Proteins feel more than they see : Fine-tuning of binding affinity by properties of the non-interacting surface

NARCIS (Netherlands)

Kastritis, Panagiotis L.|info:eu-repo/dai/nl/315886668; Garcia Lopes Maia Rodrigues, João|info:eu-repo/dai/nl/330827391; Folkers, Gert E.|info:eu-repo/dai/nl/162277202; Boelens, Rolf|info:eu-repo/dai/nl/070151407; Bonvin, Alexandre M J J|info:eu-repo/dai/nl/113691238

2014-01-01

Protein-protein complexes orchestrate most cellular processes such as transcription, signal transduction and apoptosis. The factors governing their affinity remain elusive however, especially when it comes to describing dissociation rates (koff). Here we demonstrate that, next to direct

TNU-9 Zeolite: Aluminum Distribution and Extra-Framework Sites of Divalent Cations

Czech Academy of Sciences Publication Activity Database

Karcz, R.; Dědeček, Jiří; Supronowicz, B.; Thomas, Haunani M.; Klein, Petr; Tabor, Edyta; Sazama, Petr; Pashková, Veronika; Sklenák, Štěpán

2017-01-01

Roč. 23, JUL 2017 (2017), s. 8857-8870 ISSN 1521-3765 R&D Projects: GA ČR(CZ) GA15-14007S; GA MŠk(CZ) LM2015073 Grant - others:Ga MŠk(CZ) LM2015070 Institutional support: RVO:61388955 Keywords : Aluminum * Cobalt * Density functional calculations * Structure elucidation * Zeolites Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry

Urethral advancement in hypospadias with a distal division of the corpus spongiosum: outcome in 158 cases.

Science.gov (United States)

Thiry, S; Gorduza, D; Mouriquand, P

2014-06-01

Outcome of urethral mobilization and advancement (Koff procedure) in hypospadias with a distal division of the corpus spongiosum and redo cases with distal urethral failure. From January 1999 to November 2012, 158 children with a distal hypospadias (115 primary cases and 43 redo cases) underwent surgical repair using the Koff technique with a median age at surgery of 21 months (range, 12-217 months). Mean follow-up was 19 months (median, 14 months). Thirty patients (19%) presented with a complication (13.9% in primary cases and 32.5% in redo surgery) mostly at the beginning of our experience. Meatal stenosis was the most common one (3.5% in primary case, 6% overall). Ventral curvature (>10°), which is considered as a possible long-term iatrogenic complication of the Koff procedure, was not found in patients with fully grown penis except in one redo patient who had, retrospectively, an inadequate indication for this type of repair. Of 158 patients, 33 reached the age of puberty (>14 years old) with a mean follow-up of 34 months, only one presented with a significant ventral curvature. Urethral mobilization and advancement is a reasonable alternative for anterior hypospadias and distal fistula repair in selected cases. It has two major advantages compared to other techniques: it avoids any urethroplasty with non-urethral tissue and eliminates dysplastic tissues located beyond the division of the corpus spongiosum, which may not grow at the same pace as the rest of the penis. Significant iatrogenic curvature in fully grown penis is not supported by this series. Copyright © 2013 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Stress: Specific Life Events in the Teaching Profession.

Science.gov (United States)

Martray, Carl R.; Adams, Ronald D.

This study examined the greatest stressors in teaching situations that affect teachers, and how these events vary for groups of elementary, middle, and secondary school teachers. The list of possibly stressful situations was taken from the Teaching Events Stress Inventory (TESI), developed by Cichon and Koff in 1978. Data were collected from…

Improving estimation of kinetic parameters in dynamic force spectroscopy using cluster analysis

Science.gov (United States)

Yen, Chi-Fu; Sivasankar, Sanjeevi

2018-03-01

Dynamic Force Spectroscopy (DFS) is a widely used technique to characterize the dissociation kinetics and interaction energy landscape of receptor-ligand complexes with single-molecule resolution. In an Atomic Force Microscope (AFM)-based DFS experiment, receptor-ligand complexes, sandwiched between an AFM tip and substrate, are ruptured at different stress rates by varying the speed at which the AFM-tip and substrate are pulled away from each other. The rupture events are grouped according to their pulling speeds, and the mean force and loading rate of each group are calculated. These data are subsequently fit to established models, and energy landscape parameters such as the intrinsic off-rate (koff) and the width of the potential energy barrier (xβ) are extracted. However, due to large uncertainties in determining mean forces and loading rates of the groups, errors in the estimated koff and xβ can be substantial. Here, we demonstrate that the accuracy of fitted parameters in a DFS experiment can be dramatically improved by sorting rupture events into groups using cluster analysis instead of sorting them according to their pulling speeds. We test different clustering algorithms including Gaussian mixture, logistic regression, and K-means clustering, under conditions that closely mimic DFS experiments. Using Monte Carlo simulations, we benchmark the performance of these clustering algorithms over a wide range of koff and xβ, under different levels of thermal noise, and as a function of both the number of unbinding events and the number of pulling speeds. Our results demonstrate that cluster analysis, particularly K-means clustering, is very effective in improving the accuracy of parameter estimation, particularly when the number of unbinding events are limited and not well separated into distinct groups. Cluster analysis is easy to implement, and our performance benchmarks serve as a guide in choosing an appropriate method for DFS data analysis.

Myofilament calcium sensitivity: Role in regulation of in vivo cardiac contraction and relaxation

Directory of Open Access Journals (Sweden)

Jae-Hoon Chung

2016-12-01

Full Text Available Myofilament calcium sensitivity is an often-used indicator of cardiac muscle function, often assessed in disease states such as hypertrophic cardiomyopathy (HCM and dilated cardiomyopathy (DCM. While calcium sensitivity measurement provides important insights into the mechanical force-generating capability of a muscle at steady-state, the dynamic behavior of the muscle cannot be sufficiently assessed with a force-pCa curve alone. The dissociation constant (Kd of the force-pCa curve depends on the ratio of the apparent on-rate (kon and apparent off-rate (koff of calcium on TnC and as a stand-alone parameter cannot provide an accurate depiction of the dynamic contraction and relaxation behavior without the additional quantification of kon or koff, or actually measuring dynamic twitch kinetics in an intact muscle. In this review, we examine the effect of length, frequency, and beta-adrenergic stimulation on myofilament calcium sensitivity and dynamic contraction, the effect of membrane permeabilization on calcium sensitivity, and the dynamic consequences of various myofilament protein mutations with potential implications in contractile and relaxation behavior.

Crème de la crème / Joel Sang

Index Scriptorium Estoniae

Sang, Joel, 1950-

2011-01-01

Auhinnažürii esimees iseloomustab Eesti Kultuurkapitali kirjandusauhindade laureaate: Indrek Koff (luule), Maarja Kangro (proosa), Urmas Vadi (näitekirjandus), Toomas Haug (esseistika), Lauri Sommer (vabaauhind), Amar Annus (ilukirjanduslik tõlge võõrkeelest eesti keelde), Ilmar Lehtpere (ilukirjanduslik tõlge eesti keelest võõrkeelde), Kristiina Kass (lastekirjandus), Sergei Issakov (venekeelse autori kirjandusauhind) ja Mart Velsker (artikliauhind)

Täägid paradiisis / Colin MacCabe ; tõlk. Indrek Koff

Index Scriptorium Estoniae

MacCabe, Colin

1999-01-01

Mängufilm "Peenike punane joon" ("The Thin Red Line") : režissöör ja stsenarist James Jonesi romaani põhjal Terrence Malick : Ameerika Ühendriigid 1998. Lisa : Terrence Malickþi elu- ja loominguloolised andmed

Characterization of plasma protein binding dissociation with online SPE-HPLC

OpenAIRE

Li, Ping; Fan, Yiran; Wang, Yunlong; Lu, Yaxin; Yin, Zheng

2015-01-01

A novel parameter of relative recovery (Rre) was defined and determined by online SPE-HPLC to characterize plasma protein binding (PPB) kinetics of highly plasma binding drugs. The proportional relationship of Rre with koff of PPB has been established with a new SPE model. A rapid, easy to use method could potentially be used to categorize PK properties of the drug candidates in the decision process of drug discovery and development.

Tallinna Ülikooli kirjandusauhinna nominendid tutvustavad oma loomingut / Krista Männa

Index Scriptorium Estoniae

Männa, Krista

2007-01-01

13. märtsil toimub Uus-Sadama 5 maja aatriumis autorite õhtu "Kell kuus kirjandusest", kus oma loomingut tutvustavad Tallinna Ülikooli kirjandusauhindade nominendid. Nominentideks on Kätlin Kaldmaa, Jan Kaus, Indrek Koff, Hasso Krull, Deniss Kuzmin, Kirill Maslov, Ülar Ploom, Nadežda Ptšelovodova, Daniele Monticelli ja Kaia Sisask. Kirjandusauhinna laureaadid kuulutatakse välja 16. märtsil Tallinna Ülikooli aastapäeva aktusel

Ühildamatud mälestused? / Jean-Pierre Minaudier ; tõlk. Indrek Koff

Index Scriptorium Estoniae

Minaudier, Jean-Pierre

2006-01-01

Teise maailmasõja mäletamise erinevustest Prantsusmaal ja Eestis. Artikli aluseks on autori ettekanne TLÜ Eesti Humanitaarinstituudi ja Prantsuse Kultuurikeskuse poolt korraldatud rahvusvahelisel konverentsil "Ajalugu ja mälu. Teise maailmasõja pärandid" 8. oktoobril 2005 Tallinnas

Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase. A single-molecule view of the transcription cycle.

