scholarly journals Utility of Bioluminescent Homogeneous Nucleotide Detection Assays in Measuring Activities of Nucleotide-Sugar Dependent Glycosyltransferases and Studying Their Inhibitors

Molecules ◽  
2021 ◽  
Vol 26 (20) ◽  
pp. 6230
Author(s):  
Laurie Engel ◽  
Juliano Alves ◽  
Jacquelyn Hennek ◽  
Said A. Goueli ◽  
Hicham Zegzouti
Keyword(s):  
Rapid Detection ◽  
High Throughput Screening ◽  
Firefly Luciferase ◽  
Radioactive Product ◽  
Nucleotide Sugar ◽  
Chemical Interference ◽  
Highly Sensitive ◽  
One Step

Traditional glycosyltransferase (GT) activity assays are not easily configured for rapid detection nor for high throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a typical glycosyltransferase biochemical reaction, two products are generated, a glycosylated product and a nucleotide released from the sugar donor substrate. Therefore, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each of these assays are performed in a one-step detection that relies on converting the nucleotide product to ATP, then to bioluminescence using firefly luciferase. These assays are highly sensitive, robust and resistant to chemical interference. Various applications of these assays are presented, including studies on the specificity of sugar transfer by diverse GTs and the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Furthermore, their utility in screening for specific GT inhibitors and the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large number of GTs and may have a significant impact on diverse areas of Glycobiology research.

scholarly journals Discovery and Characterization of Low-Molecular Weight Inhibitors of Erwinia tracheiphila

2020 ◽  
Vol 110 (5) ◽  
pp. 989-998
Author(s):  
Cláudio M. Vrisman ◽  
Loïc Deblais ◽  
Yosra A. Helmy ◽  
Reed Johnson ◽  
Gireesh Rajashekara ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
High Throughput Screening ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Bacillus Species ◽  
Erwinia Tracheiphila ◽  
Static Activity ◽  
Low Toxicity

Plant pathogenic bacteria in the genus Erwinia cause economically important diseases, including bacterial wilt of cucurbits caused by Erwinia tracheiphila. Conventional bactericides are insufficient to control this disease. Using high-throughput screening, 464 small molecules (SMs) with either cidal or static activity at 100 µM against a cucumber strain of E. tracheiphila were identified. Among them, 20 SMs (SM1 to SM20), composed of nine distinct chemical moiety structures, were cidal to multiple E. tracheiphila strains at 100 µM. These lead SMs had low toxicity to human cells and honey bees at 100 µM. No phytotoxicity was observed on melon plants at 100 µM, except when SM12 was either mixed with Silwet L-77 and foliar sprayed or when delivered through the roots. Lead SMs did not inhibit the growth of beneficial Pseudomonas and Enterobacter species but inhibited the growth of Bacillus species. Nineteen SMs were cidal to Xanthomonas cucurbitae and showed >50% growth inhibition against Pseudomonas syringae pv. lachrymans. In addition, 19 SMs were cidal or static against Erwinia amylovora in vitro. Five SMs demonstrated potential to suppress E. tracheiphila when foliar sprayed on melon plants at 2× the minimum bactericidal concentration. Thirteen SMs reduced Et load in melon plants when delivered via roots. Temperature and light did not affect the activity of SMs. In vitro cidal activity was observed after 3 to 10 h of exposure to these five SMs. Here, we report 19 SMs that provide chemical scaffolds for future development of bactericides against plant pathogenic bacterial species.

scholarly journals Use of Leishmania donovani Field Isolates Expressing the Luciferase Reporter Gene in In Vitro Drug Screening

2005 ◽  
Vol 49 (9) ◽  
pp. 3776-3783 ◽  
Author(s):  
Ashutosh ◽  
Suman Gupta ◽  
Ramesh ◽  
Shyam Sundar ◽  
Neena Goyal
Keyword(s):  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Leishmania Donovani ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Field Isolates

