A rapid and robust assay for the determination of the amino acid hypusine as a possible biomarker for a high-throughput screening of antimalarials and for the diagnosis and therapy of different diseases

Amino Acids ◽  
2011 ◽  
Vol 42 (5) ◽  
pp. 1651-1659 ◽  
Author(s):  
Annette Kaiser ◽  
Alex R. Khomutov ◽  
Alina Simonian ◽  
Enzo Agostinelli
Author(s):  
Ajay Iyer ◽  
Lisa Guerrier ◽  
Salomé Leveque ◽  
Charles S. Bestwick ◽  
Sylvia H. Duncan ◽  
...  

AbstractInvasive plants offer an interesting and unconventional source of protein and the considerable investment made towards their eradication can potentially be salvaged through their revalorisation. To identify viable sources, effective and high-throughput screening methods are required, as well as efficient procedures to isolate these components. Rigorous assessment of low-cost, high-throughput screening assays for total sugar, phenolics and protein was performed, and ninhydrin, Lever and Fast Blue assays were found to be most suitable owing to high reliability scores and false positive errors less than 1%. These assays were used to characterise invasive Scottish plants such as Gorse (Ulex europeans), Broom (Cystisus scoparius) and Fireweed (Chamaenerion angustifolium). Protein extraction (alkali-, heat- and enzyme assisted) were tested on these plants, and further purification (acid and ethanol precipitation, as well as ultrafiltration) procedures were tested on Gorse, based on protein recovery values. Cellulase treatment and ethanol precipitation gave the highest protein recovery (64.0 ± 0.5%) and purity (96.8 ± 0.1%) with Gorse. The amino acid profile of the purified protein revealed high levels of essential amino acids (34.8 ± 0.0%). Comparison of results with preceding literature revealed a strong association between amino acid profiles and overall protein recovery with the extraction method employed. The final purity of the protein concentrates was closely associated to the protein content of the initial plant mass. Leaf protein extraction technology can effectively raise crop harvest indices, revalorise underutilised plants and waste streams.


2019 ◽  
Vol 42 (18) ◽  
pp. 2968-2976 ◽  
Author(s):  
Mingrong Qian ◽  
Xiaoming Zhang ◽  
Huiyu Zhao ◽  
Xiaofeng Ji ◽  
Xiaodan Li ◽  
...  

1989 ◽  
Vol 35 (10) ◽  
pp. 2112-2115 ◽  
Author(s):  
N S Gerasimova ◽  
I V Steklova ◽  
T Tuuminen

Abstract We adapted the method of McCaman and Robins for fluorometry of phenylalanine to a microplate assay for routine phenylketonuria screening. Sensitivity is 15 mumol/L for the plasma assay and 30 mumol/L for the dried blood-spot assay, with CV less than 10% for both assays. Results for human plasma by microplate assay correlated well (r = 0.99) with results of amino acid analyzer determination of phenylalanine. When measurements are performed in an automated reader, the microplate assay has considerable advantages over conventional measurements in cuvettes: smaller volumes of reagents and automation enabling high throughput and convenience. Because the described method is a quantitative one, we can postulate that, compared with the semiquantitative Guthrie inhibition bioassay, this microplate assay is more reliable, easier to perform, and about twofold less costly.


2006 ◽  
Vol 11 (5) ◽  
pp. 481-487 ◽  
Author(s):  
Philip E. Brandish ◽  
Chi-Sung Chiu ◽  
Jonathan Schneeweis ◽  
Nicholas J. Brandon ◽  
Clare L. Leech ◽  
...  

Enzymes are often considered less “druggable” targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.


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