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Protein Binding (protein + binding)
Kinds of Protein Binding Terms modified by Protein Binding Selected AbstractsCandidate cis -elements for human renin gene expression in the promoter regionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2004Tadashi Konoshita Abstract The regulation of renin gene expression, the rate-limiting enzyme of the system, is thought to be fundamental to the total system. Previously, we mapped six putative cis -elements in the promoter region of the human renin gene with nuclear proteins from human chorionic cells and human renal cortex by DNase I protection assay (footprint A,F). Each footprint contains Ets motif like site (A), HOXñPBX recognition sequence (B), unknown sequence as DNA binding consensus (C), CRE (D), COUP-TFII (ARP-1) motif like site (E), and AGE3 like site (F). Footprint D has been characterized by means of functional studies as the genuine human renin gene CRE interacting with CREB in cooperation with the site of footprint B. To obtain further clues to the specific expression in the promoter region, these putative cis -elements were conducted to a consensus-specific binding assay to compare renin-producing and non-renin-producing cells by EMSA and electromobility super-shift assay. Different sequence-specific DNA/protein binding was obtained among the different cell lines with footprint B site, with COUP-TFII (ARP-1) motif like site and possibly with footprint F site. The results implicate these putative cis -elements and each corresponding trans -factor in the specific expression of the human renin gene in the promoter region. Further functional characterization of these elements would provide important data for a better understanding of human renin gene expression. © 2004 Wiley-Liss, Inc. [source] Decorating Liquid Crystal Surfaces with Proteins for Real-Time Detection of Specific Protein,Protein BindingADVANCED FUNCTIONAL MATERIALS, Issue 22 2009Deny Hartono Abstract Here, a novel method of immobilizing proteins with well-defined orientation directly on liquid crystal surfaces that allow subsequent real-time imaging of specific protein,protein binding events on these surfaces is reported. Self-assembly of nitrilotriacetic acid terminated amphiphiles loaded with Ni2+ ions at aqueous-liquid crystal interface creates a surface capable of immobilizing histidine-tagged ubiquitin through complex formation between Ni2+ and histidine. When these surfaces containing immobilized histidine-tagged ubiquitin are exposed to anti-ubiquitin antibody, the spatial and temporal of specific protein,protein binding events trigger orientational transitions of liquid crystals. As a result, sharp liquid crystal optical switching from dark to bright can readily be observed under polarized lighting. The protein,protein binding can be observed within seconds and only requires nanogram quantities of proteins. This work demonstrates a simple strategy to immobilize proteins with well-defined orientation on liquid crystal surfaces for real-time and label-free detection of specific protein,protein binding events, which may find use in biomedical diagnostics. [source] Mineral-Coated Polymer Microspheres for Controlled Protein Binding and ReleaseADVANCED MATERIALS, Issue 19 2009Leenaporn Jongpaiboonkit Polymer microspheres with a bone-like mineral coatings are generated via a biomimetic process, and this biodegradable coating is used as a carrier for delivery of biological molecules. Acidic and basic proteins are controllably bound and released from these microspheres, suggesting that this approach can be used for binding and delivery of a broad range of biologically active molecules. [source] The Kazal motifs of RECK protein inhibit MMP-9 secretion and activity and reduce metastasis of lung cancer cells in vitro and in vivoJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6b 2008Chong-Keng Chang Abstract RECK is a membrane-anchored glycoprotein which may negatively regulate matrix metalloproteinase (MMP) activity to suppress tumor invasion and metastasis. In this study, recombinant proteins corresponding to the residues 285,368 (named as CKM which contained cysteine knot motif), 605,799 (named as K123 which contained three Kazal motifs), 676,799 (named as K23 which contained the last two Kazal motifs) and full-length RECK were produced and their anti-cancer effects were tested. Full-length RECK and K23 but not K123 and CKM inhibited MMP9 secretion and activity. In addition, RECK and K23 inhibited invasion but not migration of metastatic lung cancer cells in vitro. Protein binding and kinetic study indicated that K23 physically interacted with MMP-9 and inhibited its activity by a non-competitive manner. Moreover, K23 reduced metastatic tumor growth in lungs of nude mice. Taken together, our results suggest that the K23 motifs of RECK protein can inhibit MMP-9 secretion and activity and attenuate metastasis of lung cancer cells. [source] The chemopreventive compound curcumin is an efficient inhibitor of Epstein-Barr virus BZLF1 transcription in Raji DR-LUC cells,MOLECULAR CARCINOGENESIS, Issue 3 2002Manfred Hergenhahn Abstract To characterize the effects of inhibitors of Epstein-Barr virus (EBV) reactivation, we established Raji DR-LUC cells as a new test system. These cells contain the firefly luciferase (LUC) gene under the control of an immediate-early gene promoter (duplicated right region [DR]) of EBV on a self-replicating episome. Luciferase induction thus serves as an intrinsic marker indicative