Scanning assay of β-galactosidase activity

β-galactosidase, encoded by the lacZ gene in E. coli, can cleave lactose and structurally related compounds to galactose and glucose or structurally related products. Its activity can be measured using an artificial substrate, o-nitrophenyl-β-D-galactopyranoside (ONPG). Miller firstly described the standard quantitative assay of β-galactosidase activity in the cells of bacterial cultures by disrupting the cell membrane with the permeabilization solution instead of preparing cell extracts. Therefore, β-galactosidase became one of the most widely used reporters of gene expression in molecular biology to reflect intracellular gene expression difference. But the Miller assay procedure could not monitor the β-galactosidase reaction in real time and its results were greatly influenced by some operations in the Miller procedure, such as permeabilization time, reaction time and concentration of the cell suspension. A scanning method based on the Miller method to determine the intracellular β-galactosidase activity in E. coli Tuner (DE3) expressing β-galactosidase in real time was developed and the permeabilization time of cells was optimized for that. The comparison of 3 assays of β-galactosidase activity (Miller, colorimetric and scanning) was made. The results proved that scanning method for the determination of enzyme activity with using ONPG as substrate is simple, fast and reproducible.     

A fluorescence-based microtiter plate assay for γ-glutamylcyclotransferase

γ-Glutamylcyclotransferase (GGCT) is a component of the γ-glutamyl cycle and specifically cleaves γ-glutamyl-amino acid dipeptides to release the free amino acid and 5-oxoproline. The action of GGCT in glutathione synthetase-deficient patients results in the accumulation of high levels of 5-oxoproline. In addition, GGCT may be a biomarker in some cancers. GGCT inhibitors, therefore, may be of value in the treatment of glutathione synthetase deficiency or in the elucidation of the role of GGCT in cancer cells. Published GGCT assays are not suitable for high-throughput screening for inhibitory molecules. We have developed a fluorescence-based 96-well plate assay for the determination of the rate of γ-glutamylcysteine cleavage by GGCT. After the reaction, the residual γ-glutamylcysteine is determined fluorometrically after derivatization with 2,3-naphthalenedicarboxaldehyde. The method has sufficient sensitivity to detect low-affinity competitive inhibitors and, as a result of its simplicity and microtiter plate format, can be readily used in high-throughput inhibitor screens.     

A new resorufin-based α-glucosidase assay for high-throughput screening

Mutations in α-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, α-glucosidase is a therapeutic target for type II diabetes, and α-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the α-glucosidase enzyme assay, resorufin α-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-α-d-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK_a of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the α-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.     

β-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG

β-Galactosidase (β-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) β-d-galactopyranoside (DDAOG), can be cleaved by β-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a β-gal activity assay method. The DDAO signal was stable for at least 18 h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the β-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-β-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The β-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The β-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of β-gal systems currently in use.     

Evaluation of dye-based assay for mannuronan 5′-epimerase

Alginates, ten other polysaccharides, and cell-wall preparations from Porphyra yezoensis are covalently dyed by Reactive Black 5. Dyed alginates resist cleavage by active preparations of guluronate lyase, indicating that dye-based assay cannot replace ultraviolet spectrophotometry in the guluronate lyase-linked assay for polymannuronan 5-epimerase.     

Mutagenicity of bi-, tri- and tetra-cyclic aromatic hydrocarbons in the “taped-plate assay” and in the conventional Salmonella mutagenicity assay

Aromatic hydrocarbons in the range of 1–4 nuclear rings were examined for mutagenicity in the so-called “taped-plate assay”. This modification of the Ames assay is particularly equipped for the detection of volatile mutagens. Of the many compounds tested only phenanthrene, pyrene, benzo[c]phenanthrene and benzoacenaphthylene were positive in this assay. The present data underline the exceptional behaviour of fluoranthene by being a rather potent bacterial mutagen with a volatile nature (as found in a previous study).     

Incorporation of lipids into cellular membranes—A spin-label assay

A spin-label method is described for the quantitative assay of lipid incorporation into biological membranes, using computer difference spectroscopy. The incorporation of spin-labeled sphingomyelin into synaptic plasma membranes from calf brain has been studied as a function of sonication time. The spin-label ESR spectra are able to distinguish labeled sphingomyelin which is integrated into the membrane, from the unincorporated label, even if the latter cosediments with the membranes. Spectral subtraction has been used to quantitate the degree of incorporation. The percentage of incorporation increases with increasing sonication time and also with incubation after sonication. The extent of degradation of tritium-labeled sphingomyelin by the neutral sphingomyelinase present in the membrane closely correlates with the dependence of the incorporation of the spin-labeled sphingomyelin on sonication time. This illustrates the utility of the method in the study of membrane-bound, lipid-metabolizing enzymes.     

Cell-based assay for screening 11β-hydroxysteroid dehydrogenase 1 inhibitors

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is primarily responsible for intracellular biosynthesis of active glucocorticoid, and its tissue-specific dysregulation has been implicated in the development of metabolic syndromes. We have developed a cell-based assay for measuring 11β-HSD1 activities using murine skeletal muscle cell line C2C12. We found that the messenger RNA (mRNA) expression of 11β-HSD1 increased on differentiation with enhanced enzyme activity as determined by homogeneous time-resolved fluorescence (HTRF) assay. Carbenoxolone, a well-known 11β-HSD1 inhibitor, exhibited an IC₅₀ value similar to that in in vitro microsomal assay (IC₅₀ = 0.3 μM). Unlike in vitro