetics is the 24-h steady-state area under the curve (AUC). We conducted this study to assess whether limited sampling at steady state or measurements following use of one or two OCs could provide an adequate proxy in epidemiological studies for the progestin 24-h steady-state AUC of a particular OC. We conducted a 13-sample, 24-h pharmacokinetic study on both day 1 and day 21 of the first cycle of a monophasic OC containing 30-mcg ethinyl estradiol and 150-mcg levonorgestrel (LNG) in 17 normal-weight healthy White women and a single-dose 9-sample study of the same OC after a 1-month washout. We compared the 13-sample steady-state results with several steady-state and single-dose results calculated using parsimonious sampling schemes. The 13-sample steady-state 24-h LNG AUC was highly correlated with the steady-state 24-h trough value [r=0.95; 95% confidence interval (0.85, 0.98)] and with the steady-state 6-, 8-, 12- and 16-h values (0.92≤r≤0.95). The trough values after one or two doses were moderately correlated with the steady-state 24-h AUC value [r=0.70; 95% CI (0.27, 0.90) and 0.77; 95% CI (0.40, 0.92), respectively]. Single time-point concentrations at steady state and after administration of one or two OCs gave highly to moderately correlated estimates of steady-state LNG AUC. Using such measures could facilitate prospective pharmaco-epidemiologic studies of the OC and its side effects. A single time-point LNG concentration at steady state is an excellent proxy for complete and resource-intensive steady-state AUC measurement. The trough level after two single doses is a fair proxy for steady-state AUC. These results provide practical tools to facilitate large studies to investigate the relationship between systemic LNG exposure and side effects in a real-life setting. Copyright © 2017 Elsevier Inc. All rights reserved.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

PubMed Central

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

2015-01-01

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

Modulated photophysics of a cationic DNA-staining dye inside protein bovine serum albumin: study of binding interaction and structural changes of protein.

PubMed

Samanta, Anuva; Jana, Sankar; Ray, Debarati; Guchhait, Nikhil

2014-01-01

The binding affinity of cationic DNA-staining dye, propidium iodide, with transport protein, bovine serum albumin, has been explored using UV-vis absorption, fluorescence, and circular dichroism spectroscopy. Steady state and time resolved fluorescence studies authenticate that fluorescence quenching of bovine serum albumin by propidium iodide is due to bovine serum albumin-propidium iodide complex formation. Thermodynamic parameters obtained from temperature dependent spectral studies cast light on binding interaction between the probe and protein. Site marker competitive binding has been encountered using phenylbutazone and flufenamic acid for site I and site II, respectively. Energy transfer efficiency and distance between bovine serum albumin and propidium iodide have been determined using Förster mechanism. Structural stabilization or destabilization of protein by propidium iodide has been investigated by urea denaturation study. The circular dichroism study as well as FT-IR measurement demonstrates some configurational changes of the protein in presence of the dye. Docking studies support the experimental data thereby reinforcing the binding site of the probe to the subdomain IIA of bovine serum albumin. Copyright © 2013 Elsevier B.V. All rights reserved.

Discovery of small molecules binding to the normal conformation of prion by combining virtual screening and multiple biological activity evaluation methods

NASA Astrophysics Data System (ADS)

Li, Lanlan; Wei, Wei; Jia, Wen-Juan; Zhu, Yongchang; Zhang, Yan; Chen, Jiang-Huai; Tian, Jiaqi; Liu, Huanxiang; He, Yong-Xing; Yao, Xiaojun

2017-12-01

Conformational conversion of the normal cellular prion protein, PrPC, into the misfolded isoform, PrPSc, is considered to be a central event in the development of fatal neurodegenerative diseases. Stabilization of prion protein at the normal cellular form (PrPC) with small molecules is a rational and efficient strategy for treatment of prion related diseases. However, few compounds have been identified as potent prion inhibitors by binding to the normal conformation of prion. In this work, to rational screening of inhibitors capable of stabilizing cellular form of prion protein, multiple approaches combining docking-based virtual screening, steady-state fluorescence quenching, surface plasmon resonance and thioflavin T fluorescence assay were used to discover new compounds interrupting PrPC to PrPSc conversion. Compound 3253-0207 that can bind to PrPC with micromolar affinity and inhibit prion fibrillation was identified from small molecule databases. Molecular dynamics simulation indicated that compound 3253-0207 can bind to the hotspot residues in the binding pocket composed by β1, β2 and α2, which are significant structure moieties in conversion from PrPC to PrPSc.

Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo

PubMed Central

Björk, Petra; Persson, Jan-Olov

2015-01-01

Eukaryotic gene expression requires the ordered association of numerous factors with precursor messenger RNAs (premRNAs)/messenger RNAs (mRNAs) to achieve efficiency and regulation. Here, we use the Balbiani ring (BR) genes to demonstrate the temporal and spatial association of the exon junction complex (EJC) core with gene-specific endogenous premRNAs and mRNAs. The EJC core components bind cotranscriptionally to BR premRNAs during or very rapidly after splicing. The EJC core does not recruit the nonsense-mediated decay mediaters UPF2 and UPF3 until the BR messenger RNA protein complexes (mRNPs) enter the interchromatin. Even though several known adapters for the export factor NXF1 become part of BR mRNPs already at the gene, NXF1 binds to BR mRNPs only in the interchromatin. In steady state, a subset of the BR mRNPs in the interchromatin binds NXF1, UPF2, and UPF3. This binding appears to occur stochastically, and the efficiency approximately equals synthesis and export of the BR mRNPs. Our data provide unique in vivo information on how export competent eukaryotic mRNPs are formed. PMID:26459599

Physical constraints determine the logic of bacterial promoter architectures

PubMed Central

Ezer, Daphne; Zabet, Nicolae Radu; Adryan, Boris

2014-01-01

Site-specific transcription factors (TFs) bind to their target sites on the DNA, where they regulate the rate at which genes are transcribed. Bacterial TFs undergo facilitated diffusion (a combination of 3D diffusion around and 1D random walk on the DNA) when searching for their target sites. Using computer simulations of this search process, we show that the organization of the binding sites, in conjunction with TF copy number and binding site affinity, plays an important role in determining not only the steady state of promoter occupancy, but also the order at which TFs bind. These effects can be captured by facilitated diffusion-based models, but not by standard thermodynamics. We show that the spacing of binding sites encodes complex logic, which can be derived from combinations of three basic building blocks: switches, barriers and clusters, whose response alone and in higher orders of organization we characterize in detail. Effective promoter organizations are commonly found in the E. coli genome and are highly conserved between strains. This will allow studies of gene regulation at a previously unprecedented level of detail, where our framework can create testable hypothesis of promoter logic. PMID:24476912

Enhancing emotional-based target prediction

NASA Astrophysics Data System (ADS)

Gosnell, Michael; Woodley, Robert

2008-04-01

This work extends existing agent-based target movement prediction to include key ideas of behavioral inertia, steady states, and catastrophic change from existing psychological, sociological, and mathematical work. Existing target prediction work inherently assumes a single steady state for target behavior, and attempts to classify behavior based on a single emotional state set. The enhanced, emotional-based target prediction maintains up to three distinct steady states, or typical behaviors, based on a target's operating conditions and observed behaviors. Each steady state has an associated behavioral inertia, similar to the standard deviation of behaviors within that state. The enhanced prediction framework also allows steady state transitions through catastrophic change and individual steady states could be used in an offline analysis with additional modeling efforts to better predict anticipated target reactions.

Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors.

PubMed

Athwal, G S; Lombardo, C R; Huber, J L; Masters, S C; Fu, H; Huber, S C

2000-04-01

The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in KD and an increase in steady-state binding level (Req). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.

Steady- and non-steady-state carbonate-silicate controls on atmospheric CO2

USGS Publications Warehouse

Sundquist, E.T.

1991-01-01

Two contrasting hypotheses have recently been proposed for the past long-term relation between atmospheric CO2 and the carbonate-silicate geochemical cycle. One approach (Berner, 1990) suggests that CO2 levels have varied in a manner that has maintained chemical weathering and carbonate sedimentation at a steady state with respect to tectonically controlled decarbonation reactions. A second approach (Raymo et al., 1988), applied specificlly to the late Cenozoic, suggests a decrease in CO2 caused by an uplift-induced increase in chemical weathering, without regard to the rate of decarbonation. According to the steady-state (first) hypothesis, increased weathering and carbonate sedimentation are generally associated with increasing atmospheric CO2, whereas the uplift (second) hypothesis implies decreasing CO2 under the same conditions. An ocean-atmosphere-sediment model has been used to assess the response of atmospheric CO2 and carbonate sedimentation to global perturbations in chemical weathering and decarbonation reactions. Although this assessment is theoretical and cannot yet be related to the geologic record, the model simulations compare steady-state and non-steady-state carbonate-silicate cycle response. The e-fold response time of the 'CO2-weathering' feedback mechanism is between 300 and 400 ka. The response of carbonate sedimentation is much more rapid. These response times provide a measure of the strength of steady-state assumptions, and imply that certain systematic relations are sustained throughout steady-state and non-steady-state scenarios for the carbonate-silicate cycle. The simulations suggest that feedbacks can maintain the system near a steady state, but that non-steady-state effects may contribute to long-term trends. The steady-state and uplift hypotheses are not necessarily incompatible over time scales of a few million years. ?? 1991.

The Parkinson Disease-linked LRRK2 Protein Mutation I2020T Stabilizes an Active State Conformation Leading to Increased Kinase Activity*

PubMed Central

Ray, Soumya; Bender, Samantha; Kang, Stephanie; Lin, Regina; Glicksman, Marcie A.; Liu, Min

2014-01-01

The effect of leucine-rich repeat kinase 2 (LRRK2) mutation I2020T on its kinase activity has been controversial, with both increased and decreased effects being reported. We conducted steady-state and pre-steady-state kinetic studies on LRRKtide and its analog LRRKtideS. Their phosphorylation differs by the rate-limiting steps: product release is rate-limiting for LRRKtide and phosphoryl transfer is rate-limiting for LRRKtideS. As a result, we observed that the I2020T mutant is more active than wild type (WT) LRRK2 for LRRKtideS phosphorylation, whereas it is less active than WT for LRRKtide phosphorylation. Our pre-steady-state kinetic data suggest that (i) the I2020T mutant accelerates the rates of phosphoryl transfer of both reactions by 3–7-fold; (ii) this increase is masked by a rate-limiting product release step for LRRKtide phosphorylation; and (iii) the observed lower activity of the mutant for LRRKtide phosphorylation is a consequence of its instability: the concentration of the active form of the mutant is 3-fold lower than WT. The I2020T mutant has a dramatically low KATP and therefore leads to resistance to ATP competitive inhibitors. Two well known DFG-out or type II inhibitors are also weaker toward the mutant because they inhibit the mutant in an unexpected ATP competitive mechanism. The I2020 residue lies next to the DYG motif of the activation loop of the LRRK2 kinase domain. Our modeling and metadynamic simulations suggest that the I2020T mutant stabilizes the DYG-in active conformation and creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion. PMID:24695735

Four Ca2+ Ions Activate TRPM2 Channels by Binding in Deep Crevices near the Pore but Intracellularly of the Gate

PubMed Central

Törőcsik, Beáta

2009-01-01

TRPM2 is a tetrameric Ca2+-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca2+, but the mechanism and the binding sites for Ca2+ activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca2+ dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca2+ activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca2+ activated ∼50-pS nonselective cation channels; K1/2 for ADPR was ∼1 µM at saturating Ca2+. Intracellular Ca2+ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca2+) reveals that Ca2+ activation is a consequence of tighter binding of Ca2+ in the open rather than in the closed channel conformation. Four Ca2+ ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant ∼33-fold, producing altogether 106-fold activation. Experiments in the presence of 1 mM of free Ca2+ on the extracellular side clearly show that closed channels do not sense extracellular Ca2+, but once channels have opened Ca2+ entering passively through the pore slows channel closure by keeping the “activating sites” saturated, despite rapid continuous Ca2+-free wash of the intracellular channel surface. This effect of extracellular Ca2+ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca2+ influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single brief puff of Ca2+ likely triggers prolonged, self-sustained TRPM2 activity. PMID:19171771

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase.

PubMed

Coelho, Catarina; Foti, Alessandro; Hartmann, Tobias; Santos-Silva, Teresa; Leimkühler, Silke; Romão, Maria João

2015-10-01

Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-Å resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-Å resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.

Lack of pharmacokinetic interaction between vinpocetine and oxazepam.

PubMed Central

Storm, G; Oosterhuis, B; Sollie, F A; Visscher, H W; Sommer, W; Beitinger, H; Jonkman, J H

1994-01-01

The influence of multiple doses of vinpocetine (10 mg three times daily) on the steady state plasma concentrations of oxazepam (10 mg three times daily) was studied in 16 healthy subjects. The mean (+/- s.d.) AUC (ng ml-1h-1) of oxazepam over 24 h during combined treatment was 4716 +/- 2296 and for oxazepam treatment alone it was 4737 +/- 2448 (95% confidence intervals for ratio of means = 95.4-103.7%). The degree of plasma protein binding of oxazepam was 98.11 +/- 0.32% and was not affected by vinpocetine. Independent of vinpocentine treatment a significant diurnal change in the plasma binding of oxazepam was observed; the free drug fraction was 20% higher during the night than during the day. Cmax and AUC values based on total oxazepam in plasma were 10% lower during the night. The results indicate a lack of influence of vinpocetine on oxazepam kinetics. Diurnal changes in the plasma binding of oxazepam probably have no clinical consequences. PMID:7981015

Interaction of Lysozyme with Rhodamine B: A combined analysis of spectroscopic & molecular docking.

PubMed

Millan, Sabera; Satish, Lakkoji; Kesh, Sandeep; Chaudhary, Yatendra S; Sahoo, Harekrushna

2016-09-01

The interaction of Rhodamine B (RB) with Lysozyme (Lys) was investigated by different optical spectroscopic techniques such as absorption, fluorescence, and circular-dichroism (CD), along with molecular docking studies. The fluorescence results (including steady-state and time-resolved mode) revealed that the addition of RB effectively causes strong quenching of intrinsic fluorescence in Lysozyme and mostly, by the static quenching mechanism. Different binding and thermodynamic parameters were calculated at different temperatures and the binding constant value was found to be 2963.54Lmol(-1) at 25°C. The average distance (r0) was found to be 3.31nm according to Förster's theory of non-radiative energy transfer between Lysozyme and RB. The conformational change in Lysozyme during interaction with RB was confirmed from absorbance, synchronous fluorescence, and circular dichroism measurements. Finally, molecular docking studies were done to confirm that the dye binds with Lysozyme. Copyright © 2016 Elsevier B.V. All rights reserved.

Discriminating tastes

PubMed Central

Storz, Gisela

2011-01-01

Hfq-binding small RNAs (sRNAs) are critical regulators that form limited base-pairing interactions with target mRNAs in bacteria. These sRNAs have been linked to diverse environmental responses, yet little is known how Hfq-binding sRNAs participate in the regulatory networks associated with each response. We recently described how the Hfq-binding sRNA Spot 42 in Escherichia coli contributes to catabolite repression, a regulatory phenomenon that allows bacteria to consume some carbon sources over others. Spot 42 base pairs with numerous mRNAs encoding enzymes in central and secondary metabolism, redox balancing, and the uptake and consumption of non-preferred carbon sources. Many of the corresponding genes are transcriptionally activated by the Spot 42-repressor CRP, forming a regulatory circuit called a multi-output feedforward loop. We found that this loop influences both the steady-state levels and dynamics of gene regulation. In this article, we discuss how the CRP-Spot 42 feedforward loop is integrated into encompassing networks and how this loop may benefit enteric bacteria facing uncertain and changing nutrient conditions. PMID:21788732

AFIR: A Dimensionless Potency Metric for Characterizing the Activity of Monoclonal Antibodies

PubMed Central

Ramakrishna, R

2017-01-01

For monoclonal antibody (mAb) drugs, soluble targets may accumulate several thousand fold after binding to the drug. Time course data of mAb and total target is often collected and, although free target is more closely related to clinical effect, it is difficult to measure. Therefore, mathematical models of this data are used to predict target engagement. In this article, a “potency factor” is introduced as an approximation for the model‐predicted target inhibition. This potency factor is defined to be the time‐Averaged Free target concentration to Initial target concentration Ratio (AFIR), and it depends on three key quantities: the average drug concentration at steady state; the binding affinity; and the degree of target accumulation. AFIR provides the intuition for how changes in dosing regimen and binding affinity affect target capture and AFIR can be used to predict the druggability of new targets and the expected benefits of more potent, second‐generation mAbs. PMID:28375563

An investigation about the structures, thermodynamics and kinetics of the formic acid involved molecular clusters

NASA Astrophysics Data System (ADS)

Zhang, Rui; Jiang, Shuai; Liu, Yi-Rong; Wen, Hui; Feng, Ya-Juan; Huang, Teng; Huang, Wei

2018-05-01

Despite the very important role of atmospheric aerosol nucleation in climate change and air quality, the detailed aerosol nucleation mechanism is still unclear. Here we investigated the formic acid (FA) involved multicomponent nucleation molecular clusters including sulfuric acid (SA), dimethylamine (DMA) and water (W) through a quantum chemical method. The thermodynamics and kinetics analysis was based on the global minima given by Basin-Hopping (BH) algorithm coupled with Density Functional Theory (DFT) and subsequent benchmarked calculations. Then the interaction analysis based on ElectroStatic Potential (ESP), Topological and Atomic Charges analysis was made to characterize the binding features of the clusters. The results show that FA binds weakly with the other molecules in the cluster while W binds more weakly. Further kinetic analysis about the time evolution of the clusters show that even though the formic acid's weak interaction with other nucleation precursors, its effect on sulfuric acid dimer steady state concentration cannot be neglected due to its high concentration in the atmosphere.

Steady States, Fluctuation-Dissipation Theorems and Homogenization for Reversible Diffusions in a Random Environment

NASA Astrophysics Data System (ADS)

Mathieu, P.; Piatnitski, A.

2018-04-01

Prolongating our previous paper on the Einstein relation, we study the motion of a particle diffusing in a random reversible environment when subject to a small external forcing. In order to describe the long time behavior of the particle, we introduce the notions of steady state and weak steady state. We establish the continuity of weak steady states for an ergodic and uniformly elliptic environment. When the environment has finite range of dependence, we prove the existence of the steady state and weak steady state and compute its derivative at a vanishing force. Thus we obtain a complete `fluctuation-dissipation Theorem' in this context as well as the continuity of the effective variance.

Steady state volcanism - Evidence from eruption histories of polygenetic volcanoes

NASA Technical Reports Server (NTRS)

Wadge, G.

1982-01-01

Cumulative volcano volume curves are presented as evidence for steady-state behavior at certain volcanoes and to develop a model of steady-state volcanism. A minimum criteria of five eruptions over a year was chosen to characterize a steady-state volcano. The subsequent model features a constant head of magmatic pressure from a reservoir supplied from depth, a sawtooth curve produced by the magma arrivals or discharge from the subvolcanic reservoir, large volume eruptions with long repose periods, and conditions of nonsupply of magma. The behavior of Mts. Etna, Nyamuragira, and Kilauea are described and show continuous levels of plasma output resulting in cumulative volume increases. Further discussion is made of steady-state andesitic and dacitic volcanism, long term patterns of the steady state, and magma storage, and the lack of a sufficient number of steady-state volcanoes in the world is taken as evidence that further data is required for a comprehensive model.

Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells.

PubMed

Fehres, Cynthia M; Bruijns, Sven C M; Sotthewes, Brigit N; Kalay, Hakan; Schaffer, Lana; Head, Steven R; de Gruijl, Tanja D; Garcia-Vallejo, Juan J; van Kooyk, Yvette

2015-01-01

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.

Pre-steady-state kinetic analysis of the three Escherichia coli pseudouridine synthases TruB, TruA, and RluA reveals uniformly slow catalysis

PubMed Central

Wright, Jaden R.; Keffer-Wilkes, Laura C.; Dobing, Selina R.; Kothe, Ute

2011-01-01

Pseudouridine synthases catalyze formation of the most abundant modification of functional RNAs by site-specifically isomerizing uridines to pseudouridines. While the structure and substrate specificity of these enzymes have been studied in detail, the kinetic and the catalytic mechanism of pseudouridine synthases remain unknown. Here, the first pre-steady-state kinetic analysis of three Escherichia coli pseudouridine synthases is presented. A novel stopped-flow absorbance assay revealed that substrate tRNA binding by TruB takes place in two steps with an overall rate of 6 sec−1. In order to observe catalysis of pseudouridine formation directly, the traditional tritium release assay was adapted for the quench-flow technique, allowing, for the first time, observation of a single round of pseudouridine formation. Thereby, the single-round rate constant of pseudouridylation (kΨ) by TruB was determined to be 0.5 sec−1. This rate constant is similar to the kcat obtained under multiple-turnover conditions in steady-state experiments, indicating that catalysis is the rate-limiting step for TruB. In order to investigate if pseudouridine synthases are characterized by slow catalysis in general, the rapid kinetic quench-flow analysis was also performed with two other E. coli enzymes, RluA and TruA, which displayed rate constants of pseudouridine formation of 0.7 and 0.35 sec−1, respectively. Hence, uniformly slow catalysis might be a general feature of pseudouridine synthases that share a conserved catalytic domain and supposedly use the same catalytic mechanism. PMID:21998096

Estimating Systemic Exposure to Levonorgestrel from an Oral Contraceptive

PubMed Central

Basaraba, Cale N; Westhoff, Carolyn L; Pike, Malcolm C; Nandakumar, Renu; Cremers, Serge

2017-01-01

Objective The gold standard for measuring oral contraceptive (OC) pharmacokinetics is the 24-hour steady-state area-under-the-curve (AUC). We conducted this study to assess whether limited sampling at steady state or measurements following use of one or two OCs could provide an adequate proxy in epidemiological studies for the progestin 24-hour steady-state AUC of a particular OC. Study Design We conducted a 13-sample, 24-hour pharmacokinetic study on both day 1 and day 21 of the first cycle of a monophasic OC containing 30 μg ethinyl estradiol and 150 μg levonorgestrel (LNG) in 17 normal-weight healthy white women, and a single-dose 9-sample study of the same OC after a one-month washout. We compared the 13-sample steady-state results with several steady-state and single-dose results calculated using parsimonious sampling schemes. Results The 13-sample steady-state 24-hour LNG AUC was highly correlated with the steady-state 24-hour trough value (r = 0.95; 95% CI [0.85, 0.98]) and with the steady-state 6, 8, 12 and 16-hour values (0.92 ≤ r ≤ 0.95). The trough values after one or two doses were moderately correlated with the steady-state 24-hour AUC value (r = 0.70; 95% CI [0.27, 0.90] and 0.77; 95% CI [0.40, 0.92], respectively). Conclusions Single time-point concentrations at steady-state and after administration of one or two OCs gave highly to moderately correlated estimates of steady-state LNG AUC. Using such measures could facilitate prospective pharmaco-epidemiologic studies of the OC and its side effects. PMID:28041990

Block of Inactivation-deficient Na+ Channels by Local Anesthetics in Stably Transfected Mammalian Cells

PubMed Central

Wang, Sho-Ya; Mitchell, Jane; Moczydlowski, Edward; Wang, Ging Kuo

2004-01-01

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na+ channels with higher affinities. However, an alternative view suggests that activation of Na+ channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na+ channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC50) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 ± 3.3 μM (n = 5) and 81.7 ± 10.6 μM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC50 values for resting-channel block at −140 mV were >12-fold higher than those for open-channel block. With 300 μM benzocaine, rapid time-dependent block (τ ≈ 0.8 ms) of inactivation-deficient Na+ currents occurred at +30 mV, but such a rapid time-dependent block was not evident at −30 mV. The peak current at −30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na+ channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding. PMID:15545401

Local anesthetics disrupt energetic coupling between the voltage-sensing segments of a sodium channel.

PubMed

Muroi, Yukiko; Chanda, Baron

2009-01-01

Local anesthetics block sodium channels in a state-dependent fashion, binding with higher affinity to open and/or inactivated states. Gating current measurements show that local anesthetics immobilize a fraction of the gating charge, suggesting that the movement of voltage sensors is modified when a local anesthetic binds to the pore of the sodium channel. Here, using voltage clamp fluorescence measurements, we provide a quantitative description of the effect of local anesthetics on the steady-state behavior of the voltage-sensing segments of a sodium channel. Lidocaine and QX-314 shifted the midpoints of the fluorescence-voltage (F-V) curves of S4 domain III in the hyperpolarizing direction by 57 and 65 mV, respectively. A single mutation in the S6 of domain IV (F1579A), a site critical for local anesthetic block, abolished the effect of QX-314 on the voltage sensor of domain III. Both local anesthetics modestly shifted the F-V relationships of S4 domain IV toward hyperpolarized potentials. In contrast, the F-V curve of the S4 domain I was shifted by 11 mV in the depolarizing direction upon QX-314 binding. These antagonistic effects of the local anesthetic indicate that the drug modifies the coupling between the voltage-sensing domains of the sodium channel. Our findings suggest a novel role of local anesthetics in modulating the gating apparatus of the sodium channel.

Error-based Extraction of States and Energy Landscapes from Experimental Single-Molecule Time-Series

NASA Astrophysics Data System (ADS)

Taylor, J. Nicholas; Li, Chun-Biu; Cooper, David R.; Landes, Christy F.; Komatsuzaki, Tamiki

2015-03-01

Characterization of states, the essential components of the underlying energy landscapes, is one of the most intriguing subjects in single-molecule (SM) experiments due to the existence of noise inherent to the measurements. Here we present a method to extract the underlying state sequences from experimental SM time-series. Taking into account empirical error and the finite sampling of the time-series, the method extracts a steady-state network which provides an approximation of the underlying effective free energy landscape. The core of the method is the application of rate-distortion theory from information theory, allowing the individual data points to be assigned to multiple states simultaneously. We demonstrate the method's proficiency in its application to simulated trajectories as well as to experimental SM fluorescence resonance energy transfer (FRET) trajectories obtained from isolated agonist binding domains of the AMPA receptor, an ionotropic glutamate receptor that is prevalent in the central nervous system.

Understanding the Broad Substrate Repertoire of Nitroreductase Based on Its Kinetic Mechanism*

PubMed Central

Pitsawong, Warintra; Hoben, John P.; Miller, Anne-Frances

2014-01-01

The oxygen-insensitive nitroreductase from Enterobacter cloacae (NR) catalyzes two-electron reduction of nitroaromatics to the corresponding nitroso compounds and, subsequently, to hydroxylamine products. NR has an unusually broad substrate repertoire, which may be related to protein dynamics (flexibility) and/or a simple non-selective kinetic mechanism. To investigate the possible role of mechanism in the broad substrate repertoire of NR, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4 °C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.90 ± 0.09 s−1, indicating one-step binding before the flavin oxidation step. There is no evidence for a distinct binding step in which specificity might be enforced. The reduction of p-NBA is rate-limiting in steady-state turnover (1.7 ± 0.3 s−1). The pre-steady-state reduction kinetics of NR by NADH indicate that NADH reduces the enzyme with a rate constant of 700 ± 20 s−1 and a dissociation constant of 0.51 ± 0.04 mm. Thus, we demonstrate simple transient kinetics in both the reductive and oxidative half-reactions that help to explain the broad substrate repertoire of NR. Finally, we tested the ability of NR to reduce para-hydroxylaminobenzoic acid, demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus E. cloacae NR is not a good candidate for enzymatic production of aromatic amines. PMID:24706760

Malate-mediated carbon catabolite repression in Bacillus subtilis involves the HPrK/CcpA pathway.

PubMed

Meyer, Frederik M; Jules, Matthieu; Mehne, Felix M P; Le Coq, Dominique; Landmann, Jens J; Görke, Boris; Aymerich, Stéphane; Stülke, Jörg

2011-12-01

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate.

Implementation of steady state approximation for modelling of reaction kinetic of UV catalysed hydrogen peroxide oxidation of starch

NASA Astrophysics Data System (ADS)

Kumoro, Andri Cahyo; Retnowati, Diah Susetyo; Ratnawati, Budiyati, Catarina Sri

2015-12-01

With regard to its low viscosity, high stability, clarity, film forming and binding properties, oxidised starch has been widely used in various applications specifically in the food, paper, textile, laundry finishing and binding materials industries. A number of methods have been used to produce oxidised starch through reactions with various oxidizing agents, such as hydrogen peroxide, air oxygen, ozone, bromine, chromic acid, permanganate, nitrogen dioxide and hypochlorite. Unfortunately, most of previous works reported in the literatures were focused on the study of reaction mechanism and physicochemical properties characterization of the oxidised starches produced without investigation of the reaction kinetics of the oxidation process. This work aimed to develop a simple kinetic model for UV catalysed hydrogen peroxide oxidation of starch through implementation of steady state approximation for the radical reaction rates. The model was then verified using experimental data available in the literature. The model verification revealed that the proposed model shows its good agreement with the experimental data as indicated by an average absolute relative error of only 2.45%. The model also confirmed that carboxyl groups are oxidised further by hydroxyl radical. The carbonyl production rate was found to follow first order reaction with respect to carbonyl concentration. Similarly, carboxyl production rate also followed first order reaction with respect to carbonyl concentration. The apparent reaction rate constant for carbonyl formation and oxidation were 6.24 × 104 s-1 and 1.01 × 104 M-1.s-1, respectively. While apparent reaction rate constant for carboxyl oxidation was 4.86 × 104 M-1.s-1.

Role of FAST Kinase Domains 3 (FASTKD3) in Post-transcriptional Regulation of Mitochondrial Gene Expression*

PubMed Central

Boehm, Erik; Zornoza, María; Jourdain, Alexis A.; Delmiro Magdalena, Aitor; García-Consuegra, Inés; Torres Merino, Rebeca; Orduña, Antonio; Martín, Miguel A.; Martinou, Jean-Claude; De la Fuente, Miguel A.; Simarro, María

2016-01-01

The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria. PMID:27789713

Multimode optical fibers: steady state mode exciter.

PubMed

Ikeda, M; Sugimura, A; Ikegami, T

1976-09-01

The steady state mode power distribution of the multimode graded index fiber was measured. A simple and effective steady state mode exciter was fabricated by an etching technique. Its insertion loss was 0.5 dB for an injection laser. Deviation in transmission characteristics of multimode graded index fibers can be avoided by using the steady state mode exciter.

Second-chance signal transduction explains cooperative flagellar switching.

PubMed

Zot, Henry G; Hasbun, Javier E; Minh, Nguyen Van

2012-01-01

The reversal of flagellar motion (switching) results from the interaction between a switch complex of the flagellar rotor and a torque-generating stationary unit, or stator (motor unit). To explain the steeply cooperative ligand-induced switching, present models propose allosteric interactions between subunits of the rotor, but do not address the possibility of a reaction that stimulates a bidirectional motor unit to reverse direction of torque. During flagellar motion, the binding of a ligand-bound switch complex at the dwell site could excite a motor unit. The probability that another switch complex of the rotor, moving according to steady-state rotation, will reach the same dwell site before that motor unit returns to ground state will be determined by the independent decay rate of the excited-state motor unit. Here, we derive an analytical expression for the energy coupling between a switch complex and a motor unit of the stator complex of a flagellum, and demonstrate that this model accounts for the cooperative switching response without the need for allosteric interactions. The analytical result can be reproduced by simulation when (1) the motion of the rotor delivers a subsequent ligand-bound switch to the excited motor unit, thereby providing the excited motor unit with a second chance to remain excited, and (2) the outputs from multiple independent motor units are constrained to a single all-or-none event. In this proposed model, a motor unit and switch complex represent the components of a mathematically defined signal transduction mechanism in which energy coupling is driven by steady-state and is regulated by stochastic ligand binding. Mathematical derivation of the model shows the analytical function to be a general form of the Hill equation (Hill AV (1910) The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J Physiol 40: iv-vii).

Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

PubMed Central

1984-01-01

Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites. PMID:6088662

Catalytic site interactions in yeast OMP synthase.

PubMed

Hansen, Michael Riis; Barr, Eric W; Jensen, Kaj Frank; Willemoës, Martin; Grubmeyer, Charles; Winther, Jakob R

2014-01-15

The enigmatic kinetics, half-of-the-sites binding, and structural asymmetry of the homodimeric microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) have been proposed to result from an alternating site mechanism in these domain-swapped enzymes [R.W. McClard et al., Biochemistry 45 (2006) 5330-5342]. This behavior was investigated in the yeast enzyme by mutations in the conserved catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif. Although the reaction is mechanistically sequential, the wild-type (WT) enzyme shows parallel lines in double reciprocal initial velocity plots. Replacement of Lys106, the postulated intersubunit communication device, produced intersecting lines in kinetic plots with a 2-fold reduction of kcat. Loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) mutant proteins, each without detectable enzymatic activity and ablated ability to bind PRPP, complemented to produce a heterodimer with a single fully functional active site showing intersecting initial velocity plots. Equilibrium binding of PRPP and orotidine 5'-monophosphate showed a single class of two binding sites per dimer in WT and K106S enzymes. Evidence here shows that the enzyme does not follow half-of-the-sites cooperativity; that interplay between catalytic sites is not an essential feature of the catalytic mechanism; and that parallel lines in steady-state kinetics probably arise from tight substrate binding. Copyright © 2013. Published by Elsevier Inc.

Illustrating the Steady-State Condition and the Single-Molecule Kinetic Method with the NMDA Receptor

ERIC Educational Resources Information Center

Kosman, Daniel J.

2009-01-01

The steady-state is a fundamental aspect of biochemical pathways in cells; indeed, the concept of steady-state is a definition of life itself. In a simple enzyme kinetic scheme, the steady-state condition is easy to define analytically but experimentally often difficult to capture because of its evanescent quality; the initial, constant velocity…

Steady state and a general scale law of deformation

NASA Astrophysics Data System (ADS)

Huang, Yan

2017-07-01

Steady state deformation has been characterized based on the experimental results for dilute single-phase aluminium alloys. It was found that although characteristic properties such as flow stress and grain size remained constant with time, a continuous loss of grain boundaries occurred as an essential feature at steady state. A physical model, which takes into account the activity of grain boundary dislocations, was developed to describe the kinetics of steady state deformation. According to this model, the steady state as a function of strain rate and temperature defines the limit of the conventional grain size and strength relationship, i.e., the Hall-Petch relation holds when the grain size is larger than that at the steady state, and an inverse Hall-Petch relation takes over if grain size is smaller than the steady state value. The transition between the two relationships relating grain size and strength is a phenomenon that depends on deformation conditions, rather than an intrinsic property as generally perceived. A general scale law of deformation is established accordingly.

Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase.

PubMed

Su, Q; Klinman, J P

1998-09-08

Bovine serum amine oxidase (BSAO) catalyzes the oxidative deamination of primary amines, concomitant with the reduction of molecular oxygen to hydrogen peroxide via a ping-pong mechanism. A protocol has been developed for an analysis of chemical and kinetic mechanisms in the conversion of dioxygen to hydrogen peroxide. Steady-state kinetics show that two groups need to be deprotonated to facilitate the oxidative half-reaction. The pH dependence of Vmax/Km(O2) reveals pKa's of 6.2 +/- 0.3 and 7.0 +/- 0.2, respectively. A pKa of 7.2 +/- 0.1 has been obtained from a titration of anaerobically reduced BSAO using UV-vis spectrophotometry. The near identity of the pKa obtained from the reduced enzyme titration with the second pKa from steady-state kinetics suggests that this second pKa arises from the reduced cofactor. The assignment of pKa is supported by the observed pH dependence for formation of the cofactor semiquinone signal, detected by EPR spectroscopy under anaerobic conditions. To address the nature of rate-limiting steps in the oxidative half-reaction, the solvent isotope effect, viscosity effect, and O-18 isotope effect on Vmax/Km(O2) have been determined. The solvent isotope effect is indistinguishable from unity, ruling out a proton transfer as a rate-determining step. Use of glucose as a solvent viscosogen shows no viscosity effect, indicating that binding of oxygen is not in the rate-determining step. The O-18 kinetic isotope effect is independent of pH with an average value of 18(V/K) = 1.0097 +/- 0. 0010. This has been compared to calculated equilibrium O-18 isotope effects for various dioxygen intermediate species [Tian and Klinman (1993) J. Am. Chem. Soc. 115, 8891], leading to the conclusion that either the first electron transfer to dioxygen or the desorption of product peroxide from a Cu(II)-OOH complex could be the rate-limiting step. The distribution of steady-state enzyme species was, therefore, analyzed through a combination of stopped-flow experiments and analysis of DV and D(V/K) for benzylamine oxidation. We conclude that the major species accumulating in the steady state are the oxidized cofactor-substrate Schiff base complex and the reduced, aminoquinol form of cofactor. These data rule out a slow release of product hydroperoxide from the aminoquinone form of enzyme, leading to the conclusion that the first electron transfer from substrate-reduced cofactor to dioxygen is the rate-determining step in the oxidative half-reaction. This step is also estimated to be 40% rate-limiting in kcat. An important mechanistic conclusion from this study is that dioxygen binding is a separate step from the rate-limiting electron-transfer step to form superoxide. On the basis of a recently determined X-ray structure for the active form of a yeast amine oxidase from Hansenula polymorpha [Li et al. (1998) Structure 6, 293], a hydrophobic space has been identified near the O-2 position of reduced cofactor as the putative dioxygen binding site. Movement of superoxide from this site onto the Cu(II) at the active site may occur prior to further electron transfer from cofactor to superoxide.

Phased array ghost elimination (PAGE) for segmented SSFP imaging with interrupted steady-state.

PubMed

Kellman, Peter; Guttman, Michael A; Herzka, Daniel A; McVeigh, Elliot R

2002-12-01

Steady-state free precession (SSFP) has recently proven to be valuable for cardiac imaging due to its high signal-to-noise ratio and blood-myocardium contrast. Data acquired using ECG-triggered, segmented sequences during the approach to steady-state, or return to steady-state after interruption, may have ghost artifacts due to periodic k-space distortion. Schemes involving several preparatory RF pulses have been proposed to restore steady-state, but these consume imaging time during early systole. Alternatively, the phased-array ghost elimination (PAGE) method may be used to remove ghost artifacts from the first several frames. PAGE was demonstrated for cardiac cine SSFP imaging with interrupted steady-state using a simple alpha/2 magnetization preparation and storage scheme and a spatial tagging preparation.

In vitro study on binding interaction of quinapril with bovine serum albumin (BSA) using multi-spectroscopic and molecular docking methods.

PubMed

Shi, Jie-Hua; Pan, Dong-Qi; Jiang, Min; Liu, Ting-Ting; Wang, Qi

2017-08-01

The binding interaction between quinapril (QNPL) and bovine serum albumin (BSA) in vitro has been investigated using UV absorption spectroscopy, steady-state fluorescence spectroscopic, synchronous fluorescence spectroscopy, 3D fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and molecular docking methods for obtaining the binding information of QNPL with BSA. The experimental results confirm that the quenching mechanism of the intrinsic fluorescence of BSA induced by QNPL is static quenching based on the decrease in the quenching constants of BSA in the presence of QNPL with the increase in temperature and the quenching rates of BSA larger than 10 10  L mol -1  s -1 , indicating forming QNPL-BSA complex through the intermolecular binding interaction. The binding constant for the QNPL-BSA complex is in the order of 10 5  M -1 , indicating there is stronger binding interaction of QNPL with BSA. The analysis of thermodynamic parameters together with molecular docking study reveal that the main binding forces in the binding process of QNPL with BSA are van der Waal's forces and hydrogen bonding interaction. And, the binding interaction of BSA with QNPL is an enthalpy-driven process. Based on Förster resonance energy transfer, the binding distance between QNPL and BSA is calculated to be 2.76 nm. The results of the competitive binding experiments and molecular docking confirm that QNPL binds to sub-domain IIA (site I) of BSA. It is confirmed there is a slight change in the conformation of BSA after binding QNPL, but BSA still retains its secondary structure α-helicity.

Bioaccumulation factors and the steady state assumption for cesium isotopes in aquatic foodwebs near nuclear facilities.

PubMed

Rowan, D J

2013-07-01

Steady state approaches, such as transfer coefficients or bioaccumulation factors, are commonly used to model the bioaccumulation of (137)Cs in aquatic foodwebs from routine operations and releases from nuclear generating stations and other nuclear facilities. Routine releases from nuclear generating stations and facilities, however, often consist of pulses as liquid waste is stored, analyzed to ensure regulatory compliance and then released. The effect of repeated pulse releases on the steady state assumption inherent in the bioaccumulation factor approach has not been evaluated. In this study, I examine the steady state assumption for aquatic biota by analyzing data for two cesium isotopes in the same biota, one isotope in steady state (stable (133)Cs) from geologic sources and the other released in pulses ((137)Cs) from reactor operations. I also compare (137)Cs bioaccumulation factors for similar upstream populations from the same system exposed solely to weapon test (137)Cs, and assumed to be in steady state. The steady state assumption appears to be valid for small organisms at lower trophic levels (zooplankton, rainbow smelt and 0+ yellow perch) but not for older and larger fish at higher trophic levels (walleye). Attempts to account for previous exposure and retention through a biokinetics approach had a similar effect on steady state, upstream and non-steady state, downstream populations of walleye, but were ineffective in explaining the more or less constant deviation between fish with steady state exposures and non-steady state exposures of about 2-fold for all age classes of walleye. These results suggest that for large, piscivorous fish, repeated exposure to short duration, pulse releases leads to much higher (137)Cs BAFs than expected from (133)Cs BAFs for the same fish or (137)Cs BAFs for similar populations in the same system not impacted by reactor releases. These results suggest that the steady state approach should be used with caution in any situation where reactor releases are episodic or pulse in nature, even if the magnitude of these releases is small. Copyright © 2012. Published by Elsevier Ltd.

Seasonal changes in plasma androgens, glucocorticoids and glucocorticoid-binding proteins in the marsupial sugar glider Petaurus breviceps.

PubMed

Bradley, A J; Stoddart, D M

1992-01-01

An investigation spanning two breeding seasons was carried out to examine endocrine changes associated with reproduction in a wild population of the marsupial sugar glider Petaurus breviceps, a small arboreal gliding possum. Using techniques of equilibrium dialysis and polyacrylamide gel electrophoresis at steady-state conditions, a high-affinity, low-capacity glucocorticoid-binding protein was demonstrated in the plasma of Petaurus breviceps. Equilibrium dialysis at 36 degrees C using cortisol gave a high-affinity binding constant of 95 +/- 5.2 litres/mumol for a presumed corticosteroid-binding globulin (CBG) while the binding constant for the cortisol-albumin interaction was 3.5 +/- 0.4 litres/mmol. There was no difference between the sexes in the affinity of binding of cortisol to CBG; however, the cortisol-binding capacity underwent seasonal variation in both sexes. Progesterone was bound strongly to the presumed CBG while neither oestradiol nor aldosterone appeared to be bound with high affinity to P. breviceps plasma. In the males, peaks in the plasma concentration of testosterone coincided with the July-September breeding season in both years. A significant inverse relationship was shown to exist between the plasma testosterone concentration and the CBG-binding capacity. In both sexes an increase occurred in the plasma concentration of free cortisol during the first breeding season, a pattern which was not repeated in the subsequent breeding season, possibly due to a lower population density in that year.

Study on the interactions between toxic nitroanilines and lysozyme by spectroscopic approaches and molecular modeling.

PubMed

Gu, Yunlan; Wang, Yanqing; Zhang, Hongmei

2018-05-05

Being exogenous environmental pollutants, nitroanilines (NAs) are highly toxic and have mutagenic and carcinogenic activity. Being lack of studies on interactions between NAs and lysozyme at molecular level, the binding interactions of lysozyme with o-nitroaniline (oNA), m-nitroaniline (mNA) and p-nitroaniline (pNA) were investigated by means of steady-state fluorescence, synchronous fluorescence, UV-vis absorption spectroscopy, as well as molecular modeling. The experimental results revealed that the fluorescence of lysozyme is quenched by oNA and mNA through a static quenching, while the fluorescence quenching triggered by pNA is a combined dynamic and static quenching. The number of binding sites (n) and the binding constant (K b ) corresponding thermodynamic parameters ΔH ⊖ , ΔS ⊖ , ΔG ⊖ at different temperatures were calculated. The reactions between NAs and lysozyme were spontaneous and entropy driven and the binding of NAs to lysozyme induced conformation changes of lysozyme. The difference of the position of -NO 2 group affected the binding and the binding constants K b decreased in the following pattern: K b (pNA) >K b (mNA) >K b (oNA). Molecular docking studies were performed to reveal the most favorable binding sites of NAs on lysozyme. Our recently results could offer mechanistic insights into the nature of the binding interactions between NAs and lysozyme and provide information about the toxicity risk of NAs to human health. Copyright © 2018 Elsevier B.V. All rights reserved.

A versatile assay for RNA-binding proteins in living cells

PubMed Central

Strein, Claudia; Alleaume, Anne-Marie; Rothbauer, Ulrich; Hentze, Matthias W.; Castello, Alfredo

2014-01-01

RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein–mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology. PMID:24664470

Steady shape analysis of tomographic pumping tests for characterization of aquifer heterogeneities

USGS Publications Warehouse

Bohling, Geoffrey C.; Zhan, Xiaoyong; Butler, James J.; Zheng, Li

2002-01-01

Hydraulic tomography, a procedure involving the performance of a suite of pumping tests in a tomographic format, provides information about variations in hydraulic conductivity at a level of detail not obtainable with traditional well tests. However, analysis of transient data from such a suite of pumping tests represents a substantial computational burden. Although steady state responses can be analyzed to reduce this computational burden significantly, the time required to reach steady state will often be too long for practical applications of the tomography concept. In addition, uncertainty regarding the mechanisms driving the system to steady state can propagate to adversely impact the resulting hydraulic conductivity estimates. These disadvantages of a steady state analysis can be overcome by exploiting the simplifications possible under the steady shape flow regime. At steady shape conditions, drawdown varies with time but the hydraulic gradient does not. Thus transient data can be analyzed with the computational efficiency of a steady state model. In this study, we demonstrate the value of the steady shape concept for inversion of hydraulic tomography data and investigate its robustness with respect to improperly specified boundary conditions.

Steady states and stability in metabolic networks without regulation.

PubMed

Ivanov, Oleksandr; van der Schaft, Arjan; Weissing, Franz J

2016-07-21

Metabolic networks are often extremely complex. Despite intensive efforts many details of these networks, e.g., exact kinetic rates and parameters of metabolic reactions, are not known, making it difficult to derive their properties. Considerable effort has been made to develop theory about properties of steady states in metabolic networks that are valid for any values of parameters. General results on uniqueness of steady states and their stability have been derived with specific assumptions on reaction kinetics, stoichiometry and network topology. For example, deep results have been obtained under the assumptions of mass-action reaction kinetics, continuous flow stirred tank reactors (CFSTR), concordant reaction networks and others. Nevertheless, a general theory about properties of steady states in metabolic networks is still missing. Here we make a step further in the quest for such a theory. Specifically, we study properties of steady states in metabolic networks with monotonic kinetics in relation to their stoichiometry (simple and general) and the number of metabolites participating in every reaction (single or many). Our approach is based on the investigation of properties of the Jacobian matrix. We show that stoichiometry, network topology, and the number of metabolites that participate in every reaction have a large influence on the number of steady states and their stability in metabolic networks. Specifically, metabolic networks with single-substrate-single-product reactions have disconnected steady states, whereas in metabolic networks with multiple-substrates-multiple-product reactions manifolds of steady states arise. Metabolic networks with simple stoichiometry have either a unique globally asymptotically stable steady state or asymptotically stable manifolds of steady states. In metabolic networks with general stoichiometry the steady states are not always stable and we provide conditions for their stability. In order to demonstrate the biological relevance we illustrate the results on the examples of the TCA cycle, the mevalonate pathway and the Calvin cycle. Copyright © 2016 Elsevier Ltd. All rights reserved.

Occupancy of the Zinc-binding Site by Transition Metals Decreases the Substrate Affinity of the Human Dopamine Transporter by an Allosteric Mechanism*

PubMed Central

Li, Yang; Mayer, Felix P.; Hasenhuetl, Peter S.; Burtscher, Verena; Schicker, Klaus; Sitte, Harald H.; Freissmuth, Michael; Sandtner, Walter

2017-01-01

The human dopamine transporter (DAT) has a tetrahedral Zn2+-binding site. Zn2+-binding sites are also recognized by other first-row transition metals. Excessive accumulation of manganese or of copper can lead to parkinsonism because of dopamine deficiency. Accordingly, we examined the effect of Mn2+, Co2+, Ni2+, and Cu2+ on transport-associated currents through DAT and DAT-H193K, a mutant with a disrupted Zn2+-binding site. All transition metals except Mn2+ modulated the transport cycle of wild-type DAT with affinities in the low micromolar range. In this concentration range, they were devoid of any action on DAT-H193K. The active transition metals reduced the affinity of DAT for dopamine. The affinity shift was most pronounced for Cu2+, followed by Ni2+ and Zn2+ (= Co2+). The extent of the affinity shift and the reciprocal effect of substrate on metal affinity accounted for the different modes of action: Ni2+ and Cu2+ uniformly stimulated and inhibited, respectively, the substrate-induced steady-state currents through DAT. In contrast, Zn2+ elicited biphasic effects on transport, i.e. stimulation at 1 μm and inhibition at 10 μm. A kinetic model that posited preferential binding of transition metal ions to the outward-facing apo state of DAT and a reciprocal interaction of dopamine and transition metals recapitulated all experimental findings. Allosteric activation of DAT via the Zn2+-binding site may be of interest to restore transport in loss-of-function mutants. PMID:28096460

Binding of resveratrol to the minor groove of DNA sequences with AATT and TTAA segments induces differential stability.

PubMed

Nair, Maya S; D'Mello, Samar; Pant, Rashmi; Poluri, Krishna Mohan

2017-05-01

Interactions of a natural stilbene compound, resveratrol with two DNA sequences containing AATT/TTAA segments have been studied. Resveratrol is found to interact with both the sequences. The mode of interaction has been studied using absorption, steady state fluorescence and circular dichroism spectroscopic techniques. UV-visible absorption and fluorescence studies provided the information regarding the binding constants and the stoichiometry of binding, whereas circular dichroism studies depicted the structural changes in DNA upon resveratrol binding. Our results evidenced that, though resveratrol showed similar affinity to both the sequences, the mode of interactions was different. The binding constants of resveratrol to AATT/TTAA sequences were found to be 7.55×10 5 M -1 and 5.42×10 5 M -1 respectively. Spectroscopic data evidenced for a groove binding interaction. Melting studies showed that the binding of resveratrol induces differential stability to the DNA sequences d(CGTTAACG) 2 and d(CGAATTCG) 2 . Fluorescence data showed a stoichiometry of 1:1 for d(CGAATTCG) 2 -resveratrol complex and 1:4 for d(CGTTAACG) 2 -resveratrol complex. Molecular docking studies demonstrated that resveratrol binds to the minor groove region of both the sequences to form stable complexes with varied atomic contacts to the DNA bases or backbone. Both the complexes are stabilized by hydrogen bond formation. Our results evidenced that modulation of DNA sequence within the same bases can greatly alter the binding geometry and stability of the complex upon binding to small molecule inhibitor compounds like resveratrol. Copyright © 2017 Elsevier B.V. All rights reserved.

Supramolecular complex of a fused zinc phthalocyanine-zinc porphyrin dyad assembled by two imidazole-C60 units: ultrafast photoevents.

PubMed

Follana-Berná, Jorge; Seetharaman, Sairaman; Martín-Gomis, Luis; Charalambidis, Georgios; Trapali, Adelais; Karr, Paul A; Coutsolelos, Athanassios G; Fernández-Lázaro, Fernando; D'Souza, Francis; Sastre-Santos, Ángela

2018-03-14

A new zinc phthalocyanine-zinc porphyrin dyad (ZnPc-ZnP) fused through a pyrazine ring has been synthesized as a receptor for imidazole-substituted C 60 (C 60 Im) electron acceptor. Self-assembly via metal-ligand axial coordination and the pertinent association constants in solution were determined by 1 H-NMR, UV-Vis and fluorescence titration experiments at room temperature. The designed host was able to bind up to two C 60 Im electron acceptor guest molecules to yield C 60 Im:ZnPc-ZnP:ImC 60 donor-acceptor supramolecular complex. The spectral data showed that the two binding sites behave independently with binding constants similar in magnitude. Steady-state fluorescence studies were indicative of an efficient singlet-singlet energy transfer from zinc porphyrin to zinc phthalocyanine within the fused dyad. Accordingly, the transient absorption studies covering a wide timescale of femto-to-milli seconds revealed ultrafast energy transfer from 1 ZnP* to ZnPc (k EnT ∼ 10 12 s -1 ) in the fused dyad. Further, a photo induced electron transfer was observed in the supramolecularly assembled C 60 Im:ZnPc-ZnP:ImC 60 donor-acceptor complex leading to charge separated states, which persisted for about 200 ns.

Thermally induced charge current through long molecules

NASA Astrophysics Data System (ADS)

Zimbovskaya, Natalya A.; Nitzan, Abraham

2018-01-01

In this work, we theoretically study steady state thermoelectric transport through a single-molecule junction with a long chain-like bridge. Electron transmission through the system is computed using a tight-binding model for the bridge. We analyze dependences of thermocurrent on the bridge length in unbiased and biased systems operating within and beyond the linear response regime. It is shown that the length-dependent thermocurrent is controlled by the lineshape of electron transmission in the interval corresponding to the HOMO/LUMO transport channel. Also, it is demonstrated that electron interactions with molecular vibrations may significantly affect the length-dependent thermocurrent.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding sitemore » are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.« less

Interaction of Flavonoids from Woodwardia unigemmata with Bovine Serum Albumin (BSA): Application of Spectroscopic Techniques and Molecular Modeling Methods.

