Dihydrofolate reductase (DHFR) reporter enzyme assay for Haloferax volcanii

Mapping Intimacies ◽

10.21203/rs.2.11263/v1 ◽

2019 ◽

Author(s):

Madeleine Huber ◽

Jörg Soppa

Keyword(s):

Dihydrofolate Reductase ◽

Enzyme Assay ◽

Background Level ◽

Microtiter Plate ◽

Haloferax Volcanii ◽

Expression Level ◽

Wildtype Strain ◽

Translational Fusion ◽

Reporter Enzyme ◽

Microtiter Plate Format

Abstract The dihydrofolate reductase (DHFR) is routinely used a reporter enzyme for H. volcanii. The DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate and the concomitant oxidation of NADPH to NADP+. This leads to a reduction of extinction at 340 nm, which is measured to quantify the DHFR activity. To avoid background, it is best to use an H. volcanii strain with a deletion of the chromosomal dhfr gene, which is available upon request ([email protected]). However, the expression level of the chromosomal dhfr gene is very low, so that it is also possible to use the wildtype strain and subtract the DHFR background level. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the dhfr gene in a translational fusion with the gene of interest.

Download Full-text

Beta-Glucuronidase (GusA) reporter enzyme assay for Escherichia coli

10.21203/rs.2.10596/v1 ◽

2019 ◽

Author(s):

Madeleine Huber ◽

Jörg Soppa

Keyword(s):

Escherichia Coli ◽

Translational Regulation ◽

Enzyme Assay ◽

Microtiter Plate ◽

E Coli ◽

Reporter Enzyme ◽

Keio Collection ◽

Beta Galactosidase ◽

Transcriptional Fusions ◽

Microtiter Plate Format

Abstract The beta-Glucuronidase (GusA) is a long-known reporter enzyme for many different species [1]. The E. coli gusA gene is often used in plant research because plants lack an endogenous gusA gene. In E. coli, the transcript of the gusA gene is more stable than that of the highly used reporter gene beta-galactosidase (lacZ) [2]. The GusA activity can be determined using the artificial substrate p-nitrophenyl-β-D-glucopyranosid (pNPG). pNPG is converted to glucoronic acid and para-nitrophenol (pNP), which can be quantified spectrometrically at 405 nm. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal gusA gene, which is available e.g. at the Keio collection [3]. The gusA gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the gusA gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.

Download Full-text

Glycerol-3-phosphate dehydrogenase (GlpD) reporter enzyme assay for Escherichia coli

10.21203/rs.2.10595/v1 ◽

2019 ◽

Author(s):

Madeleine Huber ◽

Jörg Soppa

Keyword(s):

Translational Regulation ◽

Enzyme Assay ◽

Microtiter Plate ◽

Dihydroxyacetone Phosphate ◽

Electron Carrier ◽

E Coli ◽

Reporter Enzyme ◽

Glycerol 3 Phosphate Dehydrogenase ◽

Transcriptional Fusions ◽

Microtiter Plate Format

Abstract Glycerol-3-phosphate dehydrogenase (GlpD) is a recently introduced reporter enzyme for E. coli [1]. GlpD calalyzes the oxidation of Glycerin-3-phosphate (G3P) to dihydroxyacetone-phosphate (DHAP). The oxidation is coupled to the reduction of the artificial yellow substrate tetrazol-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) to a blue-violet formazan, which is mediated by the electron carrier phenazin-methanosulfate (PMS). This leads to an increase in absorption at 570 nm, which is measured to quantify the GlpD activity. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal glpD gene, which is available e.g. at the Keio collection [2]. The glpD gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the glpD gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.

Download Full-text

Evaluation of UDP-glucuronosyltransferase 2B17 (UGT2B17) and dihydrofolate reductase (DHFR) genes deletion and the expression level of NGX6 mRNA in breast cancer

Molecular Biology Reports ◽

10.1007/s11033-012-1938-8 ◽

2012 ◽

Vol 39 (12) ◽

pp. 10531-10539 ◽

Cited By ~ 23

Author(s):

Ebrahim Eskandari-Nasab ◽

Mohammad Hashemi ◽

Hamzeh Rezaei ◽

Aliakbar Fazaeli ◽

Mohammad Ali Mashhadi ◽

...