for EBV reactivation from latency. The tumor promoter 12- O -tetradecanoylphorbol-13-acetate (TPA) induced the viral key activator BamH fragment Z left frame 1 (BZLF1) protein ("ZEBRA") in this system, as demonstrated by induction of the BZLF1 protein-responsive DR promoter upstream of the luciferase gene. Conversely, both BZLF1 protein and luciferase induction were inhibited effectively by the chemopreventive agent curcumin. Semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) further demonstrated that the EBV inducers TPA, sodium butyrate, and transforming growth factor-, (TGF-,) increased levels of the mRNA of BZLF1 mRNA at 12, 24, and 48 h after treatment in these cells. TPA treatment also induced luciferase mRNA with similar kinetics. Curcumin was found to be highly effective in decreasing TPA-, butyrate-, and TGF-,-induced levels of BZLF1 mRNA, and of TPA-induced luciferase mRNA, indicating that three major pathways of EBV are inhibited by curcumin. Electrophoretic mobility shift assays (EMSA) showed that activator protein 1 (AP-1) binding to a cognate AP-1 sequence was detected at 6 h and could be blocked by curcumin. Protein binding to the complete BZLF1 promoter ZIII site (ZIIIA+ZIIIB) demonstrated several specific complexes that gave weak signals at 6 h and 12 h but strong signals at 24 h, all of which were reduced after application of curcumin. Autostimulation of BZLF1 mRNA induction through binding to the ZIII site at 24 h was confirmed by antibody-induced supershift analysis. The present results confirm our previous finding that curcumin is an effective agent for inhibition of EBV reactivation in Raji DR-CAT cells (carrying DR-dependent chloramphenicol acetyltransferase), and they show for the first time that curcumin inhibits EBV reactivation mainly through inhibition of BZLF1 gene transcription. © 2002 Wiley-Liss, Inc. [source] Zinc diethyldithiocarbamate allergenicity: potential haptenation mechanismsCONTACT DERMATITIS, Issue 2 2008Itai Chipinda Background:, Zinc diethyldithiocarbamate (ZDEC) and its disulfide, tetraethylthiuram disulfide (TETD), are rubber accelerators and contact allergens that cross-react in some individuals. Objective:, This study explored potential protein haptenation mechanisms of ZDEC and its oxidation products. Methods:, ZDEC oxidation/reduction products and sites of protein binding were assessed using high-performance liquid chromatography and mass spectrometry. The murine local lymph node assay (LLNA) was employed to probe haptenation mechanisms of ZDEC by examining its allergenicity along with its oxidation products and through elimination of oxidation and chelation mechanisms by substituting cobalt for zinc [cobalt (II) dithiocarbamate, CoDEC]. Results:, Oxidation of ZDEC by hypochlorous acid (bleach, HOCl), iodine, or hydrogen peroxide resulted in production of TETD, tetraethylthiocarbamoyl disulfide, and tetraethyldicarbamoyl disulfide (TEDCD). Albumin thiols reduced TETD with subsequent mixed disulfide formation/haptenation. ZDEC directly chelated the copper ion on the active site of the superoxide dismutase, whereas CoDEC did not bind to Cu proteins or form mixed disulfides with free thiols. ZDEC, sodium diethyldithiocarbamate, TEDCD, and TETD were all positive in the LLNA except CoDEC, which was non-allergenic. Conclusion:, The thiol is the critical functional group in ZDEC's allergenicity, and haptenation is predominantly through chelation of metalloproteins and formation of mixed disulfides. [source] Hapten,protein binding: from theory to practical application in the in vitro prediction of skin sensitizationCONTACT DERMATITIS, Issue 4 2005Maja Divkovic In view of the forthcoming European Union ban on in vivo testing of cosmetic and toiletry ingredients, following the publication of the 7th amendment to the Cosmetics Directive, the search for practical, alternative, non-animal approaches is gathering pace. For the end-point of skin sensitization, the ultimate goal, i.e. the development and validation of alternative in vitro/in silico assays by 2013, may be achieved through a better understanding of the skin sensitization process on the cellular and molecular levels. One of the key molecular events in skin sensitization is protein haptenation, i.e. the chemical modification of self-skin protein(s) thus forming macromolecular immunogens. This concept is widely accepted and in theory can be used to explain the sensitizing capacity of many known skin sensitizers. Thus, the principle of protein or peptide haptenation could be used in in vitro assays to predict the sensitization potential of a new chemical entity. In this review, we consider some of the theoretical aspects of protein haptenation, how mechanisms of protein haptenation can be investigated experimentally and how we can use such knowledge in the development of novel, alternative approaches for predicting skin sensitization potential in the future. [source] Immobilization and Electrochemistry of Negatively Charged Proteins on Modified Nanocrystalline Metal Oxide ElectrodesELECTROANALYSIS, Issue 12 2005Emmanuel Topoglidis Abstract The immobilization of two acidic, low isoelectric point proteins, green fluorescence protein and ferredoxin (FRD) is investigated on nanocrystalline, mesoporous TiO2 and SnO2 electrodes. Modification of these electrodes with a cationic polypeptide (poly- L -lysine) or an aminosilane prior to protein immobilization is found to enhance protein binding at least ten fold, attributed to more favorable protein/electrode electrostatic