PubMed

Ma, Rui; Pan, Hong; Shen, Tao; Li, Peng; Chen, Yanan; Li, Zhenyu; Di, Xiaxia; Wang, Shuqi

2017-08-09

Phytochemical investigation on the methanol extract of Woodwardia unigemmata resulted in the isolation of seven flavonoids, including one new flavonol acylglycoside ( 1 ). The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis and comparison of literature data. The multidrug resistance (MDR) reversing activity was evaluated for the isolated compounds using doxorubicin-resistant K562/A02 cells model. Compound 6 showed comparable MDR reversing effect to verapamil. Furthermore, the interaction between compounds and bovine serum albumin (BSA) was investigated by spectroscopic methods, including steady-state fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopies, and molecular docking approach. The experimental results indicated that the seven flavonoids bind to BSA by static quenching mechanisms. The negative ΔH and ΔS values indicated that van der Waals interactions and hydrogen bonds contributed in the binding of compounds 2 - 6 to BSA. In the case of compounds 1 and 7 systems, the hydrophobic interactions play a major role. The binding of compounds to BSA causes slight changes in the secondary structure of BSA. There are two binding sites of compound 6 on BSA and site I is the main site according to the molecular docking studies and the site marker competitive binding assay.

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

PubMed

Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

2015-06-05

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

[Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

PubMed

Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

2012-01-01

It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range

PubMed Central

Ventura, Alejandra C.; Bush, Alan; Vasen, Gustavo; Goldín, Matías A.; Burkinshaw, Brianne; Bhattacharjee, Nirveek; Folch, Albert; Brent, Roger; Chernomoretz, Ariel; Colman-Lerner, Alejandro

2014-01-01

Cell signaling systems sense and respond to ligands that bind cell surface receptors. These systems often respond to changes in the concentration of extracellular ligand more rapidly than the ligand equilibrates with its receptor. We demonstrate, by modeling and experiment, a general “systems level” mechanism cells use to take advantage of the information present in the early signal, before receptor binding reaches a new steady state. This mechanism, pre-equilibrium sensing and signaling (PRESS), operates in signaling systems in which the kinetics of ligand-receptor binding are slower than the downstream signaling steps, and it typically involves transient activation of a downstream step. In the systems where it operates, PRESS expands and shifts the input dynamic range, allowing cells to make different responses to ligand concentrations so high as to be otherwise indistinguishable. Specifically, we show that PRESS applies to the yeast directional polarization in response to pheromone gradients. Consideration of preexisting kinetic data for ligand-receptor interactions suggests that PRESS operates in many cell signaling systems throughout biology. The same mechanism may also operate at other levels in signaling systems in which a slow activation step couples to a faster downstream step. PMID:25172920

Oligomeric Status and Nucleotide Binding Properties of the Plastid ATP/ADP Transporter 1: Toward a Molecular Understanding of the Transport Mechanism

PubMed Central

Deniaud, Aurélien; Panwar, Pankaj; Frelet-Barrand, Annie; Bernaudat, Florent; Juillan-Binard, Céline; Ebel, Christine; Rolland, Norbert; Pebay-Peyroula, Eva

2012-01-01

Background Chloroplast ATP/ADP transporters are essential to energy homeostasis in plant cells. However, their molecular mechanism remains poorly understood, primarily due to the difficulty of producing and purifying functional recombinant forms of these transporters. Methodology/Principal Findings In this work, we describe an expression and purification protocol providing good yields and efficient solubilization of NTT1 protein from Arabidopsis thaliana. By biochemical and biophysical analyses, we identified the best detergent for solubilization and purification of functional proteins, LAPAO. Purified NTT1 was found to accumulate as two independent pools of well folded, stable monomers and dimers. ATP and ADP binding properties were determined, and Pi, a co-substrate of ADP, was confirmed to be essential for nucleotide steady-state transport. Nucleotide binding studies and analysis of NTT1 mutants lead us to suggest the existence of two distinct and probably inter-dependent binding sites. Finally, fusion and deletion experiments demonstrated that the C-terminus of NTT1 is not essential for multimerization, but probably plays a regulatory role, controlling the nucleotide exchange rate. Conclusions/Significance Taken together, these data provide a comprehensive molecular characterization of a chloroplast ATP/ADP transporter. PMID:22438876

Potential toxicity and affinity of triphenylmethane dye malachite green to lysozyme.

PubMed

Ding, Fei; Li, Xiu-Nan; Diao, Jian-Xiong; Sun, Ye; Zhang, Li; Ma, Lin; Yang, Xin-Ling; Zhang, Li; Sun, Ying

2012-04-01

Malachite green is a triphenylmethane dye that is used extensively in many industrial and aquacultural processes, generating environmental concerns and health problems to human being. In this contribution, the complexation between lysozyme and malachite green was verified by means of computer-aided molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) approaches. The precise binding patch of malachite green in lysozyme has been identified from molecular modeling and ANS displacement, Trp-62, Trp-63, and Trp-108 residues of lysozyme were earmarked to possess high-affinity for this dye, the principal forces in the lysozyme-malachite green adduct are hydrophobic and π-π interactions. Steady state fluorescence proclaimed the complex of malachite green with lysozyme yields quenching through static type, which substantiates time-resolved fluorescence measurements that lysozyme-malachite green conjugation formation has an affinity of 10(3)M(-1). Moreover, via molecular modeling and also CD data, we can safely arrive at a conclusion that the polypeptide chain of lysozyme partially destabilized upon complexation with malachite green. The data emerged here will help to further understand the toxicological action of malachite green in human body. Copyright © 2012 Elsevier Inc. All rights reserved.

Computational multiple steady states for enzymatic esterification of ethanol and oleic acid in an isothermal CSTR.

PubMed

Ho, Pang-Yen; Chuang, Guo-Syong; Chao, An-Chong; Li, Hsing-Ya

2005-05-01

The capacity of complex biochemical reaction networks (consisting of 11 coupled non-linear ordinary differential equations) to show multiple steady states, was investigated. The system involved esterification of ethanol and oleic acid by lipase in an isothermal continuous stirred tank reactor (CSTR). The Deficiency One Algorithm and the Subnetwork Analysis were applied to determine the steady state multiplicity. A set of rate constants and two corresponding steady states are computed. The phenomena of bistability, hysteresis and bifurcation are discussed. Moreover, the capacity of steady state multiplicity is extended to the family of the studied reaction networks.

Low-dimensional manifold of actin polymerization dynamics

NASA Astrophysics Data System (ADS)

Floyd, Carlos; Jarzynski, Christopher; Papoian, Garegin

2017-12-01

Actin filaments are critical components of the eukaryotic cytoskeleton, playing important roles in a number of cellular functions, such as cell migration, organelle transport, and mechanosensation. They are helical polymers with a well-defined polarity, composed of globular subunits that bind nucleotides in one of three hydrolysis states (ATP, ADP-Pi, or ADP). Mean-field models of the dynamics of actin polymerization have succeeded in, among other things, determining the nucleotide profile of an average filament and resolving the mechanisms of accessory proteins. However, these models require numerical solution of a high-dimensional system of nonlinear ordinary differential equations. By truncating a set of recursion equations, the Brooks-Carlsson (BC) model reduces dimensionality to 11, but it still remains nonlinear and does not admit an analytical solution, hence, significantly hindering understanding of its resulting dynamics. In this work, by taking advantage of the fast timescales of the hydrolysis states of the filament tips, we propose two model reduction schemes: the quasi steady-state approximation model is five-dimensional and nonlinear, whereas the constant tip (CT) model is five-dimensional and linear, resulting from the approximation that the tip states are not dynamic variables. We provide an exact solution of the CT model and use it to shed light on the dynamical behaviors of the full BC model, highlighting the relative ordering of the timescales of various collective processes, and explaining some unusual dependence of the steady-state behavior on initial conditions.

An Intuitive Approach to Steady-State Kinetics.

ERIC Educational Resources Information Center

Raines, Ronald T.; Hansen, David E.

1988-01-01

Attempts to provide an intuitive understanding of steady state kinetics. Discusses the meaning of steady state and uses free energy profiles to illustrate and follow complex kinetic and thermodynamic relationships. Provides examples with explanations. (MVL)

Malate dehydrogenase of the cytosol. A kinetic investigation of the reaction mechanism and a comparison with lactate dehydrogenase.

PubMed Central

Lodola, A; Shore, J D; Parker, D M; Holbrook, J

1978-01-01

1. The mechanisms of the reduction of oxaloacetate and of 3-fluoro-oxaloacetate by NADH catalysed by cytoplasmic pig heart malate dehydrogenase (MDH) were investigated. 2. One mol of dimeric enzyme produces 1.7+/-0.4 mol of enzyme-bound NADH when mixed with saturating NAD+ and L-malate at a rate much higher than the subsequent turnover at pH 7.5. 3. Transient measurements of protein and nucleotide fluorescence show that the steady-state complex in the forward direction is MDH-NADH and in the reverse direction MDH-NADH-oxaloacetate. 4. The rate of dissociation of MDH-NADH was measured and is the same as Vmax. in the forward direction at pH 7.5. Both NADH-binding sites are kinetically equivalent. The rate of dissociation varies with pH, as does the equilibrium binding constant for NADH. 5. 3-Fluoro-oxaloacetate is composed of three forms (F1, F2 and S) of which F1 and F2 are immediately substrates for the enzyme. The third form, S, is not a substrate, but when the F forms are used up form S slowly and non-enzymically equilibrates to yield the active substrate forms. S is 2,2-dihydroxy-3-fluorosuccinate. 6. The steady-state compound during the reduction of form F1 is an enzyme form that does not contain NADH, probably MDH-NAD+-fluoromalate. The steady-state compound for form F2 is an enzyme form containing NADH, probably MDH-NADH-fluoro-oxaloacetate. 7. The rate-limiting reaction in the reduction of form F2 shows a deuterium isotope rate ratio of 4 when NADH is replaced by its deuterium analogue, and the rate-limiting reaction is concluded to be hydride transfer. 8. A novel titration was used to show that dimeric cytoplasmic malate dehydrogenase contains two sites that can rapidly reduce the F1 form of 3-fluoro-oxaloacetate. The enzyme shows 'all-of-the-sites' behaviour. 9. Partial mechanisms are proposed to explain the enzyme-catalysed transformations of the natural and the fluoro substrates. These mechanisms are similar to the mechanism of pig heart lactate dehydrogenase and this, and the structural results of others, can be explained if the two enzymes are a product of divergent evolution. PMID:217361

Effect of flaw size and temperature on the matrix cracking behavior of a brittle ceramic matrix composite

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anandakumar, U.; Webb, J.E.; Singh, R.N.

The matrix cracking behavior of a zircon matrix - uniaxial SCS 6 fiber composite was studied as a function of initial flaw size and temperature. The composites were fabricated by a tape casting and hot pressing technique. Surface flaws of controlled size were introduced using a vicker`s indenter. The composite samples were tested in three point flexure at three different temperatures to study the non steady state and steady state matrix cracking behavior. The composite samples exhibited steady state and non steady matrix cracking behavior at all temperatures. The steady state matrix cracking stress and steady state crack size increasedmore » with increasing temperature. The results of the study correlated well with the results predicted by the matrix cracking models.« less

Differences between automatically detected and steady-state fractional flow reserve.

PubMed

Härle, Tobias; Meyer, Sven; Vahldiek, Felix; Elsässer, Albrecht

2016-02-01

Measurement of fractional flow reserve (FFR) has become a standard diagnostic tool in the catheterization laboratory. FFR evaluation studies were based on pressure recordings during steady-state maximum hyperemia. Commercially available computer systems detect the lowest Pd/Pa ratio automatically, which might not always be measured during steady-state hyperemia. We sought to compare the automatically detected FFR and true steady-state FFR. Pressure measurement traces of 105 coronary lesions from 77 patients with intermediate coronary lesions or multivessel disease were reviewed. In all patients, hyperemia had been achieved by intravenous adenosine administration using a dosage of 140 µg/kg/min. In 42 lesions (40%) automatically detected FFR was lower than true steady-state FFR. Mean bias was 0.009 (standard deviation 0.015, limits of agreement -0.02, 0.037). In 4 lesions (3.8%) both methods lead to different treatment recommendations, in all 4 cases instantaneous wave-free ratio confirmed steady-state FFR. Automatically detected FFR was slightly lower than steady-state FFR in more than one-third of cases. Consequently, interpretation of automatically detected FFR values closely below the cutoff value requires special attention.

Myeloperoxidase acts as a source of free iron during steady-state catalysis by a feedback inhibitory pathway

PubMed Central

Maitra, Dhiman; Shaeib, Faten; Abdulhamid, Ibrahim; Abdulridha, Rasha M.; Saed, Ghassan M.; Diamond, Michael P.; Pennathur, Subramaniam; Abu-Soud, Husam M.

2013-01-01

Myeloperoxidase (MPO) is a heme-containing enzyme that generates hypochlorous acid (HOCl) from chloride (Cl−) and hydrogen peroxide (H2O2). It is implicated in the pathology of several chronic inflammatory conditions such as cardiovascular and pulmonary diseases and cancer. Recently we have shown that HOCl can destroy the heme prosthetic group of hemoproteins. Here, we investigated whether the HOCl formed during steady-state catalysis is able to destroy the MPO heme moiety and thereby function as a major source of free iron. UV–visible spectra and H2O2-specific electrode measurements recorded during steady-state HOCl synthesis by MPO showed that the degree of MPO heme destruction increased after multiple additions of H2O2 (10 μM), precluding the enzyme from functioning at maximum activity (80–90% inhibition). MPO heme destruction occurred only in the presence of Cl−. Stopped-flow measurements revealed that the HOCl-mediated MPO heme destruction was complex and occurred through transient ferric species whose formation and decay kinetics indicated it participates in heme destruction along with subsequent free iron release. MPO heme depletion was confirmed by the buildup of free iron utilizing the ferrozine assay. Hypochlorous acid, once generated, first equilibrates in the solution as a whole before binding to the heme iron and initiating heme destruction. Eliminating HOCl from the MPO milieu by scavenging HOCl, destabilizing the MPO–Compound I–Cl complex that could be formed during catalysis, and/or inhibiting MPO catalytic activity partially or completely protects MPO from HOCl insults. Collectively, this study elucidates the bidirectional relationship between MPO and HOCl, which highlights the potential role of MPO as a source of free iron. PMID:23624305

E3 ligase FLRF (Rnf41) regulates differentiation of hematopoietic progenitors by governing steady-state levels of cytokine and retinoic acid receptors

PubMed Central

Jing, Xin; Infante, Jorge; Nachtman, Ronald G.; Jurecic, Roland

2008-01-01

Objective FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3, and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. Methods FLRF was over-expressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF over-expression on EML cell differentiation into myelo-erythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells over-expressing FLRF were examined with Western and immunoprecipitation. Results Remarkably, over-expression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines Epo and IL-3, and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, Epo and RA receptor RARα in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, Epo and RARα receptors in EML and BaF3 cells, and that FLRF-mediated down-regulation of these receptors is ligand binding-independent. Conclusions The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myelo-erythroid lineages. PMID:18495327

E3 ligase FLRF (Rnf41) regulates differentiation of hematopoietic progenitors by governing steady-state levels of cytokine and retinoic acid receptors.

PubMed

Jing, Xin; Infante, Jorge; Nachtman, Ronald G; Jurecic, Roland

2008-09-01

FLRF (Rnf41) gene was identified through screening of subtracted cDNA libraries form murine hematopoietic stem cells and progenitors. Subsequent work has revealed that FLRF acts as E3 ubiquitin ligase, and that it regulates steady-state levels of neuregulin receptor ErbB3 and participates in degradation of IAP protein BRUCE and parkin. The objective of this study was to start exploring the role of FLRF during hematopoiesis. FLRF was overexpressed in a murine multipotent hematopoietic progenitor cell line EML, which can differentiate into almost all blood cell lineages, and in pro-B progenitor cell line BaF3. The impact of FLRF overexpression on EML cell differentiation into myeloerythroid lineages was studied using hematopoietic colony-forming assays. The interaction of FLRF with cytokine receptors and receptor levels in control cells and EML and BaF3 cells overexpressing FLRF were examined with Western and immunoprecipitation. Remarkably, overexpression of FLRF significantly attenuated erythroid and myeloid differentiation of EML cells in response to cytokines erythropoietin (EPO) and interleukin-3 (IL-3), and retinoic acid (RA), and resulted in significant and constitutive decrease of steady-state levels of IL-3, EPO, and RA receptor-alpha (RARalpha) in EML and BaF3 cells. Immunoprecipitation has revealed that FLRF interacts with IL-3, EPO, and RARalpha receptors in EML and BaF3 cells, and that FLRF-mediated downregulation of these receptors is ligand binding-independent. The results of this study have revealed new FLRF-mediated pathway for ligand-independent receptor level regulation, and support the notion that through maintaining basal levels of cytokine receptors, FLRF is involved in the control of hematopoietic progenitor cell differentiation into myeloerythroid lineages.

Calcium distribution in Amoeba proteus

PubMed Central

1979-01-01

A preliminary investigation of the distribution of cellular calcium in Amoeba proteus was undertaken. Total cellular calcium under control conditions was found to be 4.59 mmol/kg of cells. When the external Ca++ concentration is increased from the control level of 0.03 to 20 mM, a net Ca++ influx results with a new steady-state cellular calcium level being achieved in integral of 3 h. At steady state the amount of calcium per unit weight of cells is higher than the amount of calcium per unit weight of external solution when the external concentration of Ca++ is below 10 mM. At external Ca++ concentrations above this level, total cellular calcium approaches the medium level of Ca++. Steady- state calcium exchange in Amoeba proteus was determined with 45Ca. There is an immediate and rapid exchange of integral of 0.84 mmol/kg of cells or 18% of the total cellular calcium with the labelled Ca++. Following this initial exchange, there was very little if any further exchange observed. Most of this exchanged calcium could be eliminated from the cell with 1 mM La+++, suggesting that the exchanged calcium is associated with the surface of the cell. Increase in either the external Ca++ concentration of pH raise the amount of exchangeable calcium associated with the cell. Calcium may be associated with the cell surface as a co-ion in the diffuse double layer or bound to fixed negative sites on the surface of the cell. If Ca++-binding sites do exist on the cell surface, there may be more than one type and they may have different dissociation constants. The cytoplasmic Ca++ ion activity is probably maintained at very low levels. PMID:512628

On the time to steady state: insights from numerical modeling

NASA Astrophysics Data System (ADS)

Goren, L.; Willett, S.; McCoy, S. W.; Perron, J.

2013-12-01

How fast do fluvial landscapes approach steady state after an application of tectonic or climatic perturbation? While theory and some numerical models predict that the celerity of the advective wave (knickpoint) controls the response time for perturbations, experiments and other landscape evolution models demonstrate that the time to steady state is much longer than the theoretically predicted response time. We posit that the longevity of transient features and the time to steady state are controlled by the stability of the topology and geometry of channel networks. Evolution of a channel network occurs by a combination of discrete capture events and continuous migration of water divides, processes, which are difficult to represent accurately in landscape evolution models. We therefore address the question of the time to steady state using the DAC landscape evolution model that solves accurately for the location of water divides, using a combination of analytical solution for hillslopes and low-order channels together with a numerical solution for higher order channels. DAC also includes an explicit capture criterion. We have tested fundamental predictions from DAC and show that modeled networks reproduce natural network characteristics such as the Hack's exponent and coefficient and the fractal dimension. We define two steady-state criteria: a topographic steady state, defined by global, pointwise steady elevation, and a topological steady state defined as the state in which no further reorganization of the drainage network takes place. Analyzing block uplift simulations, we find that the time to achieve either topographic or topological steady state exceeds by an order of magnitude the theoretical response time of the fluvial network. The longevity of the transient state is the result of the area feedback, by which, migration of a divide changes the local contributing area. This change propagates downstream as a slope adjustment, forcing further divide migrations and area change in adjacent tributaries and basins. In order to characterize the evolution of the drainage network on its way to steady state, we define a proxy to steady state elevation, χ, which is also the characteristic parameter of the transient stream power PDE. Through simulations of tectonic tilting we find that reorganization tends to minimize moments of the χ distribution of the landscape and of Δχ across divides.

Quantitative broadband absorption and scattering spectroscopy in turbid media by combined frequency-domain and steady state methodologies

DOEpatents

Tromberg, Bruce J [Irvine, CA; Berger, Andrew J [Rochester, NY; Cerussi, Albert E [Lake Forest, CA; Bevilacqua, Frederic [Costa Mesa, CA; Jakubowski, Dorota [Irvine, CA

2008-09-23

A technique for measuring broadband near-infrared absorption spectra of turbid media that uses a combination of frequency-domain and steady-state reflectance methods. Most of the wavelength coverage is provided by a white-light steady-state measurement, whereas the frequency-domain data are acquired at a few selected wavelengths. Coefficients of absorption and reduced scattering derived from the frequency-domain data are used to calibrate the intensity of the steady-state measurements and to determine the reduced scattering coefficient at all wavelengths in the spectral window of interest. The absorption coefficient spectrum is determined by comparing the steady-state reflectance values with the predictions of diffusion theory, wavelength by wavelength. Absorption spectra of a turbid phantom and of human breast tissue in vivo, derived with the combined frequency-domain and steady-state technique, agree well with expected reference values.

Steady-state entanglement in levitated optomechanical systems coupled to a higher order excited atomic ensemble

NASA Astrophysics Data System (ADS)

Chen, Aixi; Nie, Wenjie; Li, Ling; Zeng, Wei; Liao, Qinghong; Xiao, Xianbo

2017-11-01

We investigate the steady-state entanglement in an optomechanical system with a levitated dielectric nanosphere and a higher order excited atomic ensemble. The single nanosphere is trapped by an external harmonic dipole trap and coupled to the single-mode cavity field by the effective optomechanical coupling, which depends on the steady-state position of the nanosphere. We show that the steady-state optomechanical entanglement can be generated via the effective optomechanical interaction between the mechanical motion and the cavity mode. Further, these exist an optimal effective cavity detuning that maximizes the optomechanical entanglement. We also analyze in detail the influences of the excitation number of atoms, the radius of the nanosphere and the thermal noise strength on the steady-state optomechanical entanglement. It is found that the steady-state entanglement can be enhanced by increasing the excitation number of atoms and the radius of the nanosphere.

Interaction of promethazine and adiphenine to human hemoglobin: A comparative spectroscopic and computational analysis

NASA Astrophysics Data System (ADS)

Maurya, Neha; ud din Parray, Mehraj; Maurya, Jitendra Kumar; Kumar, Amit; Patel, Rajan

2018-06-01

The binding nature of amphiphilic drugs viz. promethazine hydrochloride (PMT) and adiphenine hydrochloride (ADP), with human hemoglobin (Hb) was unraveled by fluorescence, absorbance, time resolved fluorescence, fluorescence resonance energy transfer (FRET) and circular dichroism (CD) spectral techniques in combination with molecular docking and molecular dynamic simulation methods. The steady state fluorescence spectra indicated that both PMT and ADP quenches the fluorescence of Hb through static quenching mechanism which was further confirmed by time resolved fluorescence spectra. The UV-Vis spectroscopy suggested ground state complex formation. The activation energy (Ea) was observed more in the case of Hb-ADP than Hb-PMT interaction system. The FRET result indicates the high probability of energy transfer from β Trp37 residue of Hb to the PMT (r = 2.02 nm) and ADP (r = 2.33 nm). The thermodynamic data reveal that binding of PMT with Hb are exothermic in nature involving hydrogen bonding and van der Waal interaction whereas in the case of ADP hydrophobic forces play the major role and binding process is endothermic in nature. The CD results show that both PMT and ADP, induced secondary structural changes of Hb and unfold the protein by losing a large helical content while the effect is more pronounced with ADP. Additionally, we also utilized computational approaches for deep insight into the binding of these drugs with Hb and the results are well matched with our experimental results.

Interaction of promethazine and adiphenine to human hemoglobin: A comparative spectroscopic and computational analysis.

PubMed

Maurya, Neha; Ud Din Parray, Mehraj; Maurya, Jitendra Kumar; Kumar, Amit; Patel, Rajan

2018-06-15

The binding nature of amphiphilic drugs viz. promethazine hydrochloride (PMT) and adiphenine hydrochloride (ADP), with human hemoglobin (Hb) was unraveled by fluorescence, absorbance, time resolved fluorescence, fluorescence resonance energy transfer (FRET) and circular dichroism (CD) spectral techniques in combination with molecular docking and molecular dynamic simulation methods. The steady state fluorescence spectra indicated that both PMT and ADP quenches the fluorescence of Hb through static quenching mechanism which was further confirmed by time resolved fluorescence spectra. The UV-Vis spectroscopy suggested ground state complex formation. The activation energy (E a ) was observed more in the case of Hb-ADP than Hb-PMT interaction system. The FRET result indicates the high probability of energy transfer from β Trp37 residue of Hb to the PMT (r=2.02nm) and ADP (r=2.33nm). The thermodynamic data reveal that binding of PMT with Hb are exothermic in nature involving hydrogen bonding and van der Waal interaction whereas in the case of ADP hydrophobic forces play the major role and binding process is endothermic in nature. The CD results show that both PMT and ADP, induced secondary structural changes of Hb and unfold the protein by losing a large helical content while the effect is more pronounced with ADP. Additionally, we also utilized computational approaches for deep insight into the binding of these drugs with Hb and the results are well matched with our experimental results. Copyright © 2018 Elsevier B.V. All rights reserved.

Complexation induced fluorescence and acid-base properties of dapoxyl dye with γ-cyclodextrin: a drug-binding application using displacement assays.

PubMed

Pal, Kaushik; Mallick, Suman; Koner, Apurba L

2015-06-28

Host-guest complexation of dapoxyl sodium sulphonate (DSS), an intramolecular charge transfer dye with water-soluble and non-toxic macrocycle γ-cyclodextrin (γ-CD), has been investigated in a wide pH range. Steady-state absorption, fluorescence and time-resolved fluorescence measurements confirm the positioning of DSS into the hydrophobic cavity of γ-CD. A large fluorescence enhancement ca. 30 times, due to 1 : 2 complex formation and host-assisted guest-protonation have been utilised for developing a method for the utilisation of CD based drug-delivery applications. A simple fluorescence-displacement based approach is implemented at physiological pH for the assessment of binding strength of pharmaceutically useful small drug molecules (ibuprofen, paracetamol, methyl salicylate, salicylic acid, aspirin, and piroxicam) and six important antibiotic drugs (resazurin, thiamphenicol, chloramphenicol, ampicillin, kanamycin, and sorbic acid) with γ-CD.

Amperometric Glucose Sensor Using Thermostable Co-Factor Binding Glucose Dehydrogenase

NASA Astrophysics Data System (ADS)

Nakazawa, Yukie; Yamazaki, Tomohiko; Tsugawa, Wakako; Ikebukuro, Kazunori; Sode, Koji

A thermostable mediator-type enzyme glucose sensor was constructed. The electrode was fabricated using chemically cross-linked thermostable co-factor binding glucose dehydrogenase (GDH) from thermophilic bacteria in carbon paste matrix. The electrode responded directly proportional to D-glucose concentration from 0.01 mM to 3 mM in stirred buffer containing 1 mM 1-methoxyphenazinemethosulfate as a mediator with the steady-state mode. The storage stability was examined by incubating the enzyme electrode at 50oC during the measurement. The cross-linked GDH immobilized electrode showed good storage stability. Ninety percent of its initial response was retained after incubation in buffer solution for 9 days at 50oC. The flow injection analysis (FIA) glucose sensing system was also constructed by immobilizing the cross-linked GDH and ferrocene as a mediator in the carbon paste matrix. The FIA system was able to measure 600 samples for 100 h.

Kinetic studies of the inhibition of a human liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme by bile acids and anti-inflammatory drugs.

PubMed

Miyabe, Y; Amano, T; Deyashiki, Y; Hara, A; Tsukada, F

1995-01-01

We have investigated the steady-state kinetics for a cytosolic 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase isozyme of human liver and its inhibition by several bile acids and anti-inflammatory drugs such as indomethacin, flufemanic acid and naproxen. Initial velocity and product inhibition studies performed in the NADP(+)-linked (S)-1-indanol oxidation at pH 7.4 were consistent with a sequential ordered mechanism in which NADP+ binds first and leaves last. The bile acids and drugs, competitive inhibitors with respect to the alcohol substrate, exhibited uncompetitive inhibition with respect to the coenzyme, with Ki values less than 1 microM, whereas indomethacin exhibited noncompetitive inhibition (Ki < 24 microM). The kinetics of the inhibition by a mixture of the two inhibitors suggests that bile acids and drugs, except indomethacin, bind to overlapping sites at the active center of the enzyme-coenzyme binary complex.

Effect of thyroid hormone on the levels of erythrocyte carbonic anhydrase isozymes and 2,3-diphosphoglycerate in rabbits.

PubMed

Kondo, T; Taniguchi, N; Ishikawa, N; Ide, H; Takakuwa, E; Murao, M

1978-05-01

Levels of rabbit erythrocyte carbonic anhydrase B and C isozymes were determined in experimental hyperthyroidism using a quantitative immunologic technique. Levels of erythrocyte 2,3-diphosphoglycerate and protein binding iodine were simultaneously determined. Thyroxine and 3,5,3'-triiodothyronine were administered to rabbits orally for 30 days. A significant decrease in carbonic anhydrase B type was observed after 30 days, although no significant change was observed in carbonic anhydrase C type. These findings suggest that the steady state level of carbonic anhydrase B type in red cells is affected by thyroid hormone more readily than that of carbonic anhydrase C type. The level of red cell 2,3-diphosphoglycerate increased markedly after 10 days of treatment, corresponding to the increase of protein binding iodine. The clinical or pathologic significances were discussed in relation to the changes in the levels of these isozymes and 2,3-diphosphglycerate in red cells.

Loop L5 Assumes Three Distinct Orientations during the ATPase Cycle of the Mitotic Kinesin Eg5

PubMed Central

Muretta, Joseph M.; Behnke-Parks, William M.; Major, Jennifer; Petersen, Karl J.; Goulet, Adeline; Moores, Carolyn A.; Thomas, David D.; Rosenfeld, Steven S.

2013-01-01

Members of the kinesin superfamily of molecular motors differ in several key structural domains, which probably allows these molecular motors to serve the different physiologies required of them. One of the most variable of these is a stem-loop motif referred to as L5. This loop is longest in the mitotic kinesin Eg5, and previous structural studies have shown that it can assume different conformations in different nucleotide states. However, enzymatic domains often consist of a mixture of conformations whose distribution shifts in response to substrate binding or product release, and this information is not available from the “static” images that structural studies provide. We have addressed this issue in the case of Eg5 by attaching a fluorescent probe to L5 and examining its fluorescence, using both steady state and time-resolved methods. This reveals that L5 assumes an equilibrium mixture of three orientations that differ in their local environment and segmental mobility. Combining these studies with transient state kinetics demonstrates that there is a major shift in this distribution during transitions that interconvert weak and strong microtubule binding states. Finally, in conjunction with previous cryo-EM reconstructions of Eg5·microtubule complexes, these fluorescence studies suggest a model in which L5 regulates both nucleotide and microtubule binding through a set of reversible interactions with helix α3. We propose that these features facilitate the production of sustained opposing force by Eg5, which underlies its role in supporting formation of a bipolar spindle in mitosis. PMID:24145034

Customized Steady-State Constraints for Parameter Estimation in Non-Linear Ordinary Differential Equation Models

PubMed Central

Rosenblatt, Marcus; Timmer, Jens; Kaschek, Daniel

2016-01-01

Ordinary differential equation models have become a wide-spread approach to analyze dynamical systems and understand underlying mechanisms. Model parameters are often unknown and have to be estimated from experimental data, e.g., by maximum-likelihood estimation. In particular, models of biological systems contain a large number of parameters. To reduce the dimensionality of the parameter space, steady-state information is incorporated in the parameter estimation process. For non-linear models, analytical steady-state calculation typically leads to higher-order polynomial equations for which no closed-form solutions can be obtained. This can be circumvented by solving the steady-state equations for kinetic parameters, which results in a linear equation system with comparatively simple solutions. At the same time multiplicity of steady-state solutions is avoided, which otherwise is problematic for optimization. When solved for kinetic parameters, however, steady-state constraints tend to become negative for particular model specifications, thus, generating new types of optimization problems. Here, we present an algorithm based on graph theory that derives non-negative, analytical steady-state expressions by stepwise removal of cyclic dependencies between dynamical variables. The algorithm avoids multiple steady-state solutions by construction. We show that our method is applicable to most common classes of biochemical reaction networks containing inhibition terms, mass-action and Hill-type kinetic equations. Comparing the performance of parameter estimation for different analytical and numerical methods of incorporating steady-state information, we show that our approach is especially well-tailored to guarantee a high success rate of optimization. PMID:27243005

Customized Steady-State Constraints for Parameter Estimation in Non-Linear Ordinary Differential Equation Models.

PubMed

Rosenblatt, Marcus; Timmer, Jens; Kaschek, Daniel

2016-01-01

Ordinary differential equation models have become a wide-spread approach to analyze dynamical systems and understand underlying mechanisms. Model parameters are often unknown and have to be estimated from experimental data, e.g., by maximum-likelihood estimation. In particular, models of biological systems contain a large number of parameters. To reduce the dimensionality of the parameter space, steady-state information is incorporated in the parameter estimation process. For non-linear models, analytical steady-state calculation typically leads to higher-order polynomial equations for which no closed-form solutions can be obtained. This can be circumvented by solving the steady-state equations for kinetic parameters, which results in a linear equation system with comparatively simple solutions. At the same time multiplicity of steady-state solutions is avoided, which otherwise is problematic for optimization. When solved for kinetic parameters, however, steady-state constraints tend to become negative for particular model specifications, thus, generating new types of optimization problems. Here, we present an algorithm based on graph theory that derives non-negative, analytical steady-state expressions by stepwise removal of cyclic dependencies between dynamical variables. The algorithm avoids multiple steady-state solutions by construction. We show that our method is applicable to most common classes of biochemical reaction networks containing inhibition terms, mass-action and Hill-type kinetic equations. Comparing the performance of parameter estimation for different analytical and numerical methods of incorporating steady-state information, we show that our approach is especially well-tailored to guarantee a high success rate of optimization.

Existence and instability of steady states for a triangular cross-diffusion system: A computer-assisted proof

NASA Astrophysics Data System (ADS)

Breden, Maxime; Castelli, Roberto

2018-05-01

In this paper, we present and apply a computer-assisted method to study steady states of a triangular cross-diffusion system. Our approach consist in an a posteriori validation procedure, that is based on using a fixed point argument around a numerically computed solution, in the spirit of the Newton-Kantorovich theorem. It allows to prove the existence of various non homogeneous steady states for different parameter values. In some situations, we obtain as many as 13 coexisting steady states. We also apply the a posteriori validation procedure to study the linear stability of the obtained steady states, proving that many of them are in fact unstable.

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid

PubMed Central

Castillo, Juan P.; De Giorgis, Daniela; Basilio, Daniel; Gadsby, David C.; Rosenthal, Joshua J. C.; Latorre, Ramon; Holmgren, Miguel; Bezanilla, Francisco

2011-01-01

The Na+/K+ pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na+ from the cell and import 2K+ per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na+/K+ pump that represent the kinetics of extracellular Na+ binding/release and Na+ occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na+ from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site. PMID:22143771

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid.

PubMed

Castillo, Juan P; De Giorgis, Daniela; Basilio, Daniel; Gadsby, David C; Rosenthal, Joshua J C; Latorre, Ramon; Holmgren, Miguel; Bezanilla, Francisco

2011-12-20

The Na(+)/K(+) pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na(+) from the cell and import 2K(+) per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na(+)/K(+) pump that represent the kinetics of extracellular Na(+) binding/release and Na(+) occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na(+) from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site.

Low-dimensional Representation of Error Covariance

NASA Technical Reports Server (NTRS)

Tippett, Michael K.; Cohn, Stephen E.; Todling, Ricardo; Marchesin, Dan

2000-01-01

Ensemble and reduced-rank approaches to prediction and assimilation rely on low-dimensional approximations of the estimation error covariances. Here stability properties of the forecast/analysis cycle for linear, time-independent systems are used to identify factors that cause the steady-state analysis error covariance to admit a low-dimensional representation. A useful measure of forecast/analysis cycle stability is the bound matrix, a function of the dynamics, observation operator and assimilation method. Upper and lower estimates for the steady-state analysis error covariance matrix eigenvalues are derived from the bound matrix. The estimates generalize to time-dependent systems. If much of the steady-state analysis error variance is due to a few dominant modes, the leading eigenvectors of the bound matrix approximate those of the steady-state analysis error covariance matrix. The analytical results are illustrated in two numerical examples where the Kalman filter is carried to steady state. The first example uses the dynamics of a generalized advection equation exhibiting nonmodal transient growth. Failure to observe growing modes leads to increased steady-state analysis error variances. Leading eigenvectors of the steady-state analysis error covariance matrix are well approximated by leading eigenvectors of the bound matrix. The second example uses the dynamics of a damped baroclinic wave model. The leading eigenvectors of a lowest-order approximation of the bound matrix are shown to approximate well the leading eigenvectors of the steady-state analysis error covariance matrix.

Malate-Mediated Carbon Catabolite Repression in Bacillus subtilis Involves the HPrK/CcpA Pathway ▿ §

PubMed Central

Meyer, Frederik M.; Jules, Matthieu; Mehne, Felix M. P.; Le Coq, Dominique; Landmann, Jens J.; Görke, Boris; Aymerich, Stéphane; Stülke, Jörg

2011-01-01

Most organisms can choose their preferred carbon source from a mixture of nutrients. This process is called carbon catabolite repression. The Gram-positive bacterium Bacillus subtilis uses glucose as the preferred source of carbon and energy. Glucose-mediated catabolite repression is caused by binding of the CcpA transcription factor to the promoter regions of catabolic operons. CcpA binds DNA upon interaction with its cofactors HPr(Ser-P) and Crh(Ser-P). The formation of the cofactors is catalyzed by the metabolite-activated HPr kinase/phosphorylase. Recently, it has been shown that malate is a second preferred carbon source for B. subtilis that also causes catabolite repression. In this work, we addressed the mechanism by which malate causes catabolite repression. Genetic analyses revealed that malate-dependent catabolite repression requires CcpA and its cofactors. Moreover, we demonstrate that HPr(Ser-P) is present in malate-grown cells and that CcpA and HPr interact in vivo in the presence of glucose or malate but not in the absence of a repressing carbon source. The formation of the cofactor HPr(Ser-P) could be attributed to the concentrations of ATP and fructose 1,6-bisphosphate in cells growing with malate. Both metabolites are available at concentrations that are sufficient to stimulate HPr kinase activity. The adaptation of cells to environmental changes requires dynamic metabolic and regulatory adjustments. The repression strength of target promoters was similar to that observed in steady-state growth conditions, although it took somewhat longer to reach the second steady-state of expression when cells were shifted to malate. PMID:22001508

Inhibition of human Na(v)1.5 sodium channels by strychnine and its analogs.

PubMed

Yuan, Chunhua; Sun, Lirong; Zhang, Meng; Li, Shuji; Wang, Xuemin; Gao, Tianming; Zhu, Xinhong

2011-08-15

Strychnine and brucine from the seeds of the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors. In this study, we have characterized the pharmacological properties of strychnine and its analogs on human Na(v)1.5 channels to assess their potential therapeutic advantage in certain arrhythmias. Among the eight alkaloids, only strychnine and icajine exhibited inhibition potency on the Na(v)1.5 channel with the half-maximum inhibition (IC(50)) values of 83.1μM and 104.6μM, respectively. Structure-function analysis indicated that the increased bulky methoxy groups on the phenyl ring or the negatively charged oxygen atom may account for this lack of inhibition on the Na(v)1.5 channel. Strychnine and icajine may bind to the channel by cation-π interactions. The substitution with a large side chain on the phenyl ring or the increased molecular volume may alter the optimized position for the compound close to the binding sites of the channel. Strychnine and icajine bind to the Na(v)1.5 channel with a new mechanism that is different from TTX and local anesthetics. They bind to the outer vestibule of the channel pore with fast association and dissociation rates at resting state. Strychnine and icajine had little effect on steady-state fast inactivation but markedly shifted the slow inactivation of Na(v)1.5 currents toward more hyperpolarized potentials. The property of icajine influencing slow-inactivated state of Na(v)1.5 channel would be potential therapeutic advantages in certain arrhythmias. Copyright © 2011 Elsevier Inc. All rights reserved.

Probing the stereoselective interaction of ofloxacin enantiomers with corresponding monoclonal antibodies by multiple spectrometry

NASA Astrophysics Data System (ADS)

Mu, Hongtao; Xu, Zhenlin; Liu, Yingju; Sun, Yuanming; Wang, Baoling; Sun, Xiulan; Wang, Zhanhui; Eremin, Sergei; Zherdev, Anatoly V.; Dzantiev, Boris B.; Lei, Hongtao

2018-04-01

Although stereoselective antibody has immense potential in chiral compounds detection and separation, the interaction traits between stereoselective antibody and the corresponding antigenic enantiomers are not yet fully exploited. In this study, the stereospecific interactions between ofloxacin isomers and corresponding monoclonal antibodies (McAb-WR1 and McAb-MS1) were investigated using time-resolved fluorescence, steady-state fluorescence, and circular dichroism (CD) spectroscopic methods. The chiral recognition discrepancies of antibodies with ofloxacin isomers were reflected through binding constant, number of binding sites, driving forces and conformational changes. The major interacting forces of McAb-WR1 and McAb-MS1 chiral interaction systems were hydrophobic force and van der Waals forces joined up with hydrogen bonds, respectively. Synchronous fluorescence spectra and CD spectra results showed that the disturbing of tyrosine and tryptophan micro-environments were so slightly that no obvious secondary structure changes were found during the chiral hapten binding. Clarification of stereospecific interaction of antibody will facilitate the application of immunoassay to analyze chiral contaminants in food and other areas.

Effects of nano red elemental selenium on sodium currents in rat dorsal root ganglion neurons.

PubMed

Yuan, Huijun; Lin, Jiarui; Lan, Tonghan

2006-01-01

Nano red elemental selenium (Nano-Se), was demonstrated to be useful in medical and scientific researches. Here, we investigated the effects of Nano-Se on sodium currents on rat dorsal root ganglion neurons (DRG), using the whole-cell patch clamp method. Nano-Se reversibly decrease the I(Na)(TTX-S) in a concentration-dependent, time-dependent and open-channel block manners without affecting I(Na)(TTX-R). It shifted the steady-state activation and inactivation curves for I(Na) to more negative potentials. In the research of recovery from inactivation, the recovery time constant is longer in the present of Nano-Se. Nano-Se had a weaker inhibitory effect on I(Na), compared with marked decrease caused by selenite which indicated that Nano-Se is less neurotoxic than selenite in short-term/large dose treatments and had similar bio availability to sodium selenite. The results of interaction between the effects of Nano-Se and selenite on sodium currents indicated a negative allosteric interaction between the selenite binding site and the Nano-Se binding site or that they have the same competitive binding site.

Contribution of highway capital to industry and national productivity growth

DOT National Transportation Integrated Search

1973-10-01

The report contains the authors initial efforts aimed at extending the steady state freeway model for optimizing freeway traffic flow to a non-steady state model. The steady-state model does not allow reaction to continuously changing conditions whic...

Critical threshold behavior for steady-state internal transport barriers in burning plasmas.

PubMed

García, J; Giruzzi, G; Artaud, J F; Basiuk, V; Decker, J; Imbeaux, F; Peysson, Y; Schneider, M

2008-06-27

Burning tokamak plasmas with internal transport barriers are investigated by means of integrated modeling simulations. The barrier sustainment in steady state, differently from the barrier formation process, is found to be characterized by a critical behavior, and the critical number of the phase transition is determined. Beyond a power threshold, alignment of self-generated and noninductively driven currents occurs and steady state becomes possible. This concept is applied to simulate a steady-state scenario within the specifications of the International Thermonuclear Experimental Reactor.

A Novel Chronic Opioid Monitoring Tool to Assess Prescription Drug Steady State Levels in Oral Fluid.

PubMed

Shaparin, Naum; Mehta, Neel; Kunkel, Frank; Stripp, Richard; Borg, Damon; Kolb, Elizabeth

2017-11-01

Interpretation limitations of urine drug testing and the invasiveness of blood toxicology have motivated the desire for the development of simpler methods to assess biologically active drug levels on an individualized patient basis. Oral fluid is a matrix well-suited for the challenge because collections are based on simple noninvasive procedures and drug concentrations better correlate to blood drug levels as oral fluid is a filtrate of the blood. Well-established pharmacokinetic models were utilized to generate oral fluid steady state concentration ranges to assess the interpretive value of the alternative matrix to monitor steady state plasma oxycodone levels. Paired oral fluid and plasma samples were collected from patients chronically prescribed oxycodone and quantitatively analyzed by liquid chromatography tandem mass spectrometry. Steady state plasma concentration ranges were calculated for each donor and converted to an equivalent range in oral fluid. Measured plasma and oral fluid oxycodone concentrations were compared with respective matrix-matched steady state ranges, using each plasma steady state classification as the control. A high degree of correlation was observed between matrices when classifying donors according to expected steady state oxycodone concentration. Agreement between plasma and oral fluid steady state classifications was observed in 75.6% of paired samples. This study supports novel application of basic pharmacokinetic knowledge to the pain management industry, simplifying and improving individualized drug monitoring and risk assessment through the use of oral fluid drug testing. Many benefits of established therapeutic drug monitoring in plasma can be realized in oral fluid for patients chronically prescribed oxycodone at steady state. © 2017 American Academy of Pain Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.

PubMed

Rohrback, Suzanne E; Wheatly, Michele G; Gillen, Christopher M

2015-01-01

Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

Gibiansky, Leonid; Gibiansky, Ekaterina

2018-02-01

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

A Data Filter for Identifying Steady-State Operating Points in Engine Flight Data for Condition Monitoring Applications

NASA Technical Reports Server (NTRS)

Simon, Donald L.; Litt, Jonathan S.

2010-01-01

This paper presents an algorithm that automatically identifies and extracts steady-state engine operating points from engine flight data. It calculates the mean and standard deviation of select parameters contained in the incoming flight data stream. If the standard deviation of the data falls below defined constraints, the engine is assumed to be at a steady-state operating point, and the mean measurement data at that point are archived for subsequent condition monitoring purposes. The fundamental design of the steady-state data filter is completely generic and applicable for any dynamic system. Additional domain-specific logic constraints are applied to reduce data outliers and variance within the collected steady-state data. The filter is designed for on-line real-time processing of streaming data as opposed to post-processing of the data in batch mode. Results of applying the steady-state data filter to recorded helicopter engine flight data are shown, demonstrating its utility for engine condition monitoring applications.

Tailored parameter optimization methods for ordinary differential equation models with steady-state constraints.

PubMed

Fiedler, Anna; Raeth, Sebastian; Theis, Fabian J; Hausser, Angelika; Hasenauer, Jan

2016-08-22

Ordinary differential equation (ODE) models are widely used to describe (bio-)chemical and biological processes. To enhance the predictive power of these models, their unknown parameters are estimated from experimental data. These experimental data are mostly collected in perturbation experiments, in which the processes are pushed out of steady state by applying a stimulus. The information that the initial condition is a steady state of the unperturbed process provides valuable information, as it restricts the dynamics of the process and thereby the parameters. However, implementing steady-state constraints in the optimization often results in convergence problems. In this manuscript, we propose two new methods for solving optimization problems with steady-state constraints. The first method exploits ideas from optimization algorithms on manifolds and introduces a retraction operator, essentially reducing the dimension of the optimization problem. The second method is based on the continuous analogue of the optimization problem. This continuous analogue is an ODE whose equilibrium points are the optima of the constrained optimization problem. This equivalence enables the use of adaptive numerical methods for solving optimization problems with steady-state constraints. Both methods are tailored to the problem structure and exploit the local geometry of the steady-state manifold and its stability properties. A parameterization of the steady-state manifold is not required. The efficiency and reliability of the proposed methods is evaluated using one toy example and two applications. The first application example uses published data while the second uses a novel dataset for Raf/MEK/ERK signaling. The proposed methods demonstrated better convergence properties than state-of-the-art methods employed in systems and computational biology. Furthermore, the average computation time per converged start is significantly lower. In addition to the theoretical results, the analysis of the dataset for Raf/MEK/ERK signaling provides novel biological insights regarding the existence of feedback regulation. Many optimization problems considered in systems and computational biology are subject to steady-state constraints. While most optimization methods have convergence problems if these steady-state constraints are highly nonlinear, the methods presented recover the convergence properties of optimizers which can exploit an analytical expression for the parameter-dependent steady state. This renders them an excellent alternative to methods which are currently employed in systems and computational biology.

‘Life is motion’: multiscale motility of molecular motors

NASA Astrophysics Data System (ADS)

Lipowsky, Reinhard; Klumpp, Stefan

2005-07-01

Life is intimately related to complex patterns of directed movement. It is quite remarkable that all of this movement is based on filaments and motor molecules which perform mechanical work on the nanometer scale. This article reviews recent theoretical work on the motility of molecular motors and motor particles that bind to cytoskeletal filaments and walk along these filaments in a directed fashion. It is emphasized that these systems exhibit several motility regimes which are well seperated in time. In their bound state, the motor particles move with a typical velocity of about 1 μm/s. The motor cycles underlying this bound motor movement can be understood in terms of driven Brownian ratchets and networks. On larger length and time scales, the motor particles unbind from the filaments and undergo peculiar motor walks consisting of many diffusional encounters with the filaments. If the mutual exclusion (or hardcore repulsion) of these motor particles is taken into account, one finds a variety of cooperative phenomena and self-organized processes: build-up of traffic jams; active structure formation leading to steady states with spatially nonuniform density and current patterns; and active phase transitions between different steady states far from equilibrium. A particularly simple active phase transition with spontaneous symmetry breaking is predicted to occur in systems with two species of motor particles which walk on the filaments in opposite directions.

The effects of structural variations of thiophene-containing Ru(II) complexes on the acid-base and DNA binding properties.

PubMed

Yuan, Cui-Li; Zhang, An-Guo; Zheng, Ze-Bo; Wang, Ke-Zhi

2013-03-01

A phenylthiophenyl-bearing Ru(II) complex of [Ru(bpy)₂(Hbptip)](PF₆)₂ {bpy = 2,2'-bipyridine, Hbptip = 2-(4-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline} was synthesized and characterized by elemental analysis, ¹H NMR spectroscopy, and electrospray ionization mass spectrometry. The ground- and excited-state acid-base properties of the complex were studied by UV-visible absorption and photoluminescence spectrophotometric pH titrations and the negative logarithm values of the ground-state acid ionization constants were derived to be pK(a1) = 1.31 ± 0.09 and pK(a2) = 5.71 ± 0.11 with the pK(a2) associated deprotonation/protonation process occurring over 3 pK(a) units more acidic than thiophenyl-free parent complex of [Ru(bpy)₂(Hpip)]²⁺ {Hpip = 2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline}. The calf thymus DNA-binding properties of [Ru(bpy)₂(Hbptip)]²⁺ in Tris-HCl buffer (pH 7.1 and 50 mM NaCl) were investigated by DNA viscosities and density functional theoretical calculations as well as UV-visible and emission spectroscopy techniques of UV-visible and luminescence titrations, steady-state emission quenching by [Fe(CN)₆]⁴⁻, DNA competitive binding with ethidium bromide, DNA melting experiments, and reverse salt effects. The complex was evidenced to bind to the DNA intercalatively with binding affinity being greater than those for previously reported analogs of [Ru(bpy)₂(Hip)]²⁺, [Ru(bpy)₂(Htip)]²⁺, and [Ru(bpy)₂(Haptip)]²⁺ {Hip = 1H-imidazo[4,5-f][1,10]phenanthroline, Htip = 2-thiophenimidazo[4,5-f][1,10]phenanthroline, Haptip = 2-(5-phenylthiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline}.

40 CFR 1039.505 - How do I test engines using steady-state duty cycles, including ramped-modal testing?

Code of Federal Regulations, 2010 CFR

2010-07-01

...-state duty cycles, including ramped-modal testing? 1039.505 Section 1039.505 Protection of Environment... duty cycles, including ramped-modal testing? This section describes how to test engines under steady-state conditions. In some cases, we allow you to choose the appropriate steady-state duty cycle for an...

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase*

PubMed Central

Liu, Li-Kai; Abdelwahab, Heba; Martin Del Campo, Julia S.; Mehra-Chaudhary, Ritcha; Sobrado, Pablo; Tanner, John J.

2016-01-01

Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2′-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 μm. Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed. PMID:27557658

The Markov process admits a consistent steady-state thermodynamic formalism

NASA Astrophysics Data System (ADS)

Peng, Liangrong; Zhu, Yi; Hong, Liu

2018-01-01

The search for a unified formulation for describing various non-equilibrium processes is a central task of modern non-equilibrium thermodynamics. In this paper, a novel steady-state thermodynamic formalism was established for general Markov processes described by the Chapman-Kolmogorov equation. Furthermore, corresponding formalisms of steady-state thermodynamics for the master equation and Fokker-Planck equation could be rigorously derived in mathematics. To be concrete, we proved that (1) in the limit of continuous time, the steady-state thermodynamic formalism for the Chapman-Kolmogorov equation fully agrees with that for the master equation; (2) a similar one-to-one correspondence could be established rigorously between the master equation and Fokker-Planck equation in the limit of large system size; (3) when a Markov process is restrained to one-step jump, the steady-state thermodynamic formalism for the Fokker-Planck equation with discrete state variables also goes to that for master equations, as the discretization step gets smaller and smaller. Our analysis indicated that general Markov processes admit a unified and self-consistent non-equilibrium steady-state thermodynamic formalism, regardless of underlying detailed models.