Science.gov (United States)

Skinner, Gary M; Baumann, Christoph G; Quinn, Diana M; Molloy, Justin E; Hoggett, James G

2004-01-30

A single-molecule transcription assay has been developed that allows, for the first time, the direct observation of promoter binding, initiation, and elongation by a single RNA polymerase (RNAP) molecule in real-time. To promote DNA binding and transcription initiation, a DNA molecule tethered between two optically trapped beads was held near a third immobile surface bead sparsely coated with RNAP. By driving the optical trap holding the upstream bead with a triangular oscillation while measuring the position of both trapped beads, we observed the onset of promoter binding, promoter escape (productive initiation), and processive elongation by individual RNAP molecules. After DNA template release, transcription re-initiation on the same DNA template is possible; thus, multiple enzymatic turnovers by an individual RNAP molecule can be observed. Using bacteriophage T7 RNAP, a commonly used RNAP paradigm, we observed the association and dissociation (k(off)= 2.9 s(-1)) of T7 RNAP and promoter DNA, the transition to the elongation mode (k(for) = 0.36 s(-1)), and the processive synthesis (k(pol) = 43 nt s(-1)) and release of a gene-length RNA transcript ( approximately 1200 nt). The transition from initiation to elongation is much longer than the mean lifetime of the binary T7 RNAP-promoter DNA complex (k(off) > k(for)), identifying a rate-limiting step between promoter DNA binding and promoter escape.

Identifying microRNAs that Regulate Neuroblastoma Cell Differentiation

Science.gov (United States)

2015-10-01

in Figure 3D -G, comparing to the control cells, more cells treated with miR-449a mimic or RA are negative for BrDU staining, which indicates that...predicted as miR-449a tar- gets decrease and increase in expression. The empiri- cal density curves in Fig- ure 3D further show the difference in the...Sang N, Druck T, Veron- ese ML, Allen SL, Chiorazzi N, Koff A, Heubner K, Croce CM, et al. Chromosomal mapping of members of the cdc2 family of

Magnetoresistive sensors for measurements of DNA hybridization kinetics - effect of TINA modifications

DEFF Research Database (Denmark)

Rizzi, Giovanni; Dufva, Martin; Hansen, Mikkel Fougt

2017-01-01

We present the use of magnetoresistive sensors integrated in a microfluidic system for real-time studies of the hybridization kinetics of DNA labeled with magnetic nanoparticles to an array of surface-tethered probes. The nanoparticles were magnetized by the magnetic field from the sensor current....... A local negative reference ensured that only the specific binding signal was measured. Analysis of the real-time hybridization using a two-compartment model yielded both the association and dissociation constants kon, and koff. The effect of probe modifications with ortho-Twisted Intercalating Nucleic...

Postnatal monitoring of prenatal diagnosed hydro nephrosis

International Nuclear Information System (INIS)

Bueva, A.; Stefanov, S.; Palashev, Y.

2011-01-01

Ultrasound has revolutionized pediatric nephrology in the last decades and has a major impact on management and treatment of several children's kidneys diseases. Hydronephrosis is the most common anomaly in childhood. Progress in fetal imaging in the last years has a important impact in diagnosing congenital hydronephrosis. The current study try to establish authors opinion on problem over coming in the every day practice in pediatric nephrologist - is surgery mandatory in high grade pediatric hydronephrosis. A discussion is undergoing in the literature, following communications that high grade hydronephrosis tend spontaneously to regress in more that 65% according to Koff (2000, 2008)

Modeling neutralization kinetics of HIV by broadly neutralizing monoclonal antibodies in genital secretions coating the cervicovaginal mucosa.

Directory of Open Access Journals (Sweden)

Scott A McKinley

Full Text Available Eliciting broadly neutralizing antibodies (bnAb in cervicovaginal mucus (CVM represents a promising "first line of defense" strategy to reduce vaginal HIV transmission. However, it remains unclear what levels of bnAb must be present in CVM to effectively reduce infection. We approached this complex question by modeling the dynamic tally of bnAb coverage on HIV. This analysis introduces a critical, timescale-dependent competition: to protect, bnAb must accumulate at sufficient stoichiometry to neutralize HIV faster than virions penetrate CVM and reach target cells. We developed a model that incorporates concentrations and diffusivities of HIV and bnAb in semen and CVM, kinetic rates for binding (kon and unbinding (koff of select bnAb, and physiologically relevant thicknesses of CVM and semen layers. Comprehensive model simulations lead to robust conclusions about neutralization kinetics in CVM. First, due to the limited time virions in semen need to penetrate CVM, substantially greater bnAb concentrations than in vitro estimates must be present in CVM to neutralize HIV. Second, the model predicts that bnAb with more rapid kon, almost independent of koff, should offer greater neutralization potency in vivo. These findings suggest the fastest arriving virions at target cells present the greatest likelihood of infection. It also implies the marked improvements in in vitro neutralization potency of many recently discovered bnAb may not translate to comparable reduction in the bnAb dose needed to confer protection against initial vaginal infections. Our modeling framework offers a valuable tool to gaining quantitative insights into the dynamics of mucosal immunity against HIV and other infectious diseases.

Modeling neutralization kinetics of HIV by broadly neutralizing monoclonal antibodies in genital secretions coating the cervicovaginal mucosa.

Science.gov (United States)

McKinley, Scott A; Chen, Alex; Shi, Feng; Wang, Simi; Mucha, Peter J; Forest, M Gregory; Lai, Samuel K

2014-01-01

Eliciting broadly neutralizing antibodies (bnAb) in cervicovaginal mucus (CVM) represents a promising "first line of defense" strategy to reduce vaginal HIV transmission. However, it remains unclear what levels of bnAb must be present in CVM to effectively reduce infection. We approached this complex question by modeling the dynamic tally of bnAb coverage on HIV. This analysis introduces a critical, timescale-dependent competition: to protect, bnAb must accumulate at sufficient stoichiometry to neutralize HIV faster than virions penetrate CVM and reach target cells. We developed a model that incorporates concentrations and diffusivities of HIV and bnAb in semen and CVM, kinetic rates for binding (kon) and unbinding (koff) of select bnAb, and physiologically relevant thicknesses of CVM and semen layers. Comprehensive model simulations lead to robust conclusions about neutralization kinetics in CVM. First, due to the limited time virions in semen need to penetrate CVM, substantially greater bnAb concentrations than in vitro estimates must be present in CVM to neutralize HIV. Second, the model predicts that bnAb with more rapid kon, almost independent of koff, should offer greater neutralization potency in vivo. These findings suggest the fastest arriving virions at target cells present the greatest likelihood of infection. It also implies the marked improvements in in vitro neutralization potency of many recently discovered bnAb may not translate to comparable reduction in the bnAb dose needed to confer protection against initial vaginal infections. Our modeling framework offers a valuable tool to gaining quantitative insights into the dynamics of mucosal immunity against HIV and other infectious diseases.

Quantification of cerebral nicotinic acetylcholine receptors by PET using 2-F-18 fluoro-A-85380 and the multi-injection approach

Energy Technology Data Exchange (ETDEWEB)

Gallezot, J D; Bottlaender, M A; Delforge, J; Valette, H; Saba, W; Dolle, F; Coulon, C M; Ottaviani, M P; Hinnen, F; Syrota, A [CEA, DSV, Serv Hosp Frederic Joliot, I2BM, F-91401 Orsay (France); Gregoire, M C [CEA, Serv Hosp Frederic Joliot, CNRS, URA 2210, F-91401 Orsay (France)

2008-07-01

The multi-injection approach was used to study in vivo interactions between {alpha}4{beta}2 nicotinic acetylcholine receptors and 2-[{sup 18}F] fluoro-A-85380 in baboons. The ligand kinetics was modeled by the usual nonlinear compartment model composed of three compartments (arterial plasma, free and specifically bound ligand in tissue). Arterial blood samples were collected to generate a metabolite-corrected plasma input function. The experimental protocol, which consisted of three injections of labeled or unlabeled ligand, was aiming at identifying all parameters in one experiment. Various parameters, including B'max (the binding sites density) and K{sub d}V{sub R} (the apparent in vivo affinity of 2-[{sup 18}F]fluoro-A-85380) could then be estimated in thalamus and in several receptor-poor regions. B'max estimate was 3.0 {+-} 0.3 pmol/ml in thalamus, and ranged from 0.25 to 1.58 pmol/ml in extra-thalamic regions. Although K{sub d}V{sub R} could be precisely estimated, the association and dissociation rate constants kon/V{sub R} and koff could not be identified separately. A second protocol was then used to estimate koff more precisely in the thalamus. Having estimated all model parameters, we performed simulations of 2-[{sup 18}F]fluoro-A-85380 kinetics to test equilibrium hypotheses underlying simplified approaches. These showed that a pseudo-equilibrium is quickly reached between the free and bound compartments, a favorable situation to apply Logan graphical analysis. In contrast, the pseudo-equilibrium between the plasma and free compartments is only reached after several hours. The ratio of radioligand concentration in these two compartments then overestimates the true equilibrium value, an unfavorable situation to estimate distribution volumes from late images after a bolus injection. (authors)

Quantification of cerebral nicotinic acetylcholine receptors by PET using 2-F-18 fluoro-A-85380 and the multi-injection approach

Energy Technology Data Exchange (ETDEWEB)

Gallezot, J.D.; Bottlaender, M.A.; Delforge, J.; Valette, H.; Saba, W.; Dolle, F.; Coulon, C.M.; Ottaviani, M.P.; Hinnen, F.; Syrota, A. [CEA, DSV, Serv Hosp Frederic Joliot, I2BM, F-91401 Orsay (France); Gregoire, M.C. [CEA, Serv Hosp Frederic Joliot, CNRS, URA 2210, F-91401 Orsay (France)

