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Y. Gloria Meng - One of the best experts on this subject based on the ideXlab platform.
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Real-time immuno-polymerase chain reaction in a 384-well format: detection of vascular endothelial Growth factor and epidermal Growth Factor-Like Domain 7.
Analytical Biochemistry, 2014Co-Authors: Jianhuan Zhang, Jean Michel Vernes, Jennifer Ni, Steven T. Chen, Richard Vandlen, Christopher Nelson, Anand Asundi, Anne Wong, Y. Gloria MengAbstract:Abstract Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody–DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial Growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal Growth Factor-Like Domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.
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Real-time immuno-polymerase chain reaction in a 384-well format: Detection of vascular endothelial Growth factor and epidermal Growth Factor-Like Domain 7
Analytical Biochemistry, 2014Co-Authors: Jianhuan Zhang, Jean Michel Vernes, Jennifer Ni, Steven T. Chen, Richard Vandlen, Christopher Nelson, Anand Asundi, Anne Wong, Y. Gloria MengAbstract:Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial Growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal Growth Factor-Like Domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays. © 2014 Elsevier Inc. All rights reserved.
Penny A Handford - One of the best experts on this subject based on the ideXlab platform.
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alphavbeta6 is a novel receptor for human fibrillin 1 comparative studies of molecular determinants underlying integrin rgd affinity and specificity
Journal of Biological Chemistry, 2007Co-Authors: Jelena Jovanovic, Junichi Takagi, Laurence Choulier, Nicola G A Abrescia, D I Stuart, Anton P Van Der Merwe, Helen J Mardon, Penny A HandfordAbstract:Abstract Human fibrillin-1, the major structural protein of connective tissue 10-12 nm microfibrils, contains multiple calcium binding epidermal Growth Factor-Like Domains interspersed with transforming Growth factor β-binding protein-like (TB) Domains. TB4 contains a flexible RGD loop that mediates cell adhesion via αVβ3 and α5β1 integrins. This study identifies integrin αVβ6 as a novel cellular receptor for fibrillin-1 with a Kd of ∼0.45 μm. Analyses of this interaction by surface plasmon resonance and immunocytochemistry reveal different module requirements for αVβ6 activation compared with those of αVβ3, suggesting that a covalent linkage of an N-terminal calcium binding epidermal Growth Factor-Like Domain to TB4 can modulate αV integrin binding specificity. Furthermore, our data suggest α5β1 is a low affinity fibrillin-1 receptor (Kd > 1 μm), thus providing a molecular explanation for the different α5β1 distribution patterns seen when human keratinocytes and fibroblasts are plated on recombinant fibrillin fragments versus those derived from the physiological ligand fibronectin. Non-focal contact distribution of α5β1 suggests that its engagement by fibrillin-1 may elicit a lesser degree and/or different type of intracellular signaling compared with that seen with a high affinity ligand.
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structural consequences of cysteine substitutions c1977y and c1977r in calcium binding epidermal Growth factor like Domain 30 of human fibrillin 1
Journal of Biological Chemistry, 2004Co-Authors: Sacha A. Jensen, Aileen Mcgettrick, Anthony C Willis, Pat Whiteman, Christina Redfield, Penny A HandfordAbstract:Abstract The largest group of disease-causing mutations affecting calcium-binding epidermal Growth Factor-Like (cbEGF) Domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF Domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF Domain fragment (cbEGF29–31). Limited proteolysis, 1H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking Domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intraDomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that Domain packing of cbEGF29–30, but not cbEGF30–31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant Domain, which disrupt Domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.