Stabilization of a spatially uniform steady state in two systems exhibiting Turing patterns

NASA Astrophysics Data System (ADS)

Konishi, Keiji; Hara, Naoyuki

2018-05-01

This paper deals with the stabilization of a spatially uniform steady state in two coupled one-dimensional reaction-diffusion systems with Turing instability. This stabilization corresponds to amplitude death that occurs in a coupled system with Turing instability. Stability analysis of the steady state shows that stabilization does not occur if the two reaction-diffusion systems are identical. We derive a sufficient condition for the steady state to be stable for any length of system and any boundary conditions. Our analytical results are supported with numerical examples.

Pseudo-compressibility methods for the incompressible flow equations

NASA Technical Reports Server (NTRS)

Turkel, Eli; Arnone, A.

1993-01-01

Preconditioning methods to accelerate convergence to a steady state for the incompressible fluid dynamics equations are considered. The analysis relies on the inviscid equations. The preconditioning consists of a matrix multiplying the time derivatives. Thus the steady state of the preconditioned system is the same as the steady state of the original system. The method is compared to other types of pseudo-compressibility. For finite difference methods preconditioning can change and improve the steady state solutions. An application to viscous flow around a cascade with a non-periodic mesh is presented.

Quantum thermodynamics of nanoscale steady states far from equilibrium

NASA Astrophysics Data System (ADS)

Taniguchi, Nobuhiko

2018-04-01

We develop an exact quantum thermodynamic description for a noninteracting nanoscale steady state that couples strongly with multiple reservoirs. We demonstrate that there exists a steady-state extension of the thermodynamic function that correctly accounts for the multiterminal Landauer-Büttiker formula of quantum transport of charge, energy, or heat via the nonequilibrium thermodynamic relations. Its explicit form is obtained for a single bosonic or fermionic level in the wide-band limit, and corresponding thermodynamic forces (affinities) are identified. Nonlinear generalization of the Onsager reciprocity relations are derived. We suggest that the steady-state thermodynamic function is also capable of characterizing the heat current fluctuations of the critical transport where the thermal fluctuations dominate. Also, the suggested nonequilibrium steady-state thermodynamic relations seemingly persist for a spin-degenerate single level with local interaction.

Integration of Steady-State and Temporal Gene Expression Data for the Inference of Gene Regulatory Networks

PubMed Central

Wang, Yi Kan; Hurley, Daniel G.; Schnell, Santiago; Print, Cristin G.; Crampin, Edmund J.

2013-01-01

We develop a new regression algorithm, cMIKANA, for inference of gene regulatory networks from combinations of steady-state and time-series gene expression data. Using simulated gene expression datasets to assess the accuracy of reconstructing gene regulatory networks, we show that steady-state and time-series data sets can successfully be combined to identify gene regulatory interactions using the new algorithm. Inferring gene networks from combined data sets was found to be advantageous when using noisy measurements collected with either lower sampling rates or a limited number of experimental replicates. We illustrate our method by applying it to a microarray gene expression dataset from human umbilical vein endothelial cells (HUVECs) which combines time series data from treatment with growth factor TNF and steady state data from siRNA knockdown treatments. Our results suggest that the combination of steady-state and time-series datasets may provide better prediction of RNA-to-RNA interactions, and may also reveal biological features that cannot be identified from dynamic or steady state information alone. Finally, we consider the experimental design of genomics experiments for gene regulatory network inference and show that network inference can be improved by incorporating steady-state measurements with time-series data. PMID:23967277

Oxidation and Volatilization of Silica-Formers in Water Vapor

NASA Technical Reports Server (NTRS)

Opila, E. J.; Gray, Hugh R. (Technical Monitor)

2002-01-01

At high temperatures SiC and Si3N4 react with water vapor to form a silica scale. Silica scales also react with water vapor to form a volatile Si(OH)4 species. These simultaneous reactions, one forming silica and the other removing silica, are described by paralinear kinetics. A steady state, in which these reactions occur at the same rate, is eventually achieved, After steady state is achieved, the oxide found on the surface is a constant thickness and recession of the underlying material occurs at a linear rate. The steady state oxide thickness, the time to achieve steady state, and the steady state recession rate can all be described in terms of the rate constants for the oxidation and volatilization reactions. In addition, the oxide thickness, the time to achieve steady state, and the recession rate can also be determined from parameters that describe a water vapor-containing environment. Accordingly, maps have been developed to show these steady state conditions as a function of reaction rate constants, pressure, and gas velocity. These maps can be used to predict the behavior of silica formers in water-vapor containing environments such as combustion environments. Finally, these maps are used to explore the limits of the paralinear oxidation model for SiC and Si3N4

X-Ray Spectral Analysis of the Steady States of GRS1915+105

NASA Astrophysics Data System (ADS)

Peris, Charith S.; Remillard, Ronald A.; Steiner, James F.; Vrtilek, Saeqa D.; Varnière, Peggy; Rodriguez, Jerome; Pooley, Guy

2016-05-01

We report on the X-ray spectral behavior within the steady states of GRS1915+105. Our work is based on the full data set of the source obtained using the Proportional Counter Array (PCA) on the Rossi X-ray Timing Explorer (RXTE) and 15 GHz radio data obtained using the Ryle Telescope. The steady observations within the X-ray data set naturally separated into two regions in the color-color diagram and we refer to these regions as steady-soft and steady-hard. GRS1915+105 displays significant curvature in the coronal component in both the soft and hard data within the RXTE/PCA bandpass. A majority of the steady-soft observations displays a roughly constant inner disk radius ({R}{{in}}), while the steady-hard observations display an evolving disk truncation which is correlated to the mass accretion rate through the disk. The disk flux and coronal flux are strongly correlated in steady-hard observations and very weakly correlated in the steady-soft observations. Within the steady-hard observations, we observe two particular circumstances when there are correlations between the coronal X-ray flux and the radio flux with log slopes η ˜ 0.68+/- 0.35 and η ˜ 1.12+/- 0.13. They are consistent with the upper and lower tracks of Gallo et al. (2012), respectively. A comparison of the model parameters to the state definitions shows that almost all of the steady-soft observations match the criteria of either a thermal or steep power-law state, while a large portion of the steady-hard observations match the hard-state criteria when the disk fraction constraint is neglected.

Studies on the synthesis, characterization, human serum albumin binding and biological activity of single chain surfactant-cobalt(III) complexes.

PubMed

Vignesh, G; Sugumar, K; Arunachalam, S; Vignesh, S; Arthur James, R; Arun, R; Premkumar, K

2016-03-01

The interaction of surfactant-cobalt(III) complexes [Co(bpy)(dien)TA](ClO4)3 · 3H2O (1) and [Co(dien)(phen)TA](ClO4)3 · 4H2O (2), where bpy = 2,2'-bipyridine, dien = diethylenetriamine, phen = 1,10-phenanthroline and TA = tetradecylamine with human serum albumin (HSA) under physiological conditions was analyzed using steady state, synchronous, 3D fluorescence, UV/visabsorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of HSA through a static mechanism. The binding constant (Kb ) and number of binding-sites (n) were obtained at different temperatures. The corresponding thermodynamic parameters (∆G°, ∆H° and ∆S°) and Ea were also obtained. According to Förster's non-radiation energy transfer theory, the binding distance (r) between the complexes and HSA were calculated. The results of synchronous and 3D fluorescence spectroscopy indicate that the binding process has changed considerably the polarity around the fluorophores, along with changes in the conformation of the protein. The antimicrobial and anticancer activities of the complexes were tested and the results show that the complexes have good activities against pathogenic microorganisms and cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.

Escape from neutralization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bates, John T.; Keefer, Christopher J.; Slaughter, James C.

2014-04-15

The role of binding kinetics in determining neutralizing potency for antiviral antibodies is poorly understood. While it is believed that increased steady-state affinity correlates positively with increased virus-neutralizing activity, the relationship between association or dissociation rate and neutralization potency is unclear. We investigated the effect of naturally-occurring antibody resistance mutations in the RSV F protein on the kinetics of binding to palivizumab. Escape from palivizumab-mediated neutralization of RSV occurred with reduced association rate (K{sub on}) for binding to RSV F protein, while alteration of dissociation rate (K{sub off}) did not significantly affect neutralizing activity. Interestingly, linkage of reduced K{sub on}more » with reduced potency mirrored the effect of increased K{sub on} found in a high-affinity enhanced potency palivizumab variant (motavizumab). These data suggest that association rate is the dominant factor driving neutralization potency for antibodies to RSV F protein antigenic site A and determines the potency of antibody somatic variants or efficiency of escape of viral glycoprotein variants. - Highlights: • The relationship of affinity to neutralization for virus antibodies is uncertain. • Palivizumab binds to RSV escape mutant fusion proteins, but with reduced affinity. • Association rate (K{sub on}) correlated well with the potency of neutralization.« less

Elucidation of intermolecular interaction of bovine serum albumin with Fenhexamid: A biophysical prospect.

PubMed

Shi, Jie-Hua; Lou, Yan-Yue; Zhou, Kai-Li; Pan, Dong-Qi

2018-03-01

Fenhexamid, as a hydroxyanilide, is widely applied to control Botrytis cinerea for protecting crops and fruits. But it could adversely affect human and animals health due to accumulation of residues in food production. Here, the affinity characteristics of fenhexamid on bovine serum albumin (BSA) was studied via a series of spectroscopic methods such as steady-state fluorescence spectroscopy, ultraviolet spectroscopy (UV), synchronous fluorescence spectroscopy (SFS), 3D fluorescence spectroscopy, and fourier transform infrared spectroscopy (FT-IR). The experimental results illustrated that the fluorescence quenching mechanism of BSA induced by fenhexamid was a static quenching. The binding constant (K b ) of fenhexamid with BSA was 2.399 × 10 4  M -1 at 298 K and the combination ratio was about 1:1. The competitive experiment demonstrated that fenhexamid was binding on the BSA at site II (subdomain IIIA), which was confirmed by the molecular docking studies. The negative values of thermodynamic parameter (ΔH 0 , ΔS 0 and ΔG 0 ) revealed that the reaction of fenhexamid with BSA could proceed spontaneously, the van der Waals force and hydrogen bonding interaction conducted the main effect, and the binding process was enthalpy-driven. What's more, the 8-Anilino-1-naphthalenesulfonate (ANS) and sucrose binding studies were also performed and further verified the binding force between BSA and fenhexamid. Copyright © 2018 Elsevier B.V. All rights reserved.

Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

PubMed Central

Brown, Jessica A.; Pack, Lindsey R.; Sherrer, Shanen M.; Kshetry, Ajay K.; Newmister, Sean A.; Fowler, Jason D.; Taylor, John-Stephen; Suo, Zucai

2010-01-01

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β (Pol β). Pre-steady state kinetic studies have shown that the Pol λ•DNA complex binds both correct and incorrect nucleotides 130-fold tighter on average than the Pol β•DNA complex, although, the base substitution fidelity of both polymerases is 10−4 to 10−5. To better understand Pol λ’s tight nucleotide binding affinity, we created single- and double-substitution mutants of Pol λ to disrupt interactions between active site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding with each of the common structural moieties in the following order: triphosphate ≫ base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and template base led to a moderate increase in the Kd. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template. PMID:20851705

Cooperativity and tunable excited state deactivation: modular self-assembly of depsipeptide dendrons on a Hamilton receptor modified porphyrin platform.

PubMed

Gnichwitz, Jan-Frederik; Wielopolski, Mateusz; Hartnagel, Kristine; Hartnagel, Uwe; Guldi, Dirk M; Hirsch, Andreas

2008-07-02

A series of novel supramolecular architectures were built around a tin tetraphenyl porphyrin platform 6--functionalized by a 2-fold 1-ethyl-3-3-(3-dimethylaminopropyl)carbodiimide (EDC) promoted condensation reaction--and chiral depsipeptide dendrons of different generations 1-4. Here, implementation of a Hamilton receptor provided the necessary means to keep the constituents together via strong hydrogen bonding. Characterization of all architectures has been performed, including 4 which is the fourth generation, on the basis of NMR and photophysical methods. In particular, several titration experiments were conducted suggesting positive cooperativity, an assessment that is based on association constants that tend to be higher for the second binding step than for the first step. Importantly, molecular modeling calculations reveal a significant deaggregation of the intermolecular network of 6 during the course of the first binding step. As a consequence, an improved accessibility of the second Hamilton receptor unit in 6 emerges and, in turn, facilitates the higher association constants. The features of the equilibrium, that is, the dynamic exchange of depsipeptide dendrons 1-4 with fullerene 5, was tested in photophysical reference experiments. These steady-state and time-resolved measurements showed the tunable excited-state deactivations of these complexes upon photoexcitation.

40 CFR 1048.505 - How do I test engines using steady-state duty cycles, including ramped-modal testing?

Code of Federal Regulations, 2010 CFR

2010-07-01

...-state duty cycles, including ramped-modal testing? 1048.505 Section 1048.505 Protection of Environment... SPARK-IGNITION ENGINES Test Procedures § 1048.505 How do I test engines using steady-state duty cycles... some cases, we allow you to choose the appropriate steady-state duty cycle for an engine. In these...

X-ray spectral analysis of the steady states of GRS 1915+105

NASA Astrophysics Data System (ADS)

Peris, Charith; Remillard, Ronald A.; Steiner, James F.; Vrtilek, Saeqa Dil; Varniere, Peggy; Rodriguez, Jerome; Pooley, Guy G.

2016-04-01

Of the black hole binaries (BHBs) discovered thus far, GRS 1915+105 stands out as an exceptional source primarily due to its wild X-ray variability, the diversity of which has not been replicated in any other stellar-mass black hole. Although extreme variability is commonplace in its light-curve, about half of the observations of GRS1915+105 show fairly steady X-ray intensity. We report on the X-ray spectral behavior within these steady observations. Our work is based on a vast RXTE/PCA data set obtained on GRS 1915+105 during the course of its entire mission and 10 years of radio data from the Ryle Telescope, which overlap the X-ray data. We find that the steady observations within the X-ray data set naturally separate into two regions in a color-color diagram, which we refer to as steady-soft and steady-hard. GRS 1915+105 displays significant curvature in the Comptonization component within the PCA band pass suggesting significantly heating from a hot disk present in all states. A new Comptonization model 'simplcut' was developed in order to model this curvature to best effect. A majority of the steady-soft observations display a roughly constant inner disk radius, remarkably reminiscent of canonical soft state black hole binaries. In contrast, the steady-hard observations display a growing disk truncation that is correlated to the mass accretion rate through the disk, which suggests a magnetically truncated disk. A comparison of X-ray model parameters to the canonical state definitions show that almost all steady-soft observations match the criteria of either thermal or steep power law state, while the thermal state observations dominate the constant radius branch. A large portion 80 % of the steady-hard observations matches the hard state criteria when the disk fraction constraint is neglected. These results combine to suggest that within the complexity of this source is a simpler underlying basis of states, which map to those observed in canonical BHBs.

Dynamical System Modeling to Simulate Donor T Cell Response to Whole Exome Sequencing-Derived Recipient Peptides Demonstrates Different Alloreactivity Potential in HLA-Matched and -Mismatched Donor-Recipient Pairs.

PubMed

Abdul Razzaq, Badar; Scalora, Allison; Koparde, Vishal N; Meier, Jeremy; Mahmood, Musa; Salman, Salman; Jameson-Lee, Max; Serrano, Myrna G; Sheth, Nihar; Voelkner, Mark; Kobulnicky, David J; Roberts, Catherine H; Ferreira-Gonzalez, Andrea; Manjili, Masoud H; Buck, Gregory A; Neale, Michael C; Toor, Amir A

2016-05-01

Immune reconstitution kinetics and subsequent clinical outcomes in HLA-matched recipients of allogeneic stem cell transplantation (SCT) are variable and difficult to predict. Considering SCT as a dynamical system may allow sequence differences across the exomes of the transplant donors and recipients to be used to simulate an alloreactive T cell response, which may allow better clinical outcome prediction. To accomplish this, whole exome sequencing was performed on 34 HLA-matched SCT donor-recipient pairs (DRPs) and the nucleotide sequence differences translated to peptides. The binding affinity of the peptides to the relevant HLA in each DRP was determined. The resulting array of peptide-HLA binding affinity values in each patient was considered as an operator modifying a hypothetical T cell repertoire vector, in which each T cell clone proliferates in accordance with the logistic equation of growth. Using an iterating system of matrices, each simulated T cell clone's growth was calculated with the steady-state population being proportional to the magnitude of the binding affinity of the driving HLA-peptide complex. Incorporating competition between T cell clones responding to different HLA-peptide complexes reproduces a number of features of clinically observed T cell clonal repertoire in the simulated repertoire, including sigmoidal growth kinetics of individual T cell clones and overall repertoire, Power Law clonal frequency distribution, increase in repertoire complexity over time with increasing clonal diversity, and alteration of clonal dominance when a different antigen array is encountered, such as in SCT. The simulated, alloreactive T cell repertoire was markedly different in HLA-matched DRPs. The patterns were differentiated by rate of growth and steady-state magnitude of the simulated T cell repertoire and demonstrate a possible correlation with survival. In conclusion, exome wide sequence differences in DRPs may allow simulation of donor alloreactive T cell response to recipient antigens and may provide a quantitative basis for refining donor selection and titration of immunosuppression after SCT. Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

NASA Technical Reports Server (NTRS)

McCarthy, T. L.; Casinghino, S.; Mittanck, D. W.; Ji, C. H.; Centrella, M.; Rotwein, P.

1996-01-01

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

Molecular modeling studies of substrate binding by penicillin acylase.

PubMed

Chilov, G G; Stroganov, O V; Svedas, V K

2008-01-01

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.

Combined spectroscopies and molecular docking approach to characterizing the binding interaction of enalapril with bovine serum albumin.

PubMed

Pan, Dong-Qi; Jiang, Min; Liu, Ting-Ting; Wang, Qi; Shi, Jie-Hua

2017-06-01

The binding interaction between bovine serum albumin (BSA) and enalapril (ENPL) at the imitated physiological conditions (pH = 7.4) was investigated using UV-vis absorption spectroscopy (UV-vis), fluorescence emission spectroscopy (FES), synchronous fluorescence spectroscopy (SFS), Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) and molecular docking methods. It can be deduced from the experimental results from the steady-state fluorescence spectroscopic titration that the intrinsic BSA fluorescence quenching mechanism induced by ENPL is static quenching, based on the decrease in the BSA quenching constants in the presence of ENPL with increase in temperature and BSA quenching rates >10 10  L mol -1  sec -1 . This result indicates that the ENPL-BSA complex is formed through an intermolecular interaction of ENPL with BSA. The main bonding forces for interaction of BSA and ENPL are van der Waal's forces and hydrogen bonding interaction based on negative values of Gibbs free energy change (ΔG 0 ), enthalpic change (ΔH 0 ) and entropic change (ΔS 0 ). The binding of ENPL with BSA is an enthalpy-driven process due to |ΔH°| > |TΔS°| in the binding process. The results of competitive binding experiments and molecular docking confirm that ENPL binds in BSA sub-domain IIA (site I) and results in a slight change in BSA conformation, but BSA still retains its α-helical secondary structure. Copyright © 2016 John Wiley & Sons, Ltd.

Spectroscopic and molecular docking approaches for investigating conformation and binding characteristics of clonazepam with bovine serum albumin (BSA).

PubMed

Lou, Yan-Yue; Zhou, Kai-Li; Pan, Dong-Qi; Shen, Jia-Le; Shi, Jie-Hua

2017-02-01

Clonazepam, a type of benzodiazepine, is a classical drug used to prevent and treat seizures, panic disorder, movement disorder, among others. For further clarifying the distribution of clonazepam in vivo and the pharmacodynamic and pharmacokinetic mechanisms, the binding interaction between clonazepam and bovine serum albumin (BSA) was investigated using ultraviolet spectroscopy (UV), steady-state fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional (3D) fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and molecular docking methods. The results well confirmed that clonazepam bound on the subdomain III A (Site II) of BSA through van der Waals force and hydrogen bonding interaction, and quenched the intrinsic fluorescence of BSA through a static quenching process. The number of binding sites (n) and binding constant (K b ) of clonazepam-BSA complex were about 1 and 7.94×10 4 M -1 at 308K, respectively. The binding process of clonazepam with BSA was spontaneous and enthalpy-driven process due to ΔG 0 <0 and|ΔH 0 |>T|ΔS 0 | over the studied temperature range. Meanwhile, the binding interaction of clonazepam with BSA resulted in the slight change in the conformation of BSA and the obvious change in the conformation of clonazepam, implying that the flexibility of clonazepam also played an important role in increasing the stability of the clonazepam-BSA complex. Copyright © 2016 Elsevier B.V. All rights reserved.

Dissection of structural and functional requirements that underlie the interaction of ERdj3 protein with substrates in the endoplasmic reticulum.

PubMed

Otero, Joel H; Lizák, Beata; Feige, Matthias J; Hendershot, Linda M

2014-10-03

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification and characterization of small molecule inhibitors of the calcium-dependent S100B-p53 tumor suppressor interaction.

PubMed

Markowitz, Joseph; Chen, Ijen; Gitti, Rossi; Baldisseri, Donna M; Pan, Yongping; Udan, Ryan; Carrier, France; MacKerell, Alexander D; Weber, David J

2004-10-07

The binding of S100B to p53 down-regulates wild-type p53 tumor suppressor activity in cancer cells such as malignant melanoma, so a search for small molecules that bind S100B and prevent S100B-p53 complex formation was undertaken. Chemical databases were computationally searched for potential inhibitors of S100B, and 60 compounds were selected for testing on the basis of energy scoring, commercial availability, and chemical similarity clustering. Seven of these compounds bound to S100B as determined by steady state fluorescence spectroscopy (1.0 microM < or = K(D) < or = 120 microM) and five inhibited the growth of primary malignant melanoma cells (C8146A) at comparable concentrations (1.0 microM < or = IC(50) < or = 50 microM). Additionally, saturation transfer difference (STD) NMR experiments confirmed binding and qualitatively identified protons from the small molecule at the small molecule-S100B interface. Heteronuclear single quantum coherence (HSQC) NMR titrations indicate that these compounds interact with the p53 binding site on S100B. An NMR-docked model of one such inhibitor, pentamidine, bound to Ca(2+)-loaded S100B was calculated using intermolecular NOE data between S100B and the drug, and indicates that pentamidine binds into the p53 binding site on S100B defined by helices 3 and 4 and loop 2 (termed the hinge region).

Concentration Gradient Immunoassay I. A Rapid Immunoassay Based on Interdiffusion and Surface Binding in a Microchannel

PubMed Central

Nelson, Kjell E.; Foley, Jennifer O.; Yager, Paul

2008-01-01

We describe a novel microfluidic immunoassay method based on the diffusion of a small molecule analyte into a parallel-flowing stream containing cognate antibody. This interdiffusion results in a steady-state gradient of antibody binding site occupancy transverse to convective flow. In contrast to the diffusion immunoassay (Hatch et al. Nature Biotechnology,19:461−465 (2001)), this antibody occupancy gradient is interrogated by a sensor surface coated with a functional analog of the analyte. Antibodies with at least one unoccupied binding site may specifically bind to this functionalized surface, leading to a quantifiable change in surface coverage by the antibody. SPR imaging is used to probe the spatial distribution of antibody binding to the surface and, therefore, the outcome of the assay. We show that the pattern of antibody binding to the SPR sensing surface correlates with the concentration of a model analyte (phenytoin) in the sample stream. Using an inexpensive disposable microfluidic device, we demonstrate assays for phenytoin ranging in concentration from 75 to 1000 nM in phosphate buffer. At a total volumetric flow rate of 90 nL/sec, the assays are complete within 10 minutes. Inclusion of an additional flow stream on the side of the antibody stream opposite to that of the sample enables simultaneous calibration of the assay. This assay method is suitable for rapid quantitative detection of low-molecular weight analytes for point-of-care diagnostic instrumentation. PMID:17437332

Dissection of Structural and Functional Requirements That Underlie the Interaction of ERdj3 Protein with Substrates in the Endoplasmic Reticulum*

PubMed Central

Otero, Joel H.; Lizák, Beata; Feige, Matthias J.; Hendershot, Linda M.

2014-01-01

ERdj3, a mammalian endoplasmic reticulum (ER) Hsp40/DnaJ family member, binds unfolded proteins, transfers them to BiP, and concomitantly stimulates BiP ATPase activity. However, the requirements for ERdj3 binding to and release from substrates in cells are not well understood. We found that ERdj3 homodimers that cannot stimulate the ATPase activity of BiP (QPD mutants) bound to unfolded ER proteins under steady state conditions in much greater amounts than wild-type ERdj3. This was due to reduced release from these substrates as opposed to enhanced binding, although in both cases dimerization was strictly required for substrate binding. Conversely, heterodimers consisting of one wild-type and one mutant ERdj3 subunit bound substrates at levels comparable with wild-type ERdj3 homodimers, demonstrating that release requires only one protomer to be functional in stimulating BiP ATPase activity. Co-expressing wild-type ERdj3 and a QPD mutant, which each exclusively formed homodimers, revealed that the release rate of wild-type ERdj3 varied according to the relative half-lives of substrates, suggesting that ERdj3 release is an important step in degradation of unfolded client proteins in the ER. Furthermore, pulse-chase experiments revealed that the binding of QPD mutant homodimers remained constant as opposed to increasing, suggesting that ERdj3 does not normally undergo reiterative binding cycles with substrates. PMID:25143379

Characterization of domain-specific interaction of synthesized dye with serum proteins by spectroscopic and docking approaches along with determination of in vitro cytotoxicity and antiviral activity.

PubMed

Rudra, Suparna; Dasmandal, Somnath; Patra, Chiranjit; Patel, Biman Kumar; Paul, Suvendu; Mahapatra, Ambikesh

2017-11-20

The interaction between a synthesized dye with proteins, bovine, and human serum albumin (BSA, HSA, respectively) under physiological conditions has been characterized in detail, by means of steady-state and time-resolved fluorescence, UV-vis absorption, and circular dichroism (CD) techniques. An extensive time-resolved fluorescence spectroscopic characterization of the quenching process has been undertaken in conjugation with temperature-dependent fluorescence quenching studies to divulge the actual quenching mechanism. From the thermodynamic observations, it is clear that the binding process is a spontaneous molecular interaction, in which van der Waals and hydrogen bonding interactions play the major roles. The UV-vis absorption and CD results confirm that the dye can induce conformational and micro-environmental changes of both the proteins. In addition, the dye binding provokes the functionality of the native proteins in terms of esterase-like activity. The average binding distance (r) between proteins and dye has been calculated using FRET. Cytotoxicity and antiviral effects of the dye have been found using Vero cell and HSV-1F virus by performing MTT assay. The AutoDock-based docking simulation reveals the probable binding location of dye within the sub-domain IIA of HSA and IB of BSA.

40 CFR 86.1363-2007 - Steady-state testing with a discrete-mode cycle.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 19 2010-07-01 2010-07-01 false Steady-state testing with a discrete-mode cycle. 86.1363-2007 Section 86.1363-2007 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Exhaust Test Procedures § 86.1363-2007 Steady-state testing with a discrete-mode cycle. This section...

AEROSOL GROWTH IN A STEADY-STATE, CONTINUOUS FLOW CHAMBER: APPLICATION TO STUDIES OF SECONDARY AEROSOL FORMATION

EPA Science Inventory

An analytical solution for the steady-state aerosol size distribution achieved in a steady-state, continuous flow chamber is derived, where particle growth is occurring by gas-to-particle conversion and particle loss is occurring by deposition to the walls of the chamber. The s...

Analytical Solution of Steady State Equations for Chemical Reaction Networks with Bilinear Rate Laws

PubMed Central

Halász, Ádám M.; Lai, Hong-Jian; McCabe, Meghan M.; Radhakrishnan, Krishnan; Edwards, Jeremy S.

2014-01-01

True steady states are a rare occurrence in living organisms, yet their knowledge is essential for quasi-steady state approximations, multistability analysis, and other important tools in the investigation of chemical reaction networks (CRN) used to describe molecular processes on the cellular level. Here we present an approach that can provide closed form steady-state solutions to complex systems, resulting from CRN with binary reactions and mass-action rate laws. We map the nonlinear algebraic problem of finding steady states onto a linear problem in a higher dimensional space. We show that the linearized version of the steady state equations obeys the linear conservation laws of the original CRN. We identify two classes of problems for which complete, minimally parameterized solutions may be obtained using only the machinery of linear systems and a judicious choice of the variables used as free parameters. We exemplify our method, providing explicit formulae, on CRN describing signal initiation of two important types of RTK receptor-ligand systems, VEGF and EGF-ErbB1. PMID:24334389

Investigations on the Interactions of 5-Fluorouracil with Herring Sperm DNA: Steady State/Time Resolved and Molecular Modeling Studies

NASA Astrophysics Data System (ADS)

Chinnathambi, Shanmugavel; Karthikeyan, Subramani; Velmurugan, Devadasan; Hanagata, Nobutaka; Aruna, Prakasarao; Ganesan, Singaravelu

2015-04-01

In the present study, the interaction of 5-Fluorouracil with herring sperm DNA is reported using spectroscopic and molecular modeling techniques. This binding study of 5-FU with hs-DNA is of paramount importance in understanding chemico-biological interactions for drug design, pharmacy and biochemistry without altering the original structure. The challenge of the study was to find the exact binding mode of the drug 5-Fluorouracil with hs-DNA. From the absorption studies, a hyperchromic effect was observed for the herring sperm DNA in the presence of 5-Fluorouracil and a binding constant of 6.153 × 103 M-1 for 5-Fluorouracil reveals the existence of weak interaction between the 5-Fluorouracil and herring sperm DNA. Ethidium bromide loaded herring sperm DNA showed a quenching in the fluorescence intensity after the addition of 5-Fluorouracil. The binding constants for 5-Fluorouracil stranded DNA and competitive bindings of 5-FU interacting with DNA-EB systems were examined by fluorescence spectra. The Stern-Volmer plots and fluorescence lifetime results confirm the static quenching nature of the drug-DNA complex. The binding constant Kb was 2.5 × 104 L mol-1 and the number of binding sites are 1.17. The 5-FU on DNA system was calculated using double logarithmic plot. From the Forster nonradiative energy transfer study it has been found that the distance of 5-FU from DNA was 4.24 nm. In addition to the spectroscopic results, the molecular modeling studies also revealed the major groove binding as well as the partial intercalation mode of binding between the 5-Fluorouracil and herring sperm DNA. The binding energy and major groove binding as -6.04 kcal mol-1 and -6.31 kcal mol-1 were calculated from the modeling studies. All the testimonies manifested that binding modes between 5-Fluorouracil and DNA were evidenced to be groove binding and in partial intercalative mode.

Human serum albumin stability and toxicity of anthraquinone dye alizarin complexone: an albumin-dye model.

PubMed

Ding, Fei; Zhang, Li; Diao, Jian-Xiong; Li, Xiu-Nan; Ma, Lin; Sun, Ying

2012-05-01

The complexation between the primary vector of ligands in blood plasma, human serum albumin (HSA) and a toxic anthraquinone dye alizarin complexone, was unmasked by means of circular dichroism (CD), molecular modeling, steady state and time-resolved fluorescence, and UV/vis absorption measurements. The structural investigation of the complexed HSA through far-UV CD, three-dimensional and synchronous fluorescence shown the polypeptide chain of HSA partially destabilizing with a reduction of α-helix upon conjugation. From molecular modeling and competitive ligand binding results, Sudlow's site I, which was the same as that of warfarin-azapropazone site, was appointed to retain high-affinity for alizarin complexone. Moreover, steady state fluorescence displayed that static type and Förster energy transfer is the operational mechanism for the vanish in the tryptophan (Trp)-214 fluorescence, this corroborates time-resolved fluorescence that HSA-alizarin complexone adduct formation has an affinity of 10(5) M(-1), and the driving forces were found to be chiefly π-π, hydrophobic, and hydrogen bonds, associated with an exothermic free energy change. These data should be utilized to illustrate the mechanism by which the toxicological action of anthraquinone dyes is mitigated by transporter HSA. Copyright © 2012 Elsevier Inc. All rights reserved.

Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation.

PubMed

Vitturi, Dario A; Teng, Xinjun; Toledo, José C; Matalon, Sadis; Lancaster, Jack R; Patel, Rakesh P

2009-05-01

Allosteric regulation of nitrite reduction by deoxyhemoglobin has been proposed to mediate nitric oxide (NO) formation during hypoxia. Nitrite is predominantly an anion at physiological pH, raising questions about the mechanism by which it enters the red blood cell (RBC) and whether this is regulated and coupled to deoxyhemoglobin-mediated reduction. We tested the hypothesis that nitrite transport by RBCs is regulated by fractional saturation. Using human RBCs, nitrite consumption was faster at lower fractional saturations, consistent with faster reactions with deoxyheme. A membrane-based regulation was suggested by slower nitrite consumption with intact versus lysed RBCs. Interestingly, upon nitrite addition, intracellular nitrite concentrations attained a steady state that, despite increased rates of consumption, did not change with decreasing oxygen tensions, suggesting a deoxygenation-sensitive step that either increases nitrite import or decreases the rate of nitrite export. A role for anion exchanger (AE)-1 in the control of nitrite export was suggested by increased intracellular nitrite concentrations in RBCs treated with DIDS. Moreover, deoxygenation decreased steady-state levels of intracellular nitrite in AE-1-inhibited RBCs. Based on these data, we propose a model in which deoxyhemoglobin binding to AE-1 inhibits nitrite export under low oxygen tensions allowing for the coupling between deoxygenation and nitrite reduction to NO along the arterial-to-venous gradient.

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

PubMed

Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

2014-01-01

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

Surface potential-governed cellular osteogenic differentiation on ferroelectric polyvinylidene fluoride trifluoroethylene films.

PubMed

Tang, Bolin; Zhang, Bo; Zhuang, Junjun; Wang, Qi; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

2018-07-01

Surface potential of biomaterials can dramatically influence cellular osteogenic differentiation. In this work, a wide range of surface potential on ferroelectric polyvinylidene fluoride trifluoroethylene (P(VDF-TrFE)) films was designed to get insight into the interfacial interaction of cell-charged surface. The P(VDF-TrFE) films poled by contact electric poling at various electric fields obtained well stabilized surface potential, with wide range from -3 to 915 mV. The osteogenic differentiation level of cells cultured on the films was strongly dependent on surface potential and reached the optimum at 391 mV in this system. Binding specificity assay indicated that surface potential could effectively govern the binding state of the adsorbed fibronectin (FN) with integrin. Molecular dynamic (MD) simulation further revealed that surface potential brought a significant difference in the relative distance between RGD and synergy PHSRN sites of adsorbed FN, resulting in a distinct integrin-FN binding state. These results suggest that the full binding of integrin α5β1 with both RGD and PHSRN sites of FN possesses a strong ability to activate osteogenic signaling pathway. This work sheds light on the underlying mechanism of osteogenic differentiation behavior on charged material surfaces, and also provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. The ferroelectric P(VDF-TrFE) films with steady and a wide range of surface potential were designed to understand underlying mechanism of cell-charged surface interaction. The results showed that the charged surface well favored upregulation of osteogenic differentiation of MC3T3-E1 cells, and more importantly, a highest level occurred on the film with a moderate surface potential. Experiments and molecular dynamics simulation demonstrated that the surface potential could govern fibronectin conformation and then the integrin-fibronectin binding. We propose that a full binding state of integrin α5β1 with fibronectin induces effective activation of integrin-mediated FAK/ERK signaling pathway to upregulate cellular osteogenic differentiation. This work provides a guidance for designing a reasonable charged surface to enhance osteogenic differentiation. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Binding of naringin and naringenin with hen egg white lysozyme: A spectroscopic investigation and molecular docking study

NASA Astrophysics Data System (ADS)

Das, Sourav; Ghosh, Pooja; Koley, Sudipta; Singha Roy, Atanu

2018-03-01

The interactions of naringenin (NG) and naringin (NR) with Hen Egg White Lysozyme (HEWL) in aqueous medium have been investigated using UV-vis spectroscopy, steady-state fluorescence, circular dichroism (CD), Fourier Transform infrared spectroscopy (FT-IR) and molecular docking analyses. Both NG and NR can quench the intrinsic fluorescence of HEWL via static quenching mechanism. At 300 K, the value of binding constant (Kb) of HEWL-NG complex (5.596 ± 0.063 × 104 M- 1) was found to be greater than that of HEWL-NR complex (3.404 ± 0.407 × 104 M- 1). The negative ΔG° values in cases of both the complexes specify the spontaneous binding. The binding distance between the donor (HEWL) and acceptor (NG/NR) was estimated using the Försters theory and the possibility of non-radiative energy transfer from HEWL to NG/NR was observed. The presence of metal ions (Ca2 +, Cu2 + and Fe2 +) decreased the binding affinity of NG/NR towards HEWL. Synchronous fluorescence studies indicate the change in Trp micro-environment due to the incorporation of NG/NR into HEWL. CD and FT-IR studies indicated that the α-helicity of the HEWL was slightly enhanced due to ligand binding. NG and NR inhibited the enzymatic activity of HEWL and exhibited their affinity for the active site of HEWL. Molecular docking studies revealed that both NG and NR bind in the close vicinity of Trp 62 and Trp 63 residues which is vital for the catalytic activity.

MATHEMATICAL ANALYSIS OF STEADY-STATE SOLUTIONS IN COMPARTMENT AND CONTINUUM MODELS OF CELL POLARIZATION

PubMed Central

ZHENG, ZHENZHEN; CHOU, CHING-SHAN; YI, TAU-MU; NIE, QING

2013-01-01

Cell polarization, in which substances previously uniformly distributed become asymmetric due to external or/and internal stimulation, is a fundamental process underlying cell mobility, cell division, and other polarized functions. The yeast cell S. cerevisiae has been a model system to study cell polarization. During mating, yeast cells sense shallow external spatial gradients and respond by creating steeper internal gradients of protein aligned with the external cue. The complex spatial dynamics during yeast mating polarization consists of positive feedback, degradation, global negative feedback control, and cooperative effects in protein synthesis. Understanding such complex regulations and interactions is critical to studying many important characteristics in cell polarization including signal amplification, tracking dynamic signals, and potential trade-off between achieving both objectives in a robust fashion. In this paper, we study some of these questions by analyzing several models with different spatial complexity: two compartments, three compartments, and continuum in space. The step-wise approach allows detailed characterization of properties of the steady state of the system, providing more insights for biological regulations during cell polarization. For cases without membrane diffusion, our study reveals that increasing the number of spatial compartments results in an increase in the number of steady-state solutions, in particular, the number of stable steady-state solutions, with the continuum models possessing infinitely many steady-state solutions. Through both analysis and simulations, we find that stronger positive feedback, reduced diffusion, and a shallower ligand gradient all result in more steady-state solutions, although most of these are not optimally aligned with the gradient. We explore in the different settings the relationship between the number of steady-state solutions and the extent and accuracy of the polarization. Taken together these results furnish a detailed description of the factors that influence the tradeoff between a single correctly aligned but poorly polarized stable steady-state solution versus multiple more highly polarized stable steady-state solutions that may be incorrectly aligned with the external gradient. PMID:21936604

An exact solution for the steady state phase distribution in an array of oscillators coupled on a hexagonal lattice

NASA Technical Reports Server (NTRS)

Pogorzelski, Ronald J.

2004-01-01

When electronic oscillators are coupled to nearest neighbors to form an array on a hexagonal lattice, the planar phase distributions desired for excitation of a phased array antenna are not steady state solutions of the governing non-linear equations describing the system. Thus the steady state phase distribution deviates from planar. It is shown to be possible to obtain an exact solution for the steady state phase distribution and thus determine the deviation from the desired planar distribution as a function of beam steering angle.

Preconditioning and the limit to the incompressible flow equations

NASA Technical Reports Server (NTRS)

Turkel, E.; Fiterman, A.; Vanleer, B.

1993-01-01

The use of preconditioning methods to accelerate the convergence to a steady state for both the incompressible and compressible fluid dynamic equations are considered. The relation between them for both the continuous problem and the finite difference approximation is also considered. The analysis relies on the inviscid equations. The preconditioning consists of a matrix multiplying the time derivatives. Hence, the steady state of the preconditioned system is the same as the steady state of the original system. For finite difference methods the preconditioning can change and improve the steady state solutions. An application to flow around an airfoil is presented.

Theoretical studies of solar-pumped lasers

NASA Technical Reports Server (NTRS)

Harries, W. L.

1982-01-01

Solar-pumped lasers were investigated by comparing experimental results from pulse experiments with steady state calculations. The time varying behavior of an IBr laser is studied. The analysis is only approximate, but indicates that conditions occurring in a pulsed experiment are quite different from those at steady state. The possibility of steady-state lasing in an IBr laser is determined. The effects of high temperatures on the quenching and recombination rates are examined. Although uncertainties in the values of the rate coefficients make it difficult to draw firm conclusions, it seems steady state running may be possible at high temperatures.

New Equilibrium Models of Drug-Receptor Interactions Derived from Target-Mediated Drug Disposition.

PubMed

Peletier, Lambertus A; Gabrielsson, Johan

2018-05-14

In vivo analyses of pharmacological data are traditionally based on a closed system approach not incorporating turnover of target and ligand-target kinetics, but mainly focussing on ligand-target binding properties. This study incorporates information about target and ligand-target kinetics parallel to binding. In a previous paper, steady-state relationships between target- and ligand-target complex versus ligand exposure were derived and a new expression of in vivo potency was derived for a circulating target. This communication is extending the equilibrium relationships and in vivo potency expression for (i) two separate targets competing for one ligand, (ii) two different ligands competing for a single target and (iii) a single ligand-target interaction located in tissue. The derived expressions of the in vivo potencies will be useful both in drug-related discovery projects and mechanistic studies. The equilibrium states of two targets and one ligand may have implications in safety assessment, whilst the equilibrium states of two competing ligands for one target may cast light on when pharmacodynamic drug-drug interactions are important. The proposed equilibrium expressions for a peripherally located target may also be useful for small molecule interactions with extravascularly located targets. Including target turnover, ligand-target complex kinetics and binding properties in expressions of potency and efficacy will improve our understanding of within and between-individual (and across species) variability. The new expressions of potencies highlight the fact that the level of drug-induced target suppression is very much governed by target turnover properties rather than by the target expression level as such.

Mechanism of hERG channel block by the psychoactive indole alkaloid ibogaine.

PubMed

Thurner, Patrick; Stary-Weinzinger, Anna; Gafar, Hend; Gawali, Vaibhavkumar S; Kudlacek, Oliver; Zezula, Juergen; Hilber, Karlheinz; Boehm, Stefan; Sandtner, Walter; Koenig, Xaver

2014-02-01

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go-related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells. Currents through hERG channels were blocked regardless of whether ibogaine was applied via the extracellular or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during activation of the channels and was not observed for resting channels. With increasing depolarizations, ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, whereas inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, these findings show that ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.

Quantification of the memory effect of steady-state currents from interaction-induced transport in quantum systems

NASA Astrophysics Data System (ADS)

Lai, Chen-Yen; Chien, Chih-Chun

2017-09-01

Dynamics of a system in general depends on its initial state and how the system is driven, but in many-body systems the memory is usually averaged out during evolution. Here, interacting quantum systems without external relaxations are shown to retain long-time memory effects in steady states. To identify memory effects, we first show quasi-steady-state currents form in finite, isolated Bose- and Fermi-Hubbard models driven by interaction imbalance and they become steady-state currents in the thermodynamic limit. By comparing the steady-state currents from different initial states or ramping rates of the imbalance, long-time memory effects can be quantified. While the memory effects of initial states are more ubiquitous, the memory effects of switching protocols are mostly visible in interaction-induced transport in lattices. Our simulations suggest that the systems enter a regime governed by a generalized Fick's law and memory effects lead to initial-state-dependent diffusion coefficients. We also identify conditions for enhancing memory effects and discuss possible experimental implications.

Spectroscopic investigation of the noncovalent association of the nerve agent simulant diisopropyl methylphosphonate (DIMP) with zinc(II) porphyrins.

PubMed

Maza, William A; Vetromile, Carissa M; Kim, Chungsik; Xu, Xue; Zhang, X Peter; Larsen, Randy W

2013-11-07

Organophosphonates pose a significant threat as chemical warfare agents, as well as environmental toxins in the form of pesticides. Thus, methodologies to sense and decontaminate these agents are of significant interest. Porphyrins and metalloporphyrins offer an excellent platform to develop chemical threat sensors and photochemical degradation systems. These highly conjugated planar molecules exhibit relatively long-lived singlet and triplet states with high quantum yields and also form self-associated complexes with a wide variety of molecules. A significant aspect of porphyrins is the ability to functionalize the peripheral ring system either directly to the pyrrole rings or to the bridging methine carbons. In this report, steady-state absorption and fluorescence are utilized to probe binding affinities of a series of symmetric and asymmetric zinc(II) metalloporphyrins for the nerve agent simulant diisopropyl methylphosphonate (DIMP) in hexane. The red shifts in the absorption and emission spectra observed for all of the metalloporphyrins probed are discussed in the frame of Gouterman's four orbital model and a common binding motif involving coordination between the metalloporphyrin and DIMP via interaction between the zinc metal center of the porphyrin and phosphoryl oxygen of DIMP (Zn-O═P) is proposed.

40 CFR Appendix C to Subpart S of... - Steady-State Short Test Standards

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 2 2010-07-01 2010-07-01 false Steady-State Short Test Standards C Appendix C to Subpart S of Part 51 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED.../Maintenance Program Requirements Pt. 51, Subpt. S, App. C Appendix C to Subpart S of Part 51—Steady-State...

40 CFR Appendix II to Part 1039 - Steady-State Duty Cycles

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Steady-State Duty Cycles II Appendix... Appendix II to Part 1039—Steady-State Duty Cycles (a) The following duty cycles apply for constant-speed engines: (1) The following duty cycle applies for discrete-mode testing: D2 mode number Engine speed...

40 CFR Appendix C to Subpart S of... - Steady-State Short Test Standards

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 2 2014-07-01 2014-07-01 false Steady-State Short Test Standards C Appendix C to Subpart S of Part 51 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED.../Maintenance Program Requirements Pt. 51, Subpt. S, App. C Appendix C to Subpart S of Part 51—Steady-State...

40 CFR Appendix C to Subpart S of... - Steady-State Short Test Standards

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 2 2012-07-01 2012-07-01 false Steady-State Short Test Standards C Appendix C to Subpart S of Part 51 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED.../Maintenance Program Requirements Pt. 51, Subpt. S, App. C Appendix C to Subpart S of Part 51—Steady-State...

40 CFR Appendix II to Part 1039 - Steady-State Duty Cycles

Code of Federal Regulations, 2011 CFR

2011-07-01

... Appendix II to Part 1039—Steady-State Duty Cycles (a) The following duty cycles apply for constant-speed engines: (1) The following duty cycle applies for discrete-mode testing: D2 mode number Engine speed...(seconds) Engine speed Torque(percent) 1, 2 1a Steady-state 53 Engine governed 100. 1b Transition 20 Engine...

Functional asymmetry in the lysyl-tRNA synthetase explored by molecular dynamics, free energy calculations and experiment

PubMed Central

Hughes, Samantha J; Tanner, Julian A; Hindley, Alison D; Miller, Andrew D; Gould, Ian R

2003-01-01

Background Charging of transfer-RNA with cognate amino acid is accomplished by the aminoacyl-tRNA synthetases, and proceeds through an aminoacyl adenylate intermediate. The lysyl-tRNA synthetase has evolved an active site that specifically binds lysine and ATP. Previous molecular dynamics simulations of the heat-inducible Escherichia coli lysyl-tRNA synthetase, LysU, have revealed differences in the binding of ATP and aspects of asymmetry between the nominally equivalent active sites of this dimeric enzyme. The possibility that this asymmetry results in different binding affinities for the ligands is addressed here by a parallel computational and biochemical study. Results Biochemical experiments employing isothermal calorimetry, steady-state fluorescence and circular dichroism are used to determine the order and stoichiometries of the lysine and nucleotide binding events, and the associated thermodynamic parameters. An ordered mechanism of substrate addition is found, with lysine having to bind prior to the nucleotide in a magnesium dependent process. Two lysines are found to bind per dimer, and trigger a large conformational change. Subsequent nucleotide binding causes little structural rearrangement and crucially only occurs at a single catalytic site, in accord with the simulations. Molecular dynamics based free energy calculations of the ATP binding process are used to determine the binding affinities of each site. Significant differences in ATP binding affinities are observed, with only one active site capable of realizing the experimental binding free energy. Half-of-the-sites models in which the nucleotide is only present at one active site achieve their full binding potential irrespective of the subunit choice. This strongly suggests the involvement of an anti-cooperative mechanism. Pathways for relaying information between the two active sites are proposed. Conclusions The asymmetry uncovered here appears to be a common feature of oligomeric aminoacyl-tRNA synthetases, and may play an important functional role. We suggest a manner in which catalytic efficiency could be improved by LysU operating in an alternating sites mechanism. PMID:12787471

Residues in the H+ Translocation Site Define the pKa for Sugar Binding to LacY†

PubMed Central

Smirnova, Irina; Kasho, Vladimir; Sugihara, Junichi; Choe, Jun-Yong; Kaback, H. Ronald

2009-01-01

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (Kdapp) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low Kdapp) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates koff as a major factor for the affinity change at alkaline pH and confirms the effects of pH on Kdapp inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding. PMID:19689129

Steady-state pattern electroretinogram and short-duration transient visual evoked potentials in glaucomatous and healthy eyes.

PubMed

Amarasekera, Dilru C; Resende, Arthur F; Waisbourd, Michael; Puri, Sanjeev; Moster, Marlene R; Hark, Lisa A; Katz, L Jay; Fudemberg, Scott J; Mantravadi, Anand V

2018-01-01

This study evaluates two rapid electrophysiological glaucoma diagnostic tests that may add a functional perspective to glaucoma diagnosis. This study aimed to determine the ability of two office-based electrophysiological diagnostic tests, steady-state pattern electroretinogram and short-duration transient visual evoked potentials, to discern between glaucomatous and healthy eyes. This is a cross-sectional study in a hospital setting. Forty-one patients with glaucoma and 41 healthy volunteers participated in the study. Steady-state pattern electroretinogram and short-duration transient visual evoked potential testing was conducted in glaucomatous and healthy eyes. A 64-bar-size stimulus with both a low-contrast and high-contrast setting was used to compare steady-state pattern electroretinogram parameters in both groups. A low-contrast and high-contrast checkerboard stimulus was used to measure short-duration transient visual evoked potential parameters in both groups. Steady-state pattern electroretinogram parameters compared were MagnitudeD, MagnitudeD/Magnitude ratio, and the signal-to-noise ratio. Short-duration transient visual evoked potential parameters compared were amplitude and latency. MagnitudeD was significantly lower in glaucoma patients when using a low-contrast (P = 0.001) and high-contrast (P < 0.001) 64-bar-size steady-state pattern electroretinogram stimulus. MagnitudeD/Magnitude ratio and SNR were significantly lower in the glaucoma group when using a high-contrast 64-bar-size stimulus (P < 0.001 and P = 0.010, respectively). Short-duration transient visual evoked potential amplitude and latency were not significantly different between the two groups. Steady-state pattern electroretinogram was effectively able to discern between glaucomatous and healthy eyes. Steady-state pattern electroretinogram may thus have a role as a clinically useful electrophysiological diagnostic tool. © 2017 Royal Australian and New Zealand College of Ophthalmologists.

Time density curve analysis for C-arm FDCT PBV imaging.

PubMed

Kamran, Mudassar; Byrne, James V

2016-04-01

Parenchymal blood volume (PBV) estimation using C-arm flat detector computed tomography (FDCT) assumes a steady-state contrast concentration in cerebral vasculature for the scan duration. Using time density curve (TDC) analysis, we explored if the steady-state assumption is met for C-arm CT PBV scans, and how consistent the contrast-material dynamics in cerebral vasculature are across patients. Thirty C-arm FDCT datasets of 26 patients with aneurysmal-SAH, acquired as part of a prospective study comparing C-arm CT PBV with MR-PWI, were analysed. TDCs were extracted from the 2D rotational projections. Goodness-of-fit of TDCs to a steady-state horizontal-line-model and the statistical similarity among the individual TDCs were tested. Influence of the differences in TDC characteristics on the agreement of resulting PBV measurements with MR-CBV was calculated. Despite identical scan parameters and contrast-injection-protocol, the individual TDCs were statistically non-identical (p < 0.01). Using Dunn's multiple comparisons test, of the total 435 individual comparisons among the 30 TDCs, 330 comparisons (62%) reached statistical significance for difference. All TDCs deviated significantly (p < 0.01) from the steady-state horizontal-line-model. PBV values of those datasets for which the TDCs showed largest deviations from the steady-state model demonstrated poor agreement and correlation with MR-CBV, compared with the PBV values of those datasets for which the TDCs were closer to steady-state. For clinical C-arm CT PBV examinations, the administered contrast material does not reach the assumed 'ideal steady-state' for the duration of scan. Using a prolonged injection protocol, the degree to which the TDCs approximate the ideal steady-state influences the agreement of resulting PBV measurements with MR-CBV. © The Author(s) 2016.

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

NASA Astrophysics Data System (ADS)

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

2011-10-01

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

Comparative studies on the human serum albumin binding of the clinically approved EGFR inhibitors gefitinib, erlotinib, afatinib, osimertinib and the investigational inhibitor KP2187.

