Weak Binding of Levamisole by the Cocaine-Binding Aptamer Does Not Interfere with an Aptamer-Based Detection Assay

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

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Faculty Opinions recommendation of Discovery of Highly Potent and Efficient PROTAC Degraders of Androgen Receptor (AR) by Employing Weak Binding Affinity VHL E3 Ligase Ligands.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.737032941.793568334 ◽

2019 ◽

Author(s):

John Lowe

Keyword(s):

Androgen Receptor ◽

Binding Affinity ◽

E3 Ligase ◽

Weak Binding

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IFN-α/β Detection Assay Using Sensor Cell Lines

BIO-PROTOCOL ◽

10.21769/bioprotoc.1130 ◽

2014 ◽

Vol 4 (10) ◽

Author(s):

Valeria Lulla ◽

Andres Merits ◽

Aleksei Lulla

Keyword(s):

Cell Lines ◽

Detection Assay ◽

Sensor Cell

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Interpenetrating Gels as Conducting/Adhering Matrices Enabling High-Performance Silicon Anodes

Journal of Materials Chemistry A ◽

10.1039/d1ta01733k ◽

2021 ◽

Author(s):

Tingting Xia ◽

Chengfei Xu ◽

Pengfei Dai ◽

Xiaoyun Li ◽

Riming Lin ◽

...

Keyword(s):

Energy Storage ◽

High Performance ◽

Electrode Materials ◽

Three Dimensional ◽

Electrochemical Energy Storage ◽

Active Materials ◽

Silicon Anodes ◽

Weak Binding ◽

Binding Forces

Three-dimensional (3D) conductive polymers are promising conductive matrices for electrode materials toward electrochemical energy storage. However, their fragile nature and weak binding forces with active materials could not guarantee long-term...

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Timely gene detection assay and reliable screening of genetically engineered plants using an improved direct PCR-based technology

Transgenic Research ◽

10.1007/s11248-021-00250-1 ◽

2021 ◽

Author(s):

Anis Ben-Amar ◽

Ahmed Mliki

Keyword(s):

Genetically Engineered ◽

Gene Detection ◽

Direct Pcr ◽

Detection Assay ◽

Genetically Engineered Plants

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Evaluation of Modification of the 3M™ Molecular Detection Assay (MDA) Salmonella Method (2013.09) for the Detection of Salmonella in Selected Foods: Collaborative Study

Journal of AOAC International ◽

10.5740/jaoacint.14-101 ◽

2014 ◽

Vol 97 (5) ◽

pp. 1329-1342 ◽

Cited By ~ 5

Author(s):

Patrick Bird ◽

Kiel Fisher ◽

Megan Boyle ◽

Travis Huffman ◽

M Joseph Benzinger ◽

...

Keyword(s):

Confidence Intervals ◽

Molecular Detection ◽

Collaborative Study ◽

Ground Beef ◽

Inoculum Level ◽

Test Portion ◽

Low Level ◽

Dog Food ◽

Detection Assay ◽

The U.S

Abstract The 3M™ Molecular Detection Assay (MDA) Salmonella utilizes isothermal amplification of nucleic acid sequences with high specificity, efficiency, rapidity and bioluminescence to detect amplification of Salmonella spp. in food, food-related, and environmental samples after enrichment. A method modification and matrix extension study of the previously approved AOAC Official MethodSM 2013.09 was conducted, and approval of the modification was received on March 20, 2014. Using an unpaired study design in a multilaboratory collaborative study, the 3M MDA Salmonella method was compared to the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) Microbiology Laboratory Guidebook (MLG) 4.05 (2011), Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Catfish Products for raw ground beef and the U.S. Food and Drug Administration (FDA)/Bacteriological Analytical Manual (BAM) Chapter 5, Salmonella reference method for wet dog food following the current AOAC guidelines. A total of 20 laboratories participated. For the 3M MDA Salmonella method, raw ground beef was analyzed using 25 g test portions, and wet dog food was analyzed using 375 g test portions. For the reference methods, 25 g test portions of each matrix were analyzed. Each matrix was artificially contaminated with Salmonella at three inoculation levels: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2–2 CFU/test portion), and a high inoculum level (2–5 CFU/test portion). In this study, 1512 unpaired replicate samples were analyzed. Statistical analysis was conducted according to the probability of detection (POD). For the low-level raw ground beef test portions, the following dLPOD (difference between the LPODs of the reference and candidate method) values with 95% confidence intervals were obtained: –0.01 (–0.14, +0.12). For the low-level wet dog food test portions, the following dLPOD with 95% confidence intervals were obtained: –0.04 (–0.16, +0.09). No significant differences were observed in the number of positive samples detected by the 3M MDA Salmonella method versus either the USDA/FSIS-MLG or FDA/BAM methods.

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Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6866 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6866-6875 ◽

Cited By ~ 20

Author(s):

D C Hagen ◽

L Bruhn ◽

C A Westby ◽

G F Sprague

Keyword(s):

Saccharomyces Cerevisiae ◽

Binding Site ◽

Dna Sequences ◽

Point Mutations ◽

Transcription Activation ◽

Specific Gene ◽

Binding Assays ◽

Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

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