scholarly journals Biotinylated Synthetic Chemokines: Their Use for the Development of Nonradioactive Whole-Cell Binding Assays

2003 ◽  
Vol 8 (3) ◽  
pp. 316-323 ◽  
Author(s):  
Anne-Christine Thierry ◽  
Geneviève Perrenoud ◽  
Stéphane Pinaud ◽  
Nicolas Bigler ◽  
Bérangère Denis ◽  
...  
Keyword(s):  
Receptor Binding ◽  
Binding Assay ◽  
Biologically Active ◽  
Binding Assays ◽  
Cell Binding ◽  
Chemical Route ◽  
Whole Cell ◽  
Migration Assay ◽  
Binding Characteristics ◽  
Whole Cells

A chemokine binding assay on whole cells was developed using biotinylated synthetic CCL22 as a model ligand. CCL22 analogues were produced by a chemical route, resulting in > 97% homogeneous and defined polypeptides. First, the 5 biotinylated CCL22 analogues synthesized were captured by agarose-immobilized streptavidin, indicating that the biotin molecules introduced in positions G1, K27, K49, K61, and K66 of CCL22 were accessible for binding. Then, it was established using a migration assay that the biotinylated chemokines were at least as biologically active as the unmodified CCL22 form. Subsequently, the biotinylated chemokines were evaluated in an FACS-based whole-cell binding assay. Surprisingly, only the CCL22 analogue with the biotin in position K66 constituted a suitable staining reagent for CCR4-positive cells. Finally, binding characteristics and reproducibility of the binding assay were outlined for the CCL22 analogue with the biotin in position K66. These results exemplified that biotinylated synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on whole cells, provided the position of the biotin moiety introduced along the sequence is adequately chosen. ( Journal of Biomolecular Screening 2003:316-323)

scholarly journals The VP5 Domain of VP4 Can Mediate Attachment of Rotaviruses to Cells

2000 ◽  
Vol 74 (2) ◽  
pp. 593-599 ◽  
Author(s):  
Selene Zárate ◽  
Rafaela Espinosa ◽  
Pedro Romero ◽  
Ernesto Méndez ◽  
Carlos F. Arias ◽  
...  
Keyword(s):  
Cell Surface ◽  
Binding Assay ◽  
Cell Attachment ◽  
Wild Type ◽  
Binding Assays ◽  
Virus Binding ◽  
Binding Characteristics ◽  
Surface Receptors ◽  
Infectivity Assay ◽  
Fusion Products

ABSTRACT Some animal rotaviruses require the presence of sialic acid (SA) on the cell surface to infect the cell. We have isolated variants of rhesus rotavirus (RRV) whose infectivity no longer depends on SA. Both the SA-dependent and -independent interactions of these viruses with the cell are mediated by the virus spike protein VP4, which is cleaved by trypsin into two domains, VP5 and VP8. In this work we have compared the binding characteristics of wild-type RRV and its variant nar3 to MA104 cells. In a direct nonradioactive binding assay, both viruses bound to the cells in a saturable and specific manner. When neutralizing monoclonal antibodies directed to both the VP8 and VP5 domains of VP4 were used to block virus binding, antibodies to VP8 blocked the cell attachment of wild-type RRV but not that of the variant nar3. Conversely, an antibody to VP5 inhibited the binding of nar3 but not that of RRV. These results suggest that while RRV binds to the cell through VP8, the variant does so through the VP5 domain of VP4. This observation was further sustained by the fact that recombinant VP8 and VP5 proteins, produced in bacteria as fusion products with glutathione S-transferase, were found to bind to MA104 cells in a specific and saturable manner and, when preincubated with the cell, were capable of inhibiting the binding of wild-type and variant viruses, respectively. In addition, the VP5 and VP8 recombinant proteins inhibited the infectivity of nar3 and RRV, respectively, confirming the results obtained in the binding assays. Interestingly, when the infectivity assay was performed on neuraminidase-treated cells, the VP5 fusion protein was also found to inhibit the infectivity of RRV, suggesting that RRV could bind to the cell through two sequential steps mediated by the interaction of VP8 and VP5 with SA-containing and SA-independent cell surface receptors, respectively.