2008-07-01

The multi-injection approach was used to study in vivo interactions between {alpha}4{beta}2 nicotinic acetylcholine receptors and 2-[{sup 18}F] fluoro-A-85380 in baboons. The ligand kinetics was modeled by the usual nonlinear compartment model composed of three compartments (arterial plasma, free and specifically bound ligand in tissue). Arterial blood samples were collected to generate a metabolite-corrected plasma input function. The experimental protocol, which consisted of three injections of labeled or unlabeled ligand, was aiming at identifying all parameters in one experiment. Various parameters, including B'max (the binding sites density) and K{sub d}V{sub R} (the apparent in vivo affinity of 2-[{sup 18}F]fluoro-A-85380) could then be estimated in thalamus and in several receptor-poor regions. B'max estimate was 3.0 {+-} 0.3 pmol/ml in thalamus, and ranged from 0.25 to 1.58 pmol/ml in extra-thalamic regions. Although K{sub d}V{sub R} could be precisely estimated, the association and dissociation rate constants kon/V{sub R} and koff could not be identified separately. A second protocol was then used to estimate koff more precisely in the thalamus. Having estimated all model parameters, we performed simulations of 2-[{sup 18}F]fluoro-A-85380 kinetics to test equilibrium hypotheses underlying simplified approaches. These showed that a pseudo-equilibrium is quickly reached between the free and bound compartments, a favorable situation to apply Logan graphical analysis. In contrast, the pseudo-equilibrium between the plasma and free compartments is only reached after several hours. The ratio of radioligand concentration in these two compartments then overestimates the true equilibrium value, an unfavorable situation to estimate distribution volumes from late images after a bolus injection. (authors)

Futuristic stories older than might appear: origin of ideas of science fiction screenplays

Directory of Open Access Journals (Sweden)

Carlos Alberto Machado

2013-12-01

Full Text Available The paper discusses the origin of the ideas of most movie scripts modern science fiction, and literaty concepts such as soft and hard, also present in the film. Pointed out the origin of these scripts mostly in the 1950s, 1960s and 1970s, they considered fertile periods in foreign science fiction literature. Also discusses about the casual predictions of the authors of this genre that end up bringing their ideas to contemporary unreasonably, but exciting, leading the media to call them visionary means. Some authors like Carrière, Xavier, Bez, Koff and Comparato assist in corroborating these ideas. Thus, the reader is led to reflect on the historical origin of these ideas.

Ajaloolased ja teised : ajaloolaste rollist Prantsusmaa avalikes arutlustes / Gérard Noiriel, Nicolas Offenstadt ; tlk. Indrek Koff

Index Scriptorium Estoniae

Noiriel, Gérard

2007-01-01

Artikli aluseks on ettekanne konverentsilt "Ajalugu ja poliitika. Mineviku kasutamisest ja ärakasutamisest" (Tallinn, 14. okt. 2006). Lisad: Ajaloo Avaliku Kasutamise Valvekomitee manifest (Vastu võetud 17. juunil 2005) ; Üleskutse valvsusele seoses ajaloo avaliku kasutamisega. Lk. 128-132

Image Restoration and Analysis of Influenza Virions Binding to Membrane Receptors Reveal Adhesion-Strengthening Kinetics.

Directory of Open Access Journals (Sweden)

Donald W Lee

Full Text Available With the development of single-particle tracking (SPT microscopy and host membrane mimics called supported lipid bilayers (SLBs, stochastic virus-membrane binding interactions can be studied in depth while maintaining control over host receptor type and concentration. However, several experimental design challenges and quantitative image analysis limitations prevent the widespread use of this approach. One main challenge of SPT studies is the low signal-to-noise ratio of SPT videos, which is sometimes inevitable due to small particle sizes, low quantum yield of fluorescent dyes, and photobleaching. These situations could render current particle tracking software to yield biased binding kinetic data caused by intermittent tracking error. Hence, we developed an effective image restoration algorithm for SPT applications called STAWASP that reveals particles with a signal-to-noise ratio of 2.2 while preserving particle features. We tested our improvements to the SPT binding assay experiment and imaging procedures by monitoring X31 influenza virus binding to α2,3 sialic acid glycolipids. Our interests lie in how slight changes to the peripheral oligosaccharide structures can affect the binding rate and residence times of viruses. We were able to detect viruses binding weakly to a glycolipid called GM3, which was undetected via assays such as surface plasmon resonance. The binding rate was around 28 folds higher when the virus bound to a different glycolipid called GD1a, which has a sialic acid group extending further away from the bilayer surface than GM3. The improved imaging allowed us to obtain binding residence time distributions that reflect an adhesion-strengthening mechanism via multivalent bonds. We empirically fitted these distributions using a time-dependent unbinding rate parameter, koff, which diverges from standard treatment of koff as a constant. We further explain how to convert these models to fit ensemble-averaged binding data

Prantsuse ühiskonna väljavaateid pärast valimisi / Jean-Pierre Minaudier ; tõlk. Indrek Koff

Index Scriptorium Estoniae

Minaudier, Jean-Pierre

2007-01-01

Kevadel toimunud presidendi- ja parlamendivalimistest Prantsusmaal. Prantsuse ühiskonna poliitilisest, majanduslikust ja kultuurilisest arengust. Autor peab positiivseteks tendentsideks Prantsusmaa poliitilises arengus Rahvusrinde populaarsuse langust ning Nicolas Sarkozy valimist presidendiks

Ökoloogia ja keskkonnateaduste doktorikool : täitus aasta projekti käivitumisest TLÜs / Tiiu Koff

Index Scriptorium Estoniae

Koff, Tiiu, 1955-

2006-01-01

Koostöös Ökoloogia ja keskkonnateaduste doktorikooliga toimusid Tallinna Ülikoolis Exeteri Ülikooli professor Christopher J. Caseldine ja Hulli Ülikooli lektor Jane Buntingi loengud ning Post-POLLANDCAL teadusvõrgustiku seminar Ökoloogia Instituudis

Aegade ühildumatus : mälukoorem tänapäeva Prantsusmaal / Jaques Revel ; tlk. Indrek Koff

Index Scriptorium Estoniae

Revel, Jaques

2007-01-01

Erinevatest ajalookäsitlustest, ajaloojutustuse mudelitest ja nende arengusuundadest ning muutumise vajadusest Prantsusmaal. Artikli aluseks on ettekanne konverentsilt "Ajalugu ja poliitika. Mineviku kasutamisest ja ärakasutamisest" (Tallinn, 14. okt. 2006)

Ajaloolased ja teised. Ajaloolaste rollist Prantsusmaa avalikes aruteludes / Gérard Noiriel, Nicolas Offenstadt ; prantsuse keelest tõlkinud Indrek Koff

Index Scriptorium Estoniae

Noiriel, Gérard

2007-01-01

Ajaloo avalikust kasutamisest. Artikli aluseks on Nicolas Offenstadti ettekanne TLÜ Eesti Humanitaarinstituudi ja Prantsuse Kultuurikeskuse korraldatud rahvusvahelisel konverentsil "Ajalugu ja poliitika. Mineviku kasutamisest ja ärakasutamisest" 14. oktoobril 2006 Tallinnas.

Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.

Science.gov (United States)

Brewer, Laurence R; Friddle, Raymond; Noy, Aleksandr; Baldwin, Enoch; Martin, Shelley S; Corzett, Michele; Balhorn, Rod; Baskin, Ronald J

2003-10-01

Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

Clues for discovering a new biological function of Vitreoscilla hemoglobin in organisms: potential sulfide receptor and storage.

Science.gov (United States)

Wang, Dandan; Liu, Li; Wang, Hui; Xu, Haoran; Chen, Lei; Ma, Li; Li, Zhengqiang

2016-04-01

The interaction between H2 S and Vitreoscilla hemoglobin (VHb) has been studied by UV-Vis and Resonance Raman spectroscopes to confirm the binding between the ligand and the protein. Kinetic constants, kon = 1.2 × 10(5) m(-1) ·s(-1) and koff = 2.5 × 10(-4) ·s(-1) , have been determined and compared with those for mammalian hemoglobins. Density Functional Theory study supports the binding of H2 S by modeling the configurations of HOMO dispersions. We hypothesized that VHb is involved in H2 S reception and storage. Different from Lucina pectinata HbI, a typical H2 S-binding hemoglobin, VHb, exhibits unusual properties on H2 S reactivity such as steric constraints playing an important role in modulating H2 S entry. A distinct mechanism of VHb interaction with H2 S is supported by studies of variant forms of VHb. © 2016 Federation of European Biochemical Societies.

Using chemical shift perturbation to characterise ligand binding.

