scholarly journals In vitro enzymatic activity assay for ENOLASE in mammalian cells in culture

2012 ◽  
Author(s):  
Florian Muller ◽  
Florian Muller ◽  
Elisa Aquilanti ◽  
Ronald DePinho
Keyword(s):  
Enzymatic Activity ◽  
Mammalian Cells ◽  
Activity Assay ◽  
Cells In Culture ◽  
Enzymatic Activity Assay

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.

Enzymatic activity assay of d-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

2006 ◽  
Vol 67 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Montserrat Andújar-Sánchez ◽  
Francisco Javier Las Heras-Vázquez ◽  
Josefa María Clemente-Jiménez ◽  
Sergio Martínez-Rodríguez ◽  
Ana Camara-Artigas ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Isothermal Titration Calorimetry ◽  
Activation Parameters ◽  
Titration Calorimetry ◽  
Activity Assay ◽  
Thermodynamic Activation Parameters ◽  
Hydrolysis Of ◽  
Enzymatic Activity Assay

Effects of Asbestos Fibers on Mammalian Cells in Culture

1975 ◽  
Vol 33 ◽  
pp. 420-421
Author(s):  
Brenda E. Barry ◽  
Harold W. Fisher
Keyword(s):  
Mammalian Cells ◽  
Growth Curves ◽  
Ultrastructural Changes ◽  
Asbestos Fibers ◽  
Cells In Culture ◽  
Cell Counts ◽  
Direct Cell ◽  
Amphibole Asbestos ◽  
Organic Pesticides

In previous reports from this laboratory, we examined the correlation of the toxic effects of several organic pesticides, herbicides and trace metals on the plating efficiency to the ultrastructural changes found in the treated cells in culture. Since there has been a long-term interest in the effects of exposure of humans and experimental animals to asbestos, the current study was undertaken to determine if ultrastructural changes would also be found in mammalian cells grown in vitro after exposure to asbestos. In a series of preliminary experiments, suitable fractionation by low-speed differential centrifugation and concentrations of amphibole asbestos were found in which there was at least 70% to 90% viability depending on the cell type. The viability of the cells incubated in the presence of the asbestos was determined both by growth curves established from direct cell counts and from staining with trypan blue which does not penetrate viable cells.

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 A Rapid Two-Step Iduronate-2-Sulfatatse Enzymatic Activity Assay for MPSII Pharmacokinetic Assessment

2017 ◽  
pp. 89-95 ◽  
Author(s):  
Mitra Azadeh ◽  
Luying Pan ◽  
Yongchang Qiu ◽  
Ruben Boado
Keyword(s):  
Enzymatic Activity ◽  
Activity Assay ◽  
Pharmacokinetic Assessment ◽  
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.

A radiochemical enzymatic activity assay for glycerol kinase and hexokinase

1967 ◽  
Vol 132 (2) ◽  
pp. 338-346 ◽  
Author(s):  
E.A. Newsholme ◽  
J. Robinson ◽  
K. Taylor
Keyword(s):  
Enzymatic Activity ◽  
Glycerol Kinase ◽  
Activity Assay ◽  
Enzymatic Activity Assay

scholarly journals A scalable lysyl hydroxylase 2 expression system and luciferase-based enzymatic activity assay

2017 ◽  
Vol 618 ◽  
pp. 45-51 ◽  
Author(s):  
Hou-Fu Guo ◽  
Eun Jeong Cho ◽  
Ashwini K. Devkota ◽  
Yulong Chen ◽  
William Russell ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Expression System ◽  
Activity Assay ◽  
Lysyl Hydroxylase ◽  
Enzymatic Activity Assay
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