interactions. Cyclic voltammetry studies of FRD-modified SnO2 electrodes indicate reversible protein electrochemistry with a midpoint potential of ,0.59,V (vs. Ag/AgCl) and an interfacial electron transfer rate constant of 0.45,s,1. [source] Capillary electrophoresis of liposomes functionalized for protein bindingELECTROPHORESIS, Issue 20 2006Gerhard Bilek Abstract CE enabled assessing the attachment of hexa-histidine-tagged proteins to functionalized phospholipid liposomes. The liposomes were made of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine, phosphatidyl-ethanolamine, cholesterol and distearoyl-glycero-3-phosphoethanolamine- N -methoxy(polyethylene glycol) in a molar ratio of 29:26:40:5. The unilamellar vesicles, which had an average diameter of 170,nm, were labelled by inclusion of FITC-dextran for fluorescence detection. CE was carried out in poly(vinyl alcohol) (PVA)-coated capillaries at 25°C with a BGE consisting of Tris-HCl (50,mM, pH,8.0). For conjugation of the liposomes with the proteins (soluble synthetic receptor fragments with molecular mass of 60 and 70,kDa, respectively), Ni2+ was implanted into the vesicle surface by an anchor lipid containing a nitrilotriacetate acid (NTA) group as complexation agent for the metal ions. The difference in surface charge enabled the separation of the different species of interest by CE: plain vesicles, vesicles functionalised with Ni-NTA, vesicle,protein complexes and the species formed upon removal of the Ni-ions by complexation with EDTA. Loss of the Ni-ions resulted in the release of the proteins and the reappearance of the plain Ni-free NTA-liposome species in the electropherograms. [source] Analysis of genomic dose-response information on arsenic to inform key events in a mode of action for carcinogenicityENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2010P. Robinan Gentry Abstract A comprehensive literature search was conducted to identify information on gene expression changes following exposures to inorganic arsenic compounds. This information was organized by compound, exposure, dose/concentration, species, tissue, and cell type. A concentration-related hierarchy of responses was observed, beginning with changes in gene/protein expression associated with adaptive responses (e.g., preinflammatory responses, delay of apoptosis). Between 0.1 and 10 ,M, additional gene/protein expression changes related to oxidative stress, proteotoxicity, inflammation, and proliferative signaling occur along with those related to DNA repair, cell cycle G2/M checkpoint control, and induction of apoptosis. At higher concentrations (10,100 ,M), changes in apoptotic genes dominate. Comparisons of primary cell results with those obtained from immortalized or tumor-derived cell lines were also evaluated to determine the extent to which similar responses are observed across cell lines. Although immortalized cells appear to respond similarly to primary cells, caution must be exercised in using gene expression data from tumor-derived cell lines, where inactivation or overexpression of key genes (e.g., p53, Bcl-2) may lead to altered genomic responses. Data from acute in vivo exposures are of limited value for evaluating the dose-response for gene expression, because of the transient, variable, and uncertain nature of tissue exposure in these studies. The available in vitro gene expression data, together with information on the metabolism and protein binding of arsenic compounds, provide evidence of a mode of action for inorganic arsenic carcinogenicity involving interactions with critical proteins, such as those involved in DNA repair, overlaid against a background of chemical stress, including proteotoxicity and depletion of nonprotein sulfhydryls. The inhibition of DNA repair under conditions of toxicity and proliferative pressure may compromise the ability of cells to maintain the integrity of their DNA. Environ. Mol. Mutagen., 2010. © 2009 Wiley-Liss, Inc. [source] Pharmacodynamic Analysis of the Interaction between Tiagabine and Midazolam with an Allosteric Model That Incorporates Signal TransductionEPILEPSIA, Issue 3 2003Daniël M. Jonker Summary: ,Purpose: The objective of this study was to characterize quantitatively the pharmacodynamic interaction between midazolam (MDL), an allosteric modulator of the ,-aminobutyric acid subtype A (GABAA) receptor, and tiagabine (TGB), an inhibitor of synaptic GABA uptake. Methods: The in vivo concentration,response relation of TGB was determined through pharmacokinetic/pharmacodynamic (PK/PD) modeling. Rats received a single intravenous dose of 10 mg/kg TGB in the absence and the presence of a steady-state plasma concentration of MDL. The EEG response in the 11.5- to 30-Hz frequency band was used as the pharmacodynamic end point. Results: Infusion of MDL resulted in a mean steady-state plasma concentration of 66 ± 3 ng/ml. A significant pharmacokinetic interaction with TGB was observed. MDL inhibited TGB clearance by 20 ± 7 ml/min/kg from the original value of 89 ± 6 ml/min/kg. However, no changes in plasma protein binding of both drugs were observed. The concentration,EEG relation of TGB was described by the sigmoid- Emax model. The pharmacodynamic parameter estimates of TGB were: Emax = 327 ± 10 ,V, EC50 = 392 ± 20 ng/ml, and nH = 3.1 ± 0.3. These values were not significantly different in the presence of MDL. Factors that may explain the lack of synergism were identified by a mechanism-based interaction model that separates the receptor activation from the signal-transduction