Science.gov (United States)

Williamson, Mike P

2013-08-01

Chemical shift perturbation (CSP, chemical shift mapping or complexation-induced changes in chemical shift, CIS) follows changes in the chemical shifts of a protein when a ligand is added, and uses these to determine the location of the binding site, the affinity of the ligand, and/or possibly the structure of the complex. A key factor in determining the appearance of spectra during a titration is the exchange rate between free and bound, or more specifically the off-rate koff. When koff is greater than the chemical shift difference between free and bound, which typically equates to an affinity Kd weaker than about 3μM, then exchange is fast on the chemical shift timescale. Under these circumstances, the observed shift is the population-weighted average of free and bound, which allows Kd to be determined from measurement of peak positions, provided the measurements are made appropriately. (1)H shifts are influenced to a large extent by through-space interactions, whereas (13)Cα and (13)Cβ shifts are influenced more by through-bond effects. (15)N and (13)C' shifts are influenced both by through-bond and by through-space (hydrogen bonding) interactions. For determining the location of a bound ligand on the basis of shift change, the most appropriate method is therefore usually to measure (15)N HSQC spectra, calculate the geometrical distance moved by the peak, weighting (15)N shifts by a factor of about 0.14 compared to (1)H shifts, and select those residues for which the weighted shift change is larger than the standard deviation of the shift for all residues. Other methods are discussed, in particular the measurement of (13)CH3 signals. Slow to intermediate exchange rates lead to line broadening, and make Kd values very difficult to obtain. There is no good way to distinguish changes in chemical shift due to direct binding of the ligand from changes in chemical shift due to allosteric change. Ligand binding at multiple sites can often be characterised, by

Draama 2007. Töörahu / Ene Paaver

Index Scriptorium Estoniae

Paaver, Ene, 1963-

2008-01-01

Ülevaade 2007. aasta eesti draamast. Pikemalt käsitletakse järgmisi autoreid ja nende näidendeid: Mart Kivastik "Tõnu võitlused", Mart Kivastik "Eesti asi", Mart Kivastik "Sõdur", Mart Kivastik "Kuninga viimane lahing ja ülikooli algus", Mart Kivastik, "Nokia 3310", Andrus Kivirähk "Voldemar", Andrus Kivirähk "Uljas neitsi", Hendrik Toompere "Kommunisti surm", Andres Ehin "Saatuslik kihlvedu ehk Volquin Varbola all", Jan Rahman "Jõujaam", Jaak Kõdar "Muinassaar", Karl Berg "Teekond valguse riiki", Angela Randmets "Lembelood vanas linnas", Toomas Kall "Tähtede sadu", Lauri Vahtre "Muna EÜE", Urmas Espenberg "Kestvad kiiduavaldused", Kalju Saaber "Koduvõõrad", Indrek Hirv "Pauluse kiriku kellad", Katrin Saukas "Võlg", Heidi Sarapuu "Austatud Riigikogu liikmed!", Heidi Sarapuu "Helisev viis", Heidi Sarapuu "Ankur hiivata", Jakob Karu "Vanaema juures", Jim Ashilevi "Nagu poisid vihma käes", Jim Ashilevi "Portselansuits", Eva Koff "Mirr", Jaak Prints "Karaoke", Kati Murutar "Mina, naine!", Ott Aardam "Börs ja Börsitar", Andrus Kivirähk, Jan Rahman, Contra "Kõrts", Mart Kase "Perekond", Villu Tamme "Haned võlgu"; Tiit Ojasoo ja Ene-Liis Semperi "GEP ehk Gorjatshije estonskije parni"

A new approach for the extraction of pollutants from wastewaters handled by the graphic industry.

Science.gov (United States)

Monteiro, C; Ventura, C; Martins, F

2013-06-15

It is widely recognized that the Graphic Industry handles toxic products and produces, in its various operations, toxic wastes. These wastes can cause serious environmental damages and can lead to severe health problems. In this work we report an efficient, simple and cheap to run method for the removal of some of the most common pollutants involved in the various stages of the Graphic Industry production, using a Solid-Phase Extraction (SPE) methodology. We have determined equilibrium constants, K(eq), and adsorption (k(up)) and desorption (k(off)) rate constants for the extraction of benzene, xylene, toluene and ethylbenzene (BXTE) from water, using C18 disks. The removal of these compounds was monitored by UV-vis spectroscopy, at room temperature. Average extraction efficiencies were of 60% in a mixture of BXTEs and close to 80% when pollutants were assessed separately. Since the retention mechanism in the C18 disk is essentially governed by hydrophobic interactions between the compounds and the alkyl chains of the disk, we have also shown that these pollutants' lipophilicity plays an important role in the rationalization of their behavior during the extraction process. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparative exploration of learning styles and teaching techniques between Thai and Vietnamese EFL students and instructors

Directory of Open Access Journals (Sweden)

Supalak Nakhornsri

2016-09-01

Full Text Available Learning styles have been a particular focus of a number of researchers over the past decades. Findings from various studies researching into how students learn highlight significant relationships between learners’ styles of learning and their language learning processes and achievement. This research focuses on a comparative analysis of the preferences of English learning styles and teaching techniques perceived by students from Thailand and Vietnam, and the teaching styles and techniques practiced by their instructors. The purposes were 1 to investigate the learning styles and teaching techniques students from both countries preferred, 2 to investigate the compatibility of the teaching styles and techniques practiced by instructors and those preferred by the students, 3 to specify the learning styles and teaching techniques students with high level of English proficiency preferred, and 4 to investigate the similarities of Thai and Vietnamese students’ preferences for learning styles and teaching techniques. The sample consisted of two main groups: 1 undergraduate students from King Mongkut’s University of Technology North Bangkok (KMUTNB, Thailand and Thai Nguyen University (TNU, Vietnam and 2 English instructors from both institutions. The instruments employed comprised the Students’ Preferred English Learning Style and Teaching Technique Questionnaire and the Teachers’ Practiced English Teaching Style and Technique Questionnaire. The collected data were analyzed using arithmetic means and standard deviation. The findings can contribute to the curriculum development and assist teachers to teach outside their comfort level to match the students’ preferred learning styles. In addition, the findings could better promote the courses provided for students. By understanding the learning style make-up of the students enrolled in the courses, faculty can adjust their modes of content delivery to match student preferences and maximize

Lastekirjandus vallutab uusi tippe : eesti lastekirjandus 2009 / Jaanus Vaiksoo

Index Scriptorium Estoniae

Vaiksoo, Jaanus, 1967-

2010-01-01

Ülevaade 2009. aasta eesti lastekirjandusest. Pikemalt käsitletakse järgmisi autoreid ja nende teoseid: Kadi Hinrikus "Kui emad olid väikesed", Kristiina Kass "Peeter ja mina", Mika Keränen "Varastatud oranž jalgratas", Andrus Kivirähk "Kaka ja kevad", Aino Pervik "Ühes väikses veidras linnas", Maarja Undusk "Päkapikk Ingo", Ilmar Tomusk "Vend Johannes", Indrek Koff "Enne kooli", Piret Raud "Härra Linnu lugu", Wimberg "Suur pidusöök", Artur Jurin "Puru kuningriigi lood II", Joonas Sildre "Maailma naba", kogumik "Uued eesti muinasjutud", Aino Pervik "Tirilinna lood", Aimeé Beekman "Päästekoer Walter", Eve Ernis "Karupoeg Nummi seiklused", Priit Aimla "Torisev vanatoi", Juhani Püttsepp "Väikese hundi lood" ja "Lauajupi Madonna", Epp Petrone "Siis, kui seened veel rääkisid", Heiki Vilep "Habemega nali", "Nõiutud linn", Leelo Tungal "Naljatilgad lähevad laulupeole", "Kama üks ja kama kaks", koos Peep Pedmansoniga Miriami lood"(esimene 3D-piltidega lasteraamat), Aidi Vallik "Pints ja Tutsik" 2. osa, Mare Müürsepp "Viis vaba kutsikat", Tiia Selli "Kass Kriibik", Aleksei Turovski "Kassipoeg Võilill", Kaider Vardja "Vana maja lugu", Merca "Mullivesi", Ülle Kütsen "Väike puu ja rändur kuu", Lehte Hainsalu "Mängiks midagi", Peep Veedla "Pöialpoiss"

Kinetics of protein–ligand unbinding: Predicting pathways, rates, and rate-limiting steps

Science.gov (United States)

Tiwary, Pratyush; Limongelli, Vittorio; Salvalaglio, Matteo; Parrinello, Michele

2015-01-01

The ability to predict the mechanisms and the associated rate constants of protein–ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin–benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate koff is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate kon. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein–ligand systems and a valid support for drug design. PMID:25605901

Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings

Science.gov (United States)

Lowry, Troy W.; Hariri, Hanaa; Prommapan, Plengchart; Kusi-Appiah, Aubrey; Vafai, Nicholas; Bienkiewicz, Ewa A.; Van Winkle, David H.; Stagg, Scott M.

2016-01-01

The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid–protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (KD) and kinetics (kon and koff). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached. PMID:26649649

Investigation of the heparin-thrombin interaction by dynamic force spectroscopy.

Science.gov (United States)

Wang, Congzhou; Jin, Yingzi; Desai, Umesh R; Yadavalli, Vamsi K

2015-06-01

The interaction between heparin and thrombin is a vital step in the blood (anti)coagulation process. Unraveling the molecular basis of the interactions is therefore extremely important in understanding the mechanisms of this complex biological process. In this study, we use a combination of an efficient thiolation chemistry of heparin, a self-assembled monolayer-based single molecule platform, and a dynamic force spectroscopy to provide new insights into the heparin-thrombin interaction from an energy viewpoint at the molecular scale. Well-separated single molecules of heparin covalently attached to mixed self-assembled monolayers are demonstrated, whereby interaction forces with thrombin can be measured via atomic force microscopy-based spectroscopy. Further these interactions are studied at different loading rates and salt concentrations to directly obtain kinetic parameters. An increase in the loading rate shows a higher interaction force between the heparin and thrombin, which can be directly linked to the kinetic dissociation rate constant (koff). The stability of the heparin/thrombin complex decreased with increasing NaCl concentration such that the off-rate was found to be driven primarily by non-ionic forces. These results contribute to understanding the role of specific and nonspecific forces that drive heparin-thrombin interactions under applied force or flow conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Probing the effect of the non-active-site mutation Y229W in New Delhi metallo-β-lactamase-1 by site-directed mutagenesis, kinetic studies, and molecular dynamics simulations.

