scholarly journals A fluorescent probe for monitoring PTP-PEST enzymatic activity

The Analyst ◽  
2020 ◽  
Vol 145 (20) ◽  
pp. 6713-6718
Author(s):  
Garrett R. Casey ◽  
Cliff I. Stains
Keyword(s):  
Enzymatic Activity ◽  
Fluorescent Probe ◽  
Activity Assay ◽  
Design And Optimization

Design and optimization of a direct activity assay for PTP-PEST.

scholarly journals A sensitive fluorescent probe for alkaline phosphatase and an activity assay based on the aggregation-induced emission effect

RSC Advances ◽  
2018 ◽  
Vol 8 (27) ◽  
pp. 14995-15000 ◽  
Author(s):  
Wenjuan Zhang ◽  
Hanxiao Yang ◽  
Nan Li ◽  
Na Zhao
Keyword(s):  
Alkaline Phosphatase ◽  
Enzymatic Activity ◽  
Fluorescent Probe ◽  
Activity Assay ◽  
Aggregation Induced Emission ◽  
Emission Effect

A sensitive fluorescent probe (TPEQN-P) was designed and synthesized for detecting alkaline phosphatase and monitoring its enzymatic activity based on the specific aggregation-induced emission effect.

scholarly journals Identification of Small Molecule Inhibitors of the Deubiquitinating Activity of the SARS-CoV-2 Papain-Like Protease: in silico Molecular Docking Studies and in vitro Enzymatic Activity Assay

2020 ◽  
Vol 8 ◽  
Author(s):  
Eleni Pitsillou ◽  
Julia Liang ◽  
Katherine Ververis ◽  
Kah Wai Lim ◽  
Andrew Hung ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Enzymatic Activity ◽  
Small Molecules ◽  
Binding Site ◽  
Protein Docking ◽  
Activity Assay ◽  
Epicatechin Gallate ◽  
Inhibitor Binding ◽  
Enzymatic Activity Assay

COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 virus with important political, socio-economic, and public health consequences. Inhibiting replication represents an important antiviral approach, and in this context two viral proteases, the SARS-CoV-2 main and papain-like proteases (PLpro), which cleave pp1a and pp1ab polypeptides, are critical. Along with protease activity, the PLpro possesses deubiquitinating activity, which is important in immune regulation. Naphthalene-based inhibitors, such as the well-investigated GRL-0617 compound, have been shown to possess dual effects, inhibiting both protease and deubiquitinating activity of the PLpro. Rather than binding to the canonical catalytic triad, these type of non-covalent inhibitors target an adjacent pocket, the naphthalene-inhibitor binding site. Using a high-throughput screen, we have previously identified the dietary hypericin, rutin, and cyanidin-3-O-glucoside compounds as potential protease inhibitors targeting the naphthalene-inhibitor binding site. Here, our aim was to investigate the binding characteristics of these compounds to the PLpro, and to evaluate deubiquitinating activity, by analyzing seven different PLpro crystal structures. Molecular docking highlighted the relatively high affinity of GRL-0617 and dietary compounds. In contrast binding of the small molecules was abolished in the presence of ubiquitin in the palm subdomain of the PLpro. Further, docking the small molecules in the naphthalene-inhibitor binding site, followed by protein-protein docking revealed displacement of ubiquitin in a conformation inconsistent with functional activity. Finally, the deubiquitinating activity was validated in vitro using an enzymatic activity assay. The findings indicated that the dietary compounds inhibited deubiquitinase activity in the micromolar range with an order of activity of GRL-0167, hypericin >> rutin, cyanidin-3-O-glucoside > epigallocatechin gallate, epicatechin gallate, and cefotaxime. Our findings are in accordance with mechanisms and potential antiviral effects of the naphthalene-based, GRL-0617 inhibitor, which is currently progressing in preclinical trials. Further, our findings indicate that in particular hypericin, rutin, and cyanidin-3-O-glucoside, represent suitable candidates for subsequent evaluation as PLpro inhibitors.

scholarly journals Antibacterial Activity of Caffeic Acid Combined with Ultraviolet-A Light against Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes

2021 ◽  
Author(s):  
Min-Young Park ◽  
Dong-Hyun Kang
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Listeria Monocytogenes ◽  
Enzymatic Activity ◽  
Caffeic Acid ◽  
Membrane Damage ◽  
Ultraviolet A ◽  
Activity Assay ◽  
Natural Polyphenol ◽  
Enzymatic Activity Assay