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solution structure and dynamics of a calcium binding epidermal Growth factor like Domain pair from the neonatal region of human fibrillin 1
Journal of Biological Chemistry, 2003Co-Authors: R S Smallridge, Penny A Handford, Pat Whiteman, Jorn M Werner, Iain D Campbell, A K DowningAbstract:Abstract Fibrillin-1 is a mosaic protein mainly composed of 43 calcium binding epidermal Growth Factor-Like (cbEGF) Domains arranged as multiple, tandem repeats. Mutations within the fibrillin-1 gene cause Marfan syndrome (MFS), a heritable disease of connective tissue. More than 60% of MFS-causing mutations identified are localized to cbEGFs, emphasizing that the native properties of these Domains are critical for fibrillin-1 function. The cbEGF12–13 Domain pair is within the longest run of cbEGFs, and many mutations that cluster in this region are associated with severe, neonatal MFS. The NMR solution structure of Ca2+-loaded cbEGF12–13 exhibits a near-linear, rod-like arrangement of Domains. This observation supports the hypothesis that all fibrillin-1 (cb)EGF-cbEGF pairs, characterized by a single interDomain linker residue, possess this rod-like structure. The Domain arrangement of cbEGF12–13 is stabilized by additional interDomain packing interactions to those observed for cbEGF32–33, which may help to explain the previously reported higher calcium binding affinity of cbEGF13. Based on this structure, a model of cbEGF11–15 that encompasses all known neonatal MFS missense mutations has highlighted a potential binding region. Backbone dynamics data confirm the extended structure of cbEGF12–13 and lend support to the hypothesis that a correlation exists between backbone flexibility and cbEGF Domain calcium affinity. These results provide important insight into the potential consequences of MFS-associated mutations for the assembly and biomechanical properties of connective tissue microfibrils.
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calcium binding properties of an epidermal Growth factor like Domain pair from human fibrillin 1
Journal of Molecular Biology, 1996Co-Authors: Vroni Knott, A K Downing, C M Cardy, Penny A HandfordAbstract:Ca2+binding epidermal Growth Factor-Like (EGF-like) Domains are found in a large number of extracellular proteins with diverse functions, including those involved in blood coagulation, determination of cell fate, cell adhesion and connective tissue architecture. Their importance is emphasised by the identification of mutations in these Domains in patients with haemophilia B (defective in coagulation factor IX) and the Marfan syndrome (defective in the connective tissue protein fibrillin-1). The X-ray crystal structure of a single Ca2 +binding EGF-like Domain from human coagulation factor IX has recently been solved. It shows that the Ca2+ligands form a pentagonal bipyramid, where one ligand is provided by an adjacent (N-terminal) EGF-like Domain in the crystal. The N and C termini of the neighbouring Domains are only|similar|4 A apart, hence the crystal packing has been proposed as a model for the association of contiguous EGF-like Domains in proteins. Since the adjacent EGF-like Domain in the crystal, although close, is not covalently linked to its neighbour, this model requires verification. In this study we have expressed and purified a Ca2+binding EGF-like Domain pair from human fibrillin-1 and used anin vitrorefolding system to obtain protein with the correct EGF fold. The Ca2+binding properties of the protein have been investigated by two-dimensional NMR. The affinity of the C-terminal Domain for Ca2 +is |similar|25-fold higher than that of the N-terminal Domain, consistent with the two Ca2+binding sites having different local environments. In addition, these data provide the first direct experimental evidence that Ca2+plays a major role in defining the interDomain linkage in multiple repeats of Ca2+binding EGF-like Domains.
Diego Clemente - One of the best experts on this subject based on the ideXlab platform.
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Tissue-type plasminogen activator exerts EGF-like chemokinetic effects on oligodendrocytes in white matter (re)myelination
Molecular Neurodegeneration, 2017Co-Authors: Camille Leonetti, Richard Macrez, Mathilde Pruvost, Yannick Hommet, Jérémie Bronsard, Maxime Perrigault, Isabel Machin, Denis Vivien, A. Fournier, Diego ClementeAbstract:BackgroundThe ability of oligodendrocyte progenitor cells (OPCs) to give raise to myelin forming cells during developmental myelination, normal adult physiology and post-lesion remyelination in white matter depends on factors which govern their proliferation, migration and differentiation. Tissue plasminogen activator (tPA) is a serine protease expressed in the central nervous system (CNS), where it regulates cell fate. In particular, tPA has been reported to protect oligodendrocytes from apoptosis and to facilitate the migration of neurons. Here, we investigated whether tPA can also participate in the migration of OPCs during CNS development and during remyelination after focal white matter lesion.MethodsOPC migration was estimated by immunohistological analysis in spinal cord and corpus callosum during development in mice embryos (E13 to P0) and after white matter lesion induced by the stereotactic injection of lysolecithin in adult mice (1 to 21 days post injection). Migration was compared in these conditions between wild type and tPA knock-out animals. The action of tPA was further investigated in an in vitro chemokinesis assay.ResultsOPC migration along vessels is delayed in tPA knock-out mice during development and during remyelination. tPA enhances OPC migration via an effect dependent on the activation of epidermal Growth factor receptor.ConclusionEndogenous tPA facilitates the migration of OPCs during development and during remyelination after white matter lesion by the virtue of its epidermal Growth Factor-Like Domain.