Directory of Open Access Journals (Sweden)

Jiao Chen

Full Text Available New Delhi metallo-β-lactamase-1 (NDM-1 has attracted extensive attention for its high catalytic activities of hydrolyzing almost all β-lactam antibiotics. NDM-1 shows relatively higher similarity to subclass B1 metallo-β-lactamases (MβLs, but its residue at position 229 is identical to that of B2/B3 MβLs, which is a Tyr instead of a B1-MβL-conserved Trp. To elucidate the possible role of Y229 in the bioactivity of NDM-1, we performed mutagenesis study and molecular dynamics (MD simulations. Although residue Y229 is spatially distant from the active site and not contacting directly with the substrate or zinc ions, the Y229W mutant was found to have higher kcat and Km values than those of wild-type NDM-1, resulting in 1 ∼ 7 fold increases in k(cat /K(m values against tested antibiotics. In addition, our MD simulations illustrated the enhanced flexibility of Loop 2 upon Y229W mutation, which could increase the kinetics of both substrate entrance (kon and product egress (koff. The enhanced flexibility of Loop 2 might allow the enzyme to adjust the geometry of its active site to accommodate substrates with different structures, broadening its substrate spectrum. This study indicated the possible role of the residue at position 229 in the evolution of NDM-1.

Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

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Tal Noy-Porat

2016-03-01

Full Text Available Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1 that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

Selgusid aasta parimad ja populaarsemad õppejõud

Index Scriptorium Estoniae

2002-01-01

22.02.02. anti kätte TPÜ iga-aastaste konkursside võitjate autasud ning teenetemärgid ja tänukirjad. Konkurss "Aasta parimad õppejõud" - kunstiosakonna prof U. Viik. "Aasta populaarseimad õppejõud"- psühholoogia osakonna prof A. Pulver. "Silmapaistvaim publikatsioon, õpik ja loominguline projekt": parim monograafia- kultuuri osakonna prof R. Pullat, parim humanitaarteadustealane artikkel - filoloogiateaduskonna dekaan H. Metslang, parim kasvatusteadustealane artikkel- koolipedagoogika osakonna prof M. Veisson, parim loodusteadustealane artikkel- TPÜ ökoloogia instituudi direktor J.-M. Punning ja ökoloogia eriala doktorant A. Kratovits, parim täppisteadustealane artikkel- ökoloogia eriala doktorant S. Jevrejeva, parim kõrgkooli õpik - eesti filoloogia osakonna assistent K. Kerge, parim üldhariduskooli õpik- matemaatika osakonna prof R. Kolde ja endine kasvatusteaduste osakonna ja Haapsalu Kolledži dots E. Noor, parim kunstialane projekt - kunstiosakonna lektor S. Arsas ja kunstiosakonna õpetaja L. Mosolainen. TPÜ teenetemärgi said prof M. Arvisto, prof R. Maran, prof J. Orn, TPÜ ökoloogia instituudi direktor prof J.-M. Punning ja prof emer. A. Telgmaa. TPÜ tänukirja said dots A. Adusk, prof A.-R. Hausenberg, vanemteadur T. Koff, prof R. Kolde, dots K.-K. Kuiv, teadur K. Liik, õppeprodekaan K. Marmor, dekaani abi H. Meri, dots I. Moissejenko, dots T. Pau, prof V.-R. Ruus ja dots A. Tiko

Equilibrium and kinetics of Sin Nombre hantavirus binding at DAF/CD55 functionalized bead surfaces.

Science.gov (United States)

Buranda, Tione; Swanson, Scarlett; Bondu, Virginie; Schaefer, Leah; Maclean, James; Mo, Zhenzhen; Wycoff, Keith; Belle, Archana; Hjelle, Brian

2014-03-10

Decay accelerating factor (DAF/CD55) is targeted by many pathogens for cell entry. It has been implicated as a co-receptor for hantaviruses. To examine the binding of hantaviruses to DAF, we describe the use of Protein G beads for binding human IgG Fc domain-functionalized DAF ((DAF)₂-Fc). When mixed with Protein G beads the resulting DAF beads can be used as a generalizable platform for measuring kinetic and equilibrium binding constants of DAF binding targets. The hantavirus interaction has high affinity (24-30 nM; k(on) ~ 10⁵ M⁻¹ s⁻¹, k(off) ~ 0.0045 s⁻¹). The bivalent (DAF)₂-Fc/SNV data agree with hantavirus binding to DAF expressed on Tanoue B cells (K(d) = 14.0 nM). Monovalent affinity interaction between SNV and recombinant DAF of 58.0 nM is determined from competition binding. This study serves a dual purpose of presenting a convenient and quantitative approach of measuring binding affinities between DAF and the many cognate viral and bacterial ligands and providing new data on the binding constant of DAF and Sin Nombre hantavirus. Knowledge of the equilibrium binding constant allows for the determination of the relative fractions of bound and free virus particles in cell entry assays. This is important for drug discovery assays for cell entry inhibitors.

Ühe kuupäeva ajalooline ja poliitiline tagapõhi : 8. ja 9. mai Prantsusmaal, Balti riikides ja Venemaal / Jean-Pierre Minaudier ; tõlk. Indrek Koff

Index Scriptorium Estoniae

Minaudier, Jean-Pierre

2007-01-01

Teise maailmasõja lõpu tähistamise kuupäevadega seotud ajaloolise ja poliitilise tausta käsitlemisel on autor keskendunud Venemaa, Balti riikide ja Prantsusmaa riigijuhtide ametlikele kõnedele, avaldustele ja intervjuudele, mis on olulised 2005. aasta maikuu kriisi seisukohast: kahe Balti riigi presidendid keeldusid osalemast Saksamaa kapituleerumise 60. aastapäeva üritustel Moskvas. Artikli aluseks on ettekanne rahvusvahelisel konverentsil "Ajalugu ja poliitika. Mineviku kasutamisest ja ärakasutamisest" 14. oktoobril Tallinnas

Water quality and pollution status of Chambal river in National Chambal Sanctuary, Madhya Pradesh.

Science.gov (United States)

Saksena, D N; Garg, R K; Rao, R J

2008-09-01

The physico-chemical characteristics of Chambal river water in National Chambal sanctuary (Madhya Pradesh) have been studied. The stretch of Chambal river contained in the National Chambal sanctuary (located at 25 degrees 23'-26 degrees 52'N, 76 degrees 28'-79 degrees 15'E) is extending up to 600 km downstream from Kota (Rajasthan) to the confluence of the Chambal with Yamuna river (Etawah). The river flow in Madhya Pradesh spans up to approximately 400 km. Three sampling stations viz., Station A--near Palighat, district Sheopurkalan, Station B--near Rajghat, district Morena and Station C--near Baraighat, district Bhind were established for the collection of water samples during April, 2003 to March, 2004. The water quality parameters namely transparency (12.12-110 cm), colour (transparent-very turbid), turbidity (1-178 TNU), electrical conductivity (145.60-884 microS cm(-1)), total dissolved solids (260-500 mgl(-1)), pH (7.60-9.33), dissolved oxygen (4.86-14.59 mgl(-1)), free carbon dioxide (0-16.5 mgl(-1)), total alkalinity (70-290 mgl(-1)), total hardness (42-140 mgl(-1)), chloride (15.62-80.94 mgl(-1)), nitrate (0.008-0.025 mgl(-1)), nitrite (0.002-0.022 mgl(-1)), sulphate (3.50-45 mgl(-1)), phosphate (0.004-0.050 mgl(-1)), silicate (2.80-13.80 mgl(-1)), biochemical oxygen demand (0.60-5.67 mgl(-1)), chemical oxygen demand (2.40-26.80 mgl(-1)), ammonia (nil-0.56 mgl(-1)), sodium (14.30-54.40 mgl(-1)) and potassium (2.10 mgl(-1)-6.30 mgl(-1)) reflects on the pristine nature of the river in National Chambal sanctuary. On the basis of various parameters studied, Chambal river in this stretch can be placed under the category of oligosaprobic. The water quality analysis, indicated that the riverwater in the sanctuary area is pollution free and can serve as a good habitat for many aquatic animals including endangered species.

Biophysical and physicochemical methods differentiate highly ligand-efficient human D-amino acid oxidase inhibitors.

Science.gov (United States)

Lange, Jos H M; Venhorst, Jennifer; van Dongen, Maria J P; Frankena, Jurjen; Bassissi, Firas; de Bruin, Natasja M W J; den Besten, Cathaline; de Beer, Stephanie B A; Oostenbrink, Chris; Markova, Natalia; Kruse, Chris G

2011-10-01

Many early drug research efforts are too reductionist thereby not delivering key parameters such as kinetics and thermodynamics of target-ligand binding. A set of human D-Amino Acid Oxidase (DAAO) inhibitors 1-6 was applied to demonstrate the impact of key biophysical techniques and physicochemical methods in the differentiation of chemical entities that cannot be adequately distinguished on the basis of their normalized potency (ligand efficiency) values. The resulting biophysical and physicochemical data were related to relevant pharmacodynamic and pharmacokinetic properties. Surface Plasmon Resonance data indicated prolonged target-ligand residence times for 5 and 6 as compared to 1-4, based on the observed k(off) values. The Isothermal Titration Calorimetry-derived thermodynamic binding profiles of 1-6 to the DAAO enzyme revealed favorable contributions of both ΔH and ΔS to their ΔG values. Surprisingly, the thermodynamic binding profile of 3 elicited a substantially higher favorable contribution of ΔH to ΔG in comparison with the structurally closely related fused bicyclic acid 4. Molecular dynamics simulations and free energy calculations of 1, 3, and 4 led to novel insights into the thermodynamic properties of the binding process at an atomic level and in the different thermodynamic signatures of 3 and 4. The presented holistic approach is anticipated to facilitate the identification of compounds with best-in-class properties at an early research stage. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