Keyword(s):

Breast Cancer ◽

Dihydrofolate Reductase ◽

Expression Level ◽

Udp Glucuronosyltransferase

Download Full-text

Cerebrospinal Fluid Secretory Ca2+-Dependent Phospholipase A2 Activity Is Increased in Alzheimer Disease

Clinical Chemistry ◽

10.1373/clinchem.2009.130286 ◽

2009 ◽

Vol 55 (12) ◽

pp. 2171-2179 ◽

Cited By ~ 36

Author(s):

Sonia Chalbot ◽

Henrik Zetterberg ◽

Kaj Blennow ◽

Tormod Fladby ◽

Inge Grundke-Iqbal ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Alzheimer Disease ◽

Phospholipase A2 ◽

Microtiter Plate ◽

Pla2 Activity ◽

Activity Assay ◽

Proinflammatory Mediators ◽

Spla2 Activity ◽

Phospholipase A2 Activity ◽

Microtiter Plate Format

Abstract Background: The phospholipase A2 (PLA2) family comprises multiple isoenzymes that vary in their physicochemical properties, cellular localizations, calcium sensitivities, and substrate specificities. Despite these differences, PLA2s share the ability to catalyze the synthesis of the precursors of the proinflammatory mediators. To investigate the potential of PLA2 as a biomarker in screening neuroinflammatory disorders in both clinical and research settings, we developed a PLA2 assay and determined the predominant types of PLA2 activity in cerebrospinal fluid (CSF). Methods: We used liposomes composed of a fluorescent probe (bis-Bodipy® FL C11-PC [1,2-bis-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine]) and 1,2-dioleoyl-l-α-phosphatidylcholine as a substrate to measure CSF PLA2 activity in a 96-well microtiter plate format. We established the type of CSF PLA2 activity using type-specific inhibitors of PLA2. Results: Using 5 μL CSF per assay, our PLA2 activity assay was reproducible with CVs <15% in 2 CSF samples and for recombinant secretory Ca2+-dependent PLA2 (sPLA2) in concentrations ranging from 0.25 to 1 μmol/L. This PLA2 assay allowed identification of sPLA2 activity in lumbar CSF from healthy individuals 20–77 years old that did not depend on either sex or age. Additionally, CSF sPLA2 activity was found to be increased (P = 0.0008) in patients with Alzheimer disease. Conclusions: Adult human CSF has sPLA2 activity that can be measured reliably with the assay described. This enzyme activity in the CSF is independent of both sex and age and might serve as a valuable biomarker of neuroinflammation, as we demonstrated in Alzheimer disease.

Download Full-text

Development of a fluorometric microtiter plate based enzyme assay for MPS IVA (Morquio type A) using dried blood spots

Molecular Genetics and Metabolism Reports ◽

10.1016/j.ymgmr.2014.10.004 ◽

2014 ◽

Vol 1 ◽

pp. 461-464 ◽

Cited By ~ 9

Author(s):

Anirudh J. Ullal ◽

David S. Millington ◽

Deeksha S. Bali

Keyword(s):

Enzyme Assay ◽

Type A ◽

Dried Blood Spots ◽

Microtiter Plate ◽

Blood Spots ◽

Mps Iva

Download Full-text

An amended potassium persulfate ABTS antioxidant assay used for medicinal plant extracts revealed variable antioxidant capacity based upon plant extraction process

10.1101/2020.07.15.204065 ◽

2020 ◽

Author(s):

Cody E. Mingle ◽

Anthony L. Newsome

Keyword(s):