PubMed

Dömötör, Orsolya; Pelivan, Karla; Borics, Attila; Keppler, Bernhard K; Kowol, Christian R; Enyedy, Éva A

2018-05-30

Binding interactions between human serum albumin (HSA) and four approved epidermal growth factor receptor (EGFR) inhibitors gefitinib (GEF), erlotinib (ERL), afatinib (AFA), osimertinib (OSI), as well as the experimental drug KP2187, were investigated by means of spectrofluorometric and molecular modelling methods. Steady-state and time resolved spectrofluorometric techniques were carried out, including direct quenching of protein fluorescence and site marker displacement measurements. Proton dissociation processes and solvent dependent fluorescence properties were investigated as well. The EGFR inhibitors were predominantly presented in their single protonated form (HL + ) at physiological pH except ERL, which is charge-neutral. Significant solvent dependent fluorescence properties were found for GEF, ERL and KP2187, namely their emission spectra show strong dependence on the polarity and the hydrogen bonding ability of the solvents. The inhibitors proved to be bound at site I of HSA (in subdomain IIA) in a weak-to-moderate fashion (logK' 3.9-4.9) using spectrofluorometry. OSI (logK' 4.3) and KP2187 can additionally bind in site II (in subdomain IIIA), while GEF, ERL and AFA clearly show no interaction here. Docking methods qualitatively confirmed binding site preferences of compounds GEF and KP2187, and indicated that they probably bind to HSA in their neutral forms. Binding constants calculated on the basis of the various experimental data indicate a weak-to-moderate binding on HSA, only OSI exhibits somewhat higher affinity towards this protein. However, model calculations performed at physiological blood concentrations of HSA resulted in high (ca. 90%) bound fractions for the inhibitors, highlighting the importance of plasma protein binding. Copyright © 2018 Elsevier B.V. All rights reserved.

Prediction of elemental creep. [steady state and cyclic data from regression analysis

NASA Technical Reports Server (NTRS)

Davis, J. W.; Rummler, D. R.

1975-01-01

Cyclic and steady-state creep tests were performed to provide data which were used to develop predictive equations. These equations, describing creep as a function of stress, temperature, and time, were developed through the use of a least squares regression analyses computer program for both the steady-state and cyclic data sets. Comparison of the data from the two types of tests, revealed that there was no significant difference between the cyclic and steady-state creep strains for the L-605 sheet under the experimental conditions investigated (for the same total time at load). Attempts to develop a single linear equation describing the combined steady-state and cyclic creep data resulted in standard errors of estimates higher than obtained for the individual data sets. A proposed approach to predict elemental creep in metals uses the cyclic creep equation and a computer program which applies strain and time hardening theories of creep accumulation.

Absolute Steady-State Thermal Conductivity Measurements by Use of a Transient Hot-Wire System.

PubMed

Roder, H M; Perkins, R A; Laesecke, A; Nieto de Castro, C A

2000-01-01

A transient hot-wire apparatus was used to measure the thermal conductivity of argon with both steady-state and transient methods. The effects of wire diameter, eccentricity of the wire in the cavity, axial conduction, and natural convection were accounted for in the analysis of the steady-state measurements. Based on measurements on argon, the relative uncertainty at the 95 % level of confidence of the new steady-state measurements is 2 % at low densities. Using the same hot wires, the relative uncertainty of the transient measurements is 1 % at the 95 % level of confidence. This is the first report of thermal conductivity measurements made by two different methods in the same apparatus. The steady-state method is shown to complement normal transient measurements at low densities, particularly for fluids where the thermophysical properties at low densities are not known with high accuracy.

Quasi steady-state aerodynamic model development for race vehicle simulations

NASA Astrophysics Data System (ADS)

Mohrfeld-Halterman, J. A.; Uddin, M.

2016-01-01

Presented in this paper is a procedure to develop a high fidelity quasi steady-state aerodynamic model for use in race car vehicle dynamic simulations. Developed to fit quasi steady-state wind tunnel data, the aerodynamic model is regressed against three independent variables: front ground clearance, rear ride height, and yaw angle. An initial dual range model is presented and then further refined to reduce the model complexity while maintaining a high level of predictive accuracy. The model complexity reduction decreases the required amount of wind tunnel data thereby reducing wind tunnel testing time and cost. The quasi steady-state aerodynamic model for the pitch moment degree of freedom is systematically developed in this paper. This same procedure can be extended to the other five aerodynamic degrees of freedom to develop a complete six degree of freedom quasi steady-state aerodynamic model for any vehicle.

Technical challenges in the construction of the steady-state stellarator Wendelstein 7-X

NASA Astrophysics Data System (ADS)

Bosch, H.-S.; Wolf, R. C.; Andreeva, T.; Baldzuhn, J.; Birus, D.; Bluhm, T.; Bräuer, T.; Braune, H.; Bykov, V.; Cardella, A.; Durodié, F.; Endler, M.; Erckmann, V.; Gantenbein, G.; Hartmann, D.; Hathiramani, D.; Heimann, P.; Heinemann, B.; Hennig, C.; Hirsch, M.; Holtum, D.; Jagielski, J.; Jelonnek, J.; Kasparek, W.; Klinger, T.; König, R.; Kornejew, P.; Kroiss, H.; Krom, J. G.; Kühner, G.; Laqua, H.; Laqua, H. P.; Lechte, C.; Lewerentz, M.; Maier, J.; McNeely, P.; Messiaen, A.; Michel, G.; Ongena, J.; Peacock, A.; Pedersen, T. S.; Riedl, R.; Riemann, H.; Rong, P.; Rust, N.; Schacht, J.; Schauer, F.; Schroeder, R.; Schweer, B.; Spring, A.; Stäbler, A.; Thumm, M.; Turkin, Y.; Wegener, L.; Werner, A.; Zhang, D.; Zilker, M.; Akijama, T.; Alzbutas, R.; Ascasibar, E.; Balden, M.; Banduch, M.; Baylard, Ch.; Behr, W.; Beidler, C.; Benndorf, A.; Bergmann, T.; Biedermann, C.; Bieg, B.; Biel, W.; Borchardt, M.; Borowitz, G.; Borsuk, V.; Bozhenkov, S.; Brakel, R.; Brand, H.; Brown, T.; Brucker, B.; Burhenn, R.; Buscher, K.-P.; Caldwell-Nichols, C.; Cappa, A.; Cardella, A.; Carls, A.; Carvalho, P.; Ciupiński, Ł.; Cole, M.; Collienne, J.; Czarnecka, A.; Czymek, G.; Dammertz, G.; Dhard, C. P.; Davydenko, V. I.; Dinklage, A.; Drevlak, M.; Drotziger, S.; Dudek, A.; Dumortier, P.; Dundulis, G.; Eeten, P. v.; Egorov, K.; Estrada, T.; Faugel, H.; Fellinger, J.; Feng, Y.; Fernandes, H.; Fietz, W. H.; Figacz, W.; Fischer, F.; Fontdecaba, J.; Freund, A.; Funaba, T.; Fünfgelder, H.; Galkowski, A.; Gates, D.; Giannone, L.; García Regaña, J. M.; Geiger, J.; Geißler, S.; Greuner, H.; Grahl, M.; Groß, S.; Grosman, A.; Grote, H.; Grulke, O.; Haas, M.; Haiduk, L.; Hartfuß, H.-J.; Harris, J. H.; Haus, D.; Hein, B.; Heitzenroeder, P.; Helander, P.; Heller, R.; Hidalgo, C.; Hildebrandt, D.; Höhnle, H.; Holtz, A.; Holzhauer, E.; Holzthüm, R.; Huber, A.; Hunger, H.; Hurd, F.; Ihrke, M.; Illy, S.; Ivanov, A.; Jablonski, S.; Jaksic, N.; Jakubowski, M.; Jaspers, R.; Jensen, H.; Jenzsch, H.; Kacmarczyk, J.; Kaliatk, T.; Kallmeyer, J.; Kamionka, U.; Karaleviciu, R.; Kern, S.; Keunecke, M.; Kleiber, R.; Knauer, J.; Koch, R.; Kocsis, G.; Könies, A.; Köppen, M.; Koslowski, R.; Koshurinov, J.; Krämer-Flecken, A.; Krampitz, R.; Kravtsov, Y.; Krychowiak, M.; Krzesinski, G.; Ksiazek, I.; Kubkowska, M.; Kus, A.; Langish, S.; Laube, R.; Laux, M.; Lazerson, S.; Lennartz, M.; Li, C.; Lietzow, R.; Lohs, A.; Lorenz, A.; Louche, F.; Lubyako, L.; Lumsdaine, A.; Lyssoivan, A.; Maaßberg, H.; Marek, P.; Martens, C.; Marushchenko, N.; Mayer, M.; Mendelevitch, B.; Mertens, Ph.; Mikkelsen, D.; Mishchenko, A.; Missal, B.; Mizuuchi, T.; Modrow, H.; Mönnich, T.; Morizaki, T.; Murakami, S.; Musielok, F.; Nagel, M.; Naujoks, D.; Neilson, H.; Neubauer, O.; Neuner, U.; Nocentini, R.; Noterdaeme, J.-M.; Nührenberg, C.; Obermayer, S.; Offermanns, G.; Oosterbeek, H.; Otte, M.; Panin, A.; Pap, M.; Paquay, S.; Pasch, E.; Peng, X.; Petrov, S.; Pilopp, D.; Pirsch, H.; Plaum, B.; Pompon, F.; Povilaitis, M.; Preinhaelter, J.; Prinz, O.; Purps, F.; Rajna, T.; Récsei, S.; Reiman, A.; Reiter, D.; Remmel, J.; Renard, S.; Rhode, V.; Riemann, J.; Rimkevicius, S.; Riße, K.; Rodatos, A.; Rodin, I.; Romé, M.; Roscher, H.-J.; Rummel, K.; Rummel, Th.; Runov, A.; Ryc, L.; Sachtleben, J.; Samartsev, A.; Sanchez, M.; Sano, F.; Scarabosio, A.; Schmid, M.; Schmitz, H.; Schmitz, O.; Schneider, M.; Schneider, W.; Scheibl, L.; Scholz, M.; Schröder, G.; Schröder, M.; Schruff, J.; Schumacher, H.; Shikhovtsev, I. V.; Shoji, M.; Siegl, G.; Skodzik, J.; Smirnow, M.; Speth, E.; Spong, D. A.; Stadler, R.; Sulek, Z.; Szabó, V.; Szabolics, T.; Szetefi, T.; Szökefalvi-Nagy, Z.; Tereshchenko, A.; Thomsen, H.; Thumm, M.; Timmermann, D.; Tittes, H.; Toi, K.; Tournianski, M.; Toussaint, U. v.; Tretter, J.; Tulipán, S.; Turba, P.; Uhlemann, R.; Urban, J.; Urbonavicius, E.; Urlings, P.; Valet, S.; Van Eester, D.; Van Schoor, M.; Vervier, M.; Viebke, H.; Vilbrandt, R.; Vrancken, M.; Wauters, T.; Weissgerber, M.; Weiß, E.; Weller, A.; Wendorf, J.; Wenzel, U.; Windisch, T.; Winkler, E.; Winkler, M.; Wolowski, J.; Wolters, J.; Wrochna, G.; Xanthopoulos, P.; Yamada, H.; Yokoyama, M.; Zacharias, D.; Zajac, J.; Zangl, G.; Zarnstorff, M.; Zeplien, H.; Zoletnik, S.; Zuin, M.

2013-12-01

The next step in the Wendelstein stellarator line is the large superconducting device Wendelstein 7-X, currently under construction in Greifswald, Germany. Steady-state operation is an intrinsic feature of stellarators, and one key element of the Wendelstein 7-X mission is to demonstrate steady-state operation under plasma conditions relevant for a fusion power plant. Steady-state operation of a fusion device, on the one hand, requires the implementation of special technologies, giving rise to technical challenges during the design, fabrication and assembly of such a device. On the other hand, also the physics development of steady-state operation at high plasma performance poses a challenge and careful preparation. The electron cyclotron resonance heating system, diagnostics, experiment control and data acquisition are prepared for plasma operation lasting 30 min. This requires many new technological approaches for plasma heating and diagnostics as well as new concepts for experiment control and data acquisition.

40 CFR Appendix II to Part 1042 - Steady-State Duty Cycles

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Steady-State Duty Cycles II Appendix..., App. II Appendix II to Part 1042—Steady-State Duty Cycles (a) The following duty cycles apply as specified in § 1042.505(b)(1): (1) The following duty cycle applies for discrete-mode testing: E3 mode No...

Min-By-Min Respiratory Exchange and Oxygen Uptake Kinetics During Steady-State Exercise in Subjects of High and Low Max VO2

ERIC Educational Resources Information Center

Weltman, Arthur; Katch, Victor

1976-01-01

No statistically meaningful differences in steady-state vo2 uptake for high and low max vo2 groups was indicated in this study, but a clear tendency was observed for the high max vo2 group to reach the steady-state at a faster rate. (MB)

Quantitative controls on submarine slope failure morphology

USGS Publications Warehouse

Lee, H.J.; Schwab, W.C.; Edwards, B.D.; Kayen, R.E.

1991-01-01

The concept of the steady-state of deformation can be applied to predicting the ultimate form a landslide will take. The steady-state condition, defined by a line in void ratio-effective stress space, exists at large levels of strain and remolding. Conceptually, if sediment initially exists with void ratio-effective stress conditions above the steady-state line, the sediment shear strength will decrease during a transient loading event, such as an earthquake or storm. If the reduced shear strength existing at the steady state is less than the downslope shear stress induced by gravity, then large-scale internal deformation, disintegration, and flow will occur. -from Authors

Isotopic composition of transpiration and rates of change in leaf water isotopologue storage in response to environmental variables.

PubMed

Simonin, Kevin A; Roddy, Adam B; Link, Percy; Apodaca, Randy; Tu, Kevin P; Hu, Jia; Dawson, Todd E; Barbour, Margaret M

2013-12-01

During daylight hours, the isotope composition of leaf water generally approximates steady-state leaf water isotope enrichment model predictions. However, until very recently there was little direct confirmation that isotopic steady-state (ISS) transpiration in fact exists. Using isotope ratio infrared spectroscopy (IRIS) and leaf gas exchange systems we evaluated the isotope composition of transpiration and the rate of change in leaf water isotopologue storage (isostorage) when leaves were exposed to variable environments. In doing so, we developed a method for controlling the absolute humidity entering the gas exchange cuvette for a wide range of concentrations without changing the isotope composition of water vapour. The measurement system allowed estimation of (18)O enrichment both at the evaporation site and for bulk leaf water, in the steady state and the non-steady state. We show that non-steady-state effects dominate the transpiration isoflux even when leaves are at physiological steady state. Our results suggest that a variable environment likely prevents ISS transpiration from being achieved and that this effect may be exacerbated by lengthy leaf water turnover times due to high leaf water contents. © 2013 John Wiley & Sons Ltd.

Modeling Ca2+ Feedback on a Single Inositol 1,4,5-Trisphosphate Receptor and Its Modulation by Ca2+ Buffers

PubMed Central

Shuai, Jianwei; Pearson, John E.; Parker, Ian

2008-01-01

The inositol 1,4,5-trisphosphate receptor/channel (IP3R) is a major regulator of intracellular Ca2+ signaling, and liberates Ca2+ ions from the endoplasmic reticulum in response to binding at cytosolic sites for both IP3 and Ca2+. Although the steady-state gating properties of the IP3R have been extensively studied and modeled under conditions of fixed [IP3] and [Ca2+], little is known about how Ca2+ flux through a channel may modulate the gating of that same channel by feedback onto activating and inhibitory Ca2+ binding sites. We thus simulated the dynamics of Ca2+ self-feedback on monomeric and tetrameric IP3R models. A major conclusion is that self-activation depends crucially on stationary cytosolic Ca2+ buffers that slow the collapse of the local [Ca2+] microdomain after closure. This promotes burst-like reopenings by the rebinding of Ca2+ to the activating site; whereas inhibitory actions are substantially independent of stationary buffers but are strongly dependent on the location of the inhibitory Ca2+ binding site on the IP3R in relation to the channel pore. PMID:18641077

Interaction of MreB-derived antimicrobial peptides with membranes.

PubMed

Saikia, Karabi; Chaudhary, Nitin

2018-03-25

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

Investigation of the binding affinity in vitamin B12-Bovine serum albumin system using various spectroscopic methods

NASA Astrophysics Data System (ADS)

Makarska-Bialokoz, Magdalena

2017-09-01

The binding affinity between vitamin B12 (VitB12) and bovine serum albumin (BSA) has been investigated in aqueous solution at pH = 7.4, employing UV-vis absorption and steady-state, synchronous and three-dimensional fluorescence spectra techniques. Representative effects noted for BSA intrinsic fluorescence resulting from the interactions with VitB12 confirm the formation of π-π stacked non-covalent and non-fluorescent complexes in the system VitB12-BSA. All the determined parameters, the binding, fluorescence quenching and bimolecular quenching rate constants (of the order of 104 L mol- 1, 103 L mol- 1 and 1011 L mol- 1 s- 1, respectively), as well as Förster resonance energy transfer parameters validate the mechanism of static quenching. The interaction with VitB12 induces folding of the polypeptide chains around Trp residues of BSA, resulting in a more hydrophobic surrounding. Presented outcomes suggest that the addition of VitB12 can lead to the more organized BSA conformation and its more folded tertiary structure, what could influence the physiological functions of bovine serum albumin, notably in case of its overuse or abnormal metabolism.

Protein–Protein Interactions Modulate the Docking-Dependent E3-Ubiquitin Ligase Activity of Carboxy-Terminus of Hsc70-Interacting Protein (CHIP)*

PubMed Central

Narayan, Vikram; Landré, Vivien; Ning, Jia; Hernychova, Lenka; Muller, Petr; Verma, Chandra; Walkinshaw, Malcolm D.; Blackburn, Elizabeth A.; Ball, Kathryn L.

2015-01-01

CHIP is a tetratricopeptide repeat (TPR) domain protein that functions as an E3-ubiquitin ligase. As well as linking the molecular chaperones to the ubiquitin proteasome system, CHIP also has a docking-dependent mode where it ubiquitinates native substrates, thereby regulating their steady state levels and/or function. Here we explore the effect of Hsp70 on the docking-dependent E3-ligase activity of CHIP. The TPR-domain is revealed as a binding site for allosteric modulators involved in determining CHIP's dynamic conformation and activity. Biochemical, biophysical and modeling evidence demonstrate that Hsp70-binding to the TPR, or Hsp70-mimetic mutations, regulate CHIP-mediated ubiquitination of p53 and IRF-1 through effects on U-box activity and substrate binding. HDX-MS was used to establish that conformational-inhibition-signals extended from the TPR-domain to the U-box. This underscores inter-domain allosteric regulation of CHIP by the core molecular chaperones. Defining the chaperone-associated TPR-domain of CHIP as a manager of inter-domain communication highlights the potential for scaffolding modules to regulate, as well as assemble, complexes that are fundamental to protein homeostatic control. PMID:26330542

Detection-enhanced steady state entanglement with ions.

PubMed

Bentley, C D B; Carvalho, A R R; Kielpinski, D; Hope, J J

2014-07-25

Driven dissipative steady state entanglement schemes take advantage of coupling to the environment to robustly prepare highly entangled states. We present a scheme for two trapped ions to generate a maximally entangled steady state with fidelity above 0.99, appropriate for use in quantum protocols. Furthermore, we extend the scheme by introducing detection of our dissipation process, significantly enhancing the fidelity. Our scheme is robust to anomalous heating and requires no sympathetic cooling.

Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation.

PubMed

Wang, Fang; Travins, Jeremy; DeLaBarre, Byron; Penard-Lacronique, Virginie; Schalm, Stefanie; Hansen, Erica; Straley, Kimberly; Kernytsky, Andrew; Liu, Wei; Gliser, Camelia; Yang, Hua; Gross, Stefan; Artin, Erin; Saada, Veronique; Mylonas, Elena; Quivoron, Cyril; Popovici-Muller, Janeta; Saunders, Jeffrey O; Salituro, Francesco G; Yan, Shunqi; Murray, Stuart; Wei, Wentao; Gao, Yi; Dang, Lenny; Dorsch, Marion; Agresta, Sam; Schenkein, David P; Biller, Scott A; Su, Shinsan M; de Botton, Stephane; Yen, Katharine E

2013-05-03

A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite (R)-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.

A study of the interaction between malachite green and lysozyme by steady-state fluorescence.

PubMed

Ding, Fei; Liu, Wei; Liu, Feng; Li, Zhi-Yuan; Sun, Ying

2009-09-01

The interaction of a N-methylated diaminotriphenylmethane dye, malachite green, with lysozyme was investigated by fluorescence spectroscopic techniques under physiological conditions. The binding parameters have been evaluated by fluorescence quenching methods. The results revealed that malachite green caused the fluorescence quenching of lysozyme through a static quenching procedure. The thermodynamic parameters like DeltaH and DeltaS were calculated to be -15.33 kJ mol(-1) and 19.47 J mol(-1) K(-1) according to van't Hoff equation, respectively, which proves main interaction between malachite green and lysozyme is hydrophobic forces and hydrogen bond contact. The distance r between donor (lysozyme) and acceptor (malachite green) was obtained to be 3.82 nm according to Frster's theory. The results of synchronous fluorescence, UV/vis and three-dimensional fluorescence spectra showed that binding of malachite green with lysozyme can induce conformational changes in lysozyme. In addition, the effects of common ions on the constants of lysozyme-malachite green complex were also discussed.

Espins and the actin cytoskeleton of hair cell stereocilia and sensory cell microvilli

PubMed Central

Sekerková, Gabriella; Zheng, Lili; Loomis, Patricia A.; Mugnaini, Enrico; Bartles, James R.

2008-01-01

The espins are novel actin-bundling proteins that are produced in multiple isoforms from a single gene. They are present at high concentration in the parallel actin bundle of hair cell stereocilia and are the target of deafness mutations in mice and humans. Espins are also enriched in the microvilli of taste receptor cells, solitary chemoreceptor cells, vomeronasal sensory neurons and Merkel cells, suggesting that espins play important roles in the microvillar projections of vertebrate sensory cells. Espins are potent actin-bundling proteins that are not inhibited by Ca2+. In cells, they efficiently elongate parallel actin bundles and, thereby, help determine the steady-state length of microvilli and stereocilia. Espins bind actin monomer via their WH2 domain and can assemble actin bundles in cells. Certain espin isoforms can also bind phosphatidylinositol 4,5-bisphosphate, profilins or SH3 proteins. These biological activities distinguish espins from other actin-bundling proteins and may make them well-suited to sensory cells. PMID:16909209

Chloroperoxidase-catalyzed oxidation of 4,6-dimethyldibenzothiophene as dimer complexes: evidence for kinetic cooperativity.

PubMed

Torres, Eduardo; Aburto, Jorge

2005-05-15

A sigmoidal kinetic behavior of chloroperoxidase for the oxidation of 4,6-dimethyldibenzothiophene (4,6-DMDBT) in water-miscible organic solvent is for the first time reported. Kinetics of 4,6-DMDBT oxidation showed a cooperative profile probably due to the capacity of chloroperoxidase to recognize a substrate dimer (pi-pi dimer) in its active site. Experimental evidence is given for dimer formation and its presence in the active site of chloroperoxidase. The kinetic data were adjusted for a binding site able to interact with either monomer or dimer substrates, producing a cooperative model describing a one-site binding of two related species. Determination of kinetics constants by iterative calculations of possible oxidation paths of 4,6-DMDBT suggests that kinetics oxidation of dimer substrate is preferred when compared to monomer oxidation. Steady-state fluorometry of substrate in the absence and presence of chloroperoxidase, described by the spectral center of mass, supports this last conclusion.

Contribution of cellular retinol-binding protein type 1 to retinol metabolism during mouse development.

PubMed

Matt, Nicolas; Schmidt, Carsten K; Dupé, Valérie; Dennefeld, Christine; Nau, Heinz; Chambon, Pierre; Mark, Manuel; Ghyselinck, Norbert B

2005-05-01

Within cells, retinol (ROL) is bound to cytoplasmic proteins (cellular retinol-binding proteins [CRBPs]), whose proposed function is to protect it from unspecific enzymes through channeling to retinoid-metabolizing pathways. We show that, during development, ROL and retinyl ester levels are decreased in CRBP type 1 (CRBP1) -deficient embryos and fetuses by 50% and 80%, respectively. The steady state level of retinoic acid (RA) is also decreased but to a lesser extent. However, CRBP1-null fetuses do not exhibit the abnormalities characteristic of a vitamin A-deficiency syndrome. Neither CRBP1 deficiency alters the expression patterns of RA-responding genes during development, nor does CRBP1 availability modify the expression of an RA-dependent gene in primary embryonic fibroblasts treated with ROL. Therefore, CRBP1 is required in prenatal life to maintain normal amounts of ROL and to ensure its efficient storage but seems of secondary importance for RA synthesis, at least under conditions of maternal vitamin A sufficiency. Copyright 2005 Wiley-Liss, Inc.

Steady-state MR imaging sequences: physics, classification, and clinical applications.

PubMed

Chavhan, Govind B; Babyn, Paul S; Jankharia, Bhavin G; Cheng, Hai-Ling M; Shroff, Manohar M

2008-01-01

Steady-state sequences are a class of rapid magnetic resonance (MR) imaging techniques based on fast gradient-echo acquisitions in which both longitudinal magnetization (LM) and transverse magnetization (TM) are kept constant. Both LM and TM reach a nonzero steady state through the use of a repetition time that is shorter than the T2 relaxation time of tissue. When TM is maintained as multiple radiofrequency excitation pulses are applied, two types of signal are formed once steady state is reached: preexcitation signal (S-) from echo reformation; and postexcitation signal (S+), which consists of free induction decay. Depending on the signal sampled and used to form an image, steady-state sequences can be classified as (a) postexcitation refocused (only S+ is sampled), (b) preexcitation refocused (only S- is sampled), and (c) fully refocused (both S+ and S- are sampled) sequences. All tissues with a reasonably long T2 relaxation time will show additional signals due to various refocused echo paths. Steady-state sequences have revolutionized cardiac imaging and have become the standard for anatomic functional cardiac imaging and for the assessment of myocardial viability because of their good signal-to-noise ratio and contrast-to-noise ratio and increased speed of acquisition. They are also useful in abdominal and fetal imaging and hold promise for interventional MR imaging. Because steady-state sequences are now commonly used in MR imaging, radiologists will benefit from understanding the underlying physics, classification, and clinical applications of these sequences.

Dynamics of optical matter creation and annihilation in colloidal liquids controlled by laser trapping power.

PubMed

Liu, Jin; Dai, Qiao-Feng; Huang, Xu-Guang; Wu, Li-Jun; Guo, Qi; Hu, Wei; Yang, Xiang-Bo; Lan, Sheng; Gopal, Achanta Venu; Trofimov, Vyacheslav A

2008-11-15

We investigate the dynamics of optical matter creation and annihilation in a colloidal liquid that was employed to construct an all-optical switch. It is revealed that the switching-on process can be characterized by the Fermi-Dirac distribution function, while the switching-off process can be described by a steady state followed by a single exponential decay. The phase transition times exhibit a strong dependence on trapping power. With an increasing trapping power, while the switching-on time decreases rapidly, the switch-off time increases significantly, indicating the effects of optical binding and van der Waals force on the lifetime of the optical matter.

[Passage of nonsteroidal anti-inflammatory agents across the synovial membrane].

PubMed

Netter, P; Bannwarth, B; Monot, C; Royer, R J; Gaucher, A

1983-09-24

The therapeutic effectiveness of non-steroid anti-inflammatory (NSAI) drugs is partly determined by their passage across the synovial membrane. The synovium can be compared to a double barrier the permeability of which to NSAI drugs depends on the degree of inflammation of the joint and on the pharmacokinetic properties of the drugs (lipophilia, pka, protein-binding). A few hours after one single systemic dose, concentrations in the synovial fluid are higher than in serum. During chronic administration, concentrations of NSAI drugs with a short half-life vary less in synovial fluid than in serum. During steady state, free fractions of NSAI drugs with prolonged half-life may be similar in both compartments.

Time density curve analysis for C-arm FDCT PBV imaging

PubMed Central

Byrne, James V

2016-01-01

Introduction Parenchymal blood volume (PBV) estimation using C-arm flat detector computed tomography (FDCT) assumes a steady-state contrast concentration in cerebral vasculature for the scan duration. Using time density curve (TDC) analysis, we explored if the steady-state assumption is met for C-arm CT PBV scans, and how consistent the contrast-material dynamics in cerebral vasculature are across patients. Methods Thirty C-arm FDCT datasets of 26 patients with aneurysmal-SAH, acquired as part of a prospective study comparing C-arm CT PBV with MR-PWI, were analysed. TDCs were extracted from the 2D rotational projections. Goodness-of-fit of TDCs to a steady-state horizontal-line-model and the statistical similarity among the individual TDCs were tested. Influence of the differences in TDC characteristics on the agreement of resulting PBV measurements with MR-CBV was calculated. Results Despite identical scan parameters and contrast-injection-protocol, the individual TDCs were statistically non-identical (p < 0.01). Using Dunn's multiple comparisons test, of the total 435 individual comparisons among the 30 TDCs, 330 comparisons (62%) reached statistical significance for difference. All TDCs deviated significantly (p < 0.01) from the steady-state horizontal-line-model. PBV values of those datasets for which the TDCs showed largest deviations from the steady-state model demonstrated poor agreement and correlation with MR-CBV, compared with the PBV values of those datasets for which the TDCs were closer to steady-state. Conclusion For clinical C-arm CT PBV examinations, the administered contrast material does not reach the assumed ‘ideal steady-state’ for the duration of scan. Using a prolonged injection protocol, the degree to which the TDCs approximate the ideal steady-state influences the agreement of resulting PBV measurements with MR-CBV. PMID:26769736

Characterization of nicotinamidases: steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism.

PubMed

French, Jarrod B; Cen, Yana; Vrablik, Tracy L; Xu, Ping; Allen, Eleanor; Hanna-Rose, Wendy; Sauve, Anthony A

2010-12-14

Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast, and invertebrates, but there are none found in mammals. Although recent structural work has improved our understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data show that nicotinamidases are required for the growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans regulate life span in their respective organisms, consistent with proposed roles in the regulation of NAD(+) metabolism and organismal aging. In this work, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, Sa. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme disease), and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state k(cat) values typically exceeding 1 s(-1). The K(m) values for nicotinamide are low and in the range of 2 -110 μM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low micromolar to low nanomolar range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex that is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyze exchange of (18)O into the carboxy oxygens of nicotinic acid with H(2)(18)O. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid (18)O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion, and the intermediacy of a thioester intermediate.

Nucleation, growth, and repair of a cobalt-based oxygen evolving catalyst.

PubMed

Surendranath, Yogesh; Lutterman, Daniel A; Liu, Yi; Nocera, Daniel G

2012-04-11

The mechanism of nucleation, steady-state growth, and repair is investigated for an oxygen evolving catalyst prepared by electrodeposition from Co(2+) solutions in weakly basic electrolytes (Co-OEC). Potential step chronoamperometry and atomic force microscopy reveal that nucleation of Co-OEC is progressive and reaches a saturation surface coverage of ca. 70% on highly oriented pyrolytic graphite substrates. Steady-state electrodeposition of Co-OEC exhibits a Tafel slope approximately equal to 2.3 × RT/F. The electrochemical rate law exhibits a first order dependence on Co(2+) and inverse orders on proton (third order) and proton acceptor, methylphosphonate (first order for 1.8 mM ≤ [MeP(i)] ≤ 18 mM and second order dependence for 32 mM ≤ [MeP(i)] ≤ 180 mM). These electrokinetic studies, combined with recent XAS studies of catalyst structure, suggest a mechanism for steady state growth at intermediate MeP(i) concentration (1.8-18 mM) involving a rapid solution equilibrium between aquo Co(II) and Co(III) hydroxo species accompanied with a rapid surface equilibrium involving electrolyte dissociation and deprotonation of surface bound water. These equilibria are followed by a chemical rate-limiting step for incorporation of Co(III) into the growing cobaltate clusters comprising Co-OEC. At higher concentrations of MeP(i) ([MeP(i)] ≥ 32 mM), MePO(3)(2-) equilibrium binding to Co(II) in solution is suggested by the kinetic data. Consistent with the disparate pH profiles for oxygen evolution electrocatalysis and catalyst formation, NMR-based quantification of catalyst dissolution as a function of pH demonstrates functional stability and repair at pH values >6 whereas catalyst corrosion prevails at lower pH values. These kinetic insights provide a basis for developing and operating functional water oxidation (photo)anodes under benign pH conditions. © 2012 American Chemical Society

Pressure Distribution and Performance Impacts of Aerospike Nozzles on Rotating Detonation Engines

DTIC Science & Technology

2017-06-01

design methodology at both on- and off-design conditions anticipated throughout the combustion cycle. Steady-state, non -reacting computational fluid...operation. Therefore, the nozzle contour was designed using a traditional, steady-state design methodology at both on- and off-design conditions...anticipated throughout the combustion cycle. Steady-state, non -reacting computational fluid dynamics (CFD) simulations were performed on various nozzle

Universal, computer facilitated, steady state oscillator, closed loop analysis theory and some applications to precision oscillators

NASA Technical Reports Server (NTRS)

Parzen, Benjamin

1992-01-01

The theory of oscillator analysis in the immittance domain should be read in conjunction with the additional theory presented here. The combined theory enables the computer simulation of the steady state oscillator. The simulation makes the calculation of the oscillator total steady state performance practical, including noise at all oscillator locations. Some specific precision oscillators are analyzed.

Steady state analysis of Boolean molecular network models via model reduction and computational algebra.

PubMed

Veliz-Cuba, Alan; Aguilar, Boris; Hinkelmann, Franziska; Laubenbacher, Reinhard

2014-06-26

A key problem in the analysis of mathematical models of molecular networks is the determination of their steady states. The present paper addresses this problem for Boolean network models, an increasingly popular modeling paradigm for networks lacking detailed kinetic information. For small models, the problem can be solved by exhaustive enumeration of all state transitions. But for larger models this is not feasible, since the size of the phase space grows exponentially with the dimension of the network. The dimension of published models is growing to over 100, so that efficient methods for steady state determination are essential. Several methods have been proposed for large networks, some of them heuristic. While these methods represent a substantial improvement in scalability over exhaustive enumeration, the problem for large networks is still unsolved in general. This paper presents an algorithm that consists of two main parts. The first is a graph theoretic reduction of the wiring diagram of the network, while preserving all information about steady states. The second part formulates the determination of all steady states of a Boolean network as a problem of finding all solutions to a system of polynomial equations over the finite number system with two elements. This problem can be solved with existing computer algebra software. This algorithm compares favorably with several existing algorithms for steady state determination. One advantage is that it is not heuristic or reliant on sampling, but rather determines algorithmically and exactly all steady states of a Boolean network. The code for the algorithm, as well as the test suite of benchmark networks, is available upon request from the corresponding author. The algorithm presented in this paper reliably determines all steady states of sparse Boolean networks with up to 1000 nodes. The algorithm is effective at analyzing virtually all published models even those of moderate connectivity. The problem for large Boolean networks with high average connectivity remains an open problem.

Steady state analysis of Boolean molecular network models via model reduction and computational algebra

PubMed Central

2014-01-01

Background A key problem in the analysis of mathematical models of molecular networks is the determination of their steady states. The present paper addresses this problem for Boolean network models, an increasingly popular modeling paradigm for networks lacking detailed kinetic information. For small models, the problem can be solved by exhaustive enumeration of all state transitions. But for larger models this is not feasible, since the size of the phase space grows exponentially with the dimension of the network. The dimension of published models is growing to over 100, so that efficient methods for steady state determination are essential. Several methods have been proposed for large networks, some of them heuristic. While these methods represent a substantial improvement in scalability over exhaustive enumeration, the problem for large networks is still unsolved in general. Results This paper presents an algorithm that consists of two main parts. The first is a graph theoretic reduction of the wiring diagram of the network, while preserving all information about steady states. The second part formulates the determination of all steady states of a Boolean network as a problem of finding all solutions to a system of polynomial equations over the finite number system with two elements. This problem can be solved with existing computer algebra software. This algorithm compares favorably with several existing algorithms for steady state determination. One advantage is that it is not heuristic or reliant on sampling, but rather determines algorithmically and exactly all steady states of a Boolean network. The code for the algorithm, as well as the test suite of benchmark networks, is available upon request from the corresponding author. Conclusions The algorithm presented in this paper reliably determines all steady states of sparse Boolean networks with up to 1000 nodes. The algorithm is effective at analyzing virtually all published models even those of moderate connectivity. The problem for large Boolean networks with high average connectivity remains an open problem. PMID:24965213

Binding interaction of ramipril with bovine serum albumin (BSA): Insights from multi-spectroscopy and molecular docking methods.

PubMed

Shi, Jie-Hua; Pan, Dong-Qi; Jiang, Min; Liu, Ting-Ting; Wang, Qi

2016-11-01

The binding interaction between a typical angiotensin-converting enzyme inhibitor (ACEI), ramipril, and a transport protein, bovine serum albumin (BSA), was studied in vitro using UV-vis absorption spectroscopy, steady-state fluorescence spectroscopic titration, synchronous fluorescence spectroscopy, three dimensional fluorescence spectroscopy, circular dichroism and molecular docking under the imitated physiological conditions (pH=7.4). The experimental results suggested that the intrinsic fluorescence of BSA was quenched by ramipril thought a static quenching mechanism, indicating that the stable ramipril-BSA complex was formed by the intermolecular interaction. The number of binding sites (n) and binding constant of ramipril-BSA complex were about 1 and 3.50×10 4 M -1 at 298K, respectively, suggesting that there was stronger binding interaction of ramipril with BSA. The thermodynamic parameters together with molecular docking study revealed that both van der Waal's forces and hydrogen bonding interaction dominated the formation of the ramipril-BSA complex and the binding interaction of BSA with ramipril is enthalpy-driven processes due to |ΔH°|>|TΔS°| and ΔG°<0. The spatial distance between ramipril and BSA was calculated to be 3.56nm based on Förster's non-radiative energy transfer theory. The results of the competitive displacement experiments and molecular docking confirmed that ramipril inserted into the subdomain IIA (site I) of BSA, resulting in a slight change in the conformation of BSA but BSA still retained its secondary structure α-helicity. Copyright © 2016 Elsevier B.V. All rights reserved.

Structure and gelation properties of casein micelles doped with curcumin under acidic conditions.

PubMed

Khanji, Aya N; Michaux, Florentin; Jasniewski, Jordane; Petit, Jeremy; Lahimer, Emna; Cherif, Mohamed; Salameh, Dominique; Rizk, Toufic; Banon, Sylvie

2015-12-01

In this study, the ability of micellar casein (MC) to interact with curcumin during acidification and to produce acid gel was investigated. Steady-state fluorescence spectroscopy of curcumin variation and fluorescence quenching of caseins upon binding with curcumin molecules were evidenced. Increasing the temperature from 20 to 35 °C enhanced MC-curcumin interactions as reflected by the increase in the binding constant from 0.6 ± 0.3 × 10(4) to 6.6 ± 0.6 × 10(4) M(-1). From changes in entropy, enthalpy and Gibbs free energy, hydrophobic interactions were proposed as major binding forces. Static fluorescence MC quenching was demonstrated for the MC-curcumin complex during acidification. From pH 7.4 to pH 5.0, the binding site numbers varied in the range from 1.25 ± 0.05 to 1.49 ± 0.05 and the binding constant kb varied from 3.9 ± 0.4 × 10(4) to 7.5 ± 0.7 × 10(4) M(-1). Small angle X-ray scattering profiles demonstrated that the MC internal structure was unchanged upon curcumin binding. The ζ-potential value of curcumin-doped MC indicated that curcumin did not modify the global charge of MC particles. Acid gelation studied by oscillation rheology and static multiple light scattering at 20 and 35 °C led to a similar behavior for native and curcumin-doped MC suspensions. For the first time, it was demonstrated that the colloidal and functional properties of MC were unchanged when doped with curcumin during acidification.

Selective Inhibition of Mutant Isocitrate Dehydrogenase 1 (IDH1) via Disruption of a Metal Binding Network by an Allosteric Small Molecule

PubMed Central

Deng, Gejing; Shen, Junqing; Yin, Ming; McManus, Jessica; Mathieu, Magali; Gee, Patricia; He, Timothy; Shi, Chaomei; Bedel, Olivier; McLean, Larry R.; Le-Strat, Frank; Zhang, Ying; Marquette, Jean-Pierre; Gao, Qiang; Zhang, Bailin; Rak, Alexey; Hoffmann, Dietmar; Rooney, Eamonn; Vassort, Aurelie; Englaro, Walter; Li, Yi; Patel, Vinod; Adrian, Francisco; Gross, Stefan; Wiederschain, Dmitri; Cheng, Hong; Licht, Stuart

2015-01-01

Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) confer a neomorphic enzymatic activity: the reduction of α-ketoglutarate to d-2-hydroxyglutaric acid, which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. Although selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well understood. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified compound 1, a bis-imidazole phenol that inhibits d-2-hydroxyglutaric acid production in cells. We investigated the mode of inhibition of compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site and that inhibition is competitive with respect to Mg2+. A crystal structure of compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1 and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme. PMID:25391653

SOCS2 Binds to and Regulates EphA2 through Multiple Mechanisms.

PubMed

Pilling, Carissa; Cooper, Jonathan A

2017-09-07

Suppressors of cytokine signaling (SOCS) proteins inhibit signaling by serving as substrate receptors for the Cullin5-RING E3 ubiquitin ligase (CRL5) and through a variety of CRL5-independent mechanisms. CRL5, SOCS2 and SOCS6 are implicated in suppressing transformation of epithelial cells. We identified cell proteins that interact with SOCS2 and SOCS6 using two parallel proteomics techniques: BioID and Flag affinity purification mass spectrometry. The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) was identified as a SOCS2-interacting protein. SOCS2-EphA2 binding requires the SOCS2 SH2 domain and EphA2 activation loop autophosphorylation, which is stimulated by Ephrin A1 (EfnA1) or by phosphotyrosine phosphatase inhibition. Surprisingly, EfnA1-stimulated EphA2-SOCS2 binding is delayed until EphA2 has been internalized into endosomes. This suggests that SOCS2 binds to EphA2 in the context of endosomal membranes. We also found that SOCS2 overexpression decreases steady state levels of EphA2, consistent with increased EphA2 degradation. This effect is indirect: SOCS2 induces EfnA1 expression, and EfnA1 induces EphA2 down-regulation. Other RTKs have been reported to bind, and be regulated by, over-expressed SOCS proteins. Our data suggest that SOCS protein over-expression may regulate receptor tyrosine kinases through indirect and direct mechanisms.

Characterizing the binding interaction of fungicide boscalid with bovine serum albumin (BSA): A spectroscopic study in combination with molecular docking approach.

PubMed

Lou, Yan-Yue; Zhou, Kai-Li; Shi, Jie-Hua; Pan, Dong-Qi

2017-08-01

Boscalid, a carboxamide fungicide, is used in the treatment of grey mould and powdery mildew, widely applied to a variety of crops and fruits such as rice, wheat, grapes and pears. It will become a potential risk for health due to its widely application and residue in crops and fruits. In this study, the binding interaction between boscalid and bovine serum albumin (BSA) was characterized using steady-state fluorescence spectroscopy, ultraviolet spectroscopy (UV), synchronous fluorescence spectroscopy, 3D fluorescence spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and molecular docking to ascertain the store, transport and distribution of boscalid in vivo. The experimental results indicated that the fluorescence of BSA was quenched due to the forming the static boscalid-BSA complex with the binding constant of 4.57×10 3 M -1 at 298 K and boscalid bound on the subdomain III A (site II) of BSA through van der Waals force and hydrogen bonding interaction. The binding process of boscalid with BSA was spontaneous and enthalpy-driven process based on ΔG 0 <0 and |ΔH 0 |>T|ΔS 0 | over the studied temperature range. Meanwhile, the obvious change in the conformation of boscalid was observed while the slight change in the conformation of BSA when binding boscalid to the BSA, implying that the flexibility of boscalid contributes to increasing the stability of the boscalid-BSA complex. Copyright © 2017 Elsevier B.V. All rights reserved.

Transitions between strongly correlated and random steady-states for catalytic CO-oxidation on surfaces at high-pressure

DOE PAGES

Liu, Da -Jiang; Evans, James W.

2015-04-02

We explore simple lattice-gas reaction models for CO-oxidation on 1D and 2D periodic arrays of surface adsorption sites. The models are motivated by studies of CO-oxidation on RuO 2(110) at high-pressures. Although adspecies interactions are neglected, the effective absence of adspecies diffusion results in kinetically-induced spatial correlations. A transition occurs from a random mainly CO-populated steady-state at high CO-partial pressure p CO, to a strongly-correlated near-O-covered steady-state for low p CO as noted. In addition, we identify a second transition to a random near-O-covered steady-state at very low p CO.

The steady-state visual evoked potential in vision research: A review

PubMed Central

Norcia, Anthony M.; Appelbaum, L. Gregory; Ales, Justin M.; Cottereau, Benoit R.; Rossion, Bruno

2015-01-01

Periodic visual stimulation and analysis of the resulting steady-state visual evoked potentials were first introduced over 80 years ago as a means to study visual sensation and perception. From the first single-channel recording of responses to modulated light to the present use of sophisticated digital displays composed of complex visual stimuli and high-density recording arrays, steady-state methods have been applied in a broad range of scientific and applied settings.The purpose of this article is to describe the fundamental stimulation paradigms for steady-state visual evoked potentials and to illustrate these principles through research findings across a range of applications in vision science. PMID:26024451

Study of Proton Transfer in E. Coli Photolyase

NASA Astrophysics Data System (ADS)

Zhang, Meng; Liu, Zheyun; Li, Jiang; Wang, Lijuan; Zhong, Dongping

2013-06-01

Photolyase is a flavoprotein which utilizes blue-light energy to repair UV-light damaged DNA. The catalytic cofactor of photolyase, flavin adenine dinucleotide (FAD), has five redox states. Conversions between these redox states involve intraprotein electron transfer and proton transfer, which play important role in protein function. Here we systematically studied proton transfer in E. coli photolyase in vitro by site-directed mutagenesis and steady-state UV-vis spectroscopy, and proposed the proton channel in photolyase. We found that in the mutant N378C/E363L, proton channel was completely eliminated when DNA substrate was bound to the protein. Proton is suggested to be transported from protein surface to FAD by two pathways: the proton relay pathway through E363 and surface water to N378 and then to FAD; and the proton diffusion pathway through the substrate binding pocket. In addition, reaction kinetics of conversions between the redox states was then solved and redox potentials of the redox states were determined. These results described a complete picture of FAD redox changes, which are fundamental to the functions of all flavoenzymes.

Symmetry limit theory for cantilever beam-columns subjected to cyclic reversed bending

NASA Astrophysics Data System (ADS)

Uetani, K.; Nakamura, Tsuneyoshi

THE BEHAVIOR of a linear strain-hardening cantilever beam-column subjected to completely reversed plastic bending of a new idealized program under constant axial compression consists of three stages: a sequence of symmetric steady states, a subsequent sequence of asymmetric steady states and a divergent behavior involving unbounded growth of an anti-symmetric deflection mode. A new concept "symmetry limit" is introduced here as the smallest critical value of the tip-deflection amplitude at which transition from a symmetric steady state to an asymmetric steady state can occur in the response of a beam-column. A new theory is presented for predicting the symmetry limits. Although this transition phenomenon is phenomenologically and conceptually different from the branching phenomenon on an equilibrium path, it is shown that a symmetry limit may theoretically be regarded as a branching point on a "steady-state path" defined anew. The symmetry limit theory and the fundamental hypotheses are verified through numerical analysis of hysteretic responses of discretized beam-column models.

A general theory of kinetics and thermodynamics of steady-state copolymerization.

PubMed

Shu, Yao-Gen; Song, Yong-Shun; Ou-Yang, Zhong-Can; Li, Ming

2015-06-17

Kinetics of steady-state copolymerization has been investigated since the 1940s. Irreversible terminal and penultimate models were successfully applied to a number of comonomer systems, but failed for systems where depropagation is significant. Although a general mathematical treatment of the terminal model with depropagation was established in the 1980s, a penultimate model and higher-order terminal models with depropagation have not been systematically studied, since depropagation leads to hierarchically-coupled and unclosed kinetic equations which are hard to solve analytically. In this work, we propose a truncation method to solve the steady-state kinetic equations of any-order terminal models with depropagation in a unified way, by reducing them into closed steady-state equations which give the exact solution of the original kinetic equations. Based on the steady-state equations, we also derive a general thermodynamic equality in which the Shannon entropy of the copolymer sequence is explicitly introduced as part of the free energy dissipation of the whole copolymerization system.

Molecular control of steady-state dendritic cell maturation and immune homeostasis.

PubMed

Hammer, Gianna Elena; Ma, Averil

2013-01-01

Dendritic cells (DCs) are specialized sentinels responsible for coordinating adaptive immunity. This function is dependent upon coupled sensitivity to environmental signs of inflammation and infection to cellular maturation-the programmed alteration of DC phenotype and function to enhance immune cell activation. Although DCs are thus well equipped to respond to pathogens, maturation triggers are not unique to infection. Given that immune cells are exquisitely sensitive to the biological functions of DCs, we now appreciate that multiple layers of suppression are required to restrict the environmental sensitivity, cellular maturation, and even life span of DCs to prevent aberrant immune activation during the steady state. At the same time, steady-state DCs are not quiescent but rather perform key functions that support homeostasis of numerous cell types. Here we review these functions and molecular mechanisms of suppression that control steady-state DC maturation. Corruption of these steady-state operatives has diverse immunological consequences and pinpoints DCs as potent drivers of autoimmune and inflammatory disease.

Lineage tracing of murine adult hematopoietic stem cells reveals active contribution to steady-state hematopoiesis

PubMed Central

Chapple, Richard H.; Tseng, Yu-Jung; Hu, Tianyuan; Kitano, Ayumi; Takeichi, Makiko; Hoegenauer, Kevin A.

2018-01-01

Characterization of hematopoietic stem cells (HSCs) has advanced largely owing to transplantation assays, in which the developmental potential of HSCs is assessed generally in nonhomeostatic conditions. These studies established that adult HSCs extensively contribute to multilineage hematopoietic regeneration upon transplantation. On the contrary, recent studies performing lineage tracing of HSCs under homeostatic conditions have shown that adult HSCs may contribute far less to steady-state hematopoiesis than would be anticipated based on transplantation assays. Here, we used 2 independent HSC-lineage–tracing models to examine the contribution of adult HSCs to steady-state hematopoiesis. We show that adult HSCs contribute robustly to steady-state hematopoiesis, exhibiting faster efflux toward the myeloid lineages compared with lymphoid lineages. Platelets were robustly labeled by HSCs, reaching the same level of labeling as HSCs by 1 year of chase. Our results support the view that adult HSCs contribute to the continuous influx of blood cells during steady-state hematopoiesis. PMID:29848758

Steady State Condition in the Measurement of VO2and VCO2by Indirect Calorimetry.

PubMed

Cadena, M; Sacristan, E; Infante, O; Escalante, B; Rodriguez, F

2005-01-01

Resting Metabolic Rate (RMR) is computed using VO2and VCO2short time 15-minute window measurement with Indirect Calorimetry (IC) instruments designed with mixing chamber. Steady state condition using a 10% variation coefficient criteria is the main objective to achieve metabolic long time prediction reliability. This study address how susceptible is the steady state VO2, VCO2measurement condition to the clino-orthostatic physiological maneuver. 30 young healthy subjects were analyzed. Only 18 passed the 10% variation coefficient inclusive criteria. They were exposed to 10 minutes clino-stage and 10 minutes orthostage. The hypothesis tests show not statistical significance (p< 0.1) in the average and variance analysis. It is concluded that the steady state is not influenced by the patient position IC test, probably because IC mixing chamber instruments are insensitive to detect a mayor physiological dynamics changes that can modify the steady state definition.

Einstein's steady-state theory: an abandoned model of the cosmos

NASA Astrophysics Data System (ADS)

O'Raifeartaigh, Cormac; McCann, Brendan; Nahm, Werner; Mitton, Simon

2014-09-01

We present a translation and analysis of an unpublished manuscript by Albert Einstein in which he attempted to construct a `steady-state' model of the universe. The manuscript, which appears to have been written in early 1931, demonstrates that Einstein once explored a cosmic model in which the mean density of matter in an expanding universe is maintained constant by the continuous formation of matter from empty space. This model is very different to previously known Einsteinian models of the cosmos (both static and dynamic) but anticipates the later steady-state cosmology of Hoyle, Bondi and Gold in some ways. We find that Einstein's steady-state model contains a fundamental flaw and suggest that it was abandoned for this reason. We also suggest that he declined to explore a more sophisticated version because he found such theories rather contrived. The manuscript is of historical interest because it reveals that Einstein debated between steady-state and evolving models of the cosmos decades before a similar debate took place in the cosmological community.

Carbon-11-cocaine binding compared at subpharmacological and pharmacological doses: A PET study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volkow, N.D.; Fowler, J.S.; Logan, J.

The authors have characterized cocaine binding in the brain to a high-affinity site on the dopamine transporter using PET and tracer doses of [{sup 11}C]cocaine in the baboon in vivo. The binding pattern, however, of cocaine at tracer (subpharmacological) doses may differ from that observed when the drug is taken in behaviorally active doses, particularly since in vitro studies have shown that cocaine also binds to low affinity binding sites. PET was used to compare and characterize [{sup 11}C]cocaine binding in the baboon brain at low subpharmacological (18 {mu}g average dose) and at pharmacological (8000 {mu}g) doses. Serial studies onmore » the same day in the same baboon were used to assess the reproducibility of repeated measures and to assess the effects of drugs which inhibit the dopamine, norepinephrine and serotonin transporters. Time-activity curves from brain and the arterial plasma input function were used to calculate the steady-state distribution volume (DV). At subpharmacological doses, [{sup 11}C]cocaine had a more homogeneous distribution. Bmax/Kd for sub-pharmacological [{sup 11}C]cocaine corresponded to 0.5-0.6 and for pharmacological [{sup 11}C]cocaine it corresponded to 0.1-0.2. Two-point Scatchard analysis gave Bmax = 2300 pmole/g and Kd = 3600 nM. Bmax/Kd for sub-pharmacological doses of [{sup 11}C]cocaine was decreased by cocaine and drugs that inhibit the dopamine transporter, to 0.1-0.2, but not by drugs that inhibit the serotonin or the norepinephrine transporter. None of these drugs changed Bmax/Kd for a pharmacological dose of [{sup 11}C]cocaine. At subpharmacological doses, [{sup 11}C]cocaine binds predominantly to a high-affinity site on the dopamine transporter. 36 refs., 4 figs., 5 tabs.« less

Sterol Carrier Protein-2: Binding Protein for Endocannabinoids

PubMed Central

Liedhegner, Elizabeth Sabens; Vogt, Caleb D.; Sem, Daniel S.; Cunningham, Christopher W.