The aim of this study was to evaluate the antibacterial activity of caffeic acid (CA) which is a natural polyphenol, combined with Ultraviolet-A (UV-A) light against the representative foodborne bacteria, Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes. The inactivation results were obtained depending on the CA concentration, light wavelength and light dose.E. coli O157:H7 and S. Typhimurium were reduced to the detection limit when treated with 3 mM CA and UV-A for 3 J/cm2 and 4 J/cm2 respectively, and 5 J/cm2 treatment induced 3.10 log reduction in L. monocytogenes. To investigate the inactivation mechanism, measurement of polyphenol uptake, membrane damage assessment, enzymatic activity assay, and transmission electron microscopy (TEM) were conducted against S. Typhimurium and L. monocytogenes. It was revealed that CA was significantly (P < 0.05) absorbed by bacterial cells, and UV-A light allowed a higher uptake of CA for both pathogens. Additionally, CA + UV-A treatment induced significant (P < 0.05) cell membrane damage. In the enzymatic activity assay, both pathogens showed a reduction in their activity by CA and a higher reduction occurred by CA + UV-A. Moreover, TEM images indicated that CA + UV-A treatment notably destroyed the intercellular structure. In addition, antibacterial activity was also observed in commercial apple juice, which showed similar results to those obtained from PBS, resulting in a significant (P < 0.05) reduction for all three pathogens without any changes in color parameters (L*, a*, and b*), total phenolic compounds and DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity. IMPORTANCE Photodynamic inactivation (PDI), which involves photoactivation of a photosensitizer (PS), is an emerging field of study as it effectively reduces various kinds of microorganisms. Although there are several PSs that have been used for PDI, there is a need to find naturally occurring PSs for safer application in food industry. Caffeic acid, a natural polyphenol found in most of fruits and vegetables, has recently been studied for its potential to act as a novel photosensitizer. However, no studies have been conducted regarding its antibacterial activity depending on treatment conditions and its antibacterial mechanism. In this study we closely examined the effectiveness of caffeic acid in combination with UV-A light for inactivating representative foodborne bacteria in liquid medium. Therefore, the results of this research are expected to be utilized as basic data for future application of caffeic acid in PDI, especially when controlling pathogens in liquid food processing.

Colorimetric Enzymatic Activity Assay Based on Noncrosslinking Aggregation of Gold Nanoparticles Induced by Adsorption of Substrate Peptides

2008 ◽  
Vol 9 (9) ◽  
pp. 2301-2308 ◽  
Author(s):  
Jun Oishi ◽  
Yoji Asami ◽  
Takeshi Mori ◽  
Jeong-Hun Kang ◽  
Takuro Niidome ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Enzymatic Activity ◽  
Activity Assay ◽  
Enzymatic Activity Assay

scholarly journals Validation and assessment of preanalytical factors of a fluorometric in vitro assay for glucocerebrosidase activity in human cerebrospinal fluid

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Linn Oftedal ◽  
Jodi Maple-Grødem ◽  
Marthe Gurine Gunnarsdatter Førland ◽  
Guido Alves ◽  
Johannes Lange
Keyword(s):  
Cerebrospinal Fluid ◽  
Enzymatic Activity ◽  
Lewy Bodies ◽  
Activity Assay ◽  
Limit Of Quantification ◽  
Gene Encoding ◽  
Vitro Assay ◽  
Freeze Thaw ◽  
Human Cerebrospinal Fluid

AbstractLysosomal dysfunction is an emerging feature in the pathology of Parkinson’s disease and Dementia with Lewy bodies. Mutations in the GBA gene, encoding the enzyme Glucocerebrosidase (GCase), have been identified as a genetic risk factor for these synucleinopathies. As a result, there has been a growing interest in the involvement of GCase in these diseases. This GCase activity assay is based on the catalytic hydrolysis of 4-methylumbelliferyl β-d-glucopyranoside that releases the highly fluorescent 4-methylumbelliferyl (4-MU). The final assay protocol was tested for the following parameters: Lower limit of quantification (LLOQ), precision, parallelism, linearity, spike recovery, number of freeze–thaw events, and sample handling stability. The GCase activity assay is within acceptable criteria for parallelism, precision and spike recovery. The LLOQ of this assay corresponds to an enzymatic activity of generating 0.26 pmol 4-MU/min/ml. The enzymatic activity was stable when samples were processed and frozen at − 80 °C within 4 h after the lumbar puncture procedure. Repetitive freeze–thaw events significantly decreased enzyme activity. We present the validation of an optimized in vitro GCase activity assay, based on commercially available components, to quantify its enzymatic activity in human cerebrospinal fluid and the assessment of preanalytical factors.

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