Jianhuan Zhang - One of the best experts on this subject based on the ideXlab platform.
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Real-time immuno-polymerase chain reaction in a 384-well format: detection of vascular endothelial Growth factor and epidermal Growth Factor-Like Domain 7.
Analytical Biochemistry, 2014Co-Authors: Jianhuan Zhang, Jean Michel Vernes, Jennifer Ni, Steven T. Chen, Richard Vandlen, Christopher Nelson, Anand Asundi, Anne Wong, Y. Gloria MengAbstract:Abstract Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody–DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial Growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal Growth Factor-Like Domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.
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Real-time immuno-polymerase chain reaction in a 384-well format: Detection of vascular endothelial Growth factor and epidermal Growth Factor-Like Domain 7
Analytical Biochemistry, 2014Co-Authors: Jianhuan Zhang, Jean Michel Vernes, Jennifer Ni, Steven T. Chen, Richard Vandlen, Christopher Nelson, Anand Asundi, Anne Wong, Y. Gloria MengAbstract:Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial Growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal Growth Factor-Like Domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays. © 2014 Elsevier Inc. All rights reserved.
Richard Vandlen - One of the best experts on this subject based on the ideXlab platform.
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Real-time immuno-polymerase chain reaction in a 384-well format: detection of vascular endothelial Growth factor and epidermal Growth Factor-Like Domain 7.
Analytical Biochemistry, 2014Co-Authors: Jianhuan Zhang, Jean Michel Vernes, Jennifer Ni, Steven T. Chen, Richard Vandlen, Christopher Nelson, Anand Asundi, Anne Wong, Y. Gloria MengAbstract:Abstract Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody–DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial Growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal Growth Factor-Like Domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays.
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Real-time immuno-polymerase chain reaction in a 384-well format: Detection of vascular endothelial Growth factor and epidermal Growth Factor-Like Domain 7
Analytical Biochemistry, 2014Co-Authors: Jianhuan Zhang, Jean Michel Vernes, Jennifer Ni, Steven T. Chen, Richard Vandlen, Christopher Nelson, Anand Asundi, Anne Wong, Y. Gloria MengAbstract:Immuno-polymerase chain reaction (immuno-PCR) combines the specificity of antibodies with the amplification power of PCR to detect low levels of proteins. Here, we describe the development of a 384-well immuno-PCR method that uses streptavidin coated on a PCR plate to capture complexes of biotinylated capture antibody, antigen, and DNA-labeled detection antibody. Unbound molecules are removed by a wash step using a standard plate washer. Antibody-DNA molecules in bound complexes are then detected directly on the plate using real-time PCR. Circulating human vascular endothelial Growth factor concentrations measured by this method correlated with measurements obtained from enzyme-linked immunosorbent assay (ELISA). Using this method, we developed an assay for human epidermal Growth Factor-Like Domain 7 (EGFL7), an extracellular matrix-bound angiogenic factor. EGFL7 is expressed at a higher level in certain cancers, although endogenous EGFL7 concentrations have not been reported. Our 384-well EGFL7 immuno-PCR assay can detect 0.51 pM EGFL7 in plasma, approximately 16-fold more sensitive than the ELISA, utilizing the same antibodies. This assay detected EGFL7 in lysates of non-small-cell lung cancer and hepatocellular carcinoma cell lines and also hepatocellular carcinoma, breast cancer, and ovarian cancer tissues. This 384-well immuno-PCR method can be used to develop high-throughput biomarker assays. © 2014 Elsevier Inc. All rights reserved.