2015-01-01

The endocannabinoid (eCB) system, consisting of eCB ligands and the type 1 cannabinoid receptor (CB1R), subserves retrograde, activity-dependent synaptic plasticity in the brain. eCB signaling occurs “on-demand,” thus the processes regulating synthesis, mobilization and degradation of eCBs are also primary mechanisms for the regulation of CB1R activity. The eCBs, N-arachidonylethanolamine (AEA) and 2-arachidonoylglycerol (2-AG), are poorly soluble in water. We hypothesize that their aqueous solubility, and, therefore, their intracellular and transcellular distribution, are facilitated by protein binding. Using in silico docking studies, we have identified the nonspecific lipid binding protein, sterol carrier protein 2 (SCP-2), as a potential AEA binding protein. The docking studies predict that AEA and AM404 associate with SCP-2 at a putative cholesterol binding pocket with ΔG values of −3.6 and −4.6 kcal/mol, respectively. These values are considerably higher than cholesterol (−6.62 kcal/mol) but consistent with a favorable binding interaction. In support of the docking studies, SCP-2-mediated transfer of cholesterol in vitro is inhibited by micromolar concentrations of AEA; and heterologous expression of SCP-2 in HEK 293 cells increases time-related accumulation of AEA in a temperature-dependent fashion. These results suggest that SCP-2 facilitates cellular uptake of AEA. However, there is no effect of SCP-2 transfection on the cellular accumulation of AEA determined at equilibrium or the IC50 values for AEA, AM404 or 2-AG to inhibit steady state accumulation of radiolabelled AEA. We conclude that SCP-2 is a low affinity binding protein for AEA that can facilitate its cellular uptake but does not contribute significantly to intracellular sequestration of AEA. PMID:24510313

Identification of novel modulators for ionotropic glutamate receptor, iGluA2 by in-silico screening

PubMed Central

2013-01-01

Background Ionotropic glutamate receptors (iGluAs, IUPHAR nomenclature) are the major excitatory amino acid neurotransmitter receptors in the mammalian central nervous system (CNS). iGluAs are potential therapeutic drug targets for various neurological disorders including ischemia, epilepsy, Parkinson’s and Alzheimer’s diseases. The known iGluA modulators, cyclothiazide (CTZ), IDRA-21, and other benzothiadiazide derivatives (ALTZ, HCTZ, and CLTZ) bind to the ligand-binding domain of flip-form of iGluA2 at the dimer interface, thereby increasing steady-state activation by reducing desensitization. Methods To discover new modulator compounds, we performed virtual screening for the ligand binding domain (LBD) of iGluA2 against NCI Diversity Set III library containing 1597 compounds, and subsequently performed binding-energy analysis for selected compounds. The crystal structure of rat iGluA2 S1S2J (PDB ID: 3IJO) was used for docking studies. Results and conclusion From this study, we obtained four compounds: (1) 10-2(methoxyethyl)-3-phenylbenzo[g]pteridine-2,4-dione, (2) 2-benzo[e]benzotriazol-2-yl-aniline, (3) 9-nitro-6H-indolo-(2,3,-b)quinoxaline, and (4) 1-hydroxy-n-(3-nitrophenyl)-2-napthamide. The binding mode of these four compounds is very similar to that of abovementioned established modulators: two molecules of each compound independently bind to the protein symmetrically at the dimer interface; occupy the subsites B, C, B’ and C’; potentially interact with Ser518 and Ser775. Binding energy analysis shows that all the four hits are comparable to the drug molecule, CTZ, and hence, we propose that the discovered hits may be potential molecules to develop new chemical libraries for modulating the flip form of iGluA2 function. PMID:23855825

Ligand-induced changes in 2-aminopurine fluorescence as a probe for small molecule binding to HIV-1 TAR RNA

PubMed Central

BRADRICK, THOMAS D.; MARINO, JOHN P.

2004-01-01

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated in part through an interaction between the virally encoded trans-activator protein Tat and the trans-activator responsive region (TAR) of the viral RNA genome. Because TAR is highly conserved and its interaction with Tat is required for efficient viral replication, it has received much attention as an antiviral drug target. Here, we report a 2-aminopurine (2-AP) fluorescence-based assay for evaluating potential TAR inhibitors. Through selective incorporation of 2-AP within the bulge (C23 or U24) of a truncated form of the TAR sequence (Δ TAR-ap23 and Δ TAR-ap24), binding of argininamide, a 24-residue arginine-rich peptide derived from Tat, and Neomycin has been characterized using steady-state fluorescence. Binding of argininamide to the 2-AP ΔTAR constructs results in a four- to 11-fold increase in fluorescence intensity, thus providing a sensitive reporter of that interaction (KD ~ 1 mM). Similarly, binding of the Tat peptide results in an initial 14-fold increase in fluorescence (KD ~ 25 nM), but is then followed by a slight decrease that is attributed to an additional, lower-affinity association(s). Using the ΔTAR-ap23 and TAR-ap24 constructs, two classes of Neomycin binding sites are detected; the first molecule of antibiotic binds as a noncompetitive inhibitor of Tat/argininamide (KD ~ 200 nM), whereas the second, more weakly bound molecule(s) becomes associated in a presumably nonspecific manner (KD ~ 4 μM). Taken together, the results demonstrate that the 2-AP fluorescence-detected binding assays provide accurate and general methods for quantitatively assessing TAR interactions. PMID:15273324

A double tyrosine motif in the cardiac sodium channel domain III-IV linker couples calcium-dependent calmodulin binding to inactivation gating.

PubMed

Sarhan, Maen F; Van Petegem, Filip; Ahern, Christopher A

2009-11-27

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr(1494) and Tyr(1495)) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.

Identification of novel modulators for ionotropic glutamate receptor, iGluA2 by in-silico screening.

PubMed

Padmanabhan, Balasundaram

2013-07-15

Ionotropic glutamate receptors (iGluAs, IUPHAR nomenclature) are the major excitatory amino acid neurotransmitter receptors in the mammalian central nervous system (CNS). iGluAs are potential therapeutic drug targets for various neurological disorders including ischemia, epilepsy, Parkinson's and Alzheimer's diseases. The known iGluA modulators, cyclothiazide (CTZ), IDRA-21, and other benzothiadiazide derivatives (ALTZ, HCTZ, and CLTZ) bind to the ligand-binding domain of flip-form of iGluA2 at the dimer interface, thereby increasing steady-state activation by reducing desensitization. To discover new modulator compounds, we performed virtual screening for the ligand binding domain (LBD) of iGluA2 against NCI Diversity Set III library containing 1597 compounds, and subsequently performed binding-energy analysis for selected compounds. The crystal structure of rat iGluA2 S1S2J (PDB ID: 3IJO) was used for docking studies. From this study, we obtained four compounds: (1) 10-2(methoxyethyl)-3-phenylbenzo[g]pteridine-2,4-dione, (2) 2-benzo[e]benzotriazol-2-yl-aniline, (3) 9-nitro-6H-indolo-(2,3,-b)quinoxaline, and (4) 1-hydroxy-n-(3-nitrophenyl)-2-napthamide. The binding mode of these four compounds is very similar to that of abovementioned established modulators: two molecules of each compound independently bind to the protein symmetrically at the dimer interface; occupy the subsites B, C, B' and C'; potentially interact with Ser518 and Ser775. Binding energy analysis shows that all the four hits are comparable to the drug molecule, CTZ, and hence, we propose that the discovered hits may be potential molecules to develop new chemical libraries for modulating the flip form of iGluA2 function.

Nucleotide binding properties of bovine brain uncoating ATPase.

PubMed

Gao, B; Emoto, Y; Greene, L; Eisenberg, E

1993-04-25

Many functions of the 70-kDa heat-shock proteins (hsp70s) appear to be regulated by bound nucleotide. In this study we examined the nucleotide binding properties of purified bovine brain uncoating ATPase, one of the constitutively expressed members of the hsp70 family. We found that uncoating ATPase purified by ATP-agarose column chromatography retained one ADP molecule bound per enzyme molecule which could not be removed by extensive dialysis. Since this bound ADP exchanged rapidly with free ADP or ATP, the inability to remove the bound nucleotide was not due to slow dissociation but rather to strong binding of the nucleotide to the uncoating ATPase. In confirmation of this view, equilibrium dialysis experiments suggested that the dissociation constants for both ADP and ATP were less than 0.1 microM. Schmid et al. (Schmid, S. L., Braell, W. A., and Rothman, J. E. (1985) J. Biol. Chem 260, 10057-10062) suggested that the uncoating ATPase had two sites for bound nucleotide, one specific for ATP and one binding both ATP and ATP analogues but not ADP. In contrast, we found that enzyme with bound ADP did not bind further adenosine 5'-(beta,gamma-imino)triphosphate or dATP, nor did more than one ATP molecule bind per enzyme even in 200 microM free ATP. These results strongly suggest that the enzyme has only one binding site for nucleotide. During steady-state ATP hydrolysis, 85% of the bound nucleotide at this site was determined to be ATP and 15% ADP; this is consistent with the rate of ADP release determined in the exchange experiments noted above, where ADP release was found to be six times faster than the overall rate of ATP hydrolysis.

In Silico Docking and Electrophysiological Characterization of Lacosamide Binding Sites on Collapsin Response Mediator Protein-2 Identifies a Pocket Important in Modulating Sodium Channel Slow Inactivation*

PubMed Central

Wang, Yuying; Brittain, Joel M.; Jarecki, Brian W.; Park, Ki Duk; Wilson, Sarah M.; Wang, Bo; Hale, Rachel; Meroueh, Samy O.; Cummins, Theodore R.; Khanna, Rajesh

2010-01-01

The anti-epileptic drug (R)-lacosamide ((2R)-2-(acetylamino)-N-benzyl-3-methoxypropanamide (LCM)) modulates voltage-gated sodium channels (VGSCs) by preferentially interacting with slow inactivated sodium channels, but the observation that LCM binds to collapsin response mediator protein 2 (CRMP-2) suggests additional mechanisms of action for LCM. We postulated that CRMP-2 levels affects the actions of LCM on VGSCs. CRMP-2 labeling by LCM analogs was competitively displaced by excess LCM in rat brain lysates. Manipulation of CRMP-2 levels in the neuronal model system CAD cells affected slow inactivation of VGSCs without any effects on other voltage-dependent properties. In silico docking was performed to identify putative binding sites in CRMP-2 that may modulate the effects of LCM on VGSCs. These studies identified five cavities in CRMP-2 that can accommodate LCM. CRMP-2 alanine mutants of key residues within these cavities were functionally similar to wild-type CRMP-2 as assessed by similar levels of enhancement in dendritic complexity of cortical neurons. Next, we examined the effects of expression of wild-type and mutant CRMP-2 constructs on voltage-sensitive properties of VGSCs in CAD cells: 1) steady-state voltage-dependent activation and fast-inactivation properties were not affected by LCM, 2) CRMP-2 single alanine mutants reduced the LCM-mediated effects on the ability of endogenous Na+ channels to transition to a slow inactivated state, and 3) a quintuplicate CRMP-2 alanine mutant further decreased this slow inactivated fraction. Collectively, these results identify key CRMP-2 residues that can coordinate LCM binding thus making it more effective on its primary clinical target. PMID:20538611

A Poisson-like closed-form expression for the steady-state wealth distribution in a kinetic model of gambling

NASA Astrophysics Data System (ADS)

Garcia, Jane Bernadette Denise M.; Esguerra, Jose Perico H.

2017-08-01

An approximate but closed-form expression for a Poisson-like steady state wealth distribution in a kinetic model of gambling was formulated from a finite number of its moments, which were generated from a βa,b(x) exchange distribution. The obtained steady-state wealth distributions have tails which are qualitatively similar to those observed in actual wealth distributions.

The relationship between skin aging and steady state ultraweak photon emission as an indicator of skin oxidative stress in vivo.

PubMed

Gabe, Y; Osanai, O; Takema, Y

2014-08-01

Ultraweak photon emission (UPE) is one potential method to evaluate the oxidative status of the skin in vivo. However, little is known about how the daily oxidative stress of the skin is related to skin aging-related alterations in vivo. We characterized the steady state UPE and performed a skin survey. We evaluated the skin oxidative status by UPE, skin elasticity, epidermal thickness and skin color on the inner upper arm, the outer forearm, and the buttock of 70 Japanese volunteers. The steady state UPE at the three skin sites increased with age. Correlation analysis revealed that the steady state UPE only from the buttock was related to skin elasticity, which showed age-dependent changes. Moreover, analysis by age group indicated that b* values of the inner upper arm of subjects in their 20s were inversely correlated with UPE as occurred in buttock skin. In contrast, photoaged skin did not show a clear relationship with steady state UPE because the accumulation of sun-exposure might influence the sensitivity to oxidative stress. These results suggest that steady state UPE reflects not only intrinsic skin aging and cutaneous color but also the current oxidative status independent of skin aging. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Evaporation rate of nucleating clusters.

PubMed

Zapadinsky, Evgeni

2011-11-21

The Becker-Döring kinetic scheme is the most frequently used approach to vapor liquid nucleation. In the present study it has been extended so that master equations for all cluster configurations are included into consideration. In the Becker-Döring kinetic scheme the nucleation rate is calculated through comparison of the balanced steady state and unbalanced steady state solutions of the set of kinetic equations. It is usually assumed that the balanced steady state produces equilibrium cluster distribution, and the evaporation rates are identical in the balanced and unbalanced steady state cases. In the present study we have shown that the evaporation rates are not identical in the equilibrium and unbalanced steady state cases. The evaporation rate depends on the number of clusters at the limit of the cluster definition. We have shown that the ratio of the number of n-clusters at the limit of the cluster definition to the total number of n-clusters is different in equilibrium and unbalanced steady state cases. This causes difference in evaporation rates for these cases and results in a correction factor to the nucleation rate. According to rough estimation it is 10(-1) by the order of magnitude and can be lower if carrier gas effectively equilibrates the clusters. The developed approach allows one to refine the correction factor with Monte Carlo and molecular dynamic simulations.

Perception of steady-state vowels and vowelless syllables by adults and children

NASA Astrophysics Data System (ADS)

Nittrouer, Susan

2005-04-01

Vowels can be produced as long, isolated, and steady-state, but that is not how they are found in natural speech. Instead natural speech consists of almost continuously changing (i.e., dynamic) acoustic forms from which mature listeners recover underlying phonetic form. Some theories suggest that children need steady-state information to recognize vowels (and so learn vowel systems), even though that information is sparse in natural speech. The current study examined whether young children can recover vowel targets from dynamic forms, or whether they need steady-state information. Vowel recognition was measured for adults and children (3, 5, and 7 years) for natural productions of /dæd/, /dUd/ /æ/, /U/ edited to make six stimulus sets: three dynamic (whole syllables; syllables with middle 50-percent replaced by cough; syllables with all but the first and last three pitch periods replaced by cough), and three steady-state (natural, isolated vowels; reiterated pitch periods from those vowels; reiterated pitch periods from the syllables). Adults scored nearly perfectly on all but first/last three pitch period stimuli. Children performed nearly perfectly only when the entire syllable was heard, and performed similarly (near 80%) for all other stimuli. Consequently, children need dynamic forms to perceive vowels; steady-state forms are not preferred.

A stability analysis of the power-law steady state of marine size spectra.

PubMed

Datta, Samik; Delius, Gustav W; Law, Richard; Plank, Michael J

2011-10-01

This paper investigates the stability of the power-law steady state often observed in marine ecosystems. Three dynamical systems are considered, describing the abundance of organisms as a function of body mass and time: a "jump-growth" equation, a first order approximation which is the widely used McKendrick-von Foerster equation, and a second order approximation which is the McKendrick-von Foerster equation with a diffusion term. All of these yield a power-law steady state. We derive, for the first time, the eigenvalue spectrum for the linearised evolution operator, under certain constraints on the parameters. This provides new knowledge of the stability properties of the power-law steady state. It is shown analytically that the steady state of the McKendrick-von Foerster equation without the diffusion term is always unstable. Furthermore, numerical plots show that eigenvalue spectra of the McKendrick-von Foerster equation with diffusion give a good approximation to those of the jump-growth equation. The steady state is more likely to be stable with a low preferred predator:prey mass ratio, a large diet breadth and a high feeding efficiency. The effects of demographic stochasticity are also investigated and it is concluded that these are likely to be small in real systems.

EEMD-Based Steady-State Indexes and Their Applications to Condition Monitoring and Fault Diagnosis of Railway Axle Bearings

PubMed Central

Fan, Wei; Tsui, Kwok-Leung; Lin, Jianhui

2018-01-01

Railway axle bearings are one of the most important components used in vehicles and their failures probably result in unexpected accidents and economic losses. To realize a condition monitoring and fault diagnosis scheme of railway axle bearings, three dimensionless steadiness indexes in a time domain, a frequency domain, and a shape domain are respectively proposed to measure the steady states of bearing vibration signals. Firstly, vibration data collected from some designed experiments are pre-processed by using ensemble empirical mode decomposition (EEMD). Then, the coefficient of variation is introduced to construct two steady-state indexes from pre-processed vibration data in a time domain and a frequency domain, respectively. A shape function is used to construct a steady-state index in a shape domain. At last, to distinguish normal and abnormal bearing health states, some guideline thresholds are proposed. Further, to identify axle bearings with outer race defects, a pin roller defect, a cage defect, and coupling defects, the boundaries of all steadiness indexes are experimentally established. Experimental results showed that the proposed condition monitoring and fault diagnosis scheme is effective in identifying different bearing health conditions. PMID:29495446

Bipolar pulse field for magnetic refrigeration

DOEpatents

Lubell, Martin S.

1994-01-01

A magnetic refrigeration apparatus includes first and second steady state magnets, each having a field of substantially equal strength and opposite polarity, first and second bodies made of magnetocaloric material disposed respectively in the influence of the fields of the first and second steady state magnets, and a pulsed magnet, concentric with the first and second steady state magnets, and having a field which cycles between the fields of the first and second steady state magnets, thereby cyclically magnetizing and demagnetizing and thus heating and cooling the first and second bodies. Heat exchange apparatus of suitable design can be used to expose a working fluid to the first and second bodies of magnetocaloric material. A controller is provided to synchronize the flow of working fluid with the changing states of magnetization of the first and second bodies.

Steady State Advanced Tokamak (SSAT): The mission and the machine

NASA Astrophysics Data System (ADS)

Thomassen, K.; Goldston, R.; Nevins, B.; Neilson, H.; Shannon, T.; Montgomery, B.

1992-03-01

Extending the tokamak concept to the steady state regime and pursuing advances in tokamak physics are important and complementary steps for the magnetic fusion energy program. The required transition away from inductive current drive will provide exciting opportunities for advances in tokamak physics, as well as important impetus to drive advances in fusion technology. Recognizing this, the Fusion Policy Advisory Committee and the U.S. National Energy Strategy identified the development of steady state tokamak physics and technology, and improvements in the tokamak concept, as vital elements in the magnetic fusion energy development plan. Both called for the construction of a steady state tokamak facility to address these plan elements. Advances in physics that produce better confinement and higher pressure limits are required for a similar unit size reactor. Regimes with largely self-driven plasma current are required to permit a steady-state tokamak reactor with acceptable recirculating power. Reliable techniques of disruption control will be needed to achieve the availability goals of an economic reactor. Thus the central role of this new tokamak facility is to point the way to a more attractive demonstration reactor (DEMO) than the present data base would support. To meet the challenges, we propose a new 'Steady State Advanced Tokamak' (SSAT) facility that would develop and demonstrate optimized steady state tokamak operating mode. While other tokamaks in the world program employ superconducting toroidal field coils, SSAT would be the first major tokamak to operate with a fully superconducting coil set in the elongated, divertor geometry planned for ITER and DEMO.

Is steady-state capitalism viable? A review of the issues and an answer in the affirmative.

PubMed

Lawn, Philip

2011-02-01

Most ecological economists believe that the transition to a steady-state economy is necessary to ensure ecological sustainability and to maximize a nation's economic welfare. While some observers agree with the necessity of the steady-state economy, they are nonetheless critical of the suggestion made by ecological economists-in particular, Herman Daly-that a steady-state economy is compatible with a capitalist system. First, they believe that steady-state capitalism is based on the untenable assumption that growth is an optional rather than in-built element of capitalism. Second, they argue that capitalist notions of efficient resource allocation are too restrictive to facilitate the transition to an "ecological" or steady-state economy. I believe these observers are outright wrong with their first criticism and, because they misunderstand Daly's vision of a steady-state economy, are misplaced with their second criticism. The nature of a capitalist system depends upon the institutional framework that supports and shapes it. Hence, a capitalist system can exist in a wide variety of forms. Unfortunately, many observers fail to recognize that the current "growth imperative" is the result of capitalist systems everywhere being institutionally designed to grow. They need not be designed this way to survive and thrive. Indeed, because continued growth is both existentially undesirable and ecologically unsustainable, redesigning capitalist systems through the introduction of Daly-like institutions would prove to be capitalism's savior. What's more, it would constitute humankind's best hope of achieving sustainable development. © 2011 New York Academy of Sciences.

Understanding emotional transitions: the interpersonal consequences of changing emotions in negotiations.

PubMed

Filipowicz, Allan; Barsade, Sigal; Melwani, Shimul

2011-09-01

Research on the interpersonal functions of emotions has focused primarily on steady-state emotion rather than on emotional transitions, the movement between emotion states. The authors examined the influence of emotional transitions on social interactions and found that emotional transitions led to consistently different outcomes than their corresponding steady-state emotions. Across 2 computer-mediated negotiations and a face-to-face negotiation, participants negotiating with partners who displayed a "becoming angry" (happy to angry) emotional transition accepted worse negotiation outcomes yet formed better relational impressions of their partners than participants negotiating with partners who displayed steady-state anger. This relationship was mediated through 2 mechanisms: attributional and emotional contagion processes. The "becoming happy" (angry to happy) emotional transition as compared with steady-state happiness was not significantly related to differences in negotiation outcomes but was significantly related to differences in relational impressions, where perceivers of the "becoming happy" emotional transition gave their partners lower relational impression ratings than perceivers of steady-state happiness. PsycINFO Database Record (c) 2011 APA, all rights reserved.

Quantized transport and steady states of Floquet topological insulators

NASA Astrophysics Data System (ADS)

Esin, Iliya; Rudner, Mark S.; Refael, Gil; Lindner, Netanel H.

2018-06-01

Robust electronic edge or surface modes play key roles in the fascinating quantized responses exhibited by topological materials. Even in trivial materials, topological bands and edge states can be induced dynamically by a time-periodic drive. Such Floquet topological insulators (FTIs) inherently exist out of equilibrium; the extent to which they can host quantized transport, which depends on the steady-state population of their dynamically induced edge states, remains a crucial question. In this work, we obtain the steady states of two-dimensional FTIs in the presence of the natural dissipation mechanisms present in solid state systems. We give conditions under which the steady-state distribution resembles that of a topological insulator in the Floquet basis. In this state, the distribution in the Floquet edge modes exhibits a sharp feature akin to a Fermi level, while the bulk hosts a small density of excitations. We determine the regimes where topological edge-state transport persists and can be observed in FTIs.

Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

PubMed

Jin, Liang; Zhang, Kai; Sternglanz, Rolf; Neiman, Aaron M

2017-05-01

In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80 -induced genes, PES4 and MIP6 , encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts. Copyright © 2017 American Society for Microbiology.

Evaluation of the availability of bound analyte for passive sampling in the presence of mobile binding matrix.

PubMed

Xu, Jianqiao; Huang, Shuyao; Jiang, Ruifen; Cui, Shufen; Luan, Tiangang; Chen, Guosheng; Qiu, Junlang; Cao, Chenyang; Zhu, Fang; Ouyang, Gangfeng

2016-04-21

Elucidating the availability of the bound analytes for the mass transfer through the diffusion boundary layers (DBLs) adjacent to passive samplers is important for understanding the passive sampling kinetics in complex samples, in which the lability factor of the bound analyte in the DBL is an important parameter. In this study, the mathematical expression of lability factor was deduced by assuming a pseudo-steady state during passive sampling, and the equation indicated that the lability factor was equal to the ratio of normalized concentration gradients between the bound and free analytes. Through the introduction of the mathematical expression of lability factor, the modified effective average diffusion coefficient was proven to be more suitable for describing the passive sampling kinetics in the presence of mobile binding matrixes. Thereafter, the lability factors of the bound polycyclic aromatic hydrocarbons (PAHs) with sodium dodecylsulphate (SDS) micelles as the binding matrixes were figured out according to the improved theory. The lability factors were observed to decrease with larger binding ratios and smaller micelle sizes, and were successfully used to predict the mass transfer efficiencies of PAHs through DBLs. This study would promote the understanding of the availability of bound analytes for passive sampling based on the theoretical improvements and experimental assessments. Copyright © 2016 Elsevier B.V. All rights reserved.

Kinetic and CD/MCD spectroscopic studies of the atypical, three-His-ligated, non-heme Fe2+ center in diketone dioxygenase: the role of hydrophilic outer shell residues in catalysis.

PubMed

Straganz, Grit D; Diebold, Adrienne R; Egger, Sigrid; Nidetzky, Bernd; Solomon, Edward I

2010-02-09

Diketone cleaving enzyme (Dke1) is a dioxygenase with an atypical, three-histidine-ligated, mononuclear non-heme Fe(2+) center. To assess the role in enzyme catalysis of the hydrophilic residues in the active site pocket, residues Glu98, Arg80, Tyr70, and Thr107 were subjected to mutational analysis. Steady state and pre-steady state kinetics indicated a role for Glu98 in promoting both substrate binding and O(2) reduction. Additionally, the Glu98 substitution eliminated the pH dependence of substrate binding (k(cat)(app)/K(M)(app)-pH profile) present in wild-type Dke1 (pK(a) = 6.3 +/- 0.4 and 8.4 +/- 0.4). MCD spectroscopy revealed that the Glu98 --> Gln mutation leads to the conversion of the six-coordinate (6C) resting Fe(2+) center present in the wild-type enzyme at pH 7.0 to a mixture of five-coordinate (5C) and 6C sites. The 6C geometry was restored with a pH shift to 9.5 which also resulted in ligand field (LF) energy splittings identical to that found for wild-type (WT) Dke1 at pH 9.5. In WT Dke1, these LF transitions are shifted up in energy by approximately 300 cm(-1) at pH 9.5 relative to pH 7.0. These data, combined with CD pH titrations which reveal a pK(a) of approximately 8.2 for resting WT Dke1 and the Glu98 --> Gln variant, indicate the deprotonation of a metal-ligated water. Together, the kinetic and spectroscopic data reveal a stabilizing effect of Glu98 on the 6C geometry of the metal center, priming it for substrate ligation. Arg80 and Tyr70 are shown to promote O(2) reduction, while Thr107 stabilizes the Fe(II) cofactor.

Estimation of the optimal dosing regimen of escitalopram in dogs: A dose occupancy study with [11C]DASB

PubMed Central

Polis, Ingeborgh; Dockx, Robrecht; Vlerick, Lise; Dobbeleir, Andre; Goethals, Ingeborg; Saunders, Jimmy; Sadones, Nele; Baeken, Chris; De Vos, Filip; Peremans, Kathelijne

2017-01-01

Although the favourable characteristics of escitalopram as being the most selective serotonin reuptake inhibitor and having an increased therapeutic efficacy via binding on an additional allosteric binding site of the serotonin transporter, its dosing regimen has not yet been optimized for its use in dogs. This study aimed to estimate the optimal dosing frequency and the required dose for achieving 80% occupancy of the serotonin transporters in the basal ganglia. The dosing frequency was investigated by determining the elimination half-life after a four day oral pre-treatment period with 0.83 mg/kg escitalopram (3 administrations/day) and a subsequent i.v. injection 0.83 mg/kg. Blood samples were taken up to 12 hours after i.v. injection and the concentration of escitalopram in plasma was analysed via LC-MSMS. The dose-occupancy relationship was then determined by performing two PET scans in five adult beagles: a baseline PET scan and a second scan after steady state conditions were achieved following oral treatment with a specific dose of escitalopram ranging from 0.5 to 2.5 mg/kg/day. As the elimination half-life was determined to be 6.7 hours a dosing frequency of three administrations a day was proposed for the second part of the study. Further it was opted for a treatment period of four days, which well exceeded the minimum period to achieve steady state conditions. The optimal dosing regimen to achieve 80% occupancy in the basal ganglia and elicit a therapeutic effect, was calculated to be 1.85 mg/kg/day, divided over three administrations. Under several circumstances, such as insufficient response to other SSRIs, concurrent drug intake or in research studies focused on SERT, the use of escitalopram can be preferred over the use of the already for veterinary use registered fluoxetine, however, in case of long-term treatment with escitalopram, regularly cardiac screening is recommended. PMID:28644875

Estimation of the optimal dosing regimen of escitalopram in dogs: A dose occupancy study with [11C]DASB.

PubMed

Taylor, Olivia; Van Laeken, Nick; Polis, Ingeborgh; Dockx, Robrecht; Vlerick, Lise; Dobbeleir, Andre; Goethals, Ingeborg; Saunders, Jimmy; Sadones, Nele; Baeken, Chris; De Vos, Filip; Peremans, Kathelijne

2017-01-01

Although the favourable characteristics of escitalopram as being the most selective serotonin reuptake inhibitor and having an increased therapeutic efficacy via binding on an additional allosteric binding site of the serotonin transporter, its dosing regimen has not yet been optimized for its use in dogs. This study aimed to estimate the optimal dosing frequency and the required dose for achieving 80% occupancy of the serotonin transporters in the basal ganglia. The dosing frequency was investigated by determining the elimination half-life after a four day oral pre-treatment period with 0.83 mg/kg escitalopram (3 administrations/day) and a subsequent i.v. injection 0.83 mg/kg. Blood samples were taken up to 12 hours after i.v. injection and the concentration of escitalopram in plasma was analysed via LC-MSMS. The dose-occupancy relationship was then determined by performing two PET scans in five adult beagles: a baseline PET scan and a second scan after steady state conditions were achieved following oral treatment with a specific dose of escitalopram ranging from 0.5 to 2.5 mg/kg/day. As the elimination half-life was determined to be 6.7 hours a dosing frequency of three administrations a day was proposed for the second part of the study. Further it was opted for a treatment period of four days, which well exceeded the minimum period to achieve steady state conditions. The optimal dosing regimen to achieve 80% occupancy in the basal ganglia and elicit a therapeutic effect, was calculated to be 1.85 mg/kg/day, divided over three administrations. Under several circumstances, such as insufficient response to other SSRIs, concurrent drug intake or in research studies focused on SERT, the use of escitalopram can be preferred over the use of the already for veterinary use registered fluoxetine, however, in case of long-term treatment with escitalopram, regularly cardiac screening is recommended.

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ

PubMed Central

1993-01-01

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-- >E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s- 1. PMID:8270908

Quantum mechanical hydrogen tunneling in bacterial copper amine oxidase reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murakawa, Takeshi; Okajima, Toshihide; Kuroda, Shun'ichi

A key step decisively affecting the catalytic efficiency of copper amine oxidase is stereospecific abstraction of substrate {alpha}-proton by a conserved Asp residue. We analyzed this step by pre-steady-state kinetics using a bacterial enzyme and stereospecifically deuterium-labeled substrates, 2-phenylethylamine and tyramine. A small and temperature-dependent kinetic isotope effect (KIE) was observed with 2-phenylethylamine, whereas a large and temperature-independent KIE was observed with tyramine in the {alpha}-proton abstraction step, showing that this step is driven by quantum mechanical hydrogen tunneling rather than the classical transition-state mechanism. Furthermore, an Arrhenius-type preexponential factor ratio approaching a transition-state value was obtained in the reactionmore » of a mutant enzyme lacking the critical Asp. These results provide strong evidence for enzyme-enhanced hydrogen tunneling. X-ray crystallographic structures of the reaction intermediates revealed a small difference in the binding mode of distal parts of substrates, which would modulate hydrogen tunneling proceeding through either active or passive dynamics.« less

Spurious Numerical Solutions Of Differential Equations

NASA Technical Reports Server (NTRS)

Lafon, A.; Yee, H. C.

1995-01-01

Paper presents detailed study of spurious steady-state numerical solutions of differential equations that contain nonlinear source terms. Main objectives of this study are (1) to investigate how well numerical steady-state solutions of model nonlinear reaction/convection boundary-value problem mimic true steady-state solutions and (2) to relate findings of this investigation to implications for interpretation of numerical results from computational-fluid-dynamics algorithms and computer codes used to simulate reacting flows.

Electronic transport characterization of silicon wafers by spatially resolved steady-state photocarrier radiometric imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Qian; University of the Chinese Academy of Sciences, Beijing 100039; Li, Bincheng, E-mail: bcli@ioe.ac.cn

2015-09-28

Spatially resolved steady-state photocarrier radiometric (PCR) imaging technique is developed to characterize the electronic transport properties of silicon wafers. Based on a nonlinear PCR theory, simulations are performed to investigate the effects of electronic transport parameters (the carrier lifetime, the carrier diffusion coefficient, and the front surface recombination velocity) on the steady-state PCR intensity profiles. The electronic transport parameters of an n-type silicon wafer are simultaneously determined by fitting the measured steady-state PCR intensity profiles to the three-dimensional nonlinear PCR model. The determined transport parameters are in good agreement with the results obtained by the conventional modulated PCR technique withmore » multiple pump beam radii.« less

A Waved Journal Bearing Concept-Evaluating Steady-State and Dynamic Performance with a Potential Active Control Alternative

NASA Technical Reports Server (NTRS)

Dimofte, Florin

1993-01-01

Analysis of the waved journal bearing concept featuring a waved inner bearing diameter for use with a compressible lubricant (gas) is presented. The performance of generic waved bearings having either three or four waves is predicted for air lubricated bearings. Steady-state performance is discussed in terms of bearing load capacity, while the dynamic performance is discussed in terms of fluid film stability and dynamic coefficients. It was found that the bearing wave amplitude has an important influence on both the steady-state and the dynamic performance of the waved journal bearing. For a fixed eccentricity ratio, the bearing steady-state load capacity and direct dynamic stiffness coefficient increase as the wave amplitude increases.

[Specific features in realization of the principle of minimum energy dissipation during individual development].

PubMed

Zotin, A A

2012-01-01

Realization of the principle of minimum energy dissipation (Prigogine's theorem) during individual development has been analyzed. This analysis has suggested the following reformulation of this principle for living objects: when environmental conditions are constant, the living system evolves to a current steady state in such a way that the difference between entropy production and entropy flow (psi(u) function) is positive and constantly decreases near the steady state, approaching zero. In turn, the current steady state tends to a final steady state in such a way that the difference between the specific entropy productions in an organism and its environment tends to be minimal. In general, individual development completely agrees with the law of entropy increase (second law of thermodynamics).

Fabrication and Characterization of Ultrathin-ring Electrodes for Pseudo-steady-state Amperometric Detection.

PubMed

Kitazumi, Yuki; Hamamoto, Katsumi; Noda, Tatsuo; Shirai, Osamu; Kano, Kenji

2015-01-01

The fabrication of ultrathin-ring electrodes with a diameter of 2 mm and a thickness of 100 nm is established. The ultrathin-ring electrodes provide a large density of pseudo-steady-state currents, and realize pseudo-steady-state amperometry under quiescent conditions without a Faraday cage. Under the limiting current conditions, the current response at the ultrathin-ring electrode can be well explained by the theory of the microband electrode response. Cyclic voltammograms at the ultrathin-ring electrode show sigmoidal characteristics with some hysteresis. Numerical simulation reveals that the hysteresis can be ascribed to the time-dependence of pseudo-steady-state current. The performance of amperometry with the ultrathin-ring electrode has been verified in its application to redox enzyme kinetic measurements.

Differences in the dynamics of serotonin reuptake transporter occupancy may explain superior clinical efficacy of escitalopram versus citalopram.

PubMed

Kasper, Siegfried; Sacher, Julia; Klein, Nikolas; Mossaheb, Nilufar; Attarbaschi-Steiner, Trawat; Lanzenberger, Rupert; Spindelegger, Christoph; Asenbaum, Susanne; Holik, Alexander; Dudczak, Robert

2009-05-01

Escitalopram the S-enantiomer of the racemate citalopram, is clinically more effective than citalopram in the treatment of major depressive disorder. However, the precise mechanism by which escitalopram achieves superiority over citalopram is yet to be determined. It has been hypothesized that the therapeutically inactive R-enantiomer competes with the serotonin-enhancing S-enantiomer at a low-affinity allosteric site on serotonin reuptake transporters (SERTs), and reduces the effectiveness of the S-enantiomer at the primary, high-affinity serotonin-binding site. This study summarizes the results of two recent single-photon emission computerized tomography studies measuring SERT occupancy in citalopram-treated and escitalopram-treated healthy volunteers, after a single dose and multiple doses (i.e. under steady-state conditions). The single-dose study showed no attenuating effect of R-citalopram. After multiple dosing, however, SERT occupancy was significantly reduced in the presence of R-citalopram. Under steady-state conditions, R-enantiomer concentrations were greater than for the S-enantiomer because of slower clearance of R-citalopram. A pooled analysis suggests that build-up of the R-enantiomer after repeated citalopram dosing may lead to increased inhibition of S-enantiomer occupancy of SERT. This review adds to the growing body of evidence regarding differences in the dynamics of SERT occupancy, that is, molecular mechanisms underlying the often-observed superior clinical efficacy of escitalopram compared with citalopram in major depressive disorder.

Synthetic gene circuits for metabolic control: design trade-offs and constraints

PubMed Central

Oyarzún, Diego A.; Stan, Guy-Bart V.

2013-01-01

A grand challenge in synthetic biology is to push the design of biomolecular circuits from purely genetic constructs towards systems that interface different levels of the cellular machinery, including signalling networks and metabolic pathways. In this paper, we focus on a genetic circuit for feedback regulation of unbranched metabolic pathways. The objective of this feedback system is to dampen the effect of flux perturbations caused by changes in cellular demands or by engineered pathways consuming metabolic intermediates. We consider a mathematical model for a control circuit with an operon architecture, whereby the expression of all pathway enzymes is transcriptionally repressed by the metabolic product. We address the existence and stability of the steady state, the dynamic response of the network under perturbations, and their dependence on common tuneable knobs such as the promoter characteristic and ribosome binding site (RBS) strengths. Our analysis reveals trade-offs between the steady state of the enzymes and the intermediates, together with a separation principle between promoter and RBS design. We show that enzymatic saturation imposes limits on the parameter design space, which must be satisfied to prevent metabolite accumulation and guarantee the stability of the network. The use of promoters with a broad dynamic range and a small leaky expression enlarges the design space. Simulation results with realistic parameter values also suggest that the control circuit can effectively upregulate enzyme production to compensate flux perturbations. PMID:23054953

Constrained Analysis of Fluorescence Anisotropy Decay:Application to Experimental Protein Dynamics

PubMed Central

Feinstein, Efraim; Deikus, Gintaras; Rusinova, Elena; Rachofsky, Edward L.; Ross, J. B. Alexander; Laws, William R.

2003-01-01

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay. PMID:12524313

Dissipative production of a maximally entangled steady state of two quantum bits.

PubMed

Lin, Y; Gaebler, J P; Reiter, F; Tan, T R; Bowler, R; Sørensen, A S; Leibfried, D; Wineland, D J

2013-12-19

Entangled states are a key resource in fundamental quantum physics, quantum cryptography and quantum computation. Introduction of controlled unitary processes--quantum gates--to a quantum system has so far been the most widely used method to create entanglement deterministically. These processes require high-fidelity state preparation and minimization of the decoherence that inevitably arises from coupling between the system and the environment, and imperfect control of the system parameters. Here we combine unitary processes with engineered dissipation to deterministically produce and stabilize an approximate Bell state of two trapped-ion quantum bits (qubits), independent of their initial states. Compared with previous studies that involved dissipative entanglement of atomic ensembles or the application of sequences of multiple time-dependent gates to trapped ions, we implement our combined process using trapped-ion qubits in a continuous time-independent fashion (analogous to optical pumping of atomic states). By continuously driving the system towards the steady state, entanglement is stabilized even in the presence of experimental noise and decoherence. Our demonstration of an entangled steady state of two qubits represents a step towards dissipative state engineering, dissipative quantum computation and dissipative phase transitions. Following this approach, engineered coupling to the environment may be applied to a broad range of experimental systems to achieve desired quantum dynamics or steady states. Indeed, concurrently with this work, an entangled steady state of two superconducting qubits was demonstrated using dissipation.

Poiseuille flow of soft glasses in narrow channels: from quiescence to steady state.

PubMed

Chaudhuri, Pinaki; Horbach, Jürgen

2014-10-01

Using numerical simulations, the onset of Poiseuille flow in a confined soft glass is investigated. Starting from the quiescent state, steady flow sets in at a time scale which increases with a decrease in applied forcing. At this onset time scale, a rapid transition occurs via the simultaneous fluidization of regions having different local stresses. In the absence of steady flow at long times, creep is observed even in regions where the local stress is larger than the bulk yielding threshold. Finally, we show that the time scale to attain steady flow depends strongly on the history of the initial state.

Bipolar pulse field for magnetic refrigeration

DOEpatents

Lubell, M.S.

1994-10-25

A magnetic refrigeration apparatus includes first and second steady state magnets, each having a field of substantially equal strength and opposite polarity, first and second bodies made of magnetocaloric material disposed respectively in the influence of the fields of the first and second steady state magnets, and a pulsed magnet, concentric with the first and second steady state magnets, and having a field which cycles between the fields of the first and second steady state magnets, thereby cyclically magnetizing and demagnetizing and thus heating and cooling the first and second bodies. Heat exchange apparatus of suitable design can be used to expose a working fluid to the first and second bodies of magnetocaloric material. A controller is provided to synchronize the flow of working fluid with the changing states of magnetization of the first and second bodies. 2 figs.

Steady-state kinetic modeling constrains cellular resting states and dynamic behavior.

PubMed

Purvis, Jeremy E; Radhakrishnan, Ravi; Diamond, Scott L

2009-03-01

A defining characteristic of living cells is the ability to respond dynamically to external stimuli while maintaining homeostasis under resting conditions. Capturing both of these features in a single kinetic model is difficult because the model must be able to reproduce both behaviors using the same set of molecular components. Here, we show how combining small, well-defined steady-state networks provides an efficient means of constructing large-scale kinetic models that exhibit realistic resting and dynamic behaviors. By requiring each kinetic module to be homeostatic (at steady state under resting conditions), the method proceeds by (i) computing steady-state solutions to a system of ordinary differential equations for each module, (ii) applying principal component analysis to each set of solutions to capture the steady-state solution space of each module network, and (iii) combining optimal search directions from all modules to form a global steady-state space that is searched for accurate simulation of the time-dependent behavior of the whole system upon perturbation. Importantly, this stepwise approach retains the nonlinear rate expressions that govern each reaction in the system and enforces constraints on the range of allowable concentration states for the full-scale model. These constraints not only reduce the computational cost of fitting experimental time-series data but can also provide insight into limitations on system concentrations and architecture. To demonstrate application of the method, we show how small kinetic perturbations in a modular model of platelet P2Y(1) signaling can cause widespread compensatory effects on cellular resting states.

A descriptive model of resting-state networks using Markov chains.

PubMed

Xie, H; Pal, R; Mitra, S

2016-08-01

Resting-state functional connectivity (RSFC) studies considering pairwise linear correlations have attracted great interests while the underlying functional network structure still remains poorly understood. To further our understanding of RSFC, this paper presents an analysis of the resting-state networks (RSNs) based on the steady-state distributions and provides a novel angle to investigate the RSFC of multiple functional nodes. This paper evaluates the consistency of two networks based on the Hellinger distance between the steady-state distributions of the inferred Markov chain models. The results show that generated steady-state distributions of default mode network have higher consistency across subjects than random nodes from various RSNs.

Contribution of remote substrate binding energy to the enzymatic rate acceleration for 3α-hydroxysteroid dehydrogenase/carbonyl reductase.

PubMed

Hwang, Chi-Ching; Chang, Pei-Ru; Wang, Tzu-Pin

2017-10-01

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidation of androsterone with NAD + to form androstanedione and NADH with the rate limiting step being the release of NADH. In this study, we elucidate the role of remote substrate binding interactions contributing to the rate enhancement by 3α-HSD/CR through steady-state kinetic studies with the truncated substrate analogs. No enzyme activity was detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. As compared to cyclohexanol, the activity for 2-decalol, androstenol, and androsterone increases by 0.9-, 90-, and 200-fold in k cat , and 37-, 1.9 × 10 6 -, and 1.8 × 10 6 -fold in k cat /K B , respectively. The rate limiting step is hydride transfer for 3α-HSD/CR catalyzing the reaction of cyclohexanol with NAD + based on the observed rapid equilibrium ordered mechanism and equal deuterium isotope effects of 3.9 on V and V/K for cyclohexanol. The k cat /K B value results in ΔG ‡ of 14.7, 12.6, 6.2, and 6.2 kcal/mol for the 3α-HSD/CR catalyzed reaction of cyclohexanol, 2-decalol, androstenol, and androsterone, respectively. Thus, the uniform binding energy from the B-ring of steroids with the active site of 3α-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in k cat for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3α-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3α-HSD/CR catalysis, resulting in the increases in k cat and k cat /K B . Copyright © 2017 Elsevier B.V. All rights reserved.

Results of the ETV-1 breadboard tests under steady-state and transient conditions. [conducted in the NASA-LeRC Road Load Simulator

NASA Technical Reports Server (NTRS)

Sargent, N. B.; Dustin, M. O.

1981-01-01

Steady state tests were run to characterize the system and component efficiencies over the complete speed-torque capabilities of the propulsion system in both motoring and regenerative modes of operation. The steady state data were obtained using a battery simulator to separate the effects on efficiency caused by changing battery state-of-charge and component temperature. Transient tests were performed to determine the energy profiles of the propulsion system operating over the SAE J227a driving schedules.

Experimental investigation of cryogenic oscillating heat pipes.

PubMed

Jiao, A J; Ma, H B; Critser, J K

2009-07-01

A novel cryogenic heat pipe, oscillating heat pipe (OHP), which consists of an 4 × 18.5 cm evaporator, a 6 × 18.5 cm condenser, and 10 cm length of adiabatic section, has been developed and experimental characterization conducted. Experimental results show that the maximum heat transport capability of the OHP reached 380W with average temperature difference of 49 °C between the evaporator and condenser when the cryogenic OHP was charged with liquid nitrogen at 48% (v/v) and operated in a horizontal direction. The thermal resistance decreased from 0.256 to 0.112 while the heat load increased from 22.5 to 321.8 W. When the OHP was operated at a steady state and an incremental heat load was added to it, the OHP operation changed from a steady state to an unsteady state until a new steady state was reached. This process can be divided into three regions: (I) unsteady state; (II) transient state; and (III) new steady state. In the steady state, the amplitude of temperature change in the evaporator is smaller than that of the condenser while the temperature response keeps the same frequency both in the evaporator and the condenser. The experimental results also showed that the amplitude of temperature difference between the evaporator and the condenser decreased when the heat load increased.

Experimental investigation of cryogenic oscillating heat pipes

PubMed Central

Jiao, A.J.; Ma, H.B.; Critser, J.K.

2010-01-01

A novel cryogenic heat pipe, oscillating heat pipe (OHP), which consists of an 4 × 18.5 cm evaporator, a 6 × 18.5 cm condenser, and 10 cm length of adiabatic section, has been developed and experimental characterization conducted. Experimental results show that the maximum heat transport capability of the OHP reached 380W with average temperature difference of 49 °C between the evaporator and condenser when the cryogenic OHP was charged with liquid nitrogen at 48% (v/v) and operated in a horizontal direction. The thermal resistance decreased from 0.256 to 0.112 while the heat load increased from 22.5 to 321.8 W. When the OHP was operated at a steady state and an incremental heat load was added to it, the OHP operation changed from a steady state to an unsteady state until a new steady state was reached. This process can be divided into three regions: (I) unsteady state; (II) transient state; and (III) new steady state. In the steady state, the amplitude of temperature change in the evaporator is smaller than that of the condenser while the temperature response keeps the same frequency both in the evaporator and the condenser. The experimental results also showed that the amplitude of temperature difference between the evaporator and the condenser decreased when the heat load increased. PMID:20585410

Slow and deep respiration suppresses steady-state sympathetic nerve activity in patients with chronic heart failure: from modeling to clinical application.

PubMed

Harada, Daisuke; Asanoi, Hidetsugu; Takagawa, Junya; Ishise, Hisanari; Ueno, Hiroshi; Oda, Yoshitaka; Goso, Yukiko; Joho, Shuji; Inoue, Hiroshi

2014-10-15

Influences of slow and deep respiration on steady-state sympathetic nerve activity remain controversial in humans and could vary depending on disease conditions and basal sympathetic nerve activity. To elucidate the respiratory modulation of steady-state sympathetic nerve activity, we modeled the dynamic nature of the relationship between lung inflation and muscle sympathetic nerve activity (MSNA) in 11 heart failure patients with exaggerated sympathetic outflow at rest. An autoregressive exogenous input model was utilized to simulate entire responses of MSNA to variable respiratory patterns. In another 18 patients, we determined the influence of increasing tidal volume and slowing respiratory frequency on MSNA; 10 patients underwent a 15-min device-guided slow respiration and the remaining 8 had no respiratory modification. The model predicted that a 1-liter, step increase of lung volume decreased MSNA dynamically; its nadir (-33 ± 22%) occurred at 2.4 s; and steady-state decrease (-15 ± 5%), at 6 s. Actually, in patients with the device-guided slow and deep respiration, respiratory frequency effectively fell from 16.4 ± 3.9 to 6.7 ± 2.8/min (P < 0.0001) with a concomitant increase in tidal volume from 499 ± 206 to 1,177 ± 497 ml (P < 0.001). Consequently, steady-state MSNA was decreased by 31% (P < 0.005). In patients without respiratory modulation, there were no significant changes in respiratory frequency, tidal volume, and steady-state MSNA. Thus slow and deep respiration suppresses steady-state sympathetic nerve activity in patients with high levels of resting sympathetic tone as in heart failure. Copyright © 2014 the American Physiological Society.

Spatial variability of steady-state infiltration into a two-layer soil system on burned hillslopes

USGS Publications Warehouse

Kinner, D.A.; Moody, J.A.

2010-01-01

Rainfall-runoff simulations were conducted to estimate the characteristics of the steady-state infiltration rate into 1-m2 north- and south-facing hillslope plots burned by a wildfire in October 2003. Soil profiles in the plots consisted of a two-layer system composed of an ash on top of sandy mineral soil. Multiple rainfall rates (18.4-51.2 mm h-1) were used during 14 short-duration (30 min) and 2 long-duration simulations (2-4 h). Steady state was reached in 7-26 min. Observed spatially-averaged steady-state infiltration rates ranged from 18.2 to 23.8 mm h-1 for north-facing and from 17.9 to 36.0 mm h-1 for south-facing plots. Three different theoretical spatial distribution models of steady-state infiltration rate were fit to the measurements of rainfall rate and steady-state discharge to provided estimates of the spatial average (19.2-22.2 mm h-1) and the coefficient of variation (0.11-0.40) of infiltration rates, overland flow contributing area (74-90% of the plot area), and infiltration threshold (19.0-26 mm h-1). Tensiometer measurements indicated a downward moving pressure wave and suggest that infiltration-excess overland flow is the runoff process on these burned hillslope with a two-layer system. Moreover, the results indicate that the ash layer is wettable, may restrict water flow into the underlying layer, and increase the infiltration threshold; whereas, the underlying mineral soil, though coarser, limits the infiltration rate. These results of the spatial variability of steady-state infiltration can be used to develop physically-based rainfall-runoff models for burned areas with a two-layer soil system. ?? 2010 Elsevier B.V.

Steady states of a diode with counterstreaming electron and positron beams

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ender, A. Ya.; Kuznetsov, V. I., E-mail: victor.kuznetsov@mail.ioffe.ru; Gruzdev, A. A.

2016-10-15

Steady states of a plasma layer with counterstreaming beams of oppositely charged particles moving without collisions in a self-consistent electric field are analyzed. The study is aimed at clarifying the mechanism of generation and reconstruction of pulsar radiation. Such a layer also models the processes occurring in Knudsen plasma diodes with counterstreaming electron and ion beams. The steady-state solutions are exhaustively classified. The existence of several solutions at the same external parameters is established.

Steady states of a diode with counterstreaming electron and positron beams

NASA Astrophysics Data System (ADS)

Ender, A. Ya.; Kuznetsov, V. I.; Gruzdev, A. A.

2016-10-01

Steady states of a plasma layer with counterstreaming beams of oppositely charged particles moving without collisions in a self-consistent electric field are analyzed. The study is aimed at clarifying the mechanism of generation and reconstruction of pulsar radiation. Such a layer also models the processes occurring in Knudsen plasma diodes with counterstreaming electron and ion beams. The steady-state solutions are exhaustively classified. The existence of several solutions at the same external parameters is established.

Transient and steady-state performance of a single turbojet combustor with four different fuel nozzles

NASA Technical Reports Server (NTRS)

Mccafferty, Richard J; Donlon, Richard H

1955-01-01

Acceleration and steady-state performance of a tubular combustor was evaluated at two simulated altitudes with four different fuel nozzles. Temperature response lag was observed with all the nozzles. Except for rich-limit blowout, the only combustion failures observed during acceleration were with a fuel nozzle that gave an interrupted flow delivery during the acceleration. This same nozzle, because of superior fuel atomization, gave the highest steady-state combustion efficiencies.