Antioxidant Activity ◽

Plant Extracts ◽

Biological Fluids ◽

Antioxidant Properties ◽

Cation Radical ◽

Microtiter Plate ◽

Potassium Persulfate ◽

Antioxidant Assay ◽

Medicinal Plant Extracts ◽

Microtiter Plate Format

AbstractUse of potassium persulfate (K2S208) for oxidation of 7.0 mM ABTS to a stable ABTS radical for antioxidant studies was first reported in 1999. A feature of this popular antioxidant assay has been the requirement of an overnight reaction (6 to 12 h) for the formation of a stable ABTS colored radical. It is now reported that when the concentration of ABTS is lowered to 0.7 mM, complete oxidation to the stable cation radical occurs in 30 min, thus circumventing the necessary overnight step. Using this format, it is now possible to accurately assess antioxidant activity based on the potassium persulfate/ABTS format in less than one hour which includes formation time of a stable ABTS radical. This methodology documented the presence of antioxidant properties of plant extracts used in Traditional Chinese Medicine. The degree of antioxidant activity was directly related to the extraction method. Greater antioxidant activity was associated with butanol extraction. When incorporated into a microtiter plate format, it supported rapid assessment of multiple determinations of dilutions of plant extracts in less than one hour which included time required for formation of a stable ABTS radical. The ease, improved time prerequisites, and minimal reagent needs with the microtiter plate format, makes this design attractive. It would prove of particular interest to individuals engaged in both smaller and high-volume throughput antioxidant assays of food and health products, and other biological fluids and tissues.

Download Full-text

Discovery of New Schiff Bases Tethered Pyrazole Moiety: Design, Synthesis, Biological Evaluation, and Molecular Docking Study as Dual Targeting DHFR/DNA Gyrase Inhibitors with Immunomodulatory Activity

Molecules ◽

10.3390/molecules25112593 ◽

2020 ◽

Vol 25 (11) ◽

pp. 2593 ◽

Cited By ~ 1

Author(s):

Ashraf S. Hassan ◽

Ahmed A. Askar ◽

Ahmed M. Naglah ◽

Abdulrahman A. Almehizia ◽

Ahmed Ragab

Keyword(s):

Molecular Docking ◽

Schiff Bases ◽

Dihydrofolate Reductase ◽

Enzyme Assay ◽

Dna Gyrase ◽

Docking Study ◽

Biological Evaluation ◽

Immunomodulatory Activity ◽

Molecular Docking Study ◽

Design Synthesis

A series of Bis-pyrazole Schiff bases (6a–d and 7a–d) and mono-pyrazole Schiff bases (8a–d and 9a–d) were designed and synthesized through the reaction of 5-aminopyrazoles 1a–d with aldehydes 2–5 using mild reaction condition with a good yield percentage. The chemical structure of newly formed Schiff bases tethered pyrazole core was confirmed based on spectral and experimental data. All the newly formed pyrazole Schiff bases were evaluated against eight pathogens (Gram-positive, Gram-negative, and fungi). The result exhibited that, most of them have good and broad activities. Among those, only six Schiff bases (6b, 7b, 7c, 8a, 8d, and 9b) displayed MIC values (0.97–62.5 µg/mL) compared to Tetracycline (15.62–62.5 µg/mL) and Amphotericin B (15.62–31.25 µg/mL), MBC values (1.94–87.5 µg/mL) and selectivity to tumor cell than normal cells. Immunomodulatory activities showed that the promising Schiff bases increase the immunomodulator effect of defense cell and the Schiff base 8a is the highest one by (Intra. killing activity = 136.5 ± 0.3%) having a pyrazole moiety as well as amide function (O=C-NH2) and piperidinyl core. Furthermore, the most potent one exhibited broad activity depending on both MIC and MBC values. Moreover, to study the mechanism of these pyrazole Schiff bases, two active Schiff bases 8a and 9b from six derivatives were introduced to study the enzyme assay as dihydrofolate reductase (DHFR) on E. coli organism and DNA gyrase with two different organisms, S. aureus and B. subtilis, to determine the inhibitory activities with lower values in the case of DNA gyrase (8a and 9b) or nearly as DHFR compound 9b, while pyrazole 8a showed excellent inhibitory against all enzyme assay. The molecular docking study against dihydrofolate reductase and DNA gyrase were performed to study the binding between active site in the pocket with the two Schiff bases (8a and 9b) that exhibited good binding affinity with different bond types as H-bonding, aren-aren, and arene-cation interaction as well as study the physicochemical and pharmacokinetic properties of the two active Schiff bases 8a and 9b.