212ʻ Fahrenheiti : eesti luule 2006 / Piret Bristol

Index Scriptorium Estoniae

Bristol, Piret, 1968-

2008-01-01

Järgmistest 2006. a. ilmunud luulekogudest: Ryytle, Indrek. Inglid rokijaamas. Puhja : I. Rüütle ; Runnel, Hando. Viru veri ei värise. Tartu : Ilmamaa ; Vabat, Martin. Mina olengi kirjandusklassik. Tartu : Eesti Kirjanduse Selts ; Korts, Eliina. Lööklaused murravad metsi. Tartu : Eesti Kirjanduse Selts ; Koff, Indrek. Vana laul. Luige : Verb ; Teede, Andra. Takso Tallinna taevas. Luige : Verb ; Vusser, Kaspar. Vusserduste võnked. Tallinn : P. Kukk ; Elbing, Andrus. Siin Beebilõust, tere! : häired pimelinna tänavalt = Ciao, it's Babyface : trouble from the streets of a blind city. Tallinn : Epifanio ; Hint, Miina. Vabaduse vang, ehk, Põrandaalune kirjandus. Tallinn : MR ; Jüriado, Tiiu. Luule on ime. Habaja : Kentaur ; Ligi, Katre. Naabrivalve. Tartu : Ilmamaa ; Krull, Hasso. Talv. Tallinn : Tuum ; Mets, Mae. Pühapäeval on reede. Tartu : Ilmamaa ; Ploom, Ülar. Porr ja sorry. Tallinn : Tuum ; Hirv, Indrek, Surmapõletaja. Tallinn : Tuum ; Piir, Milvi Martina. Kõrkjavaas. Tartu : Fantaasia ; Soomets, Triin. Väljas. Tallinn : Tuum ; Mathura. Kohalolu. Rapla : Allikäärne ; Afanasjev, Vahur. Katedraal Emajões. Tartu ; Brüssel : ID Salong//Sild, Ivar. Spermaga ja puha. Pärnu : Jumalikud Ilmutused ; Meiel, Kaupo. Polügrafisti käsiraamat. Pärnu : Jumalikud Ilmutused ; Kivisildnik, Sven. Vägistatud jäämägi. Pärnu : Jumalikud Ilmutused ; Arder, Ott. Luule sünnib kus sünnib kui sünnib / koost. Leelo Tungal. Tallinn : Tänapäev ; Kompus, Marko. Vallaliste jõgede tõkkejooksja. Tartu : M. Kompus

Ühe kuupäeva ajalooline ja poliitiline tagapõhi : 8. ja 9. mai Prantsusmaal, Balti riikides ja Venemaal / Jean-Pierre Minaudier ; prantsuse keelest tõlkinud Indrek Koff

Index Scriptorium Estoniae

Minaudier, Jean-Pierre

2007-01-01

Teise Maailmasõja lõpu tähistamisest 8. ja 9. mail Balti riikides, Venemaal ja Prantsusmaal. Artikli aluseks on ettekanne TLÜ Eesti Humanitaarinstituudi ja Prantsuse Kultuurikeskuse korraldatud rahvusvahelisel konverentsil "Ajalugu ja poliitika. Mineviku kasutamisest ja ärakasutamisest" 14. oktoobril 2006 Tallinnas

Hra na flétnu v předškolním věku a význam hudby v dalším životě dítěte

OpenAIRE

PAVLIŠOVÁ, Irena

2013-01-01

This bachelor thesis called "Playing a Flute at Preschool Age and the Importance of Music for Further Life of a Child" aims to find out what is the importance of music for further life of children who learnt to play a flute at preschool age. The theoretical part introduces terms such as a recorder, it also focuses on the therapeutic playing a flute and its history and it also deals with the idea of professor Václav Žilka as reflected in his book called "Veselé pískání - zdravé dýchání". Other...

Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry.

Directory of Open Access Journals (Sweden)

Steffen M Sedlak

Full Text Available The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin's tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10-6 s-1 range.

Monodisperse measurement of the biotin-streptavidin interaction strength in a well-defined pulling geometry.

Science.gov (United States)

Sedlak, Steffen M; Bauer, Magnus S; Kluger, Carleen; Schendel, Leonard C; Milles, Lukas F; Pippig, Diana A; Gaub, Hermann E

2017-01-01

The widely used interaction of the homotetramer streptavidin with the small molecule biotin has been intensively studied by force spectroscopy and has become a model system for receptor ligand interaction. However, streptavidin's tetravalency results in diverse force propagation pathways through the different binding interfaces. This multiplicity gives rise to polydisperse force spectroscopy data. Here, we present an engineered monovalent streptavidin tetramer with a single cysteine in its functional subunit that allows for site-specific immobilization of the molecule, orthogonal to biotin binding. Functionality of streptavidin and its binding properties for biotin remain unaffected. We thus created a stable and reliable molecular anchor with a unique high-affinity binding site for biotinylated molecules or nanoparticles, which we expect to be useful for many single-molecule applications. To characterize the mechanical properties of the bond between biotin and our monovalent streptavidin, we performed force spectroscopy experiments using an atomic force microscope. We were able to conduct measurements at the single-molecule level with 1:1-stoichiometry and a well-defined geometry, in which force exclusively propagates through a single subunit of the streptavidin tetramer. For different force loading rates, we obtained narrow force distributions of the bond rupture forces ranging from 200 pN at 1,500 pN/s to 230 pN at 110,000 pN/s. The data are in very good agreement with the standard Bell-Evans model with a single potential barrier at Δx0 = 0.38 nm and a zero-force off-rate koff,0 in the 10-6 s-1 range.

Molecular features of interaction between VEGFA and anti-angiogenic drugs used in retinal diseases: a computational approach

Directory of Open Access Journals (Sweden)

Chiara Bianca Maria Platania

2015-10-01

Full Text Available Anti-angiogenic agents are biological drugs used for treatment of retinal neovascular degenerative diseases. In this study, we aimed at in-silico analysis of interaction of vascular endothelial growth factor A (VEGFA, the main mediator of angiogenesis, with binding domains of anti-angiogenic agents used for treatment of retinal diseases, such as ranibizumab, bevacizumab and aflibercept. The analysis of anti-VEGF/VEGFA complexes was carried out by means of protein-protein docking and molecular dynamics (MD coupled to molecular mechanics-Poisson Boltzmann Surface Area (MM-PBSA calculation. Molecular dynamics simulation was further analyzed by protein contact networks. Rough energetic evaluation with protein-protein docking scores revealed that aflibercept/VEGFA complex was characterized by electrostatic stabilization, whereas ranibizumab and bevacizumab complexes were stabilized by Van der Waals (VdW energy term; these results were confirmed by MM-PBSA. Comparison of MM-PBSA predicted energy terms with experimental binding parameters reported in literature indicated that the high association rate (Kon of aflibercept to VEGFA was consistent with high stabilizing electrostatic energy. On the other hand, the relatively low experimental dissociation rate (Koff of ranibizumab may be attributed to lower conformational fluctuations of the ranibizumab/VEGFA complex, higher number of contacts and hydrogen bonds in comparison to bevacizumab and aflibercept. Thus, the anti-angiogenic agents have been found to be considerably different both in terms of molecular interactions and stabilizing energy. Characterization of such features can improve the design of novel biological drugs potentially useful in clinical practice.

Investigating the binding behaviour of two avidin-based testosterone binders using molecular recognition force spectroscopy.

Science.gov (United States)

Rangl, Martina; Leitner, Michael; Riihimäki, Tiina; Lehtonen, Soili; Hytönen, Vesa P; Gruber, Hermann J; Kulomaa, Markku; Hinterdorfer, Peter; Ebner, Andreas

2014-02-01

Molecular recognition force spectroscopy, a biosensing atomic force microscopy technique allows to characterise the dissociation of ligand-receptor complexes at the molecular level. Here, we used molecular recognition force spectroscopy to study the binding capability of recently developed testosterone binders. The two avidin-based proteins called sbAvd-1 and sbAvd-2 are expected to bind both testosterone and biotin but differ in their binding behaviour towards these ligands. To explore the ligand binding and dissociation energy landscape of these proteins, we tethered biotin or testosterone to the atomic force microscopy probe while the testosterone-binding protein was immobilized on the surface. Repeated formation and rupture of the ligand-receptor complex at different pulling velocities allowed determination of the loading rate dependence of the complex-rupturing force. In this way, we obtained the molecular dissociation rate (k(off)) and energy landscape distances (x(β)) of the four possible complexes: sbAvd-1-biotin, sbAvd-1-testosterone, sbAvd-2-biotin and sbAvd-2-testosterone. It was found that the kinetic off-rates for both proteins and both ligands are similar. In contrast, the x(β) values, as well as the probability of complex formations, varied considerably. In addition, competitive binding experiments with biotin and testosterone in solution differ significantly for the two testosterone-binding proteins, implying a decreased cross-reactivity of sbAvd-2. Unravelling the binding behaviour of the investigated testosterone-binding proteins is expected to improve their usability for possible sensing applications. Copyright © 2014 John Wiley & Sons, Ltd.

Development of a tetrameric streptavidin mutein with reversible biotin binding capability: engineering a mobile loop as an exit door for biotin.

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Valerie J O'Sullivan

Full Text Available A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop(3-4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop(3-4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop(7-8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop(7-8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (k(off of 4.28×10(-4 s(-1 and K(d of 1.9×10(-8 M make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible.

Development of peptide and protein based radiopharmaceuticals.