Lactate and Acrylate Metabolism by Megasphaera elsdenii under Batch and Steady-State Conditions

PubMed Central

Prabhu, Rupal; Altman, Elliot

2012-01-01

The growth of Megasphaera elsdenii on lactate with acrylate and acrylate analogues was studied under batch and steady-state conditions. Under batch conditions, lactate was converted to acetate and propionate, and acrylate was converted into propionate. Acrylate analogues 2-methyl propenoate and 3-butenoate containing a terminal double bond were similarly converted into their respective saturated acids (isobutyrate and butyrate), while crotonate and lactate analogues 3-hydroxybutyrate and (R)-2-hydroxybutyrate were not metabolized. Under carbon-limited steady-state conditions, lactate was converted to acetate and butyrate with no propionate formed. As the acrylate concentration in the feed was increased, butyrate and hydrogen formation decreased and propionate was increasingly generated, while the calculated ATP yield was unchanged. M. elsdenii metabolism differs substantially under batch and steady-state conditions. The results support the conclusion that propionate is not formed during lactate-limited steady-state growth because of the absence of this substrate to drive the formation of lactyl coenzyme A (CoA) via propionyl-CoA transferase. Acrylate and acrylate analogues are reduced under both batch and steady-state growth conditions after first being converted to thioesters via propionyl-CoA transferase. Our findings demonstrate the central role that CoA transferase activity plays in the utilization of acids by M. elsdenii and allows us to propose a modified acrylate pathway for M. elsdenii. PMID:23023753

A critical investigation of post-liquefaction strength and steady-state flow behavior of saturated soils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jong, H.L.

1988-01-01

The first objective was to perform a critical evaluation of the recently proposed steady-state analysis methodology for evaluation of post-liquefaction stability of potentially liquefiable soils. This analysis procedure is based on direct comparison between the in-situ undrained residual (steady state) strength of soils in an embankment or foundation, and the driving shear stresses in these soils. A laboratory investigation was performed to investigate factors affecting steady-state strengths, and also to evaluate the validity of assumptions involved in correcting the results of laboratory steady-state strength tests on undisturbed samples for effects of sampling disturbance in order to estimate in-situ strengths. Next,more » a field case study was performed using the steady-state analysis and testing methodologies to analyze Lower San Fernando Dam, which suffered a liquefaction-induced slope failure as a results of a 1971 earthquake. This leads to the second objective which was to extend the Lower San Fernando Dam case study to consideration of analysis methods used to evaluate the likelihood of triggering liquefaction during an earthquake. Finally, a number of the high quality undisturbed samples were subjected to undrained cyclic testing in order to repeat an earlier (1973) study of the use of cyclic tests data to predict liquefaction behavior at Lower San Fernando Dam.« less

Multiple Steady States of Buoyancy Induced Flow in Cold Water and Their Stability.

NASA Astrophysics Data System (ADS)

El-Henawy, Ibrahim Mahmoud

In Chapters 1 and 2 the physical background and the literature related to buoyancy-induced flows are reviewed. An accurate representation, based upon experimental data, of the motion-causing buoyancy force, in the vicinity of maximum density in pure water at low temperatures, is used. This representation is an accurate and quite simple formulation due to Gebhart and Mollendorf (1977). Using the representation, we study, numerically, Chapter 3, a model for the laminar, boundary-layer flow arising from natural convection adjacent to a vertical isothermal flat surface submerged in quiescent cold water. The results demonstrate for the first time the existence of multiple steady-state solutions in a natural convection flow. The existence of these new multiple steady-state solutions led to an investigation of their stability. This is carried out in Chapter 4 by a mathematical method, different from that of the usual hydrodynamic stability approach, Lin (1955) and Razinand and Reid (1982). Three real eigenvalue and eigenvector pairs corresponding to the new steady-state -solutions were found. Each of these eigenvalues changes its algebraic sign at a particular limit point (point of vertical tangency, nose, knee) in the bifurcation diagrams found in Chapter 3. The results indicate that the new steady-state solutions are unstable and that the previously found steady-state solutions, Carey, Gebhart, and Mollendorf (1980), may be stable.

Prospective evaluation of haemoglobin oxygen saturation at rest and after exercise in paediatric sickle cell disease patients

PubMed Central

Campbell, Andrew; Minniti, Caterina P.; Nouraie, Mehdi; Arteta, Manuel; Rana, Sohail; Onyekwere, Onyinye; Sable, Craig; Ensing, Gregory; Dham, Niti; Luchtman-Jones, Lori; Kato, Gregory J.; Gladwin, Mark T.; Castro, Oswaldo L.; Gordeuk, Victor R.

2009-01-01

Summary Low steady state haemoglobin oxygen saturation in patients with sickle cell anaemia has been associated with the degree of anaemia and haemolysis. How much pulmonary dysfunction contributes to low saturation is not clear. In a prospective study of children and adolescents with sickle cell disease aged 3–20 years at steady state and matched controls, 52% of 391 patients versus 24% of 63 controls had steady state oxygen saturation <99% (P < 0·0001), 9% of patients versus no controls had saturation <95% (P = 0·008) and 8% of patients versus no controls had exercise-induced reduction in saturation ≥3%. Decreasing haemoglobin concentration (P ≤ 0·001) and increasing haemolysis (P ≤ 0·003) but not pulmonary function tests were independent predictors of both lower steady-state saturation and exercise-induced reduction in saturation. Neither history of stroke nor history of acute chest syndrome was significantly associated with lower steady-state oxygen saturation or exercise-induced reduction in saturation. Tricuspid regurgitation velocity was higher in patients with lower steady state haemoglobin oxygen saturation (P = 0·003) and with greater decline in oxygen saturation during the six-minute walk (P = 0·022). In conclusion, lower haemoglobin oxygen saturation is independently associated with increasing degrees of anaemia and haemolysis but not pulmonary function abnormalities among children and adolescents with sickle cell disease. PMID:19694721

Nitrogen Can Alleviate the Inhibition of Photosynthesis Caused by High Temperature Stress under Both Steady-State and Flecked Irradiance.

PubMed

Huang, Guanjun; Zhang, Qiangqiang; Wei, Xinghai; Peng, Shaobing; Li, Yong

2017-01-01

Nitrogen is one of the most important elements for plants and is closely related to photosynthesis. High temperature stress significantly inhibits photosynthesis under both steady-state and flecked irradiance. However, it is not known whether nitrogen can affect the decrease in photosynthesis caused by high temperature, especially under flecked irradiance. In the present study, a pot experiment was conducted under two nitrogen (N) supplies with rice plants, and the steady-state and dynamic photosynthesis rates were measured under 28 and 40°C. High temperature significantly increased leaf hydraulic conductance ( K leaf ) under high N supply (HN) but not under low N supply (LN). The increased K leaf maintained a constant leaf water potential (Ψ leaf ) and steady-state stomatal conductance ( g s,sat ) under HN, while the Ψ leaf and g s,sat significantly decreased under high temperature in LN conditions. This resulted in a more severe decrease in steady-state photosynthesis ( A sat ) under high temperature in the LN conditions. After shifting from low to high light, high temperature significantly delayed the recovery of photosynthesis, which resulted in more carbon loss under flecked irradiance. These effects were obtained under HN to a lesser extent than under LN supply. Therefore, it is concluded that nitrogen can alleviate the inhibition of photosynthesis caused by high temperature stress under both steady-state and flecked irradiance.

Quasi-steady-state voltammetry of rapid electron transfer reactions at the macroscopic substrate of the scanning electrochemical microscope.

PubMed

Nioradze, Nikoloz; Kim, Jiyeon; Amemiya, Shigeru

2011-02-01

We report on a novel theory and experiment for scanning electrochemical microscopy (SECM) to enable quasi-steady-state voltammetry of rapid electron transfer (ET) reactions at macroscopic substrates. With this powerful approach, the substrate potential is cycled widely across the formal potential of a redox couple while the reactant or product of a substrate reaction is amperometrically detected at the tip in the feedback or substrate generation/tip collection mode, respectively. The plot of tip current versus substrate potential features the retraceable sigmoidal shape of a quasi-steady-state voltammogram although a transient voltammogram is obtained at the macroscopic substrate. Finite element simulations reveal that a short tip-substrate distance and a reversible substrate reaction (except under the tip) are required for quasi-steady-state voltammetry. Advantageously, a pair of quasi-steady-state voltammograms is obtained by employing both operation modes to reliably determine all transport, thermodynamic, and kinetic parameters as confirmed experimentally for rapid ET reactions of ferrocenemethanol and 7,7,8,8-tetracyanoquinodimethane at a Pt substrate with ∼0.5 μm-radius Pt tips positioned at 90 nm-1 μm distances. Standard ET rate constants of ∼7 cm/s were obtained for the latter mediator as the largest determined for a substrate reaction by SECM. Various potential applications of quasi-steady-state voltammetry are also proposed.

Role of irregular otolith afferents in the steady-state nystagmus during off-vertical axis rotation

NASA Technical Reports Server (NTRS)

Angelaki, D. E.; Perachio, A. A.; Mustari, M. J.; Strunk, C. L.

1992-01-01

1. During constant velocity off-vertical axis rotations (OVAR) in the dark a compensatory ocular nystagmus is present throughout rotation despite the lack of a maintained signal from the semicircular canals. Lesion experiments and canal plugging have attributed the steady-state ocular nystagmus during OVAR to inputs from the otolith organs and have demonstrated that it depends on an intact velocity storage mechanism. 2. To test whether irregularly discharging otolith afferents play a crucial role in the generation of the steady-state eye nystagmus during OVAR, we have used anodal (inhibitory) currents bilaterally to selectively and reversibly block irregular vestibular afferent discharge. During delivery of DC anodal currents (100 microA) bilaterally to both ears, the slow phase eye velocity of the steady-state nystagmus during OVAR was reduced or completely abolished. The disruption of the steady-state nystagmus was transient and lasted only during the period of galvanic stimulation. 3. To distinguish a possible effect of ablation of the background discharge rates of irregular vestibular afferents on the velocity storage mechanism from specific contributions of the dynamic responses from irregular otolith afferents to the circuit responsible for the generation of the steady-state nystagmus, bilateral DC anodal galvanic stimulation was applied during optokinetic nystagmus (OKN) and optokinetic afternystagmus (OKAN). No change in OKN and OKAN was observed.(ABSTRACT TRUNCATED AT 250 WORDS).

Hyperoxia, unlike phorbol ester, induces glutathione peroxidase through a protein kinase C-independent mechanism.

PubMed Central

Jornot, L; Junod, A F

1997-01-01

Human selenium-dependent glutathione peroxidase (GP) is implicated as a mechanism of resistance against oxygen free radicals. The 5' flanking sequence upstream from the coding region of GP contained an oxygen-responsive element termed ORE1 that is responsive to hypoxia, as well as several copies of the activator protein-1 (AP-1)- and AP-1-like-binding sites. In this study, we sought to define the molecular events that lead to GP gene transcription in response to hyperoxia in human umbilical-vein endothelial cells, and asked whether such induction is mimicked and sustained by activation of protein kinase C (PKC) by phorbol esters. Treatment of cells with 100 nM phorbol 12,13-dibutyrate (PdBu) induced a delayed (24-48 h) but significant (2-fold) increase in steady-state GP mRNA levels. Steady-state GP mRNA levels also rose after exposure to 95% O2, again after considerable delay (48-72 h). For both PdBu and oxygen, induction was transcriptionally regulated, as demonstrated by nuclear run-on experiments. The simulations by PdBu and oxygen were additive. In contrast with PdBu, hyperoxia did not stimulate translocation of PKC from the cytosol to the particulate fraction, although the specific activity of both cytosolic and particulate-associated PKC was increased 2-fold in cells exposed to 95% O2 for 5 days. In addition, gel mobility-shift assays using double-stranded tumour-promoting-agent-responsive element (TRE) and nuclear extracts derived from phorbol- and oxygen-treated cells revealed that PdBu, but not hyperoxia, increased AP-1 DNA-binding activity. On the other hand, the up-regulation of GP expression by oxygen could not be accounted for by the ORE1 core sequence, since no specific protein-DNA binding activity could be detected using nuclear extracts from hyperoxic cells and ORE1. Taken together, these results suggest that there may be different molecular mechanisms controlling GP expression. After exposure to PdBu, GP undergoes transcriptional activation via a process that can be readily explained by a classic AP-1 interaction with the TRE sites in the GP promoter. During hyperoxia, GP also undergoes transcriptional activity, but via a process that appears to involve neither TRE nor ORE1. PMID:9337858

Spinach chloroplast 0-acetylserine (thiol)-lyase exhibits two catalytically non-equivalent pyridoxal-5'-phosphate-containing active sites.

PubMed

Rolland, N; Ruffet, M L; Job, D; Douce, R; Droux, M

1996-02-15

A synthetic gene encoding the mature spinach- chloroplast O-acetylserine (thiol)-lyase was constructed and expressed in an Escherichia coli strain carrying the T7 RNA polymerase system. The pure recombinant protein was obtained at high yield (6 mg/l cell culture) using a new purification procedure that includes affinity chromatography on Green A agarose. Its specific activity was of the order of 1000 U/mg, and its physical properties were similar to those previously reported for the natural enzyme isolated from spinach chloroplasts. In particular the recombinant enzyme, as for the natural enzyme, behaved as a homodimer composed of two identical subunits each of Mr 35000. From steady-state kinetic studies using sulfide or 5-thio(2-nitrobenzoate) (Nbs) as alternative nucleophilic co-substrates, the enzyme exhibited positive kinetic co-operativity with respect to O-acetylserine [Ser(Ac)] in the presence of sulfide and a negative kinetic co-operativity in the presence of Nbs. Binding of Ser(Ac) to the enzyme was also investigated by absorbance and fluorescence measurements to obtain insight into the role of pyridoxal 5'-phosphate and of the single tryptophan residue (Trp176) present in the enzyme molecule. Addition of Ser(Ac) to the enzyme provoked the disappearance of the 409-nm absorbance band of the pyridoxal 5'-phosphate Schiff base and the appearance of two new absorbance bands, the one located between 320 nm and 360 nm and the other centered at 470 nm. Also, the fluorescence emission of the pyridoxal 5'-phosphate Schiff base was quenched upon addition of Ser(Ac) to the enzyme. These changes were most presumably due to the formation of a Schiff base intermediate between alpha-aminoacrylate and the pyridoxal 5'-phosphate cofactor. The fluorescence emission of Trp176 was also quenched upon Ser(Ac) binding to the enzyme. Quantitative analysis of the absorbance and fluorescence equilibrium data disclosed a co-operative behavior in Ser(Ac) binding, in agreement with the steady-state kinetic results. Fluorescence quenching experiments with the acrylamide and iodide revealed that the indole ring of Trp176 was largely exposed and located within the pyridoxal 5'-phosphate active site. These results are consistent with the finding that the native enzyme is composed of two identical subunits. Yet, presumably due to subunit-subunit interactions, the enzyme exhibits two non-equivalent pyridoxal-5'-phosphate-containing active sites.

To bind or not to bind? Different temporal binding effects from voluntary pressing and releasing actions.

PubMed

Zhao, Ke; Chen, Yu-Hsin; Yan, Wen-Jing; Fu, Xiaolan

2013-01-01

Binding effect refers to the perceptual attraction between an action and an outcome leading to a subjective compression of time. Most studies investigating binding effects exclusively employ the "pressing" action without exploring other types of actions. The present study addresses this issue by introducing another action, releasing action or the voluntary lifting of the finger/wrist, to investigate the differences between voluntary pressing and releasing actions. Results reveal that releasing actions led to robust yet short-lived temporal binding effects, whereas pressing condition had steady temporal binding effects up to super-seconds. The two actions also differ in sensitivity to changes in temporal contiguity and contingency, which could be attributed to the difference in awareness of action. Extending upon current models of "willed action," our results provide insights from a temporal point of view and support the concept of a dual system consisting of predictive motor control and top-down mechanisms.

Smooth Approximation l 0-Norm Constrained Affine Projection Algorithm and Its Applications in Sparse Channel Estimation

PubMed Central

2014-01-01

We propose a smooth approximation l 0-norm constrained affine projection algorithm (SL0-APA) to improve the convergence speed and the steady-state error of affine projection algorithm (APA) for sparse channel estimation. The proposed algorithm ensures improved performance in terms of the convergence speed and the steady-state error via the combination of a smooth approximation l 0-norm (SL0) penalty on the coefficients into the standard APA cost function, which gives rise to a zero attractor that promotes the sparsity of the channel taps in the channel estimation and hence accelerates the convergence speed and reduces the steady-state error when the channel is sparse. The simulation results demonstrate that our proposed SL0-APA is superior to the standard APA and its sparsity-aware algorithms in terms of both the convergence speed and the steady-state behavior in a designated sparse channel. Furthermore, SL0-APA is shown to have smaller steady-state error than the previously proposed sparsity-aware algorithms when the number of nonzero taps in the sparse channel increases. PMID:24790588

[Study on balance group in steady-state extraction process of Chinese medicine and experimental verification to Houttuynia cordata].

PubMed

Liu, Wenlong; Zhang, Xili; He, Fuyuan; Zhang, Ping; Wang, Haiqin; Wu, Dezhi; Chen, Zuohong

2011-11-01

To establish and experimental verification the mathematical model of the balance groups that is the steady-state of traditional Chinese medicine in extraction. Using the entropy and genetic principles of statistics, and taking the coefficient of variation of GC fingerprint which is the naphtha of the Houttuynia cordata between strains in the same GAP place as a pivot to establish and verify the mathematical model was established of the balance groups that is the steady-state of traditional Chinese medicine in extraction. A mathematical model that is suitable for the balance groups of the steady-state of traditional Chinese medicine and preparation in extraction, and the balance groups which is 29 683 strains (approximately 118.7 kg) were gained with the same origin of H. cordata as the model drug. Under the GAP of quality control model, controlling the stability of the quality through further using the Hardy-Weinberg balance groups of the H. cordata between strains, the new theory and experiment foundation is established for the steady-state of traditional Chinese medicine in extraction and quality control.

How active site protonation state influences the reactivity and ligation of the heme in chlorite dismutase

PubMed Central

Streit, Bennett R.; Blanc, Béatrice; Lukat-Rodgers, Gudrun S.; Rodgers, Kenton R.; DuBois, Jennifer L.

2010-01-01

Chlorite dismutase catalyzes O2 release from chlorite with exquisite efficiency and specificity. The spectroscopic properties, ligand binding affinities, and steady state kinetics of chlorite dismutase from Dechloromonas aromatica were examined over pH 3–11.5 to gain insight into how the protonation state of the heme environment influences dioxygen formation. An acid/base transition was observed by UV/visible and resonance Raman spectroscopy with a pKa of 8.7, 2–3 pH units below analogous transitions observed in typical His-ligated peroxidases. This transition marks the conversion of a five coordinate high spin Fe(III) to a mixed high/low spin ferric-hydroxide, as confirmed by resonance Raman (rR) spectroscopy. The two Fe–OH stretching frequencies are quite low, consistent with a weak Fe–OH bond, despite the nearly neutral imidazole side chain of the proximal histidine ligand. The hydroxide is proposed to interact strongly with a distal H-bond donor, thereby weakening the Fe–OH bond. The rR spectra of Cld-CO as a function of pH reveal two forms of the complex, one in which there is minimal interaction of distal residues with the carbonyl oxygen and another, acidic form in which the oxygen is under the influence of positive charge. Recent crystallographic data reveal arginine 183 as the lone H-bond donating residue in the distal pocket. It is likely that this Arg is the strong, positively charged H-bond donor implicated by vibrational data to interact with exogenous axial heme ligands. The same Arg in its neutral (pKa ~ 6.5) form also appears to act as the active site base in binding reactions of protonated ligands, such as HCN, to ferric Cld. The steady state profile for the rate of chlorite decomposition is characterized by these same pKas. The 5 coordinate high spin acidic Cld is more active than the alkaline hydroxide-bound form. The acid form decomposes chlorite most efficiently when the distal Arg is protonated/cationic (maximum kcat = 2.0 (±0.6) × 105 s−1, kcat/KM = 3.2 (±0.4) × 107 M−1s−1, pH 5.2, 4 °C) and to a somewhat lesser extent when it acts as a H-bond donor to the axial hydroxide ligand under alkaline conditions. PMID:20356038

Advanced continuous cultivation methods for systems microbiology.

PubMed

Adamberg, Kaarel; Valgepea, Kaspar; Vilu, Raivo

2015-09-01

Increasing the throughput of systems biology-based experimental characterization of in silico-designed strains has great potential for accelerating the development of cell factories. For this, analysis of metabolism in the steady state is essential as only this enables the unequivocal definition of the physiological state of cells, which is needed for the complete description and in silico reconstruction of their phenotypes. In this review, we show that for a systems microbiology approach, high-resolution characterization of metabolism in the steady state--growth space analysis (GSA)--can be achieved by using advanced continuous cultivation methods termed changestats. In changestats, an environmental parameter is continuously changed at a constant rate within one experiment whilst maintaining cells in the physiological steady state similar to chemostats. This increases the resolution and throughput of GSA compared with chemostats, and, moreover, enables following of the dynamics of metabolism and detection of metabolic switch-points and optimal growth conditions. We also describe the concept, challenge and necessary criteria of the systematic analysis of steady-state metabolism. Finally, we propose that such systematic characterization of the steady-state growth space of cells using changestats has value not only for fundamental studies of metabolism, but also for systems biology-based metabolic engineering of cell factories.

Bench-Scale Testing and Process Performance Projections of CO2 Capture by CO2–Binding Organic Liquids (CO2BOLs) With and Without Polarity-Swing-Assisted Regeneration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Feng; Heldebrant, David J.; Mathias, Paul M.

This manuscript provides a detailed analysis of a continuous flow, bench scale study of the CO2BOL solvent platform with and without its Polarity Swing Assisted Regeneration (PSAR). This study encompassed four months of continuous flow testing of a candidate CO2BOL with a thermal regeneration and PSAR regeneration using decane antisolvent. In both regeneration schemes, steady state capture of >90 %CO2 was achieved using simulated flue gas at acceptable L/G ratios. Aspen Plus™ modeling was performed to assess process performance compared to previous equilibrium performance projections. This paper also includes net power projections, and comparisons to DOE’s Case 10 amine baseline.

Temperature-dependent excitonic photoluminescence of hybrid organometal halide perovskite films.

PubMed

Wu, Kewei; Bera, Ashok; Ma, Chun; Du, Yuanmin; Yang, Yang; Li, Liang; Wu, Tom

2014-11-07

Organometal halide perovskites have recently attracted tremendous attention due to their potential for photovoltaic applications, and they are also considered as promising materials in light emitting and lasing devices. In this work, we investigated in detail the cryogenic steady state photoluminescence properties of a prototypical hybrid perovskite CH3NH3PbI3-xClx. The evolution of the characteristics of two excitonic peaks coincides with the structural phase transition around 160 K. Our results further revealed an exciton binding energy of 62.3 ± 8.9 meV and an optical phonon energy of 25.3 ± 5.2 meV, along with an abnormal blue-shift of the band gap in the high-temperature tetragonal phase.

Kinetic study of the processing by dipeptidyl-peptidase IV/CD26 of neuropeptides involved in pancreatic insulin secretion.

PubMed

Lambeir, A M; Durinx, C; Proost, P; Van Damme, J; Scharpé, S; De Meester, I

2001-11-02

Dipeptidyl-peptidase IV (DPPIV/CD26) metabolizes neuropeptides regulating insulin secretion. We studied the in vitro steady-state kinetics of DPPIV/CD26-mediated truncation of vasoactive intestinal peptide (VIP), pituitary adenylyl cyclase-activating peptide (PACAP27 and PACAP38), gastrin-releasing peptide (GRP) and neuropeptide Y (NPY). DPPIV/CD26 sequentially cleaves off two dipeptides of VIP, PACAP27, PACAP38 and GRP. GRP situates between the best DPPIV/CD26 substrates reported, comparable to NPY. Surprisingly, the C-terminal extension of PACAP38, distant from the scissile bond, improves both PACAP38 binding and turnover. Therefore, residues remote from the scissile bond can modulate DPPIV/CD26 substrate selectivity as well as residues flanking it.

Effect of antacids on predicted steady-state cimetidine concentrations.

PubMed

Russell, W L; Lopez, L M; Normann, S A; Doering, P L; Guild, R T

1984-05-01

The purpose of this study was to evaluate effects of antacids on predicted steady-state concentrations of cimetidine. Ten healthy volunteers received in random order one week apart, cimetidine and cimetidine and antacid suspension. Blood was obtained at specified times and analyzed for cimetidine. Bioavailability was assessed by comparison of peak concentration, time to peak concentration, area under the curve, and time spent over 0.5 micrograms/ml. Single-dose data were extrapolated to steady-state using computer simulation. Concurrent administration of antacid suspension reduced parameters of bioavailability approximately 30%. When steady-state conditions were simulated, concentrations of cimetidine greater than or equal to 0.5 micrograms/ml were maintained for the entire dosing interval in seven of 10 subjects. These data suggest that temporal separation of cimetidine and antacid suspension may be unnecessary.

Interplay of interaction and disorder in the steady state of an open quantum system

NASA Astrophysics Data System (ADS)

Xu, Xiansong; Guo, Chu; Poletti, Dario

2018-04-01

Many types of dissipative processes can be found in nature or be engineered, and their interplay with a system can give rise to interesting phases of matter. Here we study the interplay among interaction, tunneling, and disorder in the steady state of a spin chain coupled to a tailored bath. We consider a dissipation which, in contrast to disorder, tends to generate a homogeneously polarized steady state. We find that the steady state can be highly sensitive even to weak disorder. We also establish that, in the presence of such dissipation, even in the absence of interaction, a finite amount of disorder is needed for localization. Last, we show that for strong disorder the system reveals signatures of localization both in the weakly and strong interacting regimes.

Functional role of a mobile loop of Escherichia coli dihydrofolate reductase in transition-state stabilization.

PubMed

Li, L; Falzone, C J; Wright, P E; Benkovic, S J

1992-09-01

The function of a highly mobile loop in Escherichia coli dihydrofolate reductase was studied by constructing a mutant (DL1) using cassette mutagenesis that had four residues deleted in the middle section of the loop (Met16-Ala19) and a glycine inserted to seal the gap. This part of the loop involves residues 16-20 and is disordered in the X-ray crystal structures of the apoprotein and the NADP+ binary complex but forms a hairpin turn that folds over the nicotinamide moiety of NADP+ and the pteridine moiety of folate in the ternary complex [Bystroff, C., & Kraut, J. (1991) Biochemistry 30, 2227-2239]. The steady-state and pre-steady-state kinetics and two-dimensional 1H NMR spectra were analyzed and compared to the wild-type protein. The kinetics on the DL1 mutant enzyme show that the KM value for NADPH (5.3 microM), the KM for dihydrofolate (2 microM), the rate constant for the release of the product tetrahydrofolate (10.3 s-1), and the intrinsic pKa value (6.2) are similar to those exhibited by the wild-type enzyme. However, the hydride-transfer rate declines markedly from the wild-type value of 950 s-1 to 1.7 s-1 for the DL1 mutant and when taken with data for substrate binding indicates that the loop contributes to substrate flux by a factor of 3.5 x 10(4). Thus, the mobility of loop I may provide a mechanism of recruiting hydrophobic residues which can properly align the nicotinamide and pteridine rings for the hydride-transfer process (a form of transition-state stabilization).(ABSTRACT TRUNCATED AT 250 WORDS)

Exact results for Schrödinger cats in driven-dissipative systems and their feedback control

NASA Astrophysics Data System (ADS)

Minganti, Fabrizio; Bartolo, Nicola; Lolli, Jared; Casteels, Wim; Ciuti, Cristiano

2016-05-01

In quantum optics, photonic Schrödinger cats are superpositions of two coherent states with opposite phases and with a significant number of photons. Recently, these states have been observed in the transient dynamics of driven-dissipative resonators subject to engineered two-photon processes. Here we present an exact analytical solution of the steady-state density matrix for this class of systems, including one-photon losses, which are considered detrimental for the achievement of cat states. We demonstrate that the unique steady state is a statistical mixture of two cat-like states with opposite parity, in spite of significant one-photon losses. The transient dynamics to the steady state depends dramatically on the initial state and can pass through a metastable regime lasting orders of magnitudes longer than the photon lifetime. By considering individual quantum trajectories in photon-counting configuration, we find that the system intermittently jumps between two cats. Finally, we propose and study a feedback protocol based on this behaviour to generate a pure cat-like steady state.

Mathematical Analysis of Vehicle Delivery Scale of Bike-Sharing Rental Nodes

NASA Astrophysics Data System (ADS)

Zhai, Y.; Liu, J.; Liu, L.

2018-04-01

Aiming at the lack of scientific and reasonable judgment of vehicles delivery scale and insufficient optimization of scheduling decision, based on features of the bike-sharing usage, this paper analyses the applicability of the discrete time and state of the Markov chain, and proves its properties to be irreducible, aperiodic and positive recurrent. Based on above analysis, the paper has reached to the conclusion that limit state (steady state) probability of the bike-sharing Markov chain only exists and is independent of the initial probability distribution. Then this paper analyses the difficulty of the transition probability matrix parameter statistics and the linear equations group solution in the traditional solving algorithm of the bike-sharing Markov chain. In order to improve the feasibility, this paper proposes a "virtual two-node vehicle scale solution" algorithm which considered the all the nodes beside the node to be solved as a virtual node, offered the transition probability matrix, steady state linear equations group and the computational methods related to the steady state scale, steady state arrival time and scheduling decision of the node to be solved. Finally, the paper evaluates the rationality and accuracy of the steady state probability of the proposed algorithm by comparing with the traditional algorithm. By solving the steady state scale of the nodes one by one, the proposed algorithm is proved to have strong feasibility because it lowers the level of computational difficulty and reduces the number of statistic, which will help the bike-sharing companies to optimize the scale and scheduling of nodes.

Current Pressure Transducer Application of Model-based Prognostics Using Steady State Conditions

NASA Technical Reports Server (NTRS)

Teubert, Christopher; Daigle, Matthew J.

2014-01-01

Prognostics is the process of predicting a system's future states, health degradation/wear, and remaining useful life (RUL). This information plays an important role in preventing failure, reducing downtime, scheduling maintenance, and improving system utility. Prognostics relies heavily on wear estimation. In some components, the sensors used to estimate wear may not be fast enough to capture brief transient states that are indicative of wear. For this reason it is beneficial to be capable of detecting and estimating the extent of component wear using steady-state measurements. This paper details a method for estimating component wear using steady-state measurements, describes how this is used to predict future states, and presents a case study of a current/pressure (I/P) Transducer. I/P Transducer nominal and off-nominal behaviors are characterized using a physics-based model, and validated against expected and observed component behavior. This model is used to map observed steady-state responses to corresponding fault parameter values in the form of a lookup table. This method was chosen because of its fast, efficient nature, and its ability to be applied to both linear and non-linear systems. Using measurements of the steady state output, and the lookup table, wear is estimated. A regression is used to estimate the wear propagation parameter and characterize the damage progression function, which are used to predict future states and the remaining useful life of the system.

Experimental demonstration of revival of oscillations from death in coupled nonlinear oscillators.

PubMed

Senthilkumar, D V; Suresh, K; Chandrasekar, V K; Zou, Wei; Dana, Syamal K; Kathamuthu, Thamilmaran; Kurths, Jürgen

2016-04-01

We experimentally demonstrate that a processing delay, a finite response time, in the coupling can revoke the stability of the stable steady states, thereby facilitating the revival of oscillations in the same parameter space where the coupled oscillators suffered the quenching of oscillation. This phenomenon of reviving of oscillations is demonstrated using two different prototype electronic circuits. Further, the analytical critical curves corroborate that the spread of the parameter space with stable steady state is diminished continuously by increasing the processing delay. Finally, the death state is completely wiped off above a threshold value by switching the stability of the stable steady state to retrieve sustained oscillations in the same parameter space. The underlying dynamical mechanism responsible for the decrease in the spread of the stable steady states and the eventual reviving of oscillation as a function of the processing delay is explained using analytical results.

Pattern Formation in Keller-Segel Chemotaxis Models with Logistic Growth

NASA Astrophysics Data System (ADS)

Jin, Ling; Wang, Qi; Zhang, Zengyan

In this paper, we investigate pattern formation in Keller-Segel chemotaxis models over a multidimensional bounded domain subject to homogeneous Neumann boundary conditions. It is shown that the positive homogeneous steady state loses its stability as chemoattraction rate χ increases. Then using Crandall-Rabinowitz local theory with χ being the bifurcation parameter, we obtain the existence of nonhomogeneous steady states of the system which bifurcate from this homogeneous steady state. Stability of the bifurcating solutions is also established through rigorous and detailed calculations. Our results provide a selection mechanism of stable wavemode which states that the only stable bifurcation branch must have a wavemode number that minimizes the bifurcation value. Finally, we perform extensive numerical simulations on the formation of stable steady states with striking structures such as boundary spikes, interior spikes, stripes, etc. These nontrivial patterns can model cellular aggregation that develop through chemotactic movements in biological systems.

Experimental demonstration of revival of oscillations from death in coupled nonlinear oscillators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Senthilkumar, D. V., E-mail: skumarusnld@gmail.com; Centre for Nonlinear Science and Engineering, School of Electrical and Electronics Engineering, SASTRA University, Thanjavur 613 401; Suresh, K.

We experimentally demonstrate that a processing delay, a finite response time, in the coupling can revoke the stability of the stable steady states, thereby facilitating the revival of oscillations in the same parameter space where the coupled oscillators suffered the quenching of oscillation. This phenomenon of reviving of oscillations is demonstrated using two different prototype electronic circuits. Further, the analytical critical curves corroborate that the spread of the parameter space with stable steady state is diminished continuously by increasing the processing delay. Finally, the death state is completely wiped off above a threshold value by switching the stability of themore » stable steady state to retrieve sustained oscillations in the same parameter space. The underlying dynamical mechanism responsible for the decrease in the spread of the stable steady states and the eventual reviving of oscillation as a function of the processing delay is explained using analytical results.« less

Determination of the Steady State Leakage Current in Structures with Ferroelectric Ceramic Films

NASA Astrophysics Data System (ADS)

Podgornyi, Yu. V.; Vorotilov, K. A.; Sigov, A. S.

2018-03-01

Steady state leakage currents have been investigated in capacitor structures with ferroelectric solgel films of lead zirconate titanate (PZT) formed on silicon substrates with a lower Pt electrode. It is established that Pt/PZT/Hg structures, regardless of the PZT film thickness, are characterized by the presence of a rectifying contact similar to p-n junction. The steady state leakage current in the forward direction increases with a decrease in the film thickness and is determined by the ferroelectric bulk conductivity.

Comparative study of binding interactions between porphyrin systems and aromatic compounds of biological importance by multiple spectroscopic techniques: A review

NASA Astrophysics Data System (ADS)

Makarska-Bialokoz, Magdalena

2018-07-01

The specific spectroscopic and redox properties of porphyrins predestine them to fulfill the role of sensors during interacting with different biologically active substances. Monitoring of binding interactions in the systems porphyrin-biologically active compound is a key question not only in the field of physiological functions of living organisms, but also in environmental protection, notably in the light of the rapidly growing drug consumption and concurrently the production of drug effluents. Not always beneficial action of drugs on natural porphyrin systems induces to further studies, with commercially available porphyrins as the model systems. Therefore the binding process between several water-soluble porphyrins and a series of biologically active compounds (e.g. caffeine, guanine, theophylline, theobromine, xanthine, uric acid) has been studied in different aqueous solutions analyzing their absorption and steady-state fluorescence spectra, the porphyrin fluorescence lifetimes and their quantum yields. The magnitude of the binding and fluorescence quenching constants values for particular quenchers decreases in a series: uric acid > guanine > caffeine > theophylline > theobromine > xanthine. In all the systems studied there are characters of static quenching, as a consequence of the π-π-stacked non-covalent and non-fluorescent complexes formation between porphyrins and interacting compounds, accompanied simultaneously by the additional specific binding interactions. The porphyrin fluorescence quenching can be explain by the photoinduced intermolecular electron transfer from aromatic compound to the center of the porphyrin molecule, playing the role of the binding site. Presented results can be valuable for designing of new fluorescent porphyrin chemosensors or monitoring of drug traces in aqueous solutions. The obtained outcomes have also the toxicological and medical importance, providing insight into the interactions of the water-soluble porphyrins with biologically active substances.

A comparative study on the binding of single and double chain surfactant-cobalt(III) complexes with bovine serum albumin

NASA Astrophysics Data System (ADS)

Vignesh, G.; Sugumar, K.; Arunachalam, S.; Vignesh, S.; Arthur James, R.

2013-09-01

The comparative binding effect of single and double aliphatic chain containing surfactant-cobalt(III) complexes cis-[Co(bpy)2(DA)2](ClO4)3ṡ2H2O (1), cis-[Co(bpy)2(DA)Cl](ClO4)2ṡ2H2O (2), cis-[Co(phen)2(CA)2](ClO4)3ṡ2H2O (3), and cis-[Co(phen)2(CA)Cl](ClO4)2ṡ2H2O (4) with bovine serum albumin (BSA) under physiological condition was analyzed by steady state, time resolved fluorescence, synchronous, three-dimensional fluorescence, UV-Visible absorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of BSA through a static mechanism. The binding constants (Kb) and the number of binding sites were calculated and binding constant values are found in the range of 104-105 M-1. The results indicate that compared to single chain complex, double chain surfactant-cobalt(III) complex interacts strongly with BSA. Also the sign of thermodynamic parameters (ΔG°, ΔH°, and ΔS°) indicate that all the complexes interact with BSA through hydrophobic force. The binding distance (r) between complexes and BSA was calculated using Förster non-radiation energy transfer theory and found to be less than 7 nm. The results of synchronous, three dimensional fluorescence and circular dichroism spectroscopic methods indicate that the double chain surfactant-cobalt(III) complexes changed the conformation of the protein considerably than the respective single chain surfactant-cobalt(III) complexes. Antimicrobial studies of the complexes showed good activities against pathogenic microorganisms.

NEUTRALIZATION OF THE ASPARTIC ACID RESIDUE D367, BUT NOT D454, INHIBITS BINDING OF NA+ TO THE GLUTAMATE-FREE FORM AND CYCLING OF THE GLUTAMATE TRANSPORTER EAAC1

PubMed Central

Tao, Zhen; Zhang, Zhou; Grewer, Christof

2008-01-01

Substrate transport by the plasma membrane glutamate transporter EAAC1 is coupled to cotransport of three sodium ions. One of these Na+ ions binds to the transporter already in the absence of glutamate. Here, we have investigated the possible involvement of two conserved aspartic acid residues in transmembrane segments 7 and 8 of EAAC1, D367 and D454, in Na+ cotransport. In order to test the effect of charge neutralization mutations in these positions on Na+ binding to the glutamate-free transporter, we recorded the Na+-induced anion leak current to determine the Km of EAAC1 for Na+. For EAAC1WT, this Km was determined as 120 mM. When the negative charge of D367 was neutralized by mutagenesis to asparagine, Na+ activated the anion leak current with a Km of about 2 M, indicating dramatically impaired Na+ binding to the mutant transporter. In contrast, the Na+ affinity of EAAC1D454N was virtually unchanged compared to the wild type transporter (Km = 90 mM). The reduced occupancy of the Na+ binding site of EAAC1D367N resulted in a dramatic reduction in glutamate affinity (Km = 3.6 mM, 140 mM [Na+]), which could be partially overcome by increasing extracellular [Na+]. In addition to impairing Na+ binding, the D367N mutation slowed glutamate transport, as shown by pre-steady-state kinetic analysis of transport currents, by strongly decreasing the rate of a reaction step associated with glutamate translocation. Our data are consistent with a model in which D367, but not D454 is involved in coordinating the bound Na+ in the glutamate-free transporter form. PMID:16478724

Mefloquine inhibits voltage dependent Nav1.4 channel by overlapping the local anaesthetic binding site.

PubMed

Paiz-Candia, Bertin; Islas, Angel A; Sánchez-Solano, Alfredo; Mancilla-Simbro, Claudia; Scior, Thomas; Millan-PerezPeña, Lourdes; Salinas-Stefanon, Eduardo M

2017-02-05

Mefloquine constitutes a multitarget antimalaric that inhibits cation currents. However, the effect and the binding site of this compound on Na + channels is unknown. To address the mechanism of action of mefloquine, we employed two-electrode voltage clamp recordings on Xenopus laevis oocytes, site-directed mutagenesis of the rat Na + channel, and a combined in silico approach using Molecular Dynamics and docking protocols. We found that mefloquine: i) inhibited Na v 1.4 currents (IC 50 =60μM), ii) significantly delayed fast inactivation but did not affect recovery from inactivation, iii) markedly the shifted steady-state inactivation curve to more hyperpolarized potentials. The presence of the β1 subunit significantly reduced mefloquine potency, but the drug induced a significant frequency-independent rundown upon repetitive depolarisations. Computational and experimental results indicate that mefloquine overlaps the local anaesthetic binding site by docking at a hydrophobic cavity between domains DIII and DIV that communicates the local anaesthetic binding site with the selectivity filter. This is supported by the fact that mefloquine potency significantly decreased on mutant Na v 1.4 channel F1579A and significantly increased on K1237S channels. In silico this compound docked above F1579 forming stable π-π interactions with this residue. We provide structure-activity insights into how cationic amphiphilic compounds may exert inhibitory effects by docking between the local anaesthetic binding site and the selectivity filter of a mammalian Na + channel. Our proposed synergistic cycle of experimental and computational studies may be useful for elucidating binding sites of other drugs, thereby saving in vitro and in silico resources. Copyright © 2016 Elsevier B.V. All rights reserved.

The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch.

PubMed

Oberst, Andrew; Malatesta, Martina; Aqeilan, Rami I; Rossi, Mario; Salomoni, Paolo; Murillas, Rodolfo; Sharma, Prashant; Kuehn, Michael R; Oren, Moshe; Croce, Carlo M; Bernassola, Francesca; Melino, Gerry

2007-07-03

Nedd4-binding partner-1 (N4BP1) has been identified as a protein interactor and a substrate of the homologous to E6AP C terminus (HECT) domain-containing E3 ubiquitin-protein ligase (E3), Nedd4. Here, we describe a previously unrecognized functional interaction between N4BP1 and Itch, a Nedd4 structurally related E3, which contains four WW domains, conferring substrate-binding activity. We show that N4BP1 association with the second WW domain (WW2) of Itch interferes with E3 binding to its substrates. In particular, we found that N4BP1 and p73 alpha, a target of Itch-mediated ubiquitin/proteasome proteolysis, share the same binding site. By competing with p73 alpha for binding to the WW2 domain, N4BP1 reduces the ability of Itch to recruit and ubiquitylate p73 alpha and inhibits Itch autoubiquitylation activity both in in vitro and in vivo ubiquitylation assays. Similarly, both c-Jun and p63 polyubiquitylation by Itch are inhibited by N4BP1. As a consequence, genetic and RNAi knockdown of N4BP1 diminish the steady-state protein levels and significantly impair the transcriptional activity of Itch substrates. Notably, stress-induced induction of c-Jun was impaired in N4BP1(-/-) cells. These results demonstrate that N4BP1 functions as a negative regulator of Itch. In addition, because inhibition of Itch by N4BP1 results in the stabilization of crucial cell death regulators such as p73 alpha and c-Jun, it is conceivable that N4BP1 may have a role in regulating tumor progression and the response of cancer cells to chemotherapy.

Changes in complementarity-determining regions significantly alter IgG binding to the neonatal Fc receptor (FcRn) and pharmacokinetics

PubMed Central

King, Amy C.; Kavosi, Mania; Wang, Mengmeng; O'Hara, Denise M.; Tchistiakova, Lioudmila; Katragadda, Madan

2018-01-01

ABSTRACT A large body of data exists demonstrating that neonatal Fc receptor (FcRn) binding of an IgG via its Fc CH2-CH3 interface trends with the pharmacokinetics (PK) of IgG. We have observed that PK of IgG molecules vary widely, even when they share identical Fc domains. This led us to hypothesize that domains distal from the Fc could contribute to FcRn binding and affect PK. In this study, we explored the role of these IgG domains in altering the affinity between IgG and FcRn. Using a surface plasmon resonance-based assay developed to examine the steady-state binding affinity (KD) of IgG molecules to FcRn, we dissected the contributions of IgG domains in modulating the affinity between FcRn and IgG. Through analysis of a broad collection of therapeutic antibodies containing more than 50 unique IgG molecules, we demonstrated that variable domains, and in particular complementarity-determining regions (CDRs), significantly alter binding affinity to FcRn in vitro. Furthermore, a panel of IgG molecules differing only by 1–5 mutations in CDRs altered binding affinity to FcRn in vitro, by up to 79-fold, and the affinity values correlated with calculated isoelectric point values of both variable domains and CDR-L3. In addition, tighter affinity values trend with faster in vivo clearance of a set of IgG molecules differing only by 1–3 mutations in human FcRn transgenic mice. Understanding the role of CDRs in modulation of IgG affinity to FcRn in vitro and their effect on PK of IgG may have far-reaching implications in the optimization of IgG therapeutics. PMID:28991504

Revelations of X-ray spectral analysis of the enigmatic black hole binary GRS 1915+105

NASA Astrophysics Data System (ADS)

Peris, Charith; Remillard, Ronald A.; Steiner, James; Vrtilek, Saeqa Dil; Varniere, Peggy; Rodriguez, Jerome; Pooley, Guy

2016-01-01

Of the black hole binaries discovered thus far, GRS 1915+105 stands out as an exceptional source primarily due to its wild X-ray variability, the diversity of which has not been replicated in any other stellar-mass black hole. Although extreme variability is commonplace in its light-curve, about half of the observations of GRS1915+105 show fairly steady X-ray intensity. We report on the X-ray spectral behavior within these steady observations. Our work is based on a vast RXTE/PCA data set obtained on GRS 1915+105 during the course of its entire mission and 10 years of radio data from the Ryle Telescope, which overlap the X-ray data. We find that the steady observations within the X-ray data set naturally separate into two regions in a color-color diagram, which we refer to as steady-soft and steady-hard. GRS 1915+105 displays significant curvature in the Comptonization component within the PCA band pass suggesting significantly heating from a hot disk present in all states. A new Comptonization model 'simplcut' was developed in order to model this curvature to best effect. A majority of the steady-soft observations display a roughly constant inner radius; remarkably reminiscent of canonical soft state black hole binaries. In contrast, the steady-hard observations display a growing disk truncation that is correlated to the mass accretion rate through the disk, which suggests a magnetically truncated disk. A comparison of X-ray model parameters to the canonical state definitions show that almost all steady-soft observations match the criteria of either thermal or steep power law state, while the thermal state observations dominate the constant radius branch. A large portion (80%) of the steady-hard observations matches the hard state criteria when the disk fraction constraint is neglected. These results suggest that within the complexity of this source is a simpler underlying basis of states, which map to those observed in canonical black hole binaries. When represented in a color-color diagram, state assignments appear to map to ``A, B and C'' (Belloni et al. 2000) regions that govern fast variability cycles in GRS 1915+105 demonstrating a compelling link between short and long time scales in its phenomenology.

Regulation of atrial natriuretic peptide clearance receptors in mesangial cells by growth factors.

PubMed

Paul, R V; Wackym, P S; Budisavljevic, M; Everett, E; Norris, J S

1993-08-25

Rat mesangial cells can express both 130-kDa guanylyl cyclase-coupled and 66-kDa non-coupled atrial natriuretic peptide (ANP) receptors (ANPR-A and ANPR-C, respectively). Exposure of mesangial cells, grown in 20% fetal calf serum, to 0.1% serum for 24 h increased total ANP receptor density more than 2-fold (Bmax = 87 versus 37 fmol/mg of cell protein) without changing binding affinity (Kd = 94 versus 88 pM). Radioligand binding and cross-linking studies demonstrated that up-regulation of ANP binding after serum deprivation was entirely due to an increase in ANPR-C, with little or no change in ANPR-A. Inhibition of protein synthesis with cycloheximide blocked up-regulation after serum deprivation. Steady-state ANPR-C mRNA level was increased 15-fold by serum deprivation, as judged by Northern blotting. There was no change in ANPR-A mRNA. Platelet-derived growth factor and phorbol myristate acetate, when added to low serum medium, blocked or reversed the effect of serum deprivation on ANPR-C. We conclude that synthesis and expression of ANPR-C but not ANPR-A is suppressed by serum, platelet-derived growth factor, and phorbol myristate acetate. Suppression of ANPR-C in vivo could contribute to mesangial cell proliferative responses to growth factors.

Spectroscopic and thermodynamic insights into the interaction between proflavine and human telomeric G-quadruplex DNA.

PubMed

Kumar, Vivek; Sengupta, Abhigyan; Gavvala, Krishna; Koninti, Raj Kumar; Hazra, Partha

2014-09-25

The G-quadruplex (GQ-DNA), an alternative structure motif of DNA, has emerged as a novel and exciting target for anticancer drug discovery. GQ-DNA formed in the presence of monovalent cations (Na(+)/K(+)) by human telomeric DNA is a point of interest due to their direct relevance for cellular aging and abnormal cell growths. Small molecules that selectively target and stabilize G-quadruplex structures are considered to be potential therapeutic anticancer agents. Herein, we probe G-quadruplex and proflavine (a well-known DNA intercalator, hence acting as an anticarcinogen) association through steady state and time-resolved fluorescence spectroscopy to explore the effect of stabilization of GQ-DNA by this well-known DNA intercalator. The structural modifications of G-quadruplex upon binding are highlighted through circular dichroism (CD) spectra. Moreover, a detailed insight into the thermodynamics of this interaction has been provided though isothermal titration calorimetry (ITC) studies. The thermodynamic parameters obtained from ITC help to gain knowledge about the nature as well as the driving forces of binding. This present study shows that proflavine (PF) can act as a stabilizer of telomeric GQ-DNA through an entropically as well as enthalpically feasible process with high binding affinity and thereby can be considered as a potential telomerase inhibitor.

ESTIMATING SYSTEMIC EXPOSURE TO ETHINYL ESTRADIOL FROM AN ORAL CONTRACEPTIVE

PubMed Central

WESTHOFF, Carolyn L.; PIKE, Malcolm C.; TANG, Rosalind; DINAPOLI, Marianne N.; SULL, Monica; CREMERS, Serge

2015-01-01

Objectives This study was conducted to compare single-dose pharmacokinetics of ethinyl estradiol in an oral contraceptive to steady-state values, and to assess whether any simpler measures could provide an adequate proxy of the ‘gold standard’ 24-hour steady-state area-under-the-curve. Identifying a simple, less expensive, measure of systemic ethinyl estradiol exposure would be useful for larger studies designed to assess the relationship between an individual’s ethinyl estradiol exposure and her side effects. Study Design We conducted a 13 samples over 24 hours pharmacokinetic analysis on day 1 and day 21 of the first cycle of a monophasic oral contraceptive containing 30 mcg ethinyl estradiol and 150 mcg levonorgestrel in 17 non-obese healthy white women. We also conducted an abbreviated single dose 9-sample pharmacokinetic analysis after a month washout. Ethinyl estradiol was measured by liquid chromatography-tandem mass spectrometry. We compared results of full 13-sample steady-state pharmacokinetic analysis with results calculated using fewer samples (9 or 5) and following the single doses. We calculated Pearson correlation coefficients to evaluate the relationships between these estimates of systemic ethinyl estradiol exposure. Results The area-under-the-curve, maximum (Cmax), and 24-hour (C24) values were similar following the two single oral contraceptive doses (area-under-the-curve, r = 0.92). The steady-state 13-sample 24-hour area-under-the-curve was highly correlated with the average 9-sample area-under-the-curve after the two single doses (r = 0.81, p = 0.0002). This correlation remained the same if the number of samples was reduced to 4, taken at time 1, 2.5, 4 and 24 hours. The C24 at steady-state was highly correlated with the 24-hour steady-state area-under-the-curve (r = 0.92, p < 0.0001). The average of the C24 values following the two single doses was also quite highly correlated with the steady-state area-under-the-curve (r = 0.72, p = 0.0026). Conclusions Limited blood sampling, including results from two single doses, gave highly correlated estimates of an oral contraceptive user’s steady-state ethinyl estradiol exposure. PMID:25511238

Estimating systemic exposure to ethinyl estradiol from an oral contraceptive.

PubMed

Westhoff, Carolyn L; Pike, Malcolm C; Tang, Rosalind; DiNapoli, Marianne N; Sull, Monica; Cremers, Serge

2015-05-01

This study was conducted to compare single-dose pharmacokinetics of ethinyl estradiol in an oral contraceptive with steady-state values and to assess whether any simpler measures could provide an adequate proxy of the "gold standard" 24-hour steady-state area under the curve (AUC) value. Identification of a simple, less expensive measure of systemic ethinyl estradiol exposure would be useful for larger studies that are designed to assess the relationship between an individual's ethinyl estradiol exposure and side-effects. We collected 13 samples over 24 hours for pharmacokinetic analysis on days 1 and 21 of the first cycle of a monophasic oral contraceptive that contained 30 μg ethinyl estradiol and 150 μg levonorgestrel in 17 nonobese healthy white women. We also conducted an abbreviated single-dose 9-sample pharmacokinetic analysis after a month washout. Ethinyl estradiol was measured by liquid chromatography-tandem mass spectrometry. We compared results of a full 13-sample steady-state pharmacokinetic analysis with results that had been calculated with the use of fewer samples (9 or 5) and after the single doses. We calculated Pearson correlation coefficients to evaluate the relationships between these estimates of systemic ethinyl estradiol exposure. The AUC, maximum, and 24-hour values were similar after the 2 single oral contraceptive doses (AUC; r=0.92). The steady-state 13-sample 24-hour AUC value was correlated highly with the average 9-sample AUC value after the 2 single doses (r=0.81; P=.0002). This correlation remained the same if the number of single-dose samples was reduced to 4, taken at time 1, 2.5, 4, and 24 hours. The 24-hour value at steady-state was correlated highly with the 24-hour steady-state AUC value (r=0.92; P<.0001). The average of the 24-hour values after the 2 single doses was also correlated quite highly with the steady-state AUC value (r=0.72; P=.0026). Limited blood sampling, including results from 2 single doses, gave highly correlated estimates of an oral contraceptive user's steady-state ethinyl estradiol exposure. Copyright © 2015 Elsevier Inc. All rights reserved.