ABSTRACT Currently available primary screens for the selection of candidate antileishmanial compounds are not ideal. These techniques are time-consuming, laborious, and difficult to scale and require macrophages, which limit their use for high-throughput screening. We have developed Leishmania donovani field isolates that constitutively express the firefly luciferase reporter gene (luc) as a part of an episomal vector. An excellent correlation between parasite number and luciferase activity was observed. luc expression was stable, even in the absence of drug selection, for 4 weeks. The transfectants were infective to macrophages, and intracellular amastigotes exhibited luciferase activity. The suitability of these recombinant field isolates for in vitro screening of antileishmanial drugs was established. The luciferase-expressing sodium stibogluconate-resistant cell lines offer a model for the screening of compounds for resistance. The system is in routine use at the Central Drug Research Institute, Lucknow, India, for high-throughput screening of newly synthesized compounds.

Characterization of an in vitro placental transfer assay for high-throughput screening

2017 ◽  
Vol 280 ◽  
pp. S263-S264
Author(s):  
Caroline Gomes ◽  
Barbara Birk ◽  
Eric Fabian ◽  
Julian Doersam ◽  
Catharina Wilhelmina van Dongen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Placental Transfer

scholarly journals A highly-sensitive sandwich ELISA procedure based on one-step antibody immobilization for potential in vitro diagnostics

2014 ◽  
Author(s):  
Sandeep Kumar Vashist ◽  
Sandeep Kumar Vashist ◽  
E. Marion Schneider ◽  
Edmond Lam ◽  
Sabahudin Hrapovic ◽  
...  
Keyword(s):  
Sandwich Elisa ◽  
Antibody Immobilization ◽  
In Vitro Diagnostics ◽  
Highly Sensitive ◽  
One Step

scholarly journals Construction, Characterization and Application of Recombinant Porcine Deltacoronavirus Expressing Nanoluciferase

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1991
Author(s):  
Puxian Fang ◽  
Huichang Zhang ◽  
He Sun ◽  
Gang Wang ◽  
Sijin Xia ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput Screening ◽  
Reverse Genetics ◽  
Infectious Clone ◽  
Artificial Chromosome ◽  
Antiviral Drugs ◽  
Reporter Virus ◽  
Suckling Piglets ◽  
One Step

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes diarrhoea in suckling piglets and has the potential for cross-species transmission. No effective PDCoV vaccines or antiviral drugs are currently available. Here, we successfully generated an infectious clone of PDCoV strain CHN-HN-2014 using a combination of bacterial artificial chromosome (BAC)-based reverse genetics system with a one-step homologous recombination. The recued virus (rCHN-HN-2014) possesses similar growth characteristics to the parental virus in vitro. Based on the established infectious clone and CRISPR/Cas9 technology, a PDCoV reporter virus expressing nanoluciferase (Nluc) was constructed by replacing the NS6 gene. Using two drugs, lycorine and resveratrol, we found that the Nluc reporter virus exhibited high sensibility and easy quantification to rapid antiviral screening. We further used the Nluc reporter virus to test the susceptibility of different cell lines to PDCoV and found that cell lines derived from various host species, including human, swine, cattle and monkey enables PDCoV replication, broadening our understanding of the PDCoV cell tropism range. Taken together, our reporter viruses are available to high throughput screening for antiviral drugs and uncover the infectivity of PDCoV in various cells, which will accelerate our understanding of PDCoV.