scholarly journals Inhibition of fibril formation in β-amyloid peptide by a novel series of benzofurans

1999 ◽  
Vol 340 (1) ◽  
pp. 283-289 ◽  
Author(s):  
David R. HOWLETT ◽  
Amanda E. PERRY ◽  
Fiona GODFREY ◽  
Jane E. SWATTON ◽  
Kevin H. JENNINGS ◽  
...  
Keyword(s):  
Binding Assay ◽  
Close Correlation ◽  
Fibril Formation ◽  
Biologically Active ◽  
Amyloid Peptide ◽  
Binding Assays ◽  
Nmr Studies ◽  
Β Amyloid ◽  
Β Amyloid Peptide ◽  
Self Aggregation

A series of benzofuran derivatives have been identified as inhibitors of fibril formation in the β-amyloid peptide. The activity of these compounds has been assessed by a novel fibril-formation-specific immunoassay and for their effects on the production of a biologically active fibril product. The inhibition afforded by the compounds seems to be associated with their binding to β-amyloid, as identified by scintillation proximity binding assay. Binding assays and NMR studies also indicate that the inhibition is associated with self-aggregation of the compounds. There is a close correlation between the activity of the benzofurans as inhibitors of fibril formation and their ability to bind to β-amyloid. Non-benzofuran inhibitors of the fibril formation process do not seem to bind to the same site on the β-amyloid molecule as the benzofurans. Thus a specific recognition site might exist for benzofurans on β-amyloid, binding to which seems to interfere with the ability of the peptide to form fibrils.

Measurement of biologically active interleukin-1 by a soluble receptor binding assay

1990 ◽  
Vol 185 (2) ◽  
pp. 206-212 ◽  
Author(s):  
Frank Riske ◽  
Richard Chizzonite ◽  
Perla Nunes ◽  
Alvin S. Stern
Keyword(s):  
Receptor Binding ◽  
Binding Assay ◽  
Interleukin 1 ◽  
Biologically Active ◽  
Soluble Receptor ◽  
Receptor Binding Assay

A Whole-Cell Binding Assay for Testing the Targeting Potential of Cyclic Peptide Ligands

2010 ◽  
pp. 593-594
Author(s):  
Sonya Cressman ◽  
Ning Fang ◽  
David D. Y. Chen ◽  
Pieter R. Cullis
Keyword(s):  
Binding Assay ◽  
Cyclic Peptide ◽  
Peptide Ligands ◽  
Cell Binding ◽  
Whole Cell

scholarly journals 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617: synthesis, radiolabeling, stability and cell binding compared to DOTA-PSMA-617 analogues

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Jean-Philippe Sinnes ◽  
Ulrike Bauder-Wüst ◽  
Martin Schäfer ◽  
Euy Sung Moon ◽  
Klaus Kopka ◽  
...  
Keyword(s):  
Human Serum ◽  
Room Temperature ◽  
Solid Phase ◽  
Negative Influence ◽  
Binding Motif ◽  
Lncap Cells ◽  
Cell Binding ◽  
Binding Characteristics

