Home About us Contact
|
Home About us Contact | ||
|
|
||
High Binding Affinity (high + binding_affinity)
Selected AbstractsPigment epithelium-derived factor binds to cell-surface F1 -ATP synthaseFEBS JOURNAL, Issue 9 2010Luigi Notari Pigment epithelium-derived factor (PEDF), a potent blocker of angiogenesis in vivo, and of endothelial cell migration and tubule formation, binds with high affinity to an as yet unknown protein on the surfaces of endothelial cells. Given that protein fingerprinting suggested a match of a , 60 kDa PEDF-binding protein in bovine retina with Bos taurus F1 -ATP synthase ,-subunit, and that F1Fo -ATP synthase components have been identified recently as cell-surface receptors, we examined the direct binding of PEDF to F1. Size-exclusion ultrafiltration assays showed that recombinant human PEDF formed a complex with recombinant yeast F1. Real-time binding as determined by surface plasmon resonance demonstrated that yeast F1 interacted specifically and reversibly with human PEDF. Kinetic evaluations revealed high binding affinity for PEDF, in agreement with PEDF affinities for endothelial cell surfaces. PEDF blocked interactions between F1 and angiostatin, another antiangiogenic factor, suggesting overlapping PEDF-binding and angiostatin-binding sites on F1. Surfaces of endothelial cells exhibited affinity for PEDF-binding proteins of , 60 kDa. Antibodies to F1,-subunit specifically captured PEDF-binding components in endothelial plasma membranes. The extracellular ATP synthesis activity of endothelial cells was examined in the presence of PEDF. PEDF significantly reduced the amount of extracellular ATP produced by endothelial cells, in agreement with direct interactions between cell-surface ATP synthase and PEDF. In addition to demonstrating that PEDF binds to cell-surface F1, these results show that PEDF is a ligand for endothelial cell-surface F1Fo -ATP synthase. They suggest that PEDF-mediated inhibition of ATP synthase may form part of the biochemical mechanisms by which PEDF exerts its antiangiogenic activity. Structured digital abstract ,,MINT-7711286: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F-ATPase alpha subunit (uniprotkb:P07251), F-ATPase beta subunit (uniprotkb:P00830), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase delta subunit (uniprotkb:Q12165) and F-ATPase epsilon subunit (uniprotkb:P21306) by competition binding (MI:0405) ,,MINT-7711113: angiostatin (uniprotkb:P00747) physically interacts (MI:0915) with F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit(uniprotkb:P00830) and F-ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107) ,,MINT-7711060: F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit (uniprotkb:P00830), F-ATPase alpha subunit (uniprotkb:P07251) and PEDF (uniprotkb:P36955) physically interact (MI:0915) by molecular sieving (MI:0071) ,,MINT-7711313: F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), PEDF (uniprotkb:P36955), F-ATPase alpha subunit (uniprotkb:P07251), F-ATPase beta subunit (uniprotkb:P00830) and F-ATPase gamma subunit(uniprotkb:P38077) physically interact (MI:0915) by molecular sieving (MI:0071) ,,MINT-7711083: PEDF (uniprotkb:P36955) physically interacts (MI:0915) with F-ATPase epsilon subunit (uniprotkb:P21306), F-ATPase delta subunit (uniprotkb:Q12165), F-ATPase gamma subunit (uniprotkb:P38077), F-ATPase beta subunit (uniprotkb:P00830) and F-ATPase alpha subunit (uniprotkb:P07251) by surface plasmon resonance (MI:0107) [source] Novel Magnetic Hydroxyapatite Nanoparticles as Non-Viral Vectors for the Glial Cell Line-Derived Neurotrophic Factor GeneADVANCED FUNCTIONAL MATERIALS, Issue 1 2010Hsi-Chin Wu Abstract Nanoparticles (NPs) of synthetic hydroxyapatite (Hap) and natural bone mineral (NBM) are rendered magnetic by treatment with iron ions using a wet-chemical process. The magnetic NPs (mNPs), which are about 300,nm in diameter, display superparamagnetic properties in a superconducting quantum interference device, with a saturation magnetization of about 30,emu g,1. X-ray diffraction and transmission electron microscopy reveal that the magnetic properties of the NPs are the result of the hetero-epitaxial growth of magnetite on the Hap and NBM crystallites. The mNPs display a high binding affinity for plasmid DNA in contrast to magnetite NPs which do not bind the plasmid well. The mHap and mNBM NPs result in substantial increases in the transfection of rat marrow-derived mesenchymal stem cells with the gene for glial cell line-derived neurotrophic factor (GDNF), with magnetofection compared to transfection in the absence of a magnet. The amount of GDNF recovered in the medium approaches