Download Full-text

FOXM1 Drives Epithelial-Mesenchymal Transition Program Through E-Cadherin Promoter Biding Ability in Non-Small-Cell Lung Cancer Cells

10.21203/rs.3.rs-702896/v1 ◽

2021 ◽

Author(s):

Ling Wang ◽

Lin Xiaolan ◽

ZongSheng Jiang ◽

Yanzi Sun ◽

Yixuan Li ◽

...

Keyword(s):

Cell Migration ◽

Enzyme Assay ◽

Epithelial Mesenchymal Transition ◽

Small Cell ◽

Migration And Invasion ◽

Small Cell Lung ◽

Mesenchymal Transition ◽

Reporter Enzyme ◽

Tgf Β1 ◽

E Cadherin

Abstract Background: Forkhead box (FOX) gene family plays a critical role in regulating Epithelial-mesenchymal transition (EMT) program, and in which, FOXM1 can mediate multiple malignant process in many type of tumor cells. However, the modulate functions of FOXM1 on EMT in non-small-cell lung cancer (NSCLC) cells, especially the transcriptional function on E-cadherin coding gene CDH1 remains unclear. This article mainly focuses on FOXM1, exploring its mechanism in regulating EMT of NSCLC cells, and FOXM1 inhibitor thiostrepton’s effects in EMT intervention. Methods: Morphological changes of overexpressed cells were observed by HE staining. The effects of scratch test, Transwell chamber test and Western-blot analysis on cell migration and invasion ability and the expression of EMT-related markers were analyzed. Dual luciferin reporter enzyme assay and nuclear transcription factor immunoprecipitation assay (ChIP, immunofluorescence) revealed the transcriptional regulation of FOXM1 on EMT markers. MTT assay and clone formation assay were used to determine the effect of thiomycin on the viability of NSCLC cells and the ability of cell clone formation.Rusults: After overexpression of FOXM1, the cells showed intermediate epithelial-mesenchymal morphology, but not complete mesenchymal morphology, and their migration and invasion abilities were enhanced. The protein expression levels of N-cadherin,Snail1 and Vimentin were increased, while the expression levels of E-cadherin were decreased. On the contrary, knockdown of FOXM1 expression showed the opposite result. The double luciferin reporter enzyme assay showed that FOXM1 inhibited the luciferin reporter vector CDH1-2000-promoter. ChIP results confirmed that FOXM1 could bind endogenous to CDH1 gene promoter. In cells overexpressing FOXM1, knockdown of Snail further promotes FOXM1-mediated CDH1 transcription. MTT results and clone formation experiments showed that thiomycin had inhibitory effect on the proliferation of NSCLC cells. Morphological observation, cell migration assay and Transwell chamber assay showed that streptotin inhibited TGF-β1-induced enhanced cell migration and invasion. Western-blot analysis showed that thiomycin down-regulated the expression of FOXM1, N-cadherin, Snail, and Vimentin induced by TGF-β1, while blocking the expression of E-cadherin induced by TGF-β1 decreased.Conclusion: FOXM1 can directly bind to the promoter of E-cadherin encoding gene, and can indirectly inhibit E-cadherin expression by stimulating Snail. Overexpression of FOXM1 can promote EMT progression in NSCLC cells. Therefore, down-regulation of FOXM1 can inhibit this process. In addition, thiostrepton, a FOXM1 inhibitor, blocked proliferation, colony formation, and EMT progression in NSCLC cells.

Download Full-text