process. High efficiency of signal transduction and the presence of a baseline response were shown to diminish the degree of synergism. Conclusions: We conclude that the in vivo pharmacodynamic interaction between MDL and TGB is additive rather than synergistic. This strongly suggests that allosteric modulation of the antiseizure activity of a GAT-1 inhibitor by a benzodiazepine does not offer a therapeutic advantage. [source] Treatment of congestive heart failure , current status of use of digitoxinEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue S2 2001G. G. Belz Digitalis glycosides exert a positive inotropic effect, i.e. an increase in myocardial contractility associated with a prolongation of relaxation period, and glycosides lower the heart rate (negative chronotropic), impede stimulus conduction (negative dromotropic) and promote myocardial excitability (positive bathmotropic). They seem to influence the activities of both the vagal and the sympathetic systems. Digitalis glycosides that belong to different substance classes are closely comparable concerning pharmacodynamics but differ substantially in regard to pharmacokinetics. Digoxin and its derivatives are less lipophilic, show lower protein binding and shorter half-life, are mainly eliminated via the kidney and accumulate rather rapidly in cases of insufficient kidney function. Digitoxin is highly lipophilic and extensively bound to plasma proteins, has a longer half-life, is mainly eliminated in the metabolized state via urine and faeces and does not accumulate in kidney dysfunction. As a result of a more stable pharmacokinetic profile, the incidence of toxic side effects seems to be lower with digitoxin than with digoxin. Since the beginning of the 1990s, the antagonists of the RAAS qualified as the standard treatment for congestive heart failure, often in combination with diuretics, vasodilators or ,-antagonists. However, the important role of digitalis glycosides as therapeutic comedication or alternative was never denied, especially in atrial fibrillation with tachycardia. The PROVED and RADIANCE trials proved a detrimental effect of the withdrawal of digoxin therapy on exercise capacity, left-ventricular ejection fraction and clinical symptoms. The DIG trial revealed that digoxin comedication in sinus rhythm patients with congestive heart failure was associated with a lower morbidity (as taken from death or hospitalization because of worsening heart failure) and an unchanged overall mortality , being a unique feature among the available inotropic drugs. Comparable studies for digitoxin have not yet been performed but, because of its higher pharmacological stability, it might well be associated with even more advantages in this regard than digoxin. [source] Differential effects of histone deacetylase inhibitors on phorbol ester- and TGF-,1 induced murine tissue inhibitor of metalloproteinases-1 gene expressionFEBS JOURNAL, Issue 8 2005David A. Young Expression of the tissue inhibitor of metalloproteinases-1 (Timp-1) gene can be induced by either phorbol myristate acetate (PMA) or transforming growth factor ,1 (TGF-,1), although the signalling pathways involved are not clearly defined. Canonically, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate (NaB) increase total cellular histone acetylation and activate expression of susceptible genes. Remarkably, PMA and TGF-,1 stimulation of Timp-1 show a differential response to TSA or NaB. TSA or NaB potentiate PMA-induced Timp-1 expression but repress TGF-,1-induced Timp-1 expression. The repression of TGF-,1-induced Timp-1 by TSA was maximal at 5 ng·mL,1, while for the superinduction of PMA-induced Timp-1 expression, the maximal dose is >,500 ng·mL,1 TSA. A further HDACi, valproic acid, did not block TGF-,1-induced Timp-1 expression, demonstrating that different HDACs impact on the induction of Timp-1. For either PMA or TGF-,1 to induce Timp-1 expression, new protein synthesis is required, and the induction of AP-1 factors closely precedes that of Timp-1. The effects of the HDACi can be reiterated in transient transfection using Timp-1 promoter constructs. Mutation or deletion of the AP-1 motif (,59/,53) in the Timp-1 promoter diminishes PMA-induction of reporter constructs, however, the further addition of TSA still superinduces the reporter. In c-Jun,/, cells, PMA still stimulates Timp-1 expression, but TSA superinduction is lost. Transfection of a series of Timp-1 promoter constructs identified three regions through which TSA superinduces PMA-induced Timp-1 and we have demonstrated specific protein binding to two of these regions which contain either an avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets) or Sp1 binding motif. [source] A steady-state modeling approach to validate an in vivo mechanism of the GAL regulatory network in Saccharomyces cerevisiaeFEBS JOURNAL, Issue 20 2004Malkhey Verma Cellular regulation is a result of complex interactions arising from DNA,protein and protein,protein binding, autoregulation, and compartmentalization and shuttling of regulatory proteins. Experiments in molecular biology have identified these mechanisms recruited by a regulatory network. Mathematical models may be used to complement the knowledge-base provided by in vitro experimental methods. Interactions identified by in vitro experiments can lead to the hypothesis of multiple candidate models explaining the in vivo mechanism. The equilibrium dissociation constants for the various interactions and the total component concentration constitute