Abstract Background The AAZTA chelator and in particular its bifunctional derivative AAZTA5 was recently investigated to demonstrate unique capabilities to complex diagnostic and therapeutic trivalent radiometals under mild conditions. This study presents a comparison of 68Ga, 44Sc and 177Lu-labeled AAZTA5-PSMA-617 with DOTA-PSMA-617 analogues. We evaluated the radiolabeling characteristics, in vitro stability of the radiolabeled compounds and evaluated their binding affinity and internalization behavior on LNCaP tumor cells in direct comparison to the radiolabeled DOTA-conjugated PSMA-617 analogs. Results AAZTA5 was synthesized in a five-step synthesis and coupled to the PSMA-617 backbone on solid phase. Radiochemical evaluation of AAZTA5-PSMA-617 with 68Ga, 44Sc and 177Lu achieved quantitative radiolabeling of > 99% after less than 5 min at room temperature. Stabilities against human serum, PBS buffer and EDTA and DTPA solutions were analyzed. While there was a small degradation of the 68Ga complex over 2 h in human serum, PBS and EDTA/DTPA, the 44Sc and 177Lu complexes were stable at 2 h and remained stable over 8 h and 1 day. For all three compounds, i.e. [natGa]Ga-AAZTA5-PSMA-617, [natSc]Sc-AAZTA5-PSMA-617 and [natLu]Lu-AAZTA5-PSMA-617, in vitro studies on PSMA-positive LNCaP cells were performed in direct comparison to radiolabeled DOTA-PSMA-617 yielding the corresponding inhibition constants (Ki). Ki values were in the range of 8–31 nM values which correspond with those of [natGa]Ga-DOTA-PSMA-617, [natSc]Sc-DOTA-PSMA-617 and [natLu]Lu-DOTA-PSMA-617, i.e. 5–7 nM, respectively. Internalization studies demonstrated cellular membrane to internalization ratios for the radiolabeled 68Ga, 44Sc and 177Lu-AAZTA5-PSMA-617 tracers (13–20%IA/106 cells) in the same range as the ones of the three radiolabeled DOTA-PSMA-617 tracers (17–20%IA/106 cells) in the same assay. Conclusions The AAZTA5-PSMA-617 structure proved fast and quantitative radiolabeling with all three radiometal complexes at room temperature, excellent stability with 44Sc, very high stability with 177Lu and medium stability with 68Ga in human serum, PBS and EDTA/DTPA solutions. All three AAZTA5-PSMA-617 tracers showed binding affinities and internalization ratios in LNCaP cells comparable with that of radiolabeled DOTA-PSMA-617 analogues. Therefore, the exchange of the chelator DOTA with AAZTA5 within the PSMA-617 binding motif has no negative influence on in vitro LNCaP cell binding characteristics. In combination with the faster and milder radiolabeling features, AAZTA5-PSMA-617 thus demonstrates promising potential for in vivo application for theranostics of prostate cancer.

scholarly journals Altering the Stereoselectivity of Whole-Cell Biotransformations via the Physicochemical Parameters Impacting the Processes

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 781
Author(s):  
Agnieszka Raczyńska ◽  
Joanna Jadczyk ◽  
Małgorzata Brzezińska-Rodak
Keyword(s):  
Culture Conditions ◽  
Biologically Active ◽  
Enantiomerically Pure ◽  
Whole Cells ◽  
Chiral Compounds ◽  
Physicochemical Factors ◽  
Pure Compounds ◽  
Genetic Level ◽  
Prochiral Ketones ◽  
Optically Pure

The enantioselective synthesis of organic compounds is one of the great challenges in organic synthetic chemistry due to its importance for the acquisition of biologically active derivatives, e.g., pharmaceuticals, agrochemicals, and others. This is why biological systems are increasingly applied as tools for chiral compounds synthesis or modification. The use of whole cells of “wild-type” microorganisms is one possible approach, especially as some methods allow improving the conversion degrees and controlling the stereoselectivity of the reaction without the need to introduce changes at the genetic level. Simple manipulation of the culture conditions, the form of a biocatalyst, or the appropriate composition of the biotransformation medium makes it possible to obtain optically pure products in a cheap, safe, and environmentally friendly manner. This review contains selected examples of the influence of physicochemical factors on the stereochemistry of the biocatalytic preparation of enantiomerically pure compounds, which is undertaken through kinetically controlled separation of their racemic mixtures or reduction of prochiral ketones and has an effect on the final enantiomeric purity and enantioselectivity of the reaction.

scholarly journals Studies on Active Constituents in Medicinal Plants Using the Receptor Binding Assay. II. : Dopamine 2 Receptor

1988 ◽  
Vol 108 (5) ◽  
pp. 450-453
Author(s):  
TOSIHIKO SUMIDA ◽  
HIROYUKI SUGIMOTO ◽  
TOHRU FUWA ◽  
KAZUO YAMASAKI ◽  
OSAMU TAKEDA ◽  
...  
Keyword(s):  
Medicinal Plants ◽  
Receptor Binding ◽  
Binding Assay ◽  
Active Constituents ◽  
Receptor Binding Assay ◽  
Dopamine 2 Receptor
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