Magnetic-fluorescent-targeting multifunctional aptasensorfor highly sensitive and one-step rapid detection of ochratoxin A

2015 ◽  
Vol 68 ◽  
pp. 783-790 ◽  
Author(s):  
Chengquan Wang ◽  
Jing Qian ◽  
Kan Wang ◽  
Kun Wang ◽  
Qian Liu ◽  
...  
Keyword(s):  
Ochratoxin A ◽  
Rapid Detection ◽  
Highly Sensitive ◽  
One Step

scholarly journals Highly Miniaturized Formats for In Vitro Drug Metabolism Assays Using Vivid® Fluorescent Substrates and Recombinant Human Cytochrome P450 Enzymes

2005 ◽  
Vol 10 (1) ◽  
pp. 56-66 ◽  
Author(s):  
Olga V. Trubetskoy ◽  
Jasmin R. Gibson ◽  
Bryan D. Marks
Keyword(s):  
Cytochrome P450 ◽  
High Throughput Screening ◽  
Cytochrome P450 Enzymes ◽  
Fluorogenic Substrates ◽  
Detailed Characterization ◽  
Human Cytochrome ◽  
Screening Assays ◽  
Well Assay

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid® fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their reactions with Vivid® fluorogenic substrates, with a focus on the specific characteristics of each component that enable screening in a low-volume 1536-well plate assay format. The screening assays were applied for the assessment of individual cytochrome P450 inhibition profiles with a panel of selected assay modifiers, including isozyme-specific substrates and inhibitors. IC50 values obtained for the modifiers in 96- and 1536-well plate formats were similar and comparable with values obtained in assays with conventional substrates. An overall examination of the 1536-well assay statistics, such as signal-to-background ratio and Z′ factor, demonstrated that these assays are a robust, successful, and reliable tool to screen for cytochrome P450 metabolism and inhibition in an ultra-high-throughput screening format. ( Journal of Biomolecular Screening 2005:56-66)

scholarly journals Characterization of Streptococcus gordonii SecA2 as a Paralogue of SecA

2009 ◽  
Vol 191 (11) ◽  
pp. 3482-3491 ◽  
Author(s):  
Barbara A. Bensing ◽  
Paul M. Sullam
Keyword(s):  
Protein Transport ◽  
Maximal Activity ◽  
Biochemical Properties ◽  
Streptococcus Gordonii ◽  
Lower Solubility ◽  
Highly Sensitive ◽  
Sec System ◽  
High Degree

ABSTRACT The accessory Sec system of Streptococcus gordonii is essential for transport of the glycoprotein GspB to the bacterial cell surface. A key component of this dedicated transport system is SecA2. The SecA2 proteins of streptococci and staphylococci are paralogues of SecA and are presumed to have an analogous role in protein transport, but they may be specifically adapted for the transport of large, serine-rich glycoproteins. We used a combination of genetic and biochemical methods to assess whether the S. gordonii SecA2 functions similarly to SecA. Although mutational analyses demonstrated that conserved amino acids are essential for the function of SecA2, replacing such residues in one of two nucleotide binding folds had only minor effects on SecA2 function. SecA2-mediated transport is highly sensitive to azide, as is SecA-mediated transport. Comparison of the S. gordonii SecA and SecA2 proteins in vitro revealed that SecA2 can hydrolyze ATP at a rate similar to that of SecA and is comparably sensitive to azide but that the biochemical properties of these enzymes are subtly different. That is, SecA2 has a lower solubility in aqueous solutions and requires higher Mg2+ concentrations for maximal activity. In spite of the high degree of similarity between the S. gordonii paralogues, analysis of SecA-SecA2 chimeras indicates that the domains are not readily interchangeable. This suggests that specific, unique contacts between SecA2 and other components of the accessory Sec system may preclude cross-functioning with the canonical Sec system.

scholarly journals A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity

2021 ◽  
Vol 22 (3) ◽  
pp. 1438
Author(s):  
Kyong-Oh Shin ◽  
Maftuna Shamshiddinova ◽  
Jung-No Lee ◽  
Kwang-Sik Lee ◽  
Yong-Moon Lee
Keyword(s):  
Embryonal Carcinoma Cell ◽  
Carcinoma Cell Lines ◽  
Highly Sensitive ◽  
Lyase Activity ◽  
Sphingosine 1 Phosphate ◽  
Km Value ◽  
S1p Lyase ◽  
Higher Sensitivity

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 μM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.

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