Comparative effectiveness of Calabadion and sugammadex to reverse non-depolarizing neuromuscular blocking agents

PubMed Central

Haerter, Friederike; Simons, Jeroen Cedric Peter; Foerster, Urs; Duarte, Ingrid Moreno; Diaz-Gil, Daniel; Ganapati, Shweta; Eikermann-Haerter, Katharina; Ayata, Cenk; Zhang, Ben; Blobner, Manfred; Isaacs, Lyle; Eikermann, Matthias

2015-01-01

Background We evaluated the comparative effectiveness of calabadion 2 to reverse non-depolarizing neuromuscular blocking agents (NMBAs) by binding and inactivation. Methods The dose-response relationship of drugs to reverse vecuronium, rocuronium, and cisatracurium-induced neuromuscular block (NMB) was evaluated in vitro (competition binding assays and urine analysis), ex vivo (n=34; phrenic nerve hemidiaphragm preparation) and in vivo (n=108; quadriceps femoris muscle of the rat). Cumulative dose-response curves of calabadions, neostigmine, or sugammadex were created ex vivo at steady-state deep NMB. In living rats, we studied the dose-response relationship of the test drugs to reverse deep block under physiological conditions and we measured the amount of calabadion 2 excreted in the urine. Results In vitro experiments showed that calabadion 2 binds rocuronium with 89 times the affinity of sugammadex (Ka = 3.4 × 109 M−1 and Ka = 3.8 × 107 M−1). Urine analysis (proton nuclear magnetic resonance), competition binding assays and ex vivo study results obtained in the absence of metabolic deactivation are in accordance with an 1:1 binding ratio of sugammadex and calabadion 2 toward rocuronium. In living rats, calabadion 2 dose-dependently and rapidly reversed all NMBAs tested. The molar potency of calabadion 2 to reverse vecuronium and rocuronium was higher compared to sugammadex. Calabadion 2 was eliminated renally, and did not affect blood pressure or heart rate. Conclusion Calabadion 2 reverses NMB-induced by benzylisoquinolines and steroidal NMBAs in rats more effectively, i.e. faster, than sugammadex. Calabadion 2 is eliminated in the urine and well tolerated in rats. PMID:26418697

Binding of basic amphipathic peptides to neutral phospholipid membranes: a thermodynamic study applied to dansyl-labeled melittin and substance P analogues.

PubMed

Pérez-Payá, E; Porcar, I; Gómez, C M; Pedrós, J; Campos, A; Abad, C

1997-08-01

A thermodynamic approach is proposed to quantitatively analyze the binding isotherms of peptides to model membranes as a function of one adjustable parameter, the actual peptide charge in solution z(p)+. The main features of this approach are a theoretical expression for the partition coefficient calculated from the molar free energies of the peptide in the aqueous and lipid phases, an equation proposed by S. Stankowski [(1991) Biophysical Journal, Vol. 60, p. 341] to evaluate the activity coefficient of the peptide in the lipid phase, and the Debye-Hückel equation that quantifies the activity coefficient of the peptide in the aqueous phase. To assess the validity of this approach we have studied, by means of steady-state fluorescence spectroscopy, the interaction of basic amphipathic peptides such as melittin and its dansylcadaverine analogue (DNC-melittin), as well as a new fluorescent analogue of substance P, SP (DNC-SP) with neutral phospholipid membranes. A consistent quantitative analysis of each binding curve was achieved. The z(p)+ values obtained were always found to be lower than the physical charge of the peptide. These z(p)+ values can be rationalized by considering that the peptide charged groups are strongly associated with counterions in buffer solution at a given ionic strength. The partition coefficients theoretically derived using the z(p)+ values were in agreement with those deduced from the Gouy-Chapman formalism. Ultimately, from the z(p)+ values the molar free energies for the free and lipid-bound states of the peptides have been calculated.

pH-Assisted control over the binding and relocation of an acridine guest between a macrocyclic nanocarrier and natural DNA.

PubMed

Sayed, Mhejabeen; Pal, Haridas

2015-04-14

The differential binding affinity of the hydroxypropyl-β-cyclodextrin (HPβCD) macrocycle, a drug delivery vehicle, towards the protonated and deprotonated forms of the well-known DNA binder and model anticancer drug acridine has been exploited as a strategy for dye-drug transportation and pH-responsive delivery to a natural DNA target. From pH-sensitive changes in the ground state absorption and steady-state fluorescence characteristics of the studied acridine dye-HPβCD-DNA ternary system and strongly supported by fluorescence lifetime, fluorescence anisotropy, Job's plots, (1)H NMR and circular dichroism results, it is revealed that in a moderately alkaline solution (pH ∼ 8.5), the dye can be predominantly bound to the HPβCD macrocycle and when the pH is lowered to a moderately acidic region (pH ∼ 4), the dye efficiently detaches from the HPβCD cavity and almost exclusively binds to DNA. In the present study we are thus able to construct a pH-sensitive supramolecular assembly where pH acts as a simple stimulus for controlled uptake and targeted release of the dye-drug. As pH is an essential and sensitive factor in various biological processes, a simple yet reliable pH-sensitive model such as is demonstrated here can have promising applications in the host-assisted delivery of prodrug to the target sites, such as cancer or tumour microenvironments, with an enhanced stability, bioavailability and activity, and also in the design of new fluorescent probes, sensors and smart materials for applications in nano-science.

Analysis of flavin oxidation and electron-transfer inhibition in Plasmodium falciparum dihydroorotate dehydrogenase.

PubMed

Malmquist, Nicholas A; Gujjar, Ramesh; Rathod, Pradipsinh K; Phillips, Margaret A

2008-02-26

Plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) is a flavin-dependent mitochondrial enzyme that provides the only route to pyrimidine biosynthesis in the parasite. Clinically significant inhibitors of human DHODH (e.g., A77 1726) bind to a pocket on the opposite face of the flavin cofactor from dihydroorotate (DHO). This pocket demonstrates considerable sequence variability, which has allowed species-specific inhibitors of the malarial enzyme to be identified. Ubiquinone (CoQ), the physiological oxidant in the reaction, has been postulated to bind this site despite a lack of structural evidence. To more clearly define the residues involved in CoQ binding and catalysis, we undertook site-directed mutagenesis of seven residues in the structurally defined A77 1726 binding site, which we term the species-selective inhibitor site. Mutation of several of these residues (H185, F188, and F227) to Ala substantially decreased the affinity of pfDHODH-specific inhibitors (40-240-fold). In contrast, only a modest increase in the Kmapp for CoQ was observed, although mutation of Y528 in particular caused a substantial reduction in kcat (40-100-fold decrease). Pre-steady-state kinetic analysis by single wavelength stopped-flow spectroscopy showed that the mutations had no effect on the rate of the DHO-dependent reductive half-reaction, but most reduced the rate of the CoQ-dependent flavin oxidation step (3-20-fold decrease), while not significantly altering the Kdox for CoQ. As with the mutants, inhibitors that bind this site block the CoQ-dependent oxidative half-reaction without affecting the DHO-dependent step. These results identify residues involved in inhibitor binding and electron transfer to CoQ. Importantly, the data provide compelling evidence that the binding sites for CoQ and species-selective site inhibitors do not overlap, and they suggest instead that inhibitors act either by blocking the electron path between flavin and CoQ or by stabilizing a conformation that excludes CoQ binding.

Propulsion System Simulation Using the Toolbox for the Modeling and Analysis of Thermodynamic System T-MATS

NASA Technical Reports Server (NTRS)

Chapman, Jeffryes W.; Lavelle, Thomas M.; May, Ryan D.; Litt, Jonathan S.; Guo, Ten-Huei

2014-01-01

A simulation toolbox has been developed for the creation of both steady-state and dynamic thermodynamic software models. This paper describes the Toolbox for the Modeling and Analysis of Thermodynamic Systems (T-MATS), which combines generic thermodynamic and controls modeling libraries with a numerical iterative solver to create a framework for the development of thermodynamic system simulations, such as gas turbine engines. The objective of this paper is to present an overview of T-MATS, the theory used in the creation of the module sets, and a possible propulsion simulation architecture. A model comparison was conducted by matching steady-state performance results from a T-MATS developed gas turbine simulation to a well-documented steady-state simulation. Transient modeling capabilities are then demonstrated when the steady-state T-MATS model is updated to run dynamically.

Propulsion System Simulation Using the Toolbox for the Modeling and Analysis of Thermodynamic Systems (T-MATS)

NASA Technical Reports Server (NTRS)

Chapman, Jeffryes W.; Lavelle, Thomas M.; May, Ryan D.; Litt, Jonathan S.; Guo, Ten-Huei

2014-01-01

A simulation toolbox has been developed for the creation of both steady-state and dynamic thermodynamic software models. This paper describes the Toolbox for the Modeling and Analysis of Thermodynamic Systems (T-MATS), which combines generic thermodynamic and controls modeling libraries with a numerical iterative solver to create a framework for the development of thermodynamic system simulations, such as gas turbine engines. The objective of this paper is to present an overview of T-MATS, the theory used in the creation of the module sets, and a possible propulsion simulation architecture. A model comparison was conducted by matching steady-state performance results from a T-MATS developed gas turbine simulation to a well-documented steady-state simulation. Transient modeling capabilities are then demonstrated when the steady-state T-MATS model is updated to run dynamically.

Coherent quantum dynamics in steady-state manifolds of strongly dissipative systems.

PubMed

Zanardi, Paolo; Campos Venuti, Lorenzo

2014-12-12

Recently, it has been realized that dissipative processes can be harnessed and exploited to the end of coherent quantum control and information processing. In this spirit, we consider strongly dissipative quantum systems admitting a nontrivial manifold of steady states. We show how one can enact adiabatic coherent unitary manipulations, e.g., quantum logical gates, inside this steady-state manifold by adding a weak, time-rescaled, Hamiltonian term into the system's Liouvillian. The effective long-time dynamics is governed by a projected Hamiltonian which results from the interplay between the weak unitary control and the fast relaxation process. The leakage outside the steady-state manifold entailed by the Hamiltonian term is suppressed by an environment-induced symmetrization of the dynamics. We present applications to quantum-computation in decoherence-free subspaces and noiseless subsystems and numerical analysis of nonadiabatic errors.

Fluctuations, Stratification and Stability in a Liquid Fluidized Bed at Low Reynolds Number

NASA Technical Reports Server (NTRS)

Segre, P. N.; McClymer, J. P.

2004-01-01

The sedimentation dynamics of extremely low polydispersity, non-colloidal, particles are studied in a liquid fluidized bed at low Reynolds number, Re much less than 1. When fluidized, the system reaches a steady state, defined where the local average volume fraction does not vary in time. In steady state, the velocity fluctuations and the particle concentrations are found to strongly depend on height. Using our results, we test a recently developed stability model for steady state sedimentation. The model describes the data well, and shows that in steady state there is a balancing of particle fluxes due to the fluctuations and the concentration gradient. Some results are also presented for the dependence of the concentration gradient in fluidized beds on particle size; the gradients become smaller as the particles become larger and fewer in number.

Acetylcholine-activated ionic currents in parasympathetic neurons of bullfrog heart.

PubMed

Tateishi, N; Kim, D K; Akaike, N

1990-05-01

1. The electrical and pharmacologic properties of acetylcholine (ACh)-induced current (IACh) were studied in the parasympathetic neurons isolated from bullfrog heart with the use of the concentration-clamp technique, which allows intracellular perfusion and rapid change of external solution within 2 ms under the single-electrode voltage-clamp condition. 2. The IACh consisted of an initial transient peak component and a successive steady-state plateau component. Both currents increased in a sigmoidal fashion with increasing ACh concentration. The dissociation constant (Kd value) and the Hill coefficient for each component were 2.2 X 10(-5) M and 1.6, respectively. 3. In the K(+)-free solution, the reversal potential (EACh) of IACh was close to the Na+ equilibrium potential (ENa). The current-voltage (I-V) relation showed inward rectification at positive potentials. 4. Nicotine mimicked only the peak component of IACh. However both peak and steady-state components were blocked nonselectively by the nicotinic blockers d-tubocurarine and hexamethonium. 5. Carbamylcholine (CCh) mimicked the steady-state component of IACh. The steady-state component was selectively inhibited by atropine at concentrations 1,000 times lower than that required for inhibition of the peak component. The steady state was blocked equally by either pirenzepine (M1 blocker) or AF-DX-116 (M2 blocker). 6. It was concluded that the IACh consisted of a peak component having double exponential activation and inactivation, mediated through the nicotinic actions, and a steady-state component having no inactivation, mediated through the muscarinic action.

Auditory steady-state response in cochlear implant patients.

PubMed

Torres-Fortuny, Alejandro; Arnaiz-Marquez, Isabel; Hernández-Pérez, Heivet; Eimil-Suárez, Eduardo

2018-03-19

Auditory steady state responses to continuous amplitude modulated tones at rates between 70 and 110Hz, have been proposed as a feasible alternative to objective frequency specific audiometry in cochlear implant subjects. The aim of the present study is to obtain physiological thresholds by means of auditory steady-state response in cochlear implant patients (Clarion HiRes 90K), with acoustic stimulation, on free field conditions and to verify its biological origin. 11 subjects comprised the sample. Four amplitude modulated tones of 500, 1000, 2000 and 4000Hz were used as stimuli, using the multiple frequency technique. The recording of auditory steady-state response was also recorded at 0dB HL of intensity, non-specific stimulus and using a masking technique. The study enabled the electrophysiological thresholds to be obtained for each subject of the explored sample. There were no auditory steady-state responses at either 0dB or non-specific stimulus recordings. It was possible to obtain the masking thresholds. A difference was identified between behavioral and electrophysiological thresholds of -6±16, -2±13, 0±22 and -8±18dB at frequencies of 500, 1000, 2000 and 4000Hz respectively. The auditory steady state response seems to be a suitable technique to evaluate the hearing threshold in cochlear implant subjects. Copyright © 2018 Sociedad Española de Otorrinolaringología y Cirugía de Cabeza y Cuello. Publicado por Elsevier España, S.L.U. All rights reserved.

Single-dose and steady-state pharmacokinetics of tenofovir disoproxil fumarate in human immunodeficiency virus-infected children.

PubMed

Hazra, Rohan; Balis, Frank M; Tullio, Antonella N; DeCarlo, Ellen; Worrell, Carol J; Steinberg, Seth M; Flaherty, John F; Yale, Kitty; Poblenz, Marianne; Kearney, Brian P; Zhong, Lijie; Coakley, Dion F; Blanche, Stephane; Bresson, Jean Louis; Zuckerman, Judith A; Zeichner, Steven L

2004-01-01

Tenofovir disoproxil fumarate (DF) is a potent nucleotide analog reverse transcriptase inhibitor approved for the treatment of human immunodeficiency virus (HIV)-infected adults. The single-dose and steady-state pharmacokinetics of tenofovir were evaluated following administration of tenofovir DF in treatment-experienced HIV-infected children requiring a change in antiretroviral therapy. Using increments of tenofovir DF 75-mg tablets, the target dose was 175 mg/m(2); the median administered dose was 208 mg/m(2). Single-dose pharmacokinetics were evaluated in 18 subjects, and the geometric mean area under the concentration-time curve from 0 h to infinity (AUC(0- infinity )) was 2,150 ng. h/ml and the geometric mean maximum concentration (C(max)) was 266 ng/ml. Subsequently, other antiretrovirals were added to each patient's regimen based upon treatment history and baseline viral resistance results. Steady-state pharmacokinetics were evaluated in 16 subjects at week 4. The steady-state, geometric mean AUC for the 24-h dosing interval was 2,920 ng. h/ml and was significantly higher than the AUC(0- infinity ) after the first dose (P = 0.0004). The geometric mean C(max) at steady state was 302 ng/ml. Tenofovir DF was generally very well tolerated. Steady-state tenofovir exposures in children receiving tenofovir DF-containing combination antiretroviral therapy approached values seen in HIV-infected adults (AUC, approximately 3,000 ng. h/ml; C(max), approximately 300 ng/ml) treated with tenofovir DF at 300 mg.

Ultrafiltration technology with a ceramic membrane for reactive dye removal: optimization of membrane performance.

PubMed

Alventosa-deLara, E; Barredo-Damas, S; Alcaina-Miranda, M I; Iborra-Clar, M I

2012-03-30

An ultrafiltration (UF) ceramic membrane was used to decolorize Reactive Black 5 (RB5) solutions at different dye concentrations (50 and 500 mg/L). Transmembrane pressure (TMP) and cross-flow velocity (CFV) were modified to study their influence on initial and steady-state permeate flux (J(p)) and dye rejection (R). Generally, J(p) increased with higher TMP and CFV and lower feed concentration, up to a maximum steady-state J(p) of 266.81 L/(m(2)h), obtained at 3 bar, 3m/s and 50mg/L. However, there was a TMP value (which changed depending on operating CFV and concentration) beyond which slight or no further increase in steady-state J(p) was observed. Similarly, the higher the CFV was, the more slightly the steady-state J(p) increased. Furthermore, the effectiveness of ultrafiltration treatment was evaluated through dye rejection coefficient. The results showed significant dye removals, regardless of the tested conditions, with steady-state R higher than 79.8% for the 50mg/L runs and around 73.2% for the 500 mg/L runs. Finally response surface methodology (RSM) was used to optimize membrane performance. At 50mg/L, a TMP of 4 bar and a CFV of 2.53 m/s were found to be the conditions giving the highest steady-state J(p), 255.86 L/(m(2)h), and the highest R, 95.2% simultaneously. Copyright © 2012 Elsevier B.V. All rights reserved.

The incorporation of hydrophobic protein receptors and artificial lipid membranes.

PubMed

Reader, T A; Fiszer de Plazas, S; Salas, P J; de Robertis, E

1976-01-01

The mechanism of chemical synaptic transmission implies: 1) the existence of a specific protein receptor at the postsynaptic membrane, and 2) the interaction between the transmitter released and the receptor, thus producing a change in ionic permeability. Previous studies from our laboratory have shown that special hydrophobic proteins extracted from postsynpatic membranes of different tissues showed a high affinity binding for the different pharmacological agents. The present paper describes experiments in which different hydrophobic protein binding acetylcholine, noradrenaline, gamma-aminobutyric acid, and glutamate were incorporated into artificial lipid membranes, similar to those first described by Mueller et al. (19). The effect of the different pharmacological agents was tested under experimental conditions of voltage clamp and the d.c. current changes measured. The results were then compared for the different lipid-protein membranes employed during the steady state and during transient conductance changes. The specificity of the responses indicate that artificial lipid membranes containing these hydrophobic proteins from electroplax, myocardium, spleen capsule and shrimp muscle can be used as a model to study pharmacologic receptors.

A critical role for the transcription factor Scl in platelet production during stress thrombopoiesis.

PubMed

McCormack, Matthew P; Hall, Mark A; Schoenwaelder, Simone M; Zhao, Quan; Ellis, Sarah; Prentice, Julia A; Clarke, Ashleigh J; Slater, Nicholas J; Salmon, Jessica M; Jackson, Shaun P; Jane, Stephen M; Curtis, David J

2006-10-01

The generation of platelets from megakaryocytes in the steady state is regulated by a variety of cytokines and transcription factors, including thrombopoietin (TPO), GATA-1, and NF-E2. Less is known about platelet production in the setting of stress thrombopoiesis, a pivotal event in the context of cytotoxic chemotherapy. Here we show in mice that the transcription factor Scl is critical for platelet production after chemotherapy and in thrombopoiesis induced by administration of TPO. Megakaryocytes from these mice showed appropriate increases in number and ploidy but failed to shed platelets. Ultrastructural examination of Scl-null megakaryocytes revealed a disorganized demarcation membrane and reduction in platelet granules. Quantitative real-time polymerase chain reaction showed that Scl-null platelets lacked NF-E2, and chromatin immunoprecipitation analysis demonstrated Scl binding to the NF-E2 promoter in the human megakaryoblastic-cell line Meg-01, along with its binding partners E47, Lmo2, and the cofactors Ldb1 and GATA-2. These findings suggest that Scl acts up-stream of NF-E2 expression to control megakaryocyte development and platelet release in settings of thrombopoietic stress.

Interactions of hemin with bovine serum albumin and human hemoglobin: A fluorescence quenching study

NASA Astrophysics Data System (ADS)

Makarska-Bialokoz, Magdalena

2018-03-01

The binding interactions between hemin (Hmi) and bovine serum albumin (BSA) or human hemoglobin (HHb), respectively, have been examined in aqueous solution at pH = 7.4, applying UV-vis absorption, as well as steady-state, synchronous and three-dimensional fluorescence spectra techniques. Representative results received for both BSA and HHb intrinsic fluorescence proceeding from the interactions with hemin suggest the formation of stacking non-covalent and non-fluorescent complexes in both the Hmi-BSA and Hmi-HHb systems, with highly possible concurrent formation of a coordinate bond between a group on the protein surface and the metal in Hmi molecule. All the values of calculated parameters, the binding, fluorescence quenching and bimolecular quenching rate constants point to the involvement of static quenching in both the systems studied. The blue shift in the synchronous fluorescence spectra imply the participation of both tryptophan and tyrosine residues in quenching of BSA and HHb intrinsic fluorescence. Depicted outcomes suggest that hemin is supposedly able to influence the physiological functions of BSA and HHb, the most important blood proteins, particularly in case of its overuse.

Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide.

PubMed

Hiner, Alexander N P; Sidrach, Lara; Chazarra, Soledad; Varón, Ramón; Tudela, José; García-Cánovas, Francisco; Rodríguez-López, José Neptuno

2004-01-01

The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented.

Basin stability measure of different steady states in coupled oscillators

NASA Astrophysics Data System (ADS)

Rakshit, Sarbendu; Bera, Bidesh K.; Majhi, Soumen; Hens, Chittaranjan; Ghosh, Dibakar

2017-04-01

In this report, we investigate the stabilization of saddle fixed points in coupled oscillators where individual oscillators exhibit the saddle fixed points. The coupled oscillators may have two structurally different types of suppressed states, namely amplitude death and oscillation death. The stabilization of saddle equilibrium point refers to the amplitude death state where oscillations are ceased and all the oscillators converge to the single stable steady state via inverse pitchfork bifurcation. Due to multistability features of oscillation death states, linear stability theory fails to analyze the stability of such states analytically, so we quantify all the states by basin stability measurement which is an universal nonlocal nonlinear concept and it interplays with the volume of basins of attractions. We also observe multi-clustered oscillation death states in a random network and measure them using basin stability framework. To explore such phenomena we choose a network of coupled Duffing-Holmes and Lorenz oscillators which are interacting through mean-field coupling. We investigate how basin stability for different steady states depends on mean-field density and coupling strength. We also analytically derive stability conditions for different steady states and confirm by rigorous bifurcation analysis.

An optimizing start-up strategy for a bio-methanator.

PubMed

Sbarciog, Mihaela; Loccufier, Mia; Vande Wouwer, Alain

2012-05-01

This paper presents an optimizing start-up strategy for a bio-methanator. The goal of the control strategy is to maximize the outflow rate of methane in anaerobic digestion processes, which can be described by a two-population model. The methodology relies on a thorough analysis of the system dynamics and involves the solution of two optimization problems: steady-state optimization for determining the optimal operating point and transient optimization. The latter is a classical optimal control problem, which can be solved using the maximum principle of Pontryagin. The proposed control law is of the bang-bang type. The process is driven from an initial state to a small neighborhood of the optimal steady state by switching the manipulated variable (dilution rate) from the minimum to the maximum value at a certain time instant. Then the dilution rate is set to the optimal value and the system settles down in the optimal steady state. This control law ensures the convergence of the system to the optimal steady state and substantially increases its stability region. The region of attraction of the steady state corresponding to maximum production of methane is considerably enlarged. In some cases, which are related to the possibility of selecting the minimum dilution rate below a certain level, the stability region of the optimal steady state equals the interior of the state space. Aside its efficiency, which is evaluated not only in terms of biogas production but also from the perspective of treatment of the organic load, the strategy is also characterized by simplicity, being thus appropriate for implementation in real-life systems. Another important advantage is its generality: this technique may be applied to any anaerobic digestion process, for which the acidogenesis and methanogenesis are, respectively, characterized by Monod and Haldane kinetics.

Pharmacokinetic Steady-States Highlight Interesting Target-Mediated Disposition Properties.

PubMed

Gabrielsson, Johan; Peletier, Lambertus A

2017-05-01

In this paper, we derive explicit expressions for the concentrations of ligand L, target R and ligand-target complex RL at steady state for the classical model describing target-mediated drug disposition, in the presence of a constant-rate infusion of ligand. We demonstrate that graphing the steady-state values of ligand, target and ligand-target complex, we obtain striking and often singular patterns, which yield a great deal of insight and understanding about the underlying processes. Deriving explicit expressions for the dependence of L, R and RL on the infusion rate, and displaying graphs of the relations between L, R and RL, we give qualitative and quantitive information for the experimentalist about the processes involved. Understanding target turnover is pivotal for optimising these processes when target-mediated drug disposition (TMDD) prevails. By a combination of mathematical analysis and simulations, we also show that the evolution of the three concentration profiles towards their respective steady-states can be quite complex, especially for lower infusion rates. We also show how parameter estimates obtained from iv bolus studies can be used to derive steady-state concentrations of ligand, target and complex. The latter may serve as a template for future experimental designs.

STEADY-STATE MODEL OF SOLAR WIND ELECTRONS REVISITED

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Peter H.; Kim, Sunjung; Choe, G. S., E-mail: yoonp@umd.edu

2015-10-20

In a recent paper, Kim et al. put forth a steady-state model for the solar wind electrons. The model assumed local equilibrium between the halo electrons, characterized by an intermediate energy range, and the whistler-range fluctuations. The basic wave–particle interaction is assumed to be the cyclotron resonance. Similarly, it was assumed that a dynamical steady state is established between the highly energetic superhalo electrons and high-frequency Langmuir fluctuations. Comparisons with the measured solar wind electron velocity distribution function (VDF) during quiet times were also made, and reasonable agreements were obtained. In such a model, however, only the steady-state solution for themore » Fokker–Planck type of electron particle kinetic equation was considered. The present paper complements the previous analysis by considering both the steady-state particle and wave kinetic equations. It is shown that the model halo and superhalo electron VDFs, as well as the assumed wave intensity spectra for the whistler and Langmuir fluctuations, approximately satisfy the quasi-linear wave kinetic equations in an approximate sense, thus further validating the local equilibrium model constructed in the paper by Kim et al.« less

Noncontrast-enhanced renal angiography using multiple inversion recovery and alternating TR balanced steady-state free precession.

PubMed

Dong, Hattie Z; Worters, Pauline W; Wu, Holden H; Ingle, R Reeve; Vasanawala, Shreyas S; Nishimura, Dwight G

2013-08-01

Noncontrast-enhanced renal angiography techniques based on balanced steady-state free precession avoid external contrast agents, take advantage of high inherent blood signal from the T 2 / T 1 contrast mechanism, and have short steady-state free precession acquisition times. However, background suppression is limited; inflow times are inflexible; labeling region is difficult to define when tagging arterial flow; and scan times are long. To overcome these limitations, we propose the use of multiple inversion recovery preparatory pulses combined with alternating pulse repetition time balanced steady-state free precession to produce renal angiograms. Multiple inversion recovery uses selective spatial saturation followed by four nonselective inversion recovery pulses to concurrently null a wide range of background T 1 species while allowing for adjustable inflow times; alternating pulse repetition time steady-state free precession maintains vessel contrast and provides added fat suppression. The high level of suppression enables imaging in three-dimensional as well as projective two-dimensional formats, the latter of which has a scan time as short as one heartbeat. In vivo studies at 1.5 T demonstrate the superior vessel contrast of this technique. © 2012 Wiley Periodicals, Inc.

Nanosensing of Pesticides by Zinc Oxide Quantum Dot: An Optical and Electrochemical Approach for the Detection of Pesticides in Water.

PubMed

Sahoo, Dibakar; Mandal, Abhishek; Mitra, Tapas; Chakraborty, Kaushik; Bardhan, Munmun; Dasgupta, Anjan Kumar

2018-01-17

Present study reveals the low concentrations (∼4 ppm) of pesticide sensing vis-à-vis degradation of pesticides with the help of nontoxic zinc oxide quantum dots (QD). In our study, we have taken four different pesticides viz., aldrin, tetradifon, glyphosate, and atrazine, which are widely used in agriculture and have structural dissimilarities/diversity. By using optical sensing techniques such as steady state and time-resolved fluorescence, we have analyzed the detailed exciton dynamics of QD in the presence of different pesticides. It has been found that the pesticide containing good leaving groups (-Cl) can interact with QD promptly and has high binding affinity (∼10 7 M -1 ). The different binding signatures of QD with different pesticides enable us to differentiate between the pesticides. Time resolved fluorescence spectroscopy provides significant variance (∼150-300 ns) for different pesticides. Furthermore, a large variation (10 5 Ω to 7 × 10 4 Ω) in the resistance of QD in the presence of different pesticides was revealed by electrochemical sensing technique. Moreover, during the interaction with pesticides, QD can also act as a photocatalyst to degrade pesticides. Present investigation explored the fact that the rate of degradation is positively affected by the binding affinity, i.e., the greater the binding, the greater is the degradation. What is more, both optical and electrochemical measurements of QD, in tandem, as described in our study could be utilized as the pattern recognition sensor for detection of several pesticides.

Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes

PubMed Central

Sitaram, Anand; Dennis, Megan K.; Chaudhuri, Rittik; De Jesus-Rojas, Wilfredo; Tenza, Danièle; Setty, Subba Rao Gangi; Wood, Christopher S.; Sviderskaya, Elena V.; Bennett, Dorothy C.; Raposo, Graça; Bonifacino, Juan S.; Marks, Michael S.

2012-01-01

Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine–based sorting signal in the pigment cell–specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1– and AP-3–favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs. PMID:22718909

Hybrid Cascading Outage Analysis of Extreme Events with Optimized Corrective Actions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vallem, Mallikarjuna R.; Vyakaranam, Bharat GNVSR; Holzer, Jesse T.

2017-10-19

Power system are vulnerable to extreme contingencies (like an outage of a major generating substation) that can cause significant generation and load loss and can lead to further cascading outages of other transmission facilities and generators in the system. Some cascading outages are seen within minutes following a major contingency, which may not be captured exclusively using the dynamic simulation of the power system. The utilities plan for contingencies either based on dynamic or steady state analysis separately which may not accurately capture the impact of one process on the other. We address this gap in cascading outage analysis bymore » developing Dynamic Contingency Analysis Tool (DCAT) that can analyze hybrid dynamic and steady state behavior of the power system, including protection system models in dynamic simulations, and simulating corrective actions in post-transient steady state conditions. One of the important implemented steady state processes is to mimic operator corrective actions to mitigate aggravated states caused by dynamic cascading. This paper presents an Optimal Power Flow (OPF) based formulation for selecting corrective actions that utility operators can take during major contingency and thus automate the hybrid dynamic-steady state cascading outage process. The improved DCAT framework with OPF based corrective actions is demonstrated on IEEE 300 bus test system.« less

Characterization of the kinetics of Fe (II) binding by the R2 protein subunit of E. coli ribonucleotide reductase

NASA Astrophysics Data System (ADS)

Chaudhuri, Dipankar; , Joseph Martin Bollinger, Jr.

2008-07-01

The kinetics of Fe(II) binding to Escherichia coli Ribonucleotide reductase (R2) has been studied using rapid kinetics techniques including chemical quenched flow (CQF) Mössbauer spectroscopy. Based on the stopped flow absorption (SF-Abs) and CQF Mössbauer spectroscopy results, the pre-steady kinetics of binding of Fe(II) to the two sites A and B on R2 have been established with attendant conformational changes. Fe (II) binds to Site B tighter and faster and these and other results provide important information towards the di-iron cofactor assembly mechanism in R2 and could have possible implications for the development of modified and new anticancer and antiviral drugs.

Modified fluctuation-dissipation and Einstein relation at nonequilibrium steady states

NASA Astrophysics Data System (ADS)

Chaudhuri, Debasish; Chaudhuri, Abhishek

2012-02-01

Starting from the pioneering work of Agarwal [G. S. Agarwal, Zeitschrift für PhysikEPJAFV1434-600110.1007/BF01391621 252, 25 (1972)], we present a unified derivation of a number of modified fluctuation-dissipation relations (MFDR) that relate response to small perturbations around nonequilibrium steady states to steady-state correlations. Using this formalism we show the equivalence of velocity forms of MFDR derived using continuum Langevin and discrete master equation dynamics. The resulting additive correction to the Einstein relation is exemplified using a flashing ratchet model of molecular motors.

Steady-state entanglement activation in optomechanical cavities

NASA Astrophysics Data System (ADS)

Farace, Alessandro; Ciccarello, Francesco; Fazio, Rosario; Giovannetti, Vittorio

2014-02-01

Quantum discord, and related indicators, are raising a relentless interest as a novel paradigm of nonclassical correlations beyond entanglement. Here, we discover a discord-activated mechanism yielding steady-state entanglement production in a realistic continuous-variable setup. This comprises two coupled optomechanical cavities, where the optical modes (OMs) communicate through a fiber. We first use a simplified model to highlight the creation of steady-state discord between the OMs. We show next that such discord improves the level of stationary optomechanical entanglement attainable in the system, making it more robust against temperature and thermal noise.

Ultrafast Exciton Delocalization, Localization, and Excimer Formation Dynamics in a Highly Defined Perylene Bisimide Quadruple π-Stack.

PubMed

Kaufmann, Christina; Kim, Woojae; Nowak-Król, Agnieszka; Hong, Yongseok; Kim, Dongho; Würthner, Frank

2018-03-28

An adequately designed, bay-tethered perylene bisimide (PBI) dimer Bis-PBI was synthesized by Pd/Cu-catalyzed Glaser-type oxidative homocoupling of the respective PBI building block. This newly synthesized PBI dimer self-assembles exclusively and with high binding constants of up to 10 6 M -1 into a discrete π-stack of four chromophores. Steady-state absorption and emission spectra show the signatures of H-type excitonic coupling among the dye units. Broadband fluorescence upconversion spectroscopy (FLUPS) reveals an ultrafast dynamics in the optically excited state. An initially coherent Frenkel exciton state that is delocalized over the whole quadruple stack rapidly (τ = ∼200 fs) loses its coherence and relaxes into an excimer state. Comparison with Frenkel exciton dynamics in PBI dimeric and oligomeric H-aggregates demonstrates that in the quadruple stack coherent exciton propagation is absent due to its short length of aggregates, thereby it has only one relaxation pathway to the excimer state. Furthermore, the absence of pump-power dependence in transient absorption experiments suggests that multiexciton cannot be generated in the quadruple stack, which is in line with time-resolved fluorescence measurements.

A mechanical energy analysis of gait initiation

NASA Technical Reports Server (NTRS)

Miller, C. A.; Verstraete, M. C.

1999-01-01

The analysis of gait initiation (the transient state between standing and walking) is an important diagnostic tool to study pathologic gait and to evaluate prosthetic devices. While past studies have quantified mechanical energy of the body during steady-state gait, to date no one has computed the mechanical energy of the body during gait initiation. In this study, gait initiation in seven normal male subjects was studied using a mechanical energy analysis to compute total body energy. The data showed three separate states: quiet standing, gait initiation, and steady-state gait. During gait initiation, the trends in the energy data for the individual segments were similar to those seen during steady-state gait (and in Winter DA, Quanbury AO, Reimer GD. Analysis of instantaneous energy of normal gait. J Biochem 1976;9:253-257), but diminished in amplitude. However, these amplitudes increased to those seen in steady-state during the gait initiation event (GIE), with the greatest increase occurring in the second step due to the push-off of the foundation leg. The baseline level of mechanical energy was due to the potential energy of the individual segments, while the cyclic nature of the data was indicative of the kinetic energy of the particular leg in swing phase during that step. The data presented showed differences in energy trends during gait initiation from those of steady state, thereby demonstrating the importance of this event in the study of locomotion.

Dispersion of a Nanoliter Bolus in Microfluidic Co-Flow.

PubMed

Conway, A J; Saadi, W M; Sinatra, F L; Kowalski, G; Larson, D; Fiering, J

2014-03-01

Microfluidic systems enable reactions and assays on the scale of nanoliters. However, at this scale nonuniformities in sample delivery become significant. To determine the fundamental minimum sample volume required for a particular device, a detailed understanding of mass transport is required. Co-flowing laminar streams are widely used in many devices, but typically only in the steady-state. Because establishing the co-flow steady-state consumes excess sample volume and time, there is a benefit to operating devices in the transient state, which predominates as the volume of the co-flow reactor decreases. Analysis of the co-flow transient has been neglected thus far. In this work we describe the fabrication of a pneumatically controlled microfluidic injector constructed to inject a discrete 50nL bolus into one side of a two-stream co-flow reactor. Using dye for image analysis, injections were performed at a range of flow rates from 0.5-10μL/min, and for comparison we collected the co-flow steady-state data for this range. The results of the image analysis were also compared against theory and simulations for device validation. For evaluation, we established a metric that indicates how well the mass distribution in the bolus injection approximates steady-state co-flow. Using such analysis, transient-state injections can approximate steady-state conditions within predefined errors, allowing straight forward measurements to be performed with reduced reagent consumption.

Fluorescence molecular probes for sensitive point detection of amyloid fibrils and protofibrils

NASA Astrophysics Data System (ADS)

Lindgren, Mikael; Jonsson, Per; Sörgjerd, Karin; Hammarström, Per

2005-10-01

Protein based infections such as prion diseases have lately attracted a large amount of interest, primarily due to the Mad Cow Epidemic in Great Britain, and the increase of Alzheimer's disease and related diseases in the ageing Western society. Infective proteins are very stable and almost untraceable prior to infection making them ideal as biological weapons. Particularly if the used agent is of human origin, the immunoresponse can be avoided, leaving no trace of the infectious agent. The transient nature of infectious oligomeric intermediates of misfolded proteins or peptide fragments that later matures into fibrillar aggregates makes them hard to study, and methods to detect and study these species are sparse. There exist a number of fluorescent probes that bind specifically to protein amyloidic structures. Thioflavins (ThT, ThS), Congo and Nile red, 4-(dicyanovinyl)-julolidine (DCVJ), as well as derivatives amino-8-naphtalene sulphonate (ANS, Bis-ANS) which are known to bind to the fibrillar or pre-fibrillar states with dissociation constants of typically 1 - 20 μM. Here, transthyretin (TTR), insulin and lysozyme were used as model proteins to detect different amyloid precursor states for diseases such as senile systemic amyloidosis, familial amyloidotic polyneuropathy (FAP) and iatrogenic amyloidosis. Specifically, the probes were employed in static assays to characterize protofibrillar and mature amyloid fibrillar states using steady state and time-resolved fluorescence techniques. Particularly, we investigate and report on the possibility to detect protofibrillar states at low concentration levels using modern fluorescence array detector systems in conjunction with lasers operating in the blue or ultraviolett wavelengths as excitation source. Results of ANS, ThT and a ThT analogue (abbreviated ThC) are discussed.

Processivity of the Kinesin-2 KIF3A Results from Rear Head Gating and Not Front Head Gating*

PubMed Central

Chen, Geng-Yuan; Arginteanu, David F. J.; Hancock, William O.

2015-01-01

The kinesin-2 family motor KIF3A/B works together with dynein to bidirectionally transport intraflagellar particles, melanosomes, and neuronal vesicles. Compared with kinesin-1, kinesin-2 is less processive, and its processivity is more sensitive to load, suggesting that processivity may be controlled by different gating mechanisms. We used stopped-flow and steady-state kinetics experiments, along with single-molecule and multimotor assays to characterize the entire kinetic cycle of a KIF3A homodimer that exhibits motility similar to that of full-length KIF3A/B. Upon first encounter with a microtubule, the motor rapidly exchanges both mADP and mATP. When adenosine 5′-[(β,γ)-imido]triphosphate was used to entrap the motor in a two-head-bound state, exchange kinetics were unchanged, indicating that rearward strain in the two-head-bound state does not alter nucleotide binding to the front head. A similar lack of front head gating was found when intramolecular strain was enhanced by shortening the neck linker domain from 17 to 14 residues. In single-molecule assays in ADP, the motor dissociates at 2.1 s−1, 20-fold slower than the stepping rate, demonstrating the presence of rear head gating. In microtubule pelleting assays, the KDMt is similar in ADP and ATP. The data and accompanying simulations suggest that, rather than KIF3A processivity resulting from strain-dependent regulation of nucleotide binding (front head gating), the motor spends a significant fraction of its hydrolysis cycle in a low affinity state but dissociates only slowly from this state. This work provides a mechanism to explain differences in the load-dependent properties of kinesin-1 and kinesin-2. PMID:25657001

Effect of Steady-State Faldaprevir on the Pharmacokinetics of Steady-State Methadone and Buprenorphine-Naloxone in Subjects Receiving Stable Addiction Management Therapy

PubMed Central

Joseph, David; Schobelock, Michael J.; Riesenberg, Robert R.; Vince, Bradley D.; Webster, Lynn R.; Adeniji, Abidemi; Elgadi, Mabrouk

2014-01-01

The effects of steady-state faldaprevir on the safety, pharmacokinetics, and pharmacodynamics of steady-state methadone and buprenorphine-naloxone were assessed in 34 healthy male and female subjects receiving stable addiction management therapy. Subjects continued receiving a stable oral dose of either methadone (up to a maximum dose of 180 mg per day) or buprenorphine-naloxone (up to a maximum dose of 24 mg-6 mg per day) and also received oral faldaprevir (240 mg) once daily (QD) for 8 days following a 480-mg loading dose. Serial blood samples were taken for pharmacokinetic analysis. The pharmacodynamics of the opioid maintenance regimens were evaluated by the objective and subjective opioid withdrawal scales. Coadministration of faldaprevir with methadone or buprenorphine-naloxone resulted in geometric mean ratios for the steady-state area under the concentration-time curve from 0 to 24 h (AUC0–24,ss), the steady-state maximum concentration of the drug in plasma (Cmax,ss), and the steady-state concentration of the drug in plasma at 24 h (C24,ss) of 0.92 to 1.18 for (R)-methadone, (S)-methadone, buprenorphine, norbuprenorphine, and naloxone, with 90% confidence intervals including, or very close to including, 1.00 (no effect), suggesting a limited overall effect of faldaprevir. Although individual data showed moderate variability in the exposures between subjects and treatments, there was no evidence of symptoms of opiate overdose or withdrawal either during the coadministration of faldaprevir with methadone or buprenorphine-naloxone or after faldaprevir dosing was stopped. Similar faldaprevir exposures were observed in the methadone- and buprenorphine-naloxone-treated subjects. In conclusion, faldaprevir at 240 mg QD can be coadministered with methadone or buprenorphine-naloxone without dose adjustment, although given the relatively narrow therapeutic windows of these agents, monitoring for opiate overdose and withdrawal may still be appropriate. (This study has been registered at ClinicalTrials.gov under registration no. NCT01637922.) PMID:25385094

A nodally condensed SUPG formulation for free-surface computation of steady-state flows constrained by unilateral contact - Application to rolling

NASA Astrophysics Data System (ADS)

Arora, Shitij; Fourment, Lionel

2018-05-01

In the context of the simulation of industrial hot forming processes, the resultant time-dependent thermo-mechanical multi-field problem (v →,p ,σ ,ɛ ) can be sped up by 10-50 times using the steady-state methods while compared to the conventional incremental methods. Though the steady-state techniques have been used in the past, but only on simple configurations and with structured meshes, and the modern-days problems are in the framework of complex configurations, unstructured meshes and parallel computing. These methods remove time dependency from the equations, but introduce an additional unknown into the problem: the steady-state shape. This steady-state shape x → can be computed as a geometric correction t → on the domain X → by solving the weak form of the steady-state equation v →.n →(t →)=0 using a Streamline Upwind Petrov Galerkin (SUPG) formulation. There exists a strong coupling between the domain shape and the material flow, hence, a two-step fixed point iterative resolution algorithm was proposed that involves (1) the computation of flow field from the resolution of thermo-mechanical equations on a prescribed domain shape and (2) the computation of steady-state shape for an assumed velocity field. The contact equations are introduced in the penalty form both during the flow computation as well as during the free-surface correction. The fact that the contact description is inhomogeneous, i.e., it is defined in the nodal form in the former, and in the weighted residual form in the latter, is assumed to be critical to the convergence of certain problems. Thus, the notion of nodal collocation is invoked in the weak form of the surface correction equation to homogenize the contact coupling. The surface correction algorithm is tested on certain analytical test cases and the contact coupling is tested with some hot rolling problems.

Higher Accuracy of the Lactate Minimum Test Compared to Established Threshold Concepts to Determine Maximal Lactate Steady State in Running.

PubMed

Wahl, Patrick; Zwingmann, Lukas; Manunzio, Christian; Wolf, Jacob; Bloch, Wilhelm

2018-05-18

This study evaluated the accuracy of the lactate minimum test, in comparison to a graded-exercise test and established threshold concepts (OBLA and mDmax) to determine running speed at maximal lactate steady state. Eighteen subjects performed a lactate minimum test, a graded-exercise test (2.4 m·s -1 start,+0.4 m·s -1 every 5 min) and 2 or more constant-speed tests of 30 min to determine running speed at maximal lactate steady state. The lactate minimum test consisted of an initial lactate priming segment, followed by a short recovery phase. Afterwards, the initial load of the subsequent incremental segment was individually determined and was increased by 0.1 m·s -1 every 120 s. Lactate minimum was determined by the lowest measured value (LM abs ) and by a third-order polynomial (LM pol ). The mean difference to maximal lactate steady state was+0.01±0.14 m·s -1 (LM abs ), 0.04±0.15 m·s -1 (LM pol ), -0.06±0.31 m·s 1 (OBLA) and -0.08±0.21 m·s 1 (mDmax). The intraclass correlation coefficient (ICC) between running velocity at maximal lactate steady state and LM abs was highest (ICC=0.964), followed by LM pol (ICC=0.956), mDmax (ICC=0.916) and OBLA (ICC=0.885). Due to the higher accuracy of the lactate minimum test to determine maximal lactate steady state compared to OBLA and mDmax, we suggest the lactate minimum test as a valid and meaningful concept to estimate running velocity at maximal lactate steady state in a single session for moderately up to well-trained athletes. © Georg Thieme Verlag KG Stuttgart · New York.

The Effects of High Intensity Interval Training vs Steady State Training on Aerobic and Anaerobic Capacity

PubMed Central

Foster, Carl; Farland, Courtney V.; Guidotti, Flavia; Harbin, Michelle; Roberts, Brianna; Schuette, Jeff; Tuuri, Andrew; Doberstein, Scott T.; Porcari, John P.

2015-01-01

High intensity interval training (HIIT) has become an increasingly popular form of exercise due to its potentially large effects on exercise capacity and small time requirement. This study compared the effects of two HIIT protocols vs steady-state training on aerobic and anaerobic capacity following 8-weeks of training. Fifty-five untrained college-aged subjects were randomly assigned to three training groups (3x weekly). Steady-state (n = 19) exercised (cycle ergometer) 20 minutes at 90% of ventilatory threshold (VT). Tabata (n = 21) completed eight intervals of 20s at 170% VO2max/10s rest. Meyer (n = 15) completed 13 sets of 30s (20 min) @ 100% PVO2 max/ 60s recovery, average PO = 90% VT. Each subject did 24 training sessions during 8 weeks. Results: There were significant (p < 0.05) increases in VO2max (+19, +18 and +18%) and PPO (+17, +24 and +14%) for each training group, as well as significant increases in peak (+8, + 9 and +5%) & mean (+4, +7 and +6%) power during Wingate testing, but no significant differences between groups. Measures of the enjoyment of the training program indicated that the Tabata protocol was significantly less enjoyable (p < 0.05) than the steady state and Meyer protocols, and that the enjoyment of all protocols declined (p < 0.05) across the duration of the study. The results suggest that although HIIT protocols are time efficient, they are not superior to conventional exercise training in sedentary young adults. Key points Steady state training equivalent to HIIT in untrained students Mild interval training presents very similar physiologic challenge compared to steady state training HIIT (particularly very high intensity variants were less enjoyable than steady state or mild interval training Enjoyment of training decreases across the course of an 8 week experimental training program PMID:26664271

The Effects of High Intensity Interval Training vs Steady State Training on Aerobic and Anaerobic Capacity.

PubMed

Foster, Carl; Farland, Courtney V; Guidotti, Flavia; Harbin, Michelle; Roberts, Brianna; Schuette, Jeff; Tuuri, Andrew; Doberstein, Scott T; Porcari, John P

2015-12-01

High intensity interval training (HIIT) has become an increasingly popular form of exercise due to its potentially large effects on exercise capacity and small time requirement. This study compared the effects of two HIIT protocols vs steady-state training on aerobic and anaerobic capacity following 8-weeks of training. Fifty-five untrained college-aged subjects were randomly assigned to three training groups (3x weekly). Steady-state (n = 19) exercised (cycle ergometer) 20 minutes at 90% of ventilatory threshold (VT). Tabata (n = 21) completed eight intervals of 20s at 170% VO2max/10s rest. Meyer (n = 15) completed 13 sets of 30s (20 min) @ 100% PVO2 max/ 60s recovery, average PO = 90% VT. Each subject did 24 training sessions during 8 weeks. There were significant (p < 0.05) increases in VO2max (+19, +18 and +18%) and PPO (+17, +24 and +14%) for each training group, as well as significant increases in peak (+8, + 9 and +5%) & mean (+4, +7 and +6%) power during Wingate testing, but no significant differences between groups. Measures of the enjoyment of the training program indicated that the Tabata protocol was significantly less enjoyable (p < 0.05) than the steady state and Meyer protocols, and that the enjoyment of all protocols declined (p < 0.05) across the duration of the study. The results suggest that although HIIT protocols are time efficient, they are not superior to conventional exercise training in sedentary young adults. Key pointsSteady state training equivalent to HIIT in untrained studentsMild interval training presents very similar physiologic challenge compared to steady state trainingHIIT (particularly very high intensity variants were less enjoyable than steady state or mild interval trainingEnjoyment of training decreases across the course of an 8 week experimental training program.

An Operational Definition of the Steady State in Enzyme Kinetics.

ERIC Educational Resources Information Center

Barnsley, E. A.

1990-01-01

The Briggs-Haldane assumption is used as the basis for the development of a kinetic model for enzyme catalysis. An alternative definition of the steady state and examples of realistic mechanisms are provided. (KR)

Vibration testing and analysis using holography

NASA Technical Reports Server (NTRS)

1971-01-01

Time average holography is useful in recording steady state vibrational mode patterns. Phase relationships under steady state conditions are measured with real time holography and special phase shifting techniques. Data from Michelson interferometer verify vibration amplitudes from holographic data.

Limiting Forces on Transit Trucks in Steady-State Curving

DOT National Transportation Integrated Search

1981-05-01

This study develops conservative bounds on wheel/rail forces and flange forces for several types of rigid and flexible trucks in steady-state curving conditions. The approximate analysis presented provides closed-form relations for estimating forces,...

Bifurcations in the theory of current transfer to cathodes of DC discharges and observations of transitions between different modes

NASA Astrophysics Data System (ADS)

Bieniek, M. S.; Santos, D. F. N.; Almeida, P. G. C.; Benilov, M. S.

2018-04-01

General scenarios of transitions between different spot patterns on electrodes of DC gas discharges and their relation to bifurcations of steady-state solutions are analyzed. In the case of cathodes of arc discharges, it is shown that any transition between different modes of current transfer is related to a bifurcation of steady-state solutions. In particular, transitions between diffuse and spot modes on axially symmetric cathodes, frequently observed in the experiment, represent an indication of the presence of pitchfork or fold bifurcations of steady-state solutions. Experimental observations of transitions on cathodes of DC glow microdischarges are analyzed and those potentially related to bifurcations of steady-state solutions are identified. The relevant bifurcations are investigated numerically and the computed patterns are found to conform to those observed in the course of the corresponding transitions in the experiment.

The effect of solute additions on the steady-state creep behavior of dispersion-strengthened aluminum.

NASA Technical Reports Server (NTRS)

Reynolds, G. H.; Lenel, F. V.; Ansell, G. S.

1971-01-01

The effect of solute additions on the steady-state creep behavior of coarse-grained dispersion-strengthened aluminum alloys was studied. Recrystallized dispersion-strengthened solid solutions were found to have stress and temperature sensitivities quite unlike those observed in single-phase solid solutions having the same composition and grain size. The addition of magnesium or copper to the matrix of a recrystallized dispersion-strengthened aluminum causes a decrease in the steady-state creep rate which is much smaller than that caused by similar amounts of solute in single-phase solid solutions. All alloys exhibited essentially a 4.0 power stress exponent in agreement with the model of Ansell and Weertman. The activation energy for steady-state creep in dispersion-strengthened Al-Mg alloys, as well as the stress dependence, was in agreement with the physical model of dislocation climb over the dispersed particles.

Steady-state performance analysis of fiber-optic ring resonator

NASA Astrophysics Data System (ADS)

Seraji, Faramarz E.

2009-01-01

This paper presents a full steady-state analysis of a fiber-optic ring resonator (FORR). Although in the literature the steady-state response of the FORR has been described, a detailed description of the same is not available. As an understanding of the different steady-state characteristics of the FORR is required to appreciate its characteristic response, in this paper, the expressions for the output and loop intensities, phase angles of the fields, conditions for resonance, output and loop intensities at resonance and off-resonance, finesse, and group delay of the FORR are given for different ideal and practical operating conditions of the resonator. Graphical plots of all the above characteristics are given, by highlighting the important results. The information presented in this paper will be helpful in explaining and understanding the pulse response of the resonator used in different applications of FORR.