therapeutic levels despite the small amount of plasmid delivered by the NPs. [source] The role of segment 32,47 of cholecystokinin receptor type A in CCK8 binding: synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studiesJOURNAL OF PEPTIDE SCIENCE, Issue 3 2003Stefania De Luca Abstract The segment 32,47 of the N -terminal extracellular domain of the type A cholecystokinin receptor, CCKA -R(32,47), was synthesized and structurally characterized in a membrane mimicking environment by CD, NMR and molecular dynamics calculations. The region of CCKA -R(32,47) encompassing residues 39,46 adopted a well-defined secondary structure in the presence of DPC micelles, whereas the conformation of the N -terminal region (segment 32,37) could not be uniquely defined by the NOE derived distance constraints because of local flexibility. The conformation of the binding domain of CCKA -R(32,47) was different from that found for the intact N -terminal receptor tail, CCKA -R(1,47). To assess whether CCKA -R(32,47) was still able to bind the nonsulfated cholecystokinin C -terminal octapeptide, CCK8, a series of titrations was carried out in SDS and DPC micelles, and the binding interaction was followed by fluorescence spectroscopy. These titrations gave no evidence for complex formation, whereas a high binding affinity was found between CCKA -R(1,47) and CCK8. The different affinities for the ligand shown by CCKA -R(32,47) and CCKA -R(1,47) were paralleled by different interaction modes between the receptor segments and the micelles. The interaction of CCKA -R(32,47) with DPC micelles was much weaker than that of CCKA -R(1,47), because the former receptor segment lacks proper stabilizing contacts with the micelle surface. In the case of SDS micelles CCKA -R(32,47) was found to form non-micellar adducts with the detergent that prevented the onset of a functionally significant interaction between the receptor segment and the micelle. It is concluded that tertiary structure interactions brought about by the 1,31 segment play a key role in the stabilization of the membrane bound, biologically active conformation of the N -terminal extracellular tail of the CCKA receptor. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd. [source] A genome-inspired DNA ligand for the affinity capture of insulin and insulin-like growth factor-2JOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2009Junfeng Xiao Abstract The insulin-linked polymorphic region (ILPR) of the human insulin gene contains tandem repeats of similar G-rich sequences, some of which form intramolecular G-quadruplex structures in vitro. Previous work showed affinity binding of insulin to an intramolecular G-quadruplex formed by ILPR variant a. Here, we report on interactions of insulin and the highly homologous insulin-like growth factor-2 (IGF-2) with ILPR variants a, h, and i. Circular dichroism indicated intramolecular G-quadruplex formation for variants a and h. Affinity MALDI MS and surface plasmon resonance were used to compare protein capture and binding strengths. Insulin and IGF-2 exhibited high binding affinity for variants a and h but not i, indicating the involvement of intramolecular G-quadruplexes. Interaction between insulin and variant a was unique in the appearance of two binding interactions with KD , 10,13 M and KD , 10,7 M, which was not observed for insulin with variant h (KD , 10,8 M) or IGF-2 with either variant (KDs , 10,9 M). The results provide a basis for the design of DNA binding ligands for insulin and IGF-2 and support a new approach to discovery of DNA affinity binding ligands based on genome-inspired sequences rather than the traditional combinatorial selection route to aptamer discovery. [source] Photothermal antimicrobial nanotherapy and nanodiagnostics with self-assembling carbon nanotube clustersLASERS IN SURGERY AND MEDICINE, Issue 7 2007Jin-Woo Kim PhD Abstract Background and Objectives Unique properties of carbon nanotubes (CNTs) would open new avenues for addressing challenges to realize rapid and sensitive antimicrobial diagnostics and therapy for human pathogens. In this study, new CNTs' capabilities for photothermal (PT) antimicrobial nanotherapy were explored in vitro using Escherichia coli as a model bacterium. Study Design/Materials and Methods Single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs) were incubated with E. coli K12 strain. CNTs' locations in bacteria and laser-induced thermal and accompanied effects around CNTs were estimated with TEM and PT microscopy, respectively. Multi-pulse lasers at 532 and 1064 nm with 12-ns pulse duration were used for irradiating sample mixtures at different laser