Science.gov (United States)

Wynendaele, Evelien; Bracke, Nathalie; Stalmans, Sofie; De Spiegeleer, Bart

2014-01-01

Radiolabelled peptides and proteins have recently gained great interest as theranostics, due to their numerous and considerable advantages over small (organic) molecules. Developmental procedures of these radiolabelled biomolecules start with the radiolabelling process, greatly defined by the amino acid composition of the molecule and the radionuclide used. Depending on the radionuclide selection, radiolabelling starting materials are whether or not essential for efficient radiolabelling, resulting in direct or indirect radioiodination, radiometal-chelate coupling, indirect radiofluorination or (3)H/(14)C-labelling. Before preclinical investigations are performed, quality control analyses of the synthesized radiopharmaceutical are recommended to eliminate false positive or negative functionality results, e.g. changed receptor binding properties due to (radiolabelled) impurities. Therefore, radionuclidic, radiochemical and chemical purity are investigated, next to the general peptide attributes as described in the European and the United States Pharmacopeia. Moreover, in vitro and in vivo stability characteristics of the peptides and proteins also need to be explored, seen their strong sensitivity to proteinases and peptidases, together with radiolysis and trans-chelation phenomena of the radiopharmaceuticals. In vitro biomedical characterization of the radiolabelled peptides and proteins is performed by saturation, kinetic and competition binding assays, analyzing KD, Bmax, kon, koff and internalization properties, taking into account the chemical and metabolic stability and adsorption events inherent to peptides and proteins. In vivo biodistribution can be adapted by linker, chelate or radionuclide modifications, minimizing normal tissue (e.g. kidney and liver) radiation, and resulting in favorable dosimetry analyses. Finally, clinical trials are initiated, eventually leading to the marketing of radiolabelled peptides and proteins for PET/SPECT-imaging and therapy

Development of a Tetrameric Streptavidin Mutein with Reversible Biotin Binding Capability: Engineering a Mobile Loop as an Exit Door for Biotin

Science.gov (United States)

O'Sullivan, Valerie J.; Barrette-Ng, Isabelle; Hommema, Eric; Hermanson, Greg T.; Schofield, Mark; Wu, Sau-Ching; Honetschlaeger, Claudia; Ng, Kenneth K.-S.; Wong, Sui-Lam

2012-01-01

A novel form of tetrameric streptavidin has been engineered to have reversible biotin binding capability. In wild-type streptavidin, loop3–4 functions as a lid for the entry and exit of biotin. When biotin is bound, interactions between biotin and key residues in loop3–4 keep this lid in the closed state. In the engineered mutein, a second biotin exit door is created by changing the amino acid sequence of loop7–8. This door is mobile even in the presence of the bound biotin and can facilitate the release of biotin from the mutein. Since loop7–8 is involved in subunit interactions, alteration of this loop in the engineered mutein results in an 11° rotation between the two dimers in reference to wild-type streptavidin. The tetrameric state of the engineered mutein is stabilized by a H127C mutation, which leads to the formation of inter-subunit disulfide bonds. The biotin binding kinetic parameters (koff of 4.28×10−4 s−1 and Kd of 1.9×10−8 M) make this engineered mutein a superb affinity agent for the purification of biotinylated biomolecules. Affinity matrices can be regenerated using gentle procedures, and regenerated matrices can be reused at least ten times without any observable reduction in binding capacity. With the combination of both the engineered mutein and wild-type streptavidin, biotinylated biomolecules can easily be affinity purified to high purity and immobilized to desirable platforms without any leakage concerns. Other potential biotechnological applications, such as development of an automated high-throughput protein purification system, are feasible. PMID:22536357

Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.

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Khan, Mateen A; Ma, Jia; Walden, William E; Merrick, William C; Theil, Elizabeth C; Goss, Dixie J

2014-06-01

Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn(2+) decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn(2+) increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn(2+) eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

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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

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

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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

Kinetic analysis of transport and opioid receptor binding of [3H](-)-cyclofoxy in rat brain in vivo: Implications for human studies

International Nuclear Information System (INIS)

Sawada, Y.; Kawai, R.; McManaway, M.; Otsuki, H.; Rice, K.C.; Patlak, C.S.; Blasberg, R.G.

1991-01-01

[3H]Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB); estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using [18F]CF and positron emission tomography

Kinetic analysis of transport and opioid receptor binding of ( sup 3 H)(-)-cyclofoxy in rat brain in vivo: Implications for human studies

Energy Technology Data Exchange (ETDEWEB)

Sawada, Y.; Kawai, R.; McManaway, M.; Otsuki, H.; Rice, K.C.; Patlak, C.S.; Blasberg, R.G. (National Institutes of Health, Bethesda, MD (USA))

1991-03-01

(3H)Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB); estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using (18F)CF and positron emission tomography.

Probing of miniPEGγ-PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy.

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Dutta, Samrat; Armitage, Bruce A; Lyubchenko, Yuri L

2016-03-15

Peptide nucleic acids (PNA) are synthetic polymers, the neutral peptide backbone of which provides elevated stability to PNA-PNA and PNA-DNA hybrid duplexes. It was demonstrated that incorporation of diethylene glycol (miniPEG) at the γ position of the peptide backbone increased the thermal stability of the hybrid duplexes (Sahu, B. et al. J. Org. Chem. 2011, 76, 5614-5627). Here, we applied atomic force microscopy (AFM) based single molecule force spectroscopy and dynamic force spectroscopy (DFS) to test the strength and stability of the hybrid 10 bp duplex. This hybrid duplex consisted of miniPEGγ-PNA and DNA of the same length (γ(MP)PNA-DNA), which we compared to a DNA duplex with a homologous sequence. AFM force spectroscopy data obtained at the same conditions showed that the γ(MP)PNA-DNA hybrid is more stable than the DNA counterpart, 65 ± 15 pN vs 47 ± 15 pN, respectively. The DFS measurements performed in a range of pulling speeds analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ≈ 0.030 ± 0.01 s⁻¹ for γ(MP)PNA-DNA hybrid duplex vs 0.375 ± 0.18 s⁻¹ for the DNA-DNA duplex suggesting that the hybrid duplex is much more stable. Correlating the high affinity of γ(MP)PNA-DNA to slow dissociation kinetics is consistent with prior bulk characterization by surface plasmon resonance. Given the growing interest in γ(MP)PNA as well as other synthetic DNA analogues, the use of single molecule experiments along with computational analysis of force spectroscopy data will provide direct characterization of various modifications as well as higher order structures such as triplexes and quadruplexes.

Viral RNAi suppressor reversibly binds siRNA to outcompete Dicer and RISC via multiple turnover.

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Rawlings, Renata A; Krishnan, Vishalakshi; Walter, Nils G

2011-04-29

RNA interference is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response to viruses and retrotransposons. During viral infection, the RNase-III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs) 21-24 nucleotides in length and helps load them into the RNA-induced silencing complex (RISC) to guide the cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressors (RSS) that tightly, and presumably quantitatively, bind siRNAs to thwart RNA-interference-mediated degradation. Viral RSS proteins also act across kingdoms as potential immunosuppressors in gene therapeutic applications. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus, as well as by human Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding [(1.69 ± 0.07) × 10(8) M(-)(1) s(-1)] and marked dissociation (k(off)=0.062 ± 0.002 s(-1)). We also observe that p19 efficiently competes with recombinant Dicer and inhibits the formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the transient formation of a ternary complex between siRNA, human Dicer, and p19. An expanded model of RNA silencing indicates that multiple turnover by reversible binding of siRNAs potentiates the efficiency of the suppressor protein. Our predictive model is expected to be applicable to the dosing of p19 as a silencing suppressor in viral gene therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.

Viral RNAi suppressor reversibly binds siRNA to outcompete Dicer and RISC via multiple-turnover

Science.gov (United States)

Rawlings, Renata A.; Krishnan, Vishalakshi; Walter, Nils G.

2011-01-01

RNA interference (RNAi) is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response against viruses and retrotransposons. During viral infection, the RNase III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs), 21–24 nucleotides in length, and helps load them into the RNA-induced silencing complex (RISC) to guide cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressor (RSS) proteins that tightly, and presumably quantitatively, bind siRNAs to thwart RNAi-mediated degradation. Viral RSS proteins also act across kingdoms as potential immunosuppressors in gene therapeutic applications. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus (CIRV), as well as by human Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding ((1.69 ± 0.07)×108 M−1s−1) and marked dissociation (koff = 0.062 ± 0.002 s−1). We also observe that p19 efficiently competes with recombinant Dicer and inhibits formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the transient formation of a ternary complex between siRNA, human Dicer, and p19. An expanded model of RNA silencing indicates that multiple-turnover by reversible binding of siRNAs potentiates the efficiency of the suppressor protein. Our predictive model is expected to be applicable to the dosing of p19 as a silencing suppressor in viral gene therapy. PMID:21354178

Dva nové dokumenty o návštěvě prezidenta Edvarda Beneše ve Spojených státech amerických a Kanadě v květnu a červnu 1943

Czech Academy of Sciences Publication Activity Database

Dejmek, Jindřich

2008-01-01

Roč. 16, - (2008), s. 291-320 ISSN 1210-6860 Institutional research plan: CEZ:AV0Z80150510 Keywords : History of Czechoslovak Foreign Policy * History of Diplomacy * History of International Relations Subject RIV: AB - History

Interaction of nitric oxide with human heme oxygenase-1.

Science.gov (United States)

Wang, Jinling; Lu, Shen; Moënne-Loccoz, Pierre; Ortiz de Montellano, Paul R

2003-01-24

NO and CO may complement each other as signaling molecules in some physiological situations. We have examined the binding of NO to human heme oxygenase-1 (hHO-1), an enzyme that oxidizes heme to biliverdin, CO, and free iron, to determine whether inhibition of hHO-1 by NO can contribute to the signaling interplay of NO and CO. An Fe(3+)-NO hHO-1-heme complex is formed with NO or the NO donors NOC9 or 2-(N,N-diethylamino)-diazenolate-2-oxide.sodium salt. Resonance Raman spectroscopy shows that ferric hHO-1-heme forms a 6-coordinated, low spin complex with NO. The nu(N-O) vibration of this complex detected by Fourier transform IR is only 4 cm(-1) lower than that of the corresponding metmyoglobin (met-Mb) complex but is broader, suggesting a greater degree of ligand conformational freedom. The Fe(3+)-NO complex of hHO-1 is much more stable than that of met-Mb. Stopped-flow studies indicate that k(on) for formation of the hHO-1-heme Fe(3+)-NO complex is approximately 50-times faster, and k(off) 10 times slower, than for met-Mb, resulting in K(d) = 1.4 microm for NO. NO thus binds 500-fold more tightly to ferric hHO-1-heme than to met-Mb. The hHO-1 mutations E29A, G139A, D140A, S142A, G143A, G143F, and K179A/R183A do not significantly diminish the tight binding of NO, indicating that NO binding is not highly sensitive to mutations of residues that normally stabilize the distal water ligand. As expected from the K(d) value, the enzyme is reversibly inhibited upon exposure to pathologically, and possibly physiologically, relevant concentrations of NO. Inhibition of hHO-1 by NO may contribute to the pleiotropic responses to NO and CO.

Od melancholii do rozpaczy. O prozie Andrzeja Stasiuka

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Claudia Snochowska-Gonzalez

2014-06-01

Full Text Available From melancholy to despair. About Andrzej Stasiuk’s prose works In Moja Europa, Jadąc do Babadag and Fado Andrzej Stasiuk describes his travelling to the countries of the East-Central Europe: its diminished, forgotten part, lying on the margins of History and Progress. It is a land of melancholy, of the eternal emptiness and lack. To praise it means to give an ironic response to the enthusiasm of a “return to the West”, to the attempts to meet East-European stigma and to the West’s fear of East-European ferocity. What is the source of this melancholy? Stasiuk refers to Cioran, his philosophy of history, his resignation and his belief in the bankruptcy of the European civilization. We know, however, that in the case of Cioran melancholy covers the memory of philosopher’s commitment to Romanian fascism; his subsequent melancholy replaces responsibility. What are the wounds and silenced victims hiding in Stasiuk’s melancholic landscape? What kind of responsibility does he not want to accept? In his next book, Dziennik pisany później, Stasiuk comes back to the same countries, this time not trying to escape the hell of questions about the East-European ethnic carnage. The author of the article analyses his turning point, using the terminology developed in Peter Hallward’s Absolutely Post-colonial to describe the dynamics between two tendencies: singular and specific.   Od melancholii do rozpaczy. O prozie Andrzeja Stasiuka W Mojej Europie, Jadąc do Babadag i Fado Andrzej Stasiuk podróżuje po krajach Europy Środkowo-Wschodniej. To Europa pomniejsza, zapomniana, na marginesie Historii i Postępu. Jest to kraina melancholii, pustki i wiecznego braku. Opiewanie jej staje się ironiczną odpowiedzią na entuzjazm „powrotu do Zachodu”, na próby sprostania wschodnioeuropejskiemu piętnu i na zachodnie przerażenie wschodnioeuropejską dzikością. Skąd się jednak bierze spowijająca ją melancholia? Stasiuk powołuje się na

Two key residues in ephrinB3 are critical for its use as an alternative receptor for Nipah virus.

Directory of Open Access Journals (Sweden)

2006-02-01

Full Text Available EphrinB2 was recently discovered as a functional receptor for Nipah virus (NiV, a lethal emerging paramyxovirus. Ephrins constitute a class of homologous ligands for the Eph class of receptor tyrosine kinases and exhibit overlapping expression patterns. Thus, we examined whether other ephrins might serve as alternative receptors for NiV. Here, we show that of all known ephrins (ephrinA1-A5 and ephrinB1-B3, only the soluble Fc-fusion proteins of ephrinB3, in addition to ephrinB2, bound to soluble NiV attachment protein G (NiV-G. Soluble NiV-G bound to cell surface ephrinB3 and B2 with subnanomolar affinities (Kd = 0.58 nM and 0.06 nM for ephrinB3 and B2, respectively. Surface plasmon resonance analysis indicated that the relatively lower affinity of NiV-G for ephrinB3 was largely due to a faster off-rate (K(off = 1.94 x 10(-3 s(-1 versus 1.06 x 10(-4 s(-1 for ephrinB3 and B2, respectively. EphrinB3 was sufficient to allow for viral entry of both pseudotype and live NiV. Soluble ephrinB2 and B3 were able to compete for NiV-envelope-mediated viral entry on both ephrinB2- and B3-expressing cells, suggesting that NiV-G interacts with both ephrinB2 and B3 via an overlapping site. Mutational analysis indicated that the Leu-Trp residues in the solvent exposed G-H loop of ephrinB2 and B3 were critical determinants of NiV binding and entry. Indeed, replacement of the Tyr-Met residues in the homologous positions in ephrinB1 with Leu-Trp conferred NiV receptor activity to ephrinB1. Thus, ephrinB3 is a bona fide alternate receptor for NiV entry, and two residues in the G-H loop of the ephrin B-class ligands are critical determinants of NiV receptor activity.

Quantitative analysis of protein-ligand interactions by NMR.

Science.gov (United States)

Furukawa, Ayako; Konuma, Tsuyoshi; Yanaka, Saeko; Sugase, Kenji

2016-08-01

Protein-ligand interactions have been commonly studied through static structures of the protein-ligand complex. Recently, however, there has been increasing interest in investigating the dynamics of protein-ligand interactions both for fundamental understanding of the underlying mechanisms and for drug development. NMR is a versatile and powerful tool, especially because it provides site-specific quantitative information. NMR has widely been used to determine the dissociation constant (KD), in particular, for relatively weak interactions. The simplest NMR method is a chemical-shift titration experiment, in which the chemical-shift changes of a protein in response to ligand titration are measured. There are other quantitative NMR methods, but they mostly apply only to interactions in the fast-exchange regime. These methods derive the dissociation constant from population-averaged NMR quantities of the free and bound states of a protein or ligand. In contrast, the recent advent of new relaxation-based experiments, including R2 relaxation dispersion and ZZ-exchange, has enabled us to obtain kinetic information on protein-ligand interactions in the intermediate- and slow-exchange regimes. Based on R2 dispersion or ZZ-exchange, methods that can determine the association rate, kon, dissociation rate, koff, and KD have been developed. In these approaches, R2 dispersion or ZZ-exchange curves are measured for multiple samples with different protein and/or ligand concentration ratios, and the relaxation data are fitted to theoretical kinetic models. It is critical to choose an appropriate kinetic model, such as the two- or three-state exchange model, to derive the correct kinetic information. The R2 dispersion and ZZ-exchange methods are suitable for the analysis of protein-ligand interactions with a micromolar or sub-micromolar dissociation constant but not for very weak interactions, which are typical in very fast exchange. This contrasts with the NMR methods that are used

Nanointaglio fabrication of optical lipid multilayer diffraction gratings with applications in biosensing

Science.gov (United States)

Lowry, Troy Warren

supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Presented next is a nanointaglio based method for quantitative measurements of lipid-protein interactions and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1. Optical diffraction gratings composed of lipids are printed on surfaces using nanointaglio, resulting in lipid multilayer gratings. Exposure of lipid multilayer gratings to Sar1 results in the inflation of lipid multilayers into unilamellar structures, the kinetics of which can be detected in a label-free manner by monitoring the diffraction of white light through an optical microscope. Local variations in lipid multilayer volume on the surface can be used to vary substrate availability in a microarray format, allowing kinetic and thermodynamic data to be obtained from a single experiment without the need for varying enzyme concentration. A quantitative model is developed and fits to the data allow measurements of both binding affinity (KD) and kinetics (kon and koff). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1 induced inflation of single bilayers from surface supported multilayers, the semi-cylindrical grating lines are observed to remodel into semi-spherical buds when a critical radius of curvature equal to 300 nm is reached, which is explained in terms of a Rayleigh type instability.

Motor abilities, movement skills and their relationship before and after eight weeks of martial arts training in people with intellectual disability [Motorické schopnosti, pohybové dovednosti a vztah mezi nimi u osob s mentálním postižením před osmitýdenním kurzem bojových umění a po jeho ukončení

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Damir Karpljuk

2012-06-01

judokas had been noted. The effects of the training program should be verified in future studies. It is worth reiterating that by improving motor abilities and expanding the boundaries of skills, people with intellectual disability enhance their quality of life. Attention in the future and thus contributes to a higher quality of intellectually disabled people’s engagement in sport as well as in life.[VÝCHODISKA: Je známo, že část populace osob s mentálním postižením má sklon k obezitě a horšímu zdravotnímu stavu. Proto by v jejich životech měl hrát významnou roli sport. Navrhnout vědecky a profesionálně účinné tréninkové programy pro sportovní vyžití osob s mentálním postižením, sestávající z nezbytné metodiky a didaktických pokynů, je sice těžký úkol, ale stává se nezbytností pro blízkou budoucnost. CÍLE: Cílem studie je stanovit vzájemný vztah mezi vy branými motorickými schopnostmi a pohybovými dovednostmi v rámci bojových umění, jakož i to, jak a do jaké míry osoby s mentálním postižením (MP, které pravidelně trénují judo, mohou po osmitýdenním tréninkovém kurzu ve svých dovednostech v oblasti bojových umění (judo, karate, box a šerm dosáhnout zlepšení. Zajímalo nás také, zda došlo k jakýmkoli změnám ve vybraných motorických dovednostech. METODIKA: Měření motorických schopností a dovedností v oblasti vybraných bojových umění bylo provedeno dvakrát – v březnu 2008, týden před začátkem kurzu, a v květnu 2008, týden po jeho ukončení. Tréninkový kurz trval dva měsíce v rozsahu dvou lekcí týdně. Zkoumaný vzorek sestával z 5 žen a 18 mužů ve věku mezi 16 a 36 lety s mírným až středně těžkým mentálním postižením. Studie byla provedena pomocí 8 testů pro hodnocení motorických schopností a 9 testů pro hodnocení dovedností v oblasti bojových umění. VÝSLEDKY: Výsledky t-testu pro závislé vzorky prokázaly statisticky v