constraints on the candidate models. In this work, we identify the most plausible in vivo network by comparing the output response to the experimental data. We demonstrate the methodology using the GAL system of Saccharomyces cerevisiae for which the steady-state analysis reveals that Gal3p neither dimerizes nor shuttles between the cytoplasm and the nucleus. [source] Decorating Liquid Crystal Surfaces with Proteins for Real-Time Detection of Specific Protein,Protein BindingADVANCED FUNCTIONAL MATERIALS, Issue 22 2009Deny Hartono Abstract Here, a novel method of immobilizing proteins with well-defined orientation directly on liquid crystal surfaces that allow subsequent real-time imaging of specific protein,protein binding events on these surfaces is reported. Self-assembly of nitrilotriacetic acid terminated amphiphiles loaded with Ni2+ ions at aqueous-liquid crystal interface creates a surface capable of immobilizing histidine-tagged ubiquitin through complex formation between Ni2+ and histidine. When these surfaces containing immobilized histidine-tagged ubiquitin are exposed to anti-ubiquitin antibody, the spatial and temporal of specific protein,protein binding events trigger orientational transitions of liquid crystals. As a result, sharp liquid crystal optical switching from dark to bright can readily be observed under polarized lighting. The protein,protein binding can be observed within seconds and only requires nanogram quantities of proteins. This work demonstrates a simple strategy to immobilize proteins with well-defined orientation on liquid crystal surfaces for real-time and label-free detection of specific protein,protein binding events, which may find use in biomedical diagnostics. [source] Functional Hydrogel Surfaces: Binding Kinesin-Based Molecular Motor Proteins to Selected Patterned Sites,ADVANCED FUNCTIONAL MATERIALS, Issue 8 2005T. Yu Abstract Hydrogel microstructures with micrometer-scale topography and controllable functionality have great potential for numerous nanobiotechnology applications including, for example, three-dimensional structures that exhibit controlled interactions with proteins and cells. Taking advantage of the strong affinity of histidine (His) residues for metal-ion,nitrilotriacetic acid (NTA) complexes, we have chemically modified hydrogels to enable protein immobilization with retention of activity by incorporating 2-methacrylamidobutyl nitrilotriacetic acid, an NTA-containing monomer that can be copolymerized with a series of monomers to form NTA-containing hydrogels. By varying the NTA-monomer composition in the hydrogels, it is possible to control the amount of protein bound to the hydrogel surface. The retention of biological activity was demonstrated by microtubule gliding assays. Normally, hydrogels are resistant to protein binding, but we have selected these materials because of their porous nature. Bringing together hydrogel functionalization and soft-lithography patterning techniques, it was possible to create a hybrid hydrogel superstructure that possesses binding specificity to His-tagged protein in selected sites. This type of surface and microstructure is not only advantageous for motor protein integration, but it can also be generally applied to the formation of His-tagged molecules for sensors and biochip applications. [source] Uremic Toxins: Removal with Different TherapiesHEMODIALYSIS INTERNATIONAL, Issue 2 2003Raymond C. Vanholder A convenient way to classify uremic solutes is to subdivide them according to the physicochemical characteristics influencing their dialytic removal into small water-soluble compounds (<500 Da), protein-bound compounds, and middle molecules (>500 Da). The prototype of small water-soluble solutes remains urea although the proof of its toxicity is scanty. Only a few other water-soluble compounds exert toxicity (e.g., the guanidines, the purines), but most of these are characterized by an intra-dialytic behavior, which is different from that of urea. In addition, the protein-bound compounds and the middle molecules behave in a different way from urea, due to their protein binding and their molecular weights, respectively. Because of these specific removal patterns, it is suggested that new approaches of influencing uremic solute concentration should be explored, such as specific adsorptive systems, alternative dialytic timeframes, removal by intestinal adsorption, modification of toxin, or general metabolism by drug administration. Middle molecule removal has been improved by the introduction of large pore, high-flux membranes, but this approach seems to have come close to its maximal removal capacity, whereas multicompartmental behavior might become an additional factor hampering attempts to decrease toxin concentration. Hence, further enhancement of uremic toxin removal should be pursued by the introduction of alternative concepts of elimination. [source] Back to the Future: Middle Molecules, High Flux Membranes, and Optimal DialysisHEMODIALYSIS INTERNATIONAL, Issue 1 2003Raymond C. Vanholder Middle molecules can be defined as compounds with a molecular weight (MW) above 500 Da. An even broader definition includes those molecules that do not cross the membranes of standard low-flux dialyzers, not only because of molecular weight, but also because of protein binding and/or multicompartmental behavior. Recently, several of these middle molecules have been linked to the increased tendency of uremic patients to develop inflammation, malnutrition, and atheromatosis. Other toxic actions can