fluences. Cell viability was evaluated using a bacterial viability test kit and epi-fluorescence microscopy. Results This study revealed CNTs' high binding affinity to bacteria, their capability to self-assemble as clusters at bacteria surfaces, and their inherent near-infrared (NIR) laser responsiveness. Cell viability was affected neither by CNTs alone nor by NIR irradiations alone. Notable changes in bacteria viability, caused by local thermal and accompanied bubble-formation phenomena, were observed starting at laser fluences of 0.1,0.5 J/cm2 with complete bacteria disintegration at 2,3 J/cm2 at both wavelengths. Furthermore, ethanol in reaction mixtures significantly (more than one order) enhanced bubble formation phenomena. Conclusion This first application of laser-activated CNTs as PT contrast antimicrobial agents demonstrated its great potential to cause irreparable damages to disease-causing pathogens as well as to detect the pathogens at single bacterium level. This unique integration of laser and nanotechnology may also be used for drinking water treatment, food processing, disinfection of medical instrumentation, and purification of grafts and implants. Furthermore, the significant ethanol-induced enhancement of bubble formation provides another unique possibility to improve the efficiency of selective nanophotothermolysis for treating cancers, wounds, and vascular legions. Lesers Surg. Med. 39:622,634, 2007. © 2007 Wiley-Liss, Inc. [source] Folate receptor , as a potential delivery route for novel folate antagonists to macrophages in the synovial tissue of rheumatoid arthritis patientsARTHRITIS & RHEUMATISM, Issue 1 2009Joost W. Van Der Heijden Objective To determine the expression of folate receptor , (FR,) in synovial biopsy tissues and peripheral blood lymphocytes from rheumatoid arthritis (RA) patients and to identify novel folate antagonists that are more selective in the targeting and internalization of FR, than methotrexate (MTX). Methods Immunohistochemistry and computer-assisted digital imaging analyses were used for the detection of FR, protein expression on immunocompetent cells in synovial biopsy samples from RA patients with active disease and in noninflammatory control synovial tissues. FR, messenger RNA (mRNA) levels were determined by reverse transcription,polymerase chain reaction analysis. Binding affinities of FR, for folate antagonists were assessed by competition experiments for 3H-folic acid binding on FR,-transfected cells. Efficacy of FR,-mediated internalization of folate antagonists was evaluated by assessment of antiproliferative effects against FR,-transfected cells. Results Immunohistochemical staining of RA synovial tissue showed high expression of FR, on macrophages in the intimal lining layer and synovial sublining, whereas no staining was observed in T cell areas or in control synovial tissue. Consistently, FR, mRNA levels were highest in synovial tissue extracts and RA monocyte-derived macrophages, but low in peripheral blood T cells and monocytes. Screening of 10 new-generation folate antagonists revealed 4 compounds for which FR, had a high binding affinity (20,77-fold higher than for MTX). One of these, the thymidylate synthase inhibitor BCG 945, displayed selective targeting against FR,-transfected cells. Conclusion Abundant FR, expression on activated macrophages in synovial tissue from RA patients deserves further exploration for selective therapeutic interventions with high-affinity,binding folate antagonists, of which BCG 945 may be a prototypical representative. [source] High-affinity human leucocyte antigen class I binding variola-derived peptides induce CD4+ T cell responses more than 30 years post-vaccinia virus vaccinationCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2009M. Wang Summary Interferon-, secreting T lymphocytes against pox virus-derived synthetic 9-mer peptides were tested by enzyme-linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high-affinity human leucocyte antigen (HLA) class I binders (KD , 5 nM). However, five of the individuals tested did not show typical CD8+ T cell-mediated HLA class I-restricted responses. Instead, these donors showed CD4+ T cell-dependent responses against four of a total of eight antigenic 9-mer peptides discovered recently by our group. These latter responses were blocked specifically in the presence of anti-HLA class II antibody. We conclude that long-lived memory responses against pox virus-derived 9-mer peptides, with high binding affinity for HLA class I molecules, are mediated in some cases by CD4+ T cells and apparently restricted by HLA class II molecules. [source] | |||||||||||||