Poissonian steady states: from stationary densities to stationary intensities.

PubMed

Eliazar, Iddo

2012-10-01

Markov dynamics are the most elemental and omnipresent form of stochastic dynamics in the sciences, with applications ranging from physics to chemistry, from biology to evolution, and from economics to finance. Markov dynamics can be either stationary or nonstationary. Stationary Markov dynamics represent statistical steady states and are quantified by stationary densities. In this paper, we generalize the notion of steady state to the case of general Markov dynamics. Considering an ensemble of independent motions governed by common Markov dynamics, we establish that the entire ensemble attains Poissonian steady states which are quantified by stationary Poissonian intensities and which hold valid also in the case of nonstationary Markov dynamics. The methodology is applied to a host of Markov dynamics, including Brownian motion, birth-death processes, random walks, geometric random walks, renewal processes, growth-collapse dynamics, decay-surge dynamics, Ito diffusions, and Langevin dynamics.

Reduction of Simulation Times for High-Q Structures using the Resonance Equation

DOE PAGES

Hall, Thomas Wesley; Bandaru, Prabhakar R.; Rees, Daniel Earl

2015-11-17

Simulating steady state performance of high quality factor (Q) resonant RF structures is computationally difficult for structures with sizes on the order of more than a few wavelengths because of the long times (on the order of ~ 0.1 ms) required to achieve steady state in comparison with maximum time step that can be used in the simulation (typically, on the order of ~ 1 ps). This paper presents analytical and computational approaches that can be used to accelerate the simulation of the steady state performance of such structures. The basis of the proposed approach is the utilization of amore » larger amplitude signal at the beginning to achieve steady state earlier relative to the nominal input signal. Finally, the methodology for finding the necessary input signal is then discussed in detail, and the validity of the approach is evaluated.« less

Steady-state solutions of a diffusive energy-balance climate model and their stability

NASA Technical Reports Server (NTRS)

Ghil, M.

1975-01-01

A diffusive energy-balance climate model, governed by a nonlinear parabolic partial differential equation, was studied. Three positive steady-state solutions of this equation are found; they correspond to three possible climates of our planet: an interglacial (nearly identical to the present climate), a glacial, and a completely ice-covered earth. Models similar to the main one are considered, and the number of their steady states was determined. All the models have albedo continuously varying with latitude and temperature, and entirely diffusive horizontal heat transfer. The stability under small perturbations of the main model's climates was investigated. A stability criterion is derived, and its application shows that the present climate and the deep freeze are stable, whereas the model's glacial is unstable. The dependence was examined of the number of steady states and of their stability on the average solar radiation.

SUPRATHERMAL SOLAR WIND ELECTRONS AND LANGMUIR TURBULENCE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sunjung; Yoon, Peter H.; Choe, G. S.

2016-09-01

The steady-state model recently put forth for the solar wind electron velocity distribution function during quiet time conditions, was originally composed of three population electrons (core, halo, and superhalo) with the core remaining nonresonant with any plasma waves while the halo and superhalo separately maintained steady-state resonance with whistler- and Langmuir-frequency range fluctuations, respectively. However, a recent paper demonstrates that whistler-range fluctuations in fact have no significant contribution. The present paper represents a consummation of the model in that a self-consistent model of the suprathermal electron population, which encompasses both the halo and the superhalo, is constructed solely on themore » basis of the Langmuir fluctuation spectrum. Numerical solutions to steady-state particle and wave kinetic equations are obtained on the basis of an initial trial electron distribution and Langmuir wave spectrum. Such a finding offers a self-consistent explanation for the observed steady-state electron distribution in the solar wind.« less

Poissonian steady states: From stationary densities to stationary intensities

NASA Astrophysics Data System (ADS)

Eliazar, Iddo

2012-10-01

Markov dynamics are the most elemental and omnipresent form of stochastic dynamics in the sciences, with applications ranging from physics to chemistry, from biology to evolution, and from economics to finance. Markov dynamics can be either stationary or nonstationary. Stationary Markov dynamics represent statistical steady states and are quantified by stationary densities. In this paper, we generalize the notion of steady state to the case of general Markov dynamics. Considering an ensemble of independent motions governed by common Markov dynamics, we establish that the entire ensemble attains Poissonian steady states which are quantified by stationary Poissonian intensities and which hold valid also in the case of nonstationary Markov dynamics. The methodology is applied to a host of Markov dynamics, including Brownian motion, birth-death processes, random walks, geometric random walks, renewal processes, growth-collapse dynamics, decay-surge dynamics, Ito diffusions, and Langevin dynamics.

The Effect of Impeller Type on Floc Size and Structure during Shear-Induced Flocculation

PubMed

Spicer; Keller; Pratsinis

1996-12-01

The effect of impeller type and shear rate on the evolution of floc size and structure during shear-induced flocculation of polystyrene particles with aluminum sulfate is investigated by image analysis. One radial flow (six-blade Rushton turbine) and two axial flow (three-blade fluid foil, four-blade 45° pitch) impeller configurations are examined. The steady state average floc size is shown to depend on the frequency of recirculation to the impeller zone and its characteristic velocity gradient. The concepts of fractal geometry are used to characterize the floc structure. For all impellers, the two-dimensional floc fractal dimension, Dpf, increases during floc growth, indicating formation of more open structures. Later on, Dpf levels off at a steady state value as breakage becomes significant and the floc size distribution approaches steady state. The shear rate does not affect the steady state Dpf of the flocs within experimental uncertainty.

Topological properties of a self-assembled electrical network via ab initio calculation

NASA Astrophysics Data System (ADS)

Stephenson, C.; Lyon, D.; Hübler, A.

2017-02-01

Interacting electrical conductors self-assemble to form tree like networks in the presence of applied voltages or currents. Experiments have shown that the degree distribution of the steady state networks are identical over a wide range of network sizes. In this work we develop a new model of the self-assembly process starting from the underlying physical interaction between conductors. In agreement with experimental results we find that for steady state networks, our model predicts that the fraction of endpoints is a constant of 0.252, and the fraction of branch points is 0.237. We find that our model predicts that these scaling properties also hold for the network during the approach to the steady state as well. In addition, we also reproduce the experimental distribution of nodes with a given Strahler number for all steady state networks studied.

The Steady-State Transport of Oxygen through Hemoglobin Solutions

PubMed Central

Keller, K. H.; Friedlander, S. K.

1966-01-01

The steady-state transport of oxygen through hemoglobin solutions was studied to identify the mechanism of the diffusion augmentation observed at low oxygen tensions. A novel technique employing a platinum-silver oxygen electrode was developed to measure the effective diffusion coefficient of oxygen in steady-state transport. The measurements were made over a wider range of hemoglobin and oxygen concentrations than previously reported. Values of the Brownian motion diffusion coefficient of oxygen in hemoglobin solution were obtained as well as measurements of facilitated transport at low oxygen tensions. Transport rates up to ten times greater than ordinary diffusion rates were found. Predictions of oxygen flux were made assuming that the oxyhemoglobin transport coefficient was equal to the Brownian motion diffusivity which was measured in a separate set of experiments. The close correlation between prediction and experiment indicates that the diffusion of oxyhemoglobin is the mechanism by which steady-state oxygen transport is facilitated. PMID:5943608

Realizing steady-state tokamak operation for fusion energy

NASA Astrophysics Data System (ADS)

Luce, T. C.

2011-03-01

Continuous operation of a tokamak for fusion energy has clear engineering advantages but requires conditions beyond those sufficient for a burning plasma. The fusion reactions and external sources must support both the pressure and the current equilibrium without inductive current drive, leading to demands on stability, confinement, current drive, and plasma-wall interactions that exceed those for pulsed tokamaks. These conditions have been met individually, and significant progress has been made in the past decade to realize scenarios where the required conditions are obtained simultaneously. Tokamaks are operated routinely without disruptions near pressure limits, as needed for steady-state operation. Fully noninductive sustainment with more than half of the current from intrinsic currents has been obtained for a resistive time with normalized pressure and confinement approaching those needed for steady-state conditions. One remaining challenge is handling the heat and particle fluxes expected in a steady-state tokamak without compromising the core plasma performance.

Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases

PubMed Central

Su, Yan; Guengerich, F. Peter

2016-01-01

Pre-steady-state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step-by-step procedure for pre-steady-state kinetic analysis of single-nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF-3 rapid quench-flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre-steady-state assays on an RQF-3 rapid quench-flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. PMID:27248785

Efficient steady-state solver for hierarchical quantum master equations

NASA Astrophysics Data System (ADS)

Zhang, Hou-Dao; Qiao, Qin; Xu, Rui-Xue; Zheng, Xiao; Yan, YiJing

2017-07-01

Steady states play pivotal roles in many equilibrium and non-equilibrium open system studies. Their accurate evaluations call for exact theories with rigorous treatment of system-bath interactions. Therein, the hierarchical equations-of-motion (HEOM) formalism is a nonperturbative and non-Markovian quantum dissipation theory, which can faithfully describe the dissipative dynamics and nonlinear response of open systems. Nevertheless, solving the steady states of open quantum systems via HEOM is often a challenging task, due to the vast number of dynamical quantities involved. In this work, we propose a self-consistent iteration approach that quickly solves the HEOM steady states. We demonstrate its high efficiency with accurate and fast evaluations of low-temperature thermal equilibrium of a model Fenna-Matthews-Olson pigment-protein complex. Numerically exact evaluation of thermal equilibrium Rényi entropies and stationary emission line shapes is presented with detailed discussion.

Marginal states in a cubic autocatalytic reaction

NASA Astrophysics Data System (ADS)

Das, Debojyoti; Ghosh, Pushpita; Ray, Deb Shankar

2011-09-01

Marginal steady state belongs to a special class of states in nonlinear dynamics. To realize this state we consider a cubic autocatalytic reaction A + 2B → 3B in a continuous-stirred-tank-reactor, where the flow rate of the reactant A can be controlled to manipulate the dynamical behavior of the open system. We demonstrate that when the flow rate is weakly noisy the autocatalytic reaction admits of a steady state which is marginal in nature and is surrounded by infinite number of periodic trajectories. When the uncatalyzed reaction A → B is included in the reaction scheme, there exists a marginal steady state which is a critical state corresponding to the point of transition between the flow branch and the equilibrium branch, similar to gas-liquid critical point of transition. This state loses its stability in the weak noise limit.

Influence of the hypercycle on the error threshold: a stochastic approach.

PubMed

García-Tejedor, A; Sanz-Nuño, J C; Olarrea, J; Javier de la Rubia, F; Montero, F

1988-10-21

The role of fluctuations on the error threshold of the hypercycle has been studied by a stochastic approach on a very simplified model. For this model, the master equation was derived and its unique steady state calculated. This state implies the extinction of the system. But the actual time necessary to reach the steady state may be astronomically long whereas for times of experimental interest the system could be near some quasi-stationary states. In order to explore this possibility a Gillespie simulation of the stochastic process has been carried out. These quasi-stationary states correspond to the deterministic steady states of the system. The error threshold shifts towards higher values of the quality factor Q. Moreover, information about the fluctuations around the quasi-stationary states is obtained. The results are discussed in relation to the deterministic states.

A Steady State and Quasi-Steady Interface Between the Generalized Fluid System Simulation Program and the SINDA/G Thermal Analysis Program

NASA Technical Reports Server (NTRS)

Schallhorn, Paul; Majumdar, Alok; Tiller, Bruce

2001-01-01

A general purpose, one dimensional fluid flow code is currently being interfaced with the thermal analysis program SINDA/G. The flow code, GFSSP, is capable of analyzing steady state and transient flow in a complex network. The flow code is capable of modeling several physical phenomena including compressibility effects, phase changes, body forces (such as gravity and centrifugal) and mixture thermodynamics for multiple species. The addition of GFSSP to SINDA/G provides a significant improvement in convective heat transfer modeling for SINDA/G. The interface development is conducted in multiple phases. This paper describes the first phase of the interface which allows for steady and quasisteady (unsteady solid, steady fluid) conjugate heat transfer modeling.

Steady-state entanglement and thermalization of coupled qubits in two common heat baths

NASA Astrophysics Data System (ADS)

Hu, Li-Zhen; Man, Zhong-Xiao; Xia, Yun-Jie

2018-03-01

In this work, we study the steady-state entanglement and thermalization of two coupled qubits embedded in two common baths with different temperatures. The common bath is relevant when the two qubits are difficult to be isolated to only contact with their local baths. With the quantum master equation constructed in the eigenstate representation of the coupled qubits, we have demonstrated the variations of steady-state entanglement with respect to various parameters of the qubits' system in both equilibrium and nonequilibrium cases of the baths. The coupling strength and energy detuning of the qubits as well as the temperature gradient of the baths are found to be beneficial to the enhancement of the entanglement. We note a dark state of the qubits that is free from time-evolution and its initial population can greatly influence the steady-state entanglement. By virtues of effective temperatures, we also study the thermalization of the coupled qubits and their variations with energy detuning.

Steady state numerical solutions for determining the location of MEMS on projectile

NASA Astrophysics Data System (ADS)

Abiprayu, K.; Abdigusna, M. F. F.; Gunawan, P. H.

2018-03-01

This paper is devoted to compare the numerical solutions for the steady and unsteady state heat distribution model on projectile. Here, the best location for installing of the MEMS on the projectile based on the surface temperature is investigated. Numerical iteration methods, Jacobi and Gauss-Seidel have been elaborated to solve the steady state heat distribution model on projectile. The results using Jacobi and Gauss-Seidel are shown identical but the discrepancy iteration cost for each methods is gained. Using Jacobi’s method, the iteration cost is 350 iterations. Meanwhile, using Gauss-Seidel 188 iterations are obtained, faster than the Jacobi’s method. The comparison of the simulation by steady state model and the unsteady state model by a reference is shown satisfying. Moreover, the best candidate for installing MEMS on projectile is observed at pointT(10, 0) which has the lowest temperature for the other points. The temperature using Jacobi and Gauss-Seidel for scenario 1 and 2 atT(10, 0) are 307 and 309 Kelvin respectively.

Azadioxatriangulenium: exploring the effect of a 20 ns fluorescence lifetime in fluorescence anisotropy measurements

NASA Astrophysics Data System (ADS)

Bogh, Sidsel A.; Bora, Ilkay; Rosenberg, Martin; Thyrhaug, Erling; Laursen, Bo W.; Just Sørensen, Thomas

2015-12-01

Azaoxatriangulenium (ADOTA) has been shown to be highly emissive despite a moderate molar absorption coefficient of the primary electronic transition. As a result, the fluorescence lifetime is ~20 ns, longer than all commonly used red fluorescent organic probes. The electronic transitions in ADOTA are highly polarised (r 0  =  0.38), which in combination with the long fluorescence lifetime extents the size-range of biomolecular weights that can be detected in fluorescence polarisation-based experiments. Here, the rotational dynamics of bovine serum albumin (BSA) are monitored with three different ADOTA derivatives, differing only in constitution of the reactive linker. A detailed study of the degree of labelling, the steady-state anisotropy, and the time-resolved anisotropy of the three different ADOTA-BSA conjugates are reported. The fluorescence quantum yields (ϕ fl) of the free dyes in PBS solution are determined to be ~55%, which is reduced to ~20% in the ADOTA-BSA conjugates. Despite the reduction in ϕ fl, a ~20 ns intensity averaged lifetime is maintained, allowing for the rotational dynamics of BSA to be monitored for up to 100 ns. Thus, ADOTA can be used in fluorescence polarisation assays to fill the gap between commonly used organic dyes and the long luminescence lifetime transition metal complexes. This allows for efficient steady-state fluorescence polarisation assays for detecting binding of analytes with molecular weights of up to 100 kDa.

Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion

PubMed Central

Nacarelli, Timothy; Azar, Ashley; Sell, Christian

2014-01-01

The regulation of mitochondrial mass and DNA content involves a complex interaction between mitochondrial DNA replication machinery, functional components of the electron transport chain, selective clearance of mitochondria, and nuclear gene expression. In order to gain insight into cellular responses to mitochondrial stress, we treated human diploid fibroblasts with ethidium bromide at concentrations that induced loss of mitochondrial DNA over a period of 7 days. The decrease in mitochondrial DNA was accompanied by a reduction in steady state levels of the mitochondrial DNA binding protein, TFAM, a reduction in several electron transport chain protein levels, increased mitochondrial and total cellular ROS, and activation of p38 MAPK. However, there was an increase in mitochondrial mass and voltage dependent anion channel levels. In addition, mechanistic target of rapamycin (mTOR) activity, as judged by p70S6K targets, was decreased while steady state levels of p62/SQSTM1 and Parkin were increased. Treatment of cells with rapamycin created a situation in which cells were better able to adapt to the mitochondrial dysfunction, resulting in decreased ROS and increased cell viability but did not prevent the reduction in mitochondrial DNA. These effects may be due to a more efficient flux through the electron transport chain, increased autophagy, or enhanced AKT signaling, coupled with a reduced growth rate. Together, the results suggest that mTOR activity is affected by mitochondrial stress, which may be part of the retrograde signal system required for normal mitochondrial homeostasis. PMID:25104948

Role of spatial inhomogenity in GPCR dimerisation predicted by receptor association-diffusion models

NASA Astrophysics Data System (ADS)

Deshpande, Sneha A.; Pawar, Aiswarya B.; Dighe, Anish; Athale, Chaitanya A.; Sengupta, Durba

2017-06-01

G protein-coupled receptor (GPCR) association is an emerging paradigm with far reaching implications in the regulation of signalling pathways and therapeutic interventions. Recent super resolution microscopy studies have revealed that receptor dimer steady state exhibits sub-second dynamics. In particular the GPCRs, muscarinic acetylcholine receptor M1 (M1MR) and formyl peptide receptor (FPR), have been demonstrated to exhibit a fast association/dissociation kinetics, independent of ligand binding. In this work, we have developed a spatial kinetic Monte Carlo model to investigate receptor homo-dimerisation at a single receptor resolution. Experimentally measured association/dissociation kinetic parameters and diffusion coefficients were used as inputs to the model. To test the effect of membrane spatial heterogeneity on the simulated steady state, simulations were compared to experimental statistics of dimerisation. In the simplest case the receptors are assumed to be diffusing in a spatially homogeneous environment, while spatial heterogeneity is modelled to result from crowding, membrane micro-domains and cytoskeletal compartmentalisation or ‘corrals’. We show that a simple association-diffusion model is sufficient to reproduce M1MR association statistics, but fails to reproduce FPR statistics despite comparable kinetic constants. A parameter sensitivity analysis is required to reproduce the association statistics of FPR. The model reveals the complex interplay between cytoskeletal components and their influence on receptor association kinetics within the features of the membrane landscape. These results constitute an important step towards understanding the factors modulating GPCR organisation.

Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex

PubMed Central

Sage, Jay M.

2014-01-01

GLUT1, the primary glucose transport protein in human erythrocytes [red blood cells (RBCs)], also transports oxidized vitamin C [dehydroascorbic acid (DHA)]. A recent study suggests that RBC GLUT1 transports DHA as its primary substrate and that only a subpopulation of GLUT1 transports sugars. This conclusion is based on measurements of cellular glucose and DHA equilibrium spaces, rather than steady-state transport rates. We have characterized RBC transport of DHA and 3-O-methylglucose (3-OMG), a transported, nonmetabolizable sugar. Steady-state 3-OMG and DHA uptake in the absence of intracellular substrate are characterized by similar Vmax (0.16 ± 0.01 and 0.13 ± 0.02 mmol·l−1·min−1, respectively) and apparent Km (1.4 ± 0.2 and 1.6 ± 0.7 mM, respectively). 3-OMG and DHA compete for uptake, with Ki(app) of 0.7 ± 0.4 and 1.1 ± 0.1 mM, respectively. Uptake measurements using RBC inside-out-membrane vesicles demonstrate that 3-OMG and DHA compete at the cytoplasmic surface of the membrane, with Ki(app) of 0.7 ± 0.1 and 0.6 ± 0.1 mM, respectively. Intracellular 3-OMG stimulates unidirectional uptake of 3-OMG and DHA. These findings indicate that DHA and 3-OMG bind at mutually exclusive sites at exo- and endofacial surfaces of GLUT1 and are transported via the same GLUT1 complex. PMID:24598365

Human erythrocytes transport dehydroascorbic acid and sugars using the same transporter complex.

PubMed

Sage, Jay M; Carruthers, Anthony

2014-05-15

GLUT1, the primary glucose transport protein in human erythrocytes [red blood cells (RBCs)], also transports oxidized vitamin C [dehydroascorbic acid (DHA)]. A recent study suggests that RBC GLUT1 transports DHA as its primary substrate and that only a subpopulation of GLUT1 transports sugars. This conclusion is based on measurements of cellular glucose and DHA equilibrium spaces, rather than steady-state transport rates. We have characterized RBC transport of DHA and 3-O-methylglucose (3-OMG), a transported, nonmetabolizable sugar. Steady-state 3-OMG and DHA uptake in the absence of intracellular substrate are characterized by similar Vmax (0.16 ± 0.01 and 0.13 ± 0.02 mmol·l(-1)·min(-1), respectively) and apparent Km (1.4 ± 0.2 and 1.6 ± 0.7 mM, respectively). 3-OMG and DHA compete for uptake, with Ki(app) of 0.7 ± 0.4 and 1.1 ± 0.1 mM, respectively. Uptake measurements using RBC inside-out-membrane vesicles demonstrate that 3-OMG and DHA compete at the cytoplasmic surface of the membrane, with Ki(app) of 0.7 ± 0.1 and 0.6 ± 0.1 mM, respectively. Intracellular 3-OMG stimulates unidirectional uptake of 3-OMG and DHA. These findings indicate that DHA and 3-OMG bind at mutually exclusive sites at exo- and endofacial surfaces of GLUT1 and are transported via the same GLUT1 complex. Copyright © 2014 the American Physiological Society.

Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency.

PubMed

Taylor, Mark R; Conrad, John A; Wahl, Daniel; O'Brien, Patrick J

2011-07-01

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg(2+) ions for catalysis and that an essential Mg(2+) ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg(2+) changes along the reaction coordinate. At saturating concentrations of ATP and Mg(2+) ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg(2+), the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5'-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Glick, R.E.; Schlagnhaufer, C.D.; Arteca, R.N.

The relationships among O{sub 3}-induced accelerated senescence, induction of ethylene, and changes in specific mRNA and protein levels were investigated in potato (Solanum tuberosum L. cv Norland) plants. When plants were exposed to 0.08 {mu}L L{sup -1} O{sub 3} for 5 h d{sup -1}, steady-state levels of rbcS mRNA declined at least 5-fold in expanding leaves after 3 d of O{sub 3} exposure and ethylene levels increased 6- to 10-fold. The expression of OIP-1, a 1-aminocyclo-propane-1-carboxylate synthase cDNA from potato, correlated with increased production of ethylene and decreased levels of rbcS mRNA in foliage of plants treated with O{sub 3}.more » In plants exposed to 0.30 {mu}L L{sup -1} O{sub 3} for 4 h, rbcS transcript levels were reduced 4-fold, whereas nuclear run-on experiments revealed that rbcS mRNA may be due, in part, to posttranscriptional regulation. The levels of transcripts for other chloroplast proteins, glyceraldehyde-3-phosphate dehydrogenase, and a photosystem II chlorophyll a/b-binding protein decreased in O{sub 3}-treated plants, in parallel with the decrease in rbcS mRNA. The steady-state mRNA level of a cytosolic glyceraldehyde-3-phosphate dehydrogenase increased in O{sub 3}-treated plants. The induction of ethylene and changes in transcript levels preceded visible leaf damage and decreases in ribulose-1,5-biphosphate carboxylase/oxygenase protein levels. 40 refs., 6 figs.« less

Discovery of talatisamine as a novel specific blocker for the delayed rectifier K+ channels in rat hippocampal neurons.

PubMed

Song, M-K; Liu, H; Jiang, H-L; Yue, J-M; Hu, G-Y; Chen, H-Z

2008-08-13

Blocking specific K+ channels has been proposed as a promising strategy for the treatment of neurodegenerative diseases. Using a computational virtual screening approach and electrophysiological testing, we found four Aconitum alkaloids are potent blockers of the delayed rectifier K+ channel in rat hippocampal neurons. In the present study, we first tested the action of the four alkaloids on the voltage-gated K+, Na+ and Ca2+ currents in rat hippocampal neurons, and then identified that talatisamine is a specific blocker for the delayed rectifier K+ channel. External application of talatisamine reversibly inhibited the delayed rectifier K+ current (IK) with an IC50 value of 146.0+/-5.8 microM in a voltage-dependent manner, but exhibited very slight blocking effect on the voltage-gated Na+ and Ca2+ currents even at the high concentration of 1-3 mM. Moreover, talatisamine exerted a significant hyperpolarizing shift of the steady-state activation, but did not influence the steady state inactivation of IK and its recovery from inactivation, suggesting that talatisamine had no allosteric action on IK channel and was a pure blocker binding to the external pore entry of the channel. Our present study made the first discovery of potent and specific IK channel blocker from Aconitum alkaloids. It has been argued that suppressing K+ efflux by blocking IK channel may be favorable for Alzheimer's disease therapy. Talatisamine can therefore be considered as a leading compound worthy of further investigations.

Using steady-state equations for transient flow calculation in natural gas pipelines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maddox, R.N.; Zhou, P.

1984-04-02

Maddox and Zhou have extended their technique for calculating the unsteady-state behavior of straight gas pipelines to complex pipeline systems and networks. After developing the steady-state flow rate and pressure profile for each pipe in the network, analysts can perform the transient-state analysis in the real-time step-wise manner described for this technique.

Simultaneous measurement of glucose transport and utilization in the human brain

PubMed Central

Shestov, Alexander A.; Emir, Uzay E.; Kumar, Anjali; Henry, Pierre-Gilles; Seaquist, Elizabeth R.

2011-01-01

Glucose is the primary fuel for brain function, and determining the kinetics of cerebral glucose transport and utilization is critical for quantifying cerebral energy metabolism. The kinetic parameters of cerebral glucose transport, KMt and Vmaxt, in humans have so far been obtained by measuring steady-state brain glucose levels by proton (1H) NMR as a function of plasma glucose levels and fitting steady-state models to these data. Extraction of the kinetic parameters for cerebral glucose transport necessitated assuming a constant cerebral metabolic rate of glucose (CMRglc) obtained from other tracer studies, such as 13C NMR. Here we present new methodology to simultaneously obtain kinetic parameters for glucose transport and utilization in the human brain by fitting both dynamic and steady-state 1H NMR data with a reversible, non-steady-state Michaelis-Menten model. Dynamic data were obtained by measuring brain and plasma glucose time courses during glucose infusions to raise and maintain plasma concentration at ∼17 mmol/l for ∼2 h in five healthy volunteers. Steady-state brain vs. plasma glucose concentrations were taken from literature and the steady-state portions of data from the five volunteers. In addition to providing simultaneous measurements of glucose transport and utilization and obviating assumptions for constant CMRglc, this methodology does not necessitate infusions of expensive or radioactive tracers. Using this new methodology, we found that the maximum transport capacity for glucose through the blood-brain barrier was nearly twofold higher than maximum cerebral glucose utilization. The glucose transport and utilization parameters were consistent with previously published values for human brain. PMID:21791622

Prediction of dendrite arm spacings in unsteady-and steady-state heat flow of unidirectionally solidified binary alloys

NASA Astrophysics Data System (ADS)

Bouchard, Dominique; Kirkaldy, John S.

1997-08-01

Various theoretical dendrite and cell spacing formulas have been tested against experimental data obtained in unsteady- and steady-state heat flow conditions. An iterative assessment strategy satisfactorily overcomes the circumstances that certain constitutive parameters are inadequately established and/or highly variable and that many of the data sets, in terms of gradients, velocities, and/or cooling rates, are unreliable. The accessed unsteady- and steady-state observations on near-terminal binary alloys for primary and secondary spacings were first examined within conventional power law representations, the deduced exponents and confidence limits for each alloy being tabularly recorded. Through this analysis, it became clear that to achieve predictive generality the many constitutive parameters must be included in a rational way, this being achievable only through extant or new theoretical formulations. However, in the case of primary spacings, all formulas, including our own, failed within the unsteady heat flow algorithm while performing adequately within their steady-state context. An earlier untested, heuristically derived steady-state formula after modification, λ _1 = 120 ( {{16X_0^{{1/2}} G_0 (\\varepsilon σ )T_M D}/{(1 - k)mΔ H G R}} )^{{1/2}} ultimately proved its utility in the unsteady regime, and so it is recommended for purposes of predictions for general terminal alloys. For secondary spacings, a Mullins and Sekerka type formula proved from the start to be adequate in both unsteady- and steady-state heat flows, and so it recommends itself in calibrated form, λ _2 = 12π ( {{4σ }/{X_0 (1 - k)^2 Δ H}( {D/R} )^2 } )^{{1/3}}

Energy cost of walking: solving the paradox of steady state in the presence of variable walking speed.

PubMed

Plasschaert, Frank; Jones, Kim; Forward, Malcolm

2009-02-01

Measurement of the energy cost of walking in children with cerebral palsy is used for baseline and outcome assessment. However, such testing relies on the establishment of steady state that is deemed present when oxygen consumption is stable. This is often assumed when walking speed is constant but in practice, speed can and does vary naturally. Whilst constant speed is achievable on a treadmill, this is often impractical clinically, thus rendering an energy cost test to an element of subjectivity. This paper attempts to address this issue by presenting a new method for calculating energy cost of walking that automatically applies a mathematically defined threshold for steady state within a (non-treadmill) walking trial and then strips out all of the non-steady state events within that trial. The method is compared with a generic approach that does not remove non-steady state data but rather uses an average value over a complete walking trial as is often used in the clinical environment. Both methods were applied to the calculation of several energy cost of walking parameters of self-selected walking speed in a cohort of unimpaired subjects and children with cerebral palsy. The results revealed that both methods were strongly correlated for each parameter but showed systematic significant differences. It is suggested that these differences are introduced by the rejection of non-steady state data that would otherwise have incorrectly been incorporated into the calculation of the energy cost of walking indices during self-selected walking with its inherent speed variation.

Reconstructing metabolic flux vectors from extreme pathways: defining the alpha-spectrum.

PubMed

Wiback, Sharon J; Mahadevan, Radhakrishnan; Palsson, Bernhard Ø

2003-10-07

The move towards genome-scale analysis of cellular functions has necessitated the development of analytical (in silico) methods to understand such large and complex biochemical reaction networks. One such method is extreme pathway analysis that uses stoichiometry and thermodynamic irreversibly to define mathematically unique, systemic metabolic pathways. These extreme pathways form the edges of a high-dimensional convex cone in the flux space that contains all the attainable steady state solutions, or flux distributions, for the metabolic network. By definition, any steady state flux distribution can be described as a nonnegative linear combination of the extreme pathways. To date, much effort has been focused on calculating, defining, and understanding these extreme pathways. However, little work has been performed to determine how these extreme pathways contribute to a given steady state flux distribution. This study represents an initial effort aimed at defining how physiological steady state solutions can be reconstructed from a network's extreme pathways. In general, there is not a unique set of nonnegative weightings on the extreme pathways that produce a given steady state flux distribution but rather a range of possible values. This range can be determined using linear optimization to maximize and minimize the weightings of a particular extreme pathway in the reconstruction, resulting in what we have termed the alpha-spectrum. The alpha-spectrum defines which extreme pathways can and cannot be included in the reconstruction of a given steady state flux distribution and to what extent they individually contribute to the reconstruction. It is shown that accounting for transcriptional regulatory constraints can considerably shrink the alpha-spectrum. The alpha-spectrum is computed and interpreted for two cases; first, optimal states of a skeleton representation of core metabolism that include transcriptional regulation, and second for human red blood cell metabolism under various physiological, non-optimal conditions.

Environment sensitive fluorescent analogue of biologically active oxazoles differentially recognizes human serum albumin and bovine serum albumin: Photophysical and molecular modeling studies

NASA Astrophysics Data System (ADS)

Maiti, Jyotirmay; Biswas, Suman; Chaudhuri, Ankur; Chakraborty, Sandipan; Chakraborty, Sibani; Das, Ranjan

2017-03-01

An environment sensitive fluorophore, 4-(5-(4-(dimethylamino)phenyl)oxazol-2-yl)benzoic acid (DMOBA), that closely mimics biologically active 2,5-disubstituited oxazoles has been designed to probe two homologous serum proteins, human serum albumin (HSA) and bovine serum albumin (BSA) by means of photophysical and molecular modeling studies. This fluorescent analogue exhibits solvent polarity sensitive fluorescence due to an intramolecular charge transfer in the excited state. In comparison to water, the steady state emission spectra of DMOBA in BSA is characterized by a greater blue shift ( 10 nm) and smaller Stokes' shift ( 5980 cm- 1) in BSA than HSA (Stokes'shift 6600 cm- 1), indicating less polar and more hydrophobic environment of the dye in the former than the latter. The dye-protein binding interactions are remarkably stronger for BSA than HSA which is evident from higher value of the association constant for the DMOBA-BSA complex (Ka 5.2 × 106 M- 1) than the DMOBA-HSA complex (Ka 1.0 × 106 M- 1). Fӧrster resonance energy transfer studies revealed remarkably less efficient energy transfer (8%) between the donor tryptophans in BSA and the acceptor DMOBA dye than that (30%) between the single tryptophan moiety in HSA and the dye, which is consistent with a much larger distance between the donor (tryptophan)-acceptor (dye) pair in BSA (34.5 Å) than HSA (25.4 Å). Site specific competitive binding assays have confirmed on the location of the dye in Sudlow's site II of BSA and in Sudlow's site I of HSA, respectively. Molecular modeling studies have shown that the fluorescent analogue is tightly packed in the binding site of BSA due to strong steric complementarity, where, binding of DMOBA to BSA is primarily dictated by the van der Waals and hydrogen bonding interactions. In contrast, in HSA the steric complementarity is less significant and binding is primarily guided by polar interactions and van der Waals interactions appear to be less significant in the formation of the HSA-DMOBA complex. Electrostatic interactions contribute significantly in the binding of DMOBA to HSA (- 2.09 kcal/mol) compared to BSA (- 0.47 kcal/mol). Electrostatic surface potential calculation reveals that the DMOBA binding site within HSA is highly charged compared to BSA.

Calibration of steady-state car-following models using macroscopic loop detector data.

DOT National Transportation Integrated Search

2010-05-01

The paper develops procedures for calibrating the steady-state component of various car following models using : macroscopic loop detector data. The calibration procedures are developed for a number of commercially available : microscopic traffic sim...

40 CFR Appendix A to Subpart S of... - Calibrations, Adjustments and Quality Control

Code of Federal Regulations, 2012 CFR

2012-07-01

... average of the pre-test and post-test ambient background levels shall be compared to the permissible...—Calibrations, Adjustments and Quality Control (I) Steady-State Test Equipment States may opt to use transient emission test equipment for steady-state tests and follow the quality control requirements in paragraph (II...

40 CFR Appendix A to Subpart S of... - Calibrations, Adjustments and Quality Control

Code of Federal Regulations, 2010 CFR

2010-07-01

... average of the pre-test and post-test ambient background levels shall be compared to the permissible...—Calibrations, Adjustments and Quality Control (I) Steady-State Test Equipment States may opt to use transient emission test equipment for steady-state tests and follow the quality control requirements in paragraph (II...

40 CFR Appendix A to Subpart S of... - Calibrations, Adjustments and Quality Control

Code of Federal Regulations, 2011 CFR

2011-07-01

... average of the pre-test and post-test ambient background levels shall be compared to the permissible...—Calibrations, Adjustments and Quality Control (I) Steady-State Test Equipment States may opt to use transient emission test equipment for steady-state tests and follow the quality control requirements in paragraph (II...

40 CFR Appendix A to Subpart S of... - Calibrations, Adjustments and Quality Control

Code of Federal Regulations, 2013 CFR

2013-07-01

... average of the pre-test and post-test ambient background levels shall be compared to the permissible...—Calibrations, Adjustments and Quality Control (I) Steady-State Test Equipment States may opt to use transient emission test equipment for steady-state tests and follow the quality control requirements in paragraph (II...

40 CFR Appendix A to Subpart S of... - Calibrations, Adjustments and Quality Control

Code of Federal Regulations, 2014 CFR

2014-07-01

... average of the pre-test and post-test ambient background levels shall be compared to the permissible...—Calibrations, Adjustments and Quality Control (I) Steady-State Test Equipment States may opt to use transient emission test equipment for steady-state tests and follow the quality control requirements in paragraph (II...

Catalytic reduction of O2 by cytochrome C using a synthetic model of cytochrome C oxidase.

PubMed

Collman, James P; Ghosh, Somdatta; Dey, Abhishek; Decréau, Richard A; Yang, Ying

2009-04-15

Cytochrome c oxidase (CcO) catalyzes the four-electron reduction of oxygen to water, the one-electron reductant Cytochrome c (Cytc) being the source of electrons. Recently we reported a functional model of CcO that electrochemically catalyzes the four-electron reduction of O(2) to H(2)O (Collman et al. Science 2007, 315, 1565). The current paper shows that the same functional CcO model catalyzes the four-electron reduction of O(2) using the actual biological reductant Cytc in a homogeneous solution. Both single and steady-state turnover kinetics studies indicate that O(2) binding is rate-determining and that O-O bond cleavage and electron transfer from reduced Cytc to the oxidized model complex are relatively fast.

Target mediated drug disposition with drug–drug interaction, Part II: competitive and uncompetitive cases

PubMed Central

Jusko, William J.; Schropp, Johannes

2017-01-01

We present competitive and uncompetitive drug–drug interaction (DDI) with target mediated drug disposition (TMDD) equations and investigate their pharmacokinetic DDI properties. For application of TMDD models, quasi-equilibrium (QE) or quasi-steady state (QSS) approximations are necessary to reduce the number of parameters. To realize those approximations of DDI TMDD models, we derive an ordinary differential equation (ODE) representation formulated in free concentration and free receptor variables. This ODE formulation can be straightforward implemented in typical PKPD software without solving any non-linear equation system arising from the QE or QSS approximation of the rapid binding assumptions. This manuscript is the second in a series to introduce and investigate DDI TMDD models and to apply the QE or QSS approximation. PMID:28074396

Regimes of radiative and nonradiative transitions in transport through an electronic system in a photon cavity reaching a steady state

NASA Astrophysics Data System (ADS)

Gudmundsson, Vidar; Jonsson, Thorsteinn H.; Bernodusson, Maria Laura; Abdullah, Nzar Rauf; Sitek, Anna; Goan, Hsi-Sheng; Tang, Chi-Shung; Manolescu, Andrei

2017-01-01

We analyze how a multilevel many-electron system in a photon cavity approaches the steady state when coupled to external leads. When a plunger gate is used to lower cavity photon dressed one- and two-electron states below the bias window defined by the external leads, we can identify one regime with nonradiative transitions dominating the electron transport, and another regime with radiative transitions. Both transitions trap the electrons in the states below the bias bringing the system into a steady state. The order of the two regimes and their relative strength depends on the location of the bias window in the energy spectrum of the system and the initial conditions.

CLASSICAL AREAS OF PHENOMENOLOGY: Temporal behaviour of open-circuit photovoltaic solitons

NASA Astrophysics Data System (ADS)

Zhang, Mei-Zhi; Lu, Ke-Qing; Cheng, Guang-Hua; Li, Ke-Hao; Zhang, Yi-Qi; Zhang, Yu-Hong; Zhang, Yan-Peng

2009-07-01

Based on the time-dependent band-transport model in a photorefractive medium, dark open-circuit photovoltaic (PV) solitons are investigated both theoretically and experimentally. Compared with those of the time-independent models, our theoretical results revealed that quasi-steady-state and steady-state PV solitons can both be obtained. Our results also revealed that when r < 1 (r is the normalized intensity at infinity), the full width at half maximum (FWHM) of solitons decreases monotonically to a constant value; when r > 1, however, the FWHM of solitons first decreases to a minimum before it increases to a constant value. Moreover, the FWHM of steady solitons decreases with increasing intensity ratio for r < 1, and increases with increasing intensity ratio for r > 1. We further observed dark PV solitons in experiments, and recorded their evolution. These results indicated that steady solitons can be observed at low optical power, while quasi-steady-state solitons can only be generated at higher optical power. Good agreement is found between theory and experiment.

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

PubMed

Gibiansky, Leonid; Gibiansky, Ekaterina

2017-10-01

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

Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis*

PubMed Central

Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh; Zaiss, Anne K.; Bunce, Matthew W.; Camire, Rodney M.; Wu, Ling; Evseenko, Denis; Herschman, Harvey R.; Bajaj, Madhu S.; Bajaj, S. Paul

2014-01-01

Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis. PMID:25262870

Comparative Study of the Fatty Acid Binding Process of a New FABP from Cherax quadricarinatus by Fluorescence Intensity, Lifetime and Anisotropy

PubMed Central

Li, Jiayao; Henry, Etienne; Wang, Lanmei; Delelis, Olivier; Wang, Huan; Simon, Françoise; Tauc, Patrick; Brochon, Jean-Claude; Zhao, Yunlong; Deprez, Eric

2012-01-01

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty acids closely parallel their prevalences in the hepatopancreas of C. quadricarinatus as measured under specific diet conditions. PMID:23284658

The Politics of the Steady State

ERIC Educational Resources Information Center

Taylor, Charles

1978-01-01

A steady state society has limits pertaining to population size, non-renewable resources, and production which emits heat or substances into soil, water, or the atmosphere. Respecting these limits means renouncing exponential quantitative growth and accepting a universally available consumption standard. (SW)

Information on estimating local government highway bonds

DOT National Transportation Integrated Search

1973-06-01

The theory of traffic flow following a lane blockage on a multi-lane freeway has been developed. Numerical results have been obtained and are presented both for the steady state case where the traffic density remains constant and the non-steady state...

Mapping current fluctuations of stochastic pumps to nonequilibrium steady states.

PubMed

Rotskoff, Grant M

2017-03-01

We show that current fluctuations in a stochastic pump can be robustly mapped to fluctuations in a corresponding time-independent nonequilibrium steady state. We thus refine a recently proposed mapping so that it ensures equivalence of not only the averages, but also optimal representation of fluctuations in currents and density. Our mapping leads to a natural decomposition of the entropy production in stochastic pumps similar to the "housekeeping" heat. As a consequence of the decomposition of entropy production, the current fluctuations in weakly perturbed stochastic pumps are shown to satisfy a universal bound determined by the steady state entropy production.

Efficiency trade-offs of steady-state methods using FEM and FDM. [iterative solutions for nonlinear flow equations

NASA Technical Reports Server (NTRS)

Gartling, D. K.; Roache, P. J.

1978-01-01

The efficiency characteristics of finite element and finite difference approximations for the steady-state solution of the Navier-Stokes equations are examined. The finite element method discussed is a standard Galerkin formulation of the incompressible, steady-state Navier-Stokes equations. The finite difference formulation uses simple centered differences that are O(delta x-squared). Operation counts indicate that a rapidly converging Newton-Raphson-Kantorovitch iteration scheme is generally preferable over a Picard method. A split NOS Picard iterative algorithm for the finite difference method was most efficient.

Arbitrary Steady-State Solutions with the K-epsilon Model

NASA Technical Reports Server (NTRS)

Rumsey, Christopher L.; Pettersson Reif, B. A.; Gatski, Thomas B.

2006-01-01

Widely-used forms of the K-epsilon turbulence model are shown to yield arbitrary steady-state converged solutions that are highly dependent on numerical considerations such as initial conditions and solution procedure. These solutions contain pseudo-laminar regions of varying size. By applying a nullcline analysis to the equation set, it is possible to clearly demonstrate the reasons for the anomalous behavior. In summary, the degenerate solution acts as a stable fixed point under certain conditions, causing the numerical method to converge there. The analysis also suggests a methodology for preventing the anomalous behavior in steady-state computations.

Response of a small-turboshaft-engine compression system to inlet temperature distortion

NASA Technical Reports Server (NTRS)

Biesiadny, T. J.; Klann, G. A.; Little, J. K.

1984-01-01

An experimental investigation was conducted into the response of a small-turboshaft-engine compression system to steady-state and transient inlet temperature distortions. Transient temperature ramps range from less than 100 K/sec to above 610 K/sec and generated instantaneous temperatures to 420 K above ambient. Steady-state temperature distortion levels were limited by the engine hardware temperature list. Simple analysis of the steady-state distortion data indicated that a particle separator at the engine inlet permitted higher levels of temperature distortion before onset of compressor surge than would be expected without the separator.

Mean field treatment of heterogeneous steady state kinetics

NASA Astrophysics Data System (ADS)

Geva, Nadav; Vaissier, Valerie; Shepherd, James; Van Voorhis, Troy

2017-10-01

We propose a method to quickly compute steady state populations of species undergoing a set of chemical reactions whose rate constants are heterogeneous. Using an average environment in place of an explicit nearest neighbor configuration, we obtain a set of equations describing a single fluctuating active site in the presence of an averaged bath. We apply this Mean Field Steady State (MFSS) method to a model of H2 production on a disordered surface for which the activation energy for the reaction varies from site to site. The MFSS populations quantitatively reproduce the KMC results across the range of rate parameters considered.

Nonthermal steady states after an interaction quench in the Falicov-Kimball model.

PubMed

Eckstein, Martin; Kollar, Marcus

2008-03-28

We present the exact solution of the Falicov-Kimball model after a sudden change of its interaction parameter using nonequilibrium dynamical mean-field theory. For different interaction quenches between the homogeneous metallic and insulating phases the system relaxes to a nonthermal steady state on time scales on the order of variant Planck's over 2pi/bandwidth, showing collapse and revival with an approximate period of h/interaction if the interaction is large. We discuss the reasons for this behavior and provide a statistical description of the final steady state by means of generalized Gibbs ensembles.

Steady state phosphorus mass balance model during hemodialysis based on a pseudo one-compartment kinetic model.

PubMed

Leypoldt, John K; Agar, Baris U; Akonur, Alp; Gellens, Mary E; Culleton, Bruce F

2012-11-01

Mathematical models of phosphorus kinetics and mass balance during hemodialysis are in early development. We describe a theoretical phosphorus steady state mass balance model during hemodialysis based on a novel pseudo one-compartment kinetic model. The steady state mass balance model accounted for net intestinal absorption of phosphorus and phosphorus removal by both dialysis and residual kidney function. Analytical mathematical solutions were derived to describe time-dependent intradialytic and interdialytic serum phosphorus concentrations assuming hemodialysis treatments were performed symmetrically throughout a week. Results from the steady state phosphorus mass balance model are described for thrice weekly hemodialysis treatment prescriptions only. The analysis predicts 1) a minimal impact of dialyzer phosphorus clearance on predialysis serum phosphorus concentration using modern, conventional hemodialysis technology, 2) variability in the postdialysis-to-predialysis phosphorus concentration ratio due to differences in patient-specific phosphorus mobilization, and 3) the importance of treatment time in determining the predialysis serum phosphorus concentration. We conclude that a steady state phosphorus mass balance model can be developed based on a pseudo one-compartment kinetic model and that predictions from this model are consistent with previous clinical observations. The predictions from this mass balance model are theoretical and hypothesis-generating only; additional prospective clinical studies will be required for model confirmation.

Dynamical modelling of haematopoiesis: an integrated view over the system in homeostasis and under perturbation.

PubMed

Manesso, Erica; Teles, José; Bryder, David; Peterson, Carsten

2013-03-06

A very high number of different types of blood cells must be generated daily through a process called haematopoiesis in order to meet the physiological requirements of the organism. All blood cells originate from a population of relatively few haematopoietic stem cells residing in the bone marrow, which give rise to specific progenitors through different lineages. Steady-state dynamics are governed by cell division and commitment rates as well as by population sizes, while feedback components guarantee the restoration of steady-state conditions. In this study, all parameters governing these processes were estimated in a computational model to describe the haematopoietic hierarchy in adult mice. The model consisted of ordinary differential equations and included negative feedback regulation. A combination of literature data, a novel divide et impera approach for steady-state calculations and stochastic optimization allowed one to reduce possible configurations of the system. The model was able to recapitulate the fundamental steady-state features of haematopoiesis and simulate the re-establishment of steady-state conditions after haemorrhage and bone marrow transplantation. This computational approach to the haematopoietic system is novel and provides insight into the dynamics and the nature of possible solutions, with potential applications in both fundamental and clinical research.

Can high fields save the tokamak? The challenge of steady-state operation for low cost compact reactors

NASA Astrophysics Data System (ADS)

Freidberg, Jeffrey; Dogra, Akshunna; Redman, William; Cerfon, Antoine

2016-10-01

The development of high field, high temperature superconductors is thought to be a game changer for the development of fusion power based on the tokamak concept. We test the validity of this assertion for pilot plant scale reactors (Q 10) for two different but related missions: pulsed operation and steady-state operation. Specifically, we derive a set of analytic criteria that determines the basic design parameters of a given fusion reactor mission. As expected there are far more constraints than degrees of freedom in any given design application. However, by defining the mission of the reactor under consideration, we have been able to determine the subset of constraints that drive the design, and calculate the values for the key parameters characterizing the tokamak. Our conclusions are as follows: 1) for pulsed reactors, high field leads to more compact designs and thus cheaper reactors - high B is the way to go; 2) steady-state reactors with H-mode like transport are large, even with high fields. The steady-state constraint is hard to satisfy in compact designs - high B helps but is not enough; 3) I-mode like transport, when combined with high fields, yields relatively compact steady-state reactors - why is there not more research on this favorable transport regime?

Steady-state hydrodynamic instabilities of active liquid crystals: hybrid lattice Boltzmann simulations.

PubMed

Marenduzzo, D; Orlandini, E; Cates, M E; Yeomans, J M

2007-09-01

We report hybrid lattice Boltzmann (HLB) simulations of the hydrodynamics of an active nematic liquid crystal sandwiched between confining walls with various anchoring conditions. We confirm the existence of a transition between a passive phase and an active phase, in which there is spontaneous flow in the steady state. This transition is attained for sufficiently "extensile" rods, in the case of flow-aligning liquid crystals, and for sufficiently "contractile" ones for flow-tumbling materials. In a quasi-one-dimensional geometry, deep in the active phase of flow-aligning materials, our simulations give evidence of hysteresis and history-dependent steady states, as well as of spontaneous banded flow. Flow-tumbling materials, in contrast, rearrange themselves so that only the two boundary layers flow in steady state. Two-dimensional simulations, with periodic boundary conditions, show additional instabilities, with the spontaneous flow appearing as patterns made up of "convection rolls." These results demonstrate a remarkable richness (including dependence on anchoring conditions) in the steady-state phase behavior of active materials, even in the absence of external forcing; they have no counterpart for passive nematics. Our HLB methodology, which combines lattice Boltzmann for momentum transport with a finite difference scheme for the order parameter dynamics, offers a robust and efficient method for probing the complex hydrodynamic behavior of active nematics.

Stable long-term blood formation by stem cells in murine steady-state hematopoiesis.

PubMed

Zavidij, Oksana; Ball, Claudia R; Herbst, Friederike; Oppel, Felix; Fessler, Sylvia; Schmidt, Manfred; von Kalle, Christof; Glimm, Hanno

2012-09-01

Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady-state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady-state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification-mediated-polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV-injected animals for up to 24 months, indicating stable marking of murine steady-state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long-term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short-lived progenitor cells. Myelosuppressive treatment of mice prior to LV-injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady-state hematopoiesis. Copyright © 2012 AlphaMed Press.

High fidelity quasi steady-state aerodynamic model effects on race vehicle performance predictions using multi-body simulation

NASA Astrophysics Data System (ADS)

Mohrfeld-Halterman, J. A.; Uddin, M.

2016-07-01

We described in this paper the development of a high fidelity vehicle aerodynamic model to fit wind tunnel test data over a wide range of vehicle orientations. We also present a comparison between the effects of this proposed model and a conventional quasi steady-state aerodynamic model on race vehicle simulation results. This is done by implementing both of these models independently in multi-body quasi steady-state simulations to determine the effects of the high fidelity aerodynamic model on race vehicle performance metrics. The quasi steady state vehicle simulation is developed with a multi-body NASCAR Truck vehicle model, and simulations are conducted for three different types of NASCAR race tracks, a short track, a one and a half mile intermediate track, and a higher speed, two mile intermediate race track. For each track simulation, the effects of the aerodynamic model on handling, maximum corner speed, and drive force metrics are analysed. The accuracy of the high-fidelity model is shown to reduce the aerodynamic model error relative to the conventional aerodynamic model, and the increased accuracy of the high fidelity aerodynamic model is found to have realisable effects on the performance metric predictions on the intermediate tracks resulting from the quasi steady-state simulation.

System and method for generating steady state confining current for a toroidal plasma fusion reactor

DOEpatents

Fisch, Nathaniel J.

1981-01-01

A system for generating steady state confining current for a toroidal plasma fusion reactor providing steady-state generation of the thermonuclear power. A dense, hot toroidal plasma is initially prepared with a confining magnetic field with toroidal and poloidal components. Continuous wave RF energy is injected into said plasma to establish a spectrum of traveling waves in the plasma, where the traveling waves have momentum components substantially either all parallel, or all anti-parallel to the confining magnetic field. The injected RF energy is phased to couple to said traveling waves with both a phase velocity component and a wave momentum component in the direction of the plasma traveling wave components. The injected RF energy has a predetermined spectrum selected so that said traveling waves couple to plasma electrons having velocities in a predetermined range .DELTA.. The velocities in the range are substantially greater than the thermal electron velocity of the plasma. In addition, the range is sufficiently broad to produce a raised plateau having width .DELTA. in the plasma electron velocity distribution so that the plateau electrons provide steady-state current to generate a poloidal magnetic field component sufficient for confining the plasma. In steady state operation of the fusion reactor, the fusion power density in the plasma exceeds the power dissipated in the plasma.