also be attributed to the middle molecules. In the present publication we will consider whether improved removal of middle molecules by large pore membranes has an impact on clinical conditions related to the uremic syndrome. The clinical benefits of large pore membranes are reduction of uremia-related amyloidosis; maintenance of residual renal function; and reduction of inflammation, malnutrition, anemia, dyslipidemia, and mortality. It is concluded that middle molecules play a role in uremic toxicity and especially in the processes related to inflammation, atherogenesis, and malnutrition. Their removal seems to be related to a better outcome, although better biocompatibility of membranes might be a confounding factor. [source] Specific Protein Detection Using Thermally Reduced Graphene Oxide Sheet Decorated with Gold Nanoparticle-Antibody ConjugatesADVANCED MATERIALS, Issue 32 2010Shun Mao A highly sensitive and selectivefield-effect transistor biosensor using thermally reduced graphene oxide (TRGO) sheet decorated with gold nanoparticle-antibody conjugates is demonstrated. Probe antibody (anti-Immunoglobulin G) is labeled on the surface of the TRGO sheet through gold nanoparticles and electrical detection of the protein binding (Immunoglobulin G and anti-Immunoglobulin G) is accomplished by FET and dc measurements. [source] Lopinavir protein binding in HIV-1-infected pregnant womenHIV MEDICINE, Issue 4 2010FT Aweeka Background Pregnancy may alter protein binding (PB) of highly bound protease inhibitors due to changes in plasma concentrations of albumin and ,-1 acid glycoprotein (AAG). Small changes in PB can greatly impact the fraction of drug unbound (FU) exerting pharmacological effect. We report lopinavir (LPV) PB during third trimester (antepartum, AP) compared to ,1.7 weeks postpartum (PP) to determine if FU changes compensate for reduced total concentrations reported previously. Methods P1026s enrolled women receiving LPV/ritonavir, soft gel capsules 400/100 mg or 533/133 mg twice daily. LPV FU, albumin and AAG were determined AP and PP. Results AP/PP samples were available from 29/25 women respectively with all but one woman receiving the same dose AP/PP. LPV FU was increased 18% AP vs. PP (mean 0.96±0.16% AP vs. 0.82±0.21% PP, P=0.001). Mean protein concentrations were reduced AP (AAG=477 mg/L; albumin=3.28 mg/dL) vs. PP (AAG=1007 mg/L; albumin=3.85 mg/dL) (P<0.0001 for each comparison). AAG concentration correlated with LPV binding. Total LPV concentration did not correlate with LPV FU AP or PP. However, higher LPV concentration PP was associated with reduced PB and higher FU after adjustment for AAG. Conclusions LPV FU was higher and AAG lower AP vs. PP. The 18% increase in LPV FU AP is smaller than the reduction in total LPV concentration reported previously and is not of sufficient magnitude to eliminate the need for an increased dose during pregnancy. [source] No pharmacokinetic interaction between paliperidone extended-release tablets and trimethoprim in healthy subjects,HUMAN PSYCHOPHARMACOLOGY: CLINICAL AND EXPERIMENTAL, Issue 7 2009An Thyssen Abstract Objective The effect of trimethoprim, a potent organic cation transport inhibitor, on the pharmacokinetics (PK) of paliperidone extended-release tablets (paliperidone ER), an organic cation mainly eliminated via renal excretion, was assessed. Methods Open-label, two-period, randomized, crossover study in 30 healthy males. Single dose of paliperidone ER 6,mg was administered either alone on day 1 or day 5 during an 8-day treatment period of trimethoprim 200,mg twice daily. Serial blood and urine samples were collected for PK and plasma protein binding of paliperidone and its enantiomers. The 90% confidence interval (CI) of ratios with/without trimethoprim for PK parameters of paliperidone and its enantiomers calculated. Results Creatinine clearance decreased from 119 to 102,mL,min,1 with trimethoprim. Addition of trimethoprim increased unbound fraction of paliperidone by 16%, renal clearance by 13%, AUC, by 9%, and t½ by 19%. The 90% CIs for ratios with/without trimethoprim were within the 80,125% range for Cmax, AUClast, and renal clearance. For AUC,, 90% CI was 79.37,101.51, marginally below the lower bound of the acceptance range. Paliperidone did not affect steady-state plasma concentrations of trimethoprim. Conclusions No clinically important drug interactions are expected when paliperidone ER is administered with organic cation transport inhibitors. Copyright © 2009 John Wiley & Sons, Ltd. [source] Bioinspired Design of Dynamic MaterialsADVANCED MATERIALS, Issue 23 2009Javeed Shaikh Mohammed Abstract An emerging approach for design of dynamic materials involves mimicking natural systems, which are adept at changing their structure and function in response to their environment. Biological systems possess a diverse range of dynamic mechanisms, including competitive ligand,protein binding, enzyme-catalyzed remodeling, and allosteric protein conformational changes. These dynamic mechanisms are now being exploited by materials scientists and engineers to design "bioinspired" synthetic materials that undergo responsive assembly and disassembly as well as dynamic volume and shape changes. The purpose of this review is to describe recent progress in design and development of bioinspired dynamic materials, with a particular emphasis on hydrogel networks. We specifically focus on emerging approaches that use biological phenomena as an inspiration for design of materials. [source] Genomic location analysis by ChIP-SeqJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2009Artem Barski Abstract The interaction of a multitude of transcription factors and other chromatin proteins with the genome can influence gene expression and subsequently cell differentiation and function. Thus systematic identification of binding targets of transcription factors is key to unraveling gene regulation networks. The recent development of ChIP-Seq has revolutionized mapping of DNA,protein interactions. Now protein binding can be mapped in a truly genome-wide manner with extremely high resolution. This review discusses ChIP-Seq technology, its possible pitfalls, data analysis and several early applications. J. Cell. Biochem. 107: 11,18, 2009. © 2009 Wiley-Liss, Inc. [source] Drug,herb interactions: unexpected suppression of free Danshen concentrations by salicylateJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 6 2002Deepali Gupta Abstract The general population of the U.S. uses over-the-counter herbal medicines. Danshen is a Chinese herbal product used for the treatment of cardiovascular disease. In a previous study we showed that Danshen has significant digoxin-like immunoreactivity, and we used this parameter to monitor total and free Danshen activities in sera (10). In this report we demonstrated strong protein binding of Danshen (50,70%), and we also identified albumin as the major serum protein that binds Danshen. Because salicylate, which is also strongly bound to albumin, is a widely used over-the-counter medicine in the U.S., we studied Danshen,salicylate interaction in vitro. We observed no significant change in free Danshen concentrations as measured by free-digoxin-like activity when salicylate concentrations were subtherapeutic (,100 ,g/mL). With therapeutic concentrations of salicylate (,150 ,g/mL), the free Danshen concentrations significantly decreased from the control. On the other hand, Danshen can displace salicylate from protein binding, thereby increasing the free salicylate concentration. We conclude that salicylate in therapeutic concentration can significantly decrease free Danshen concentrations, and Danshen can displace salicylate. J. Clin. Lab. Anal. 16:290,294, 2002. © 2002 Wiley-Liss, Inc. [source] Protein,protein docking dealing with the unknownJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 2 2010Irina S. Moreira Abstract Protein,protein binding is one of the critical events in biology, and knowledge of proteic complexes three-dimensional structures is of fundamental importance for the biochemical study of pharmacologic compounds. In the past two decades there was an emergence of a large variety of algorithms designed to predict the structures of protein,protein complexes,a procedure named docking. Computational methods, if accurate and reliable, could play an important role, both to infer functional properties and to guide new experiments. Despite the outstanding progress of the methodologies developed in this area, a few problems still prevent protein,protein docking to be a widespread practice in the structural study of proteins. In this review we focus our attention on the principles that govern docking, namely the algorithms used for searching and scoring, which are usually referred as the docking problem. We also focus our attention on the use of a flexible description of the proteins under study and the use of biological information as the localization of the hot spots, the important residues for protein,protein binding. The most common docking softwares are described too. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2010 [source] Different mechanisms influencing permeation of PDGF-AA and PDGF-BB across the blood,brain barrierJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Abba J. Kastin Abstract Platelet-derived growth factor (PDGF) exerts neurotrophic and neuromodulatory effects on the CNS. To determine the permeability of the blood,brain barrier (BBB) to PDGF, we examined the blood-to-brain influx of radioactively labeled PDGF isoforms (PDGF-AA and PDGF-BB) by multiple-time regression analysis after intravenous (i.v.) injection and by in-situ perfusion, and also determined the physicochemical characteristics which affect their permeation across the BBB, including lipophilicity (measured by octanol:buffer partition coefficient), hydrogen bonding (measured by differences in octanol : buffer and isooctane : buffer partition coefficients), serum protein binding (measured by capillary electrophoresis), and stability of PDGF in blood 10 min after i.v. injection (measured by HPLC). After i.v. bolus injection, neither 125I-PDGF-AA nor 125I-PDGF-BB crossed the BBB, their influx rates being similar to that of the vascular marker 99mTc-albumin. 125I-PDGF-AA degraded significantly faster in blood than 125I-PDGF-BB. PDGF-BB, however, was completely bound to a large protein in serum whereas PDGF-AA showed no binding. Thus, degradation might explain the poor blood-to-brain influx of PDGF-AA, whereas protein binding could explain the poor influx of circulating PDGF-BB. Despite their lack of permeation in the intact mouse, both 125I-PDGF-AA and 125I-PDGF-BB entered the brain by perfusion in blood-free buffer, and the significantly faster rate of 125I-PDGF-AA than 125I-PDGF-BB may be explained by the lower hydrogen bonding potential of 125I-PDGF-AA. Thus, the lack of significant distribution of PDGF from blood to brain is not because of the intrinsic barrier function of the BBB but probably because of degradation and protein binding. Information from these studies could be useful in the design of analogues for delivery of PDGF as a therapeutic agent. [source] On the possibility of self-induction of drug protein bindingJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2010Leonid M. Berezhkovskiy Abstract The equilibrium unbound drug fraction (fu) is an important pharmacokinetic parameter, which influences drug elimination and distribution in the body. Commonly the drug plasma concentration is substantially less then that of drug binding proteins, so that fu can be assumed constant independent of drug concentration. A general consideration of protein binding based on the mass-action law provides that the unbound drug fraction increases with the increase of drug concentration, which is also a usual experimental observation. For several drugs, though, a seemingly unusual sharp decrease of the unbound drug fraction with the increase of total drug concentration (Ro) in the interval 0,<,Ro,,,5,µM was experimentally observed. A possible explanation of this apparently strange phenomenon is presented. The explanation is based on the consideration of a two-step mechanism of drug protein binding. The first step occurs as a drug binding to the site with relatively low affinity. Consequently this binding leads to the activation of a high affinity site, which otherwise is not available for binding. The suggested binding scheme yields the curves for fu dependence on the total drug concentration that are in good agreement with experimental measurements. The interpretation of pharmacokinetic data for the drugs with such unusual concentration dependence of fu appears to be a formidable problem. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:4400,4405, 2010 [source] Ionization-specific prediction of blood,brain permeabilityJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 1 2009Kiril Lanevskij Abstract This study presents a mechanistic QSAR analysis of passive blood,brain barrier permeability of drugs and drug-like compounds in rats and mice. The experimental data represented in vivo log,PS (permeability-surface area product) from in situ perfusion, brain uptake index, and intravenous administration studies. A data set of 280 log,PS values was compiled. A subset of 178 compounds was assumed to be driven by passive transport that is free of plasma protein binding and carrier-mediated effects. This subset was described in terms of nonlinear lipophilicity and ionization dependences, that account for multiple kinetic and thermodynamic effects: (i) drug's kinetic diffusion, (ii) ion-specific partitioning between plasma and brain capillary endothelial cell membranes, and (iii) hydrophobic entrapment in phospholipid bilayer. The obtained QSAR model provides both good statistical significance (RMSE,<,0.5) and simple physicochemical interpretations (log,P and pKa), thus providing a clear route towards property-based design of new CNS drugs. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:122,134, 2009 [source] Effects of salts on protein,surface interactions: applications for column chromatographyJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2007Kouhei Tsumoto Abstract Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein,protein or protein,surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein,protein or protein,surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 1677,1690, 2007 [source] High-throughput determination of the free fraction of drugs strongly bound to plasma proteinsJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 4 2004Joachim Schuhmacher Abstract Quantification of protein binding of new chemical entities is an important early screening step during drug discovery and is of fundamental interest for the estimation of safety margins during drug development. In this publication, we describe the development of a new high-throughput assay for the determination of the free drug fraction in plasma (fu). The new technique is an enhancement of the previously published erythrocytes partition method. It is based on the distribution of drugs between plasma water, plasma proteins, and solid-supported lipid membranes (Transil®). The execution of protein binding studies by partitioning is dramatically simplified by substituting erythrocytes with commercially available Transil® beads, and makes the method particularly suitable for high-throughput studies. Eight Bayer compounds from different compound classes covering a wide range of lipophilicities (log P,=,1.9,5.6) and fu values (0.018,35%) were selected for validation of the assay. The results obtained by the new method and by either the erythrocytes partitioning technique or more conventional methods (ultrafiltration and equilibrium dialysis) are identical, confirming that the new method produces valid results even for drugs that are strongly bound to plasma proteins. Precision and accuracy of the data in the cases of very low and high fu values are comparable, indicating that the method is especially suited for highly lipophilic drugs that tend to adsorb to surfaces compared with other methods, like ultrafiltration or equilibrium dialysis, that may produce biased data. The method is also useful for the determination of binding parameters and the pH dependence of fu. In summary, this assay is well suited for high-throughput determination of protein binding during drug discovery and for extended protein binding studies during drug development. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93: 816,830, 2004 [source] | |||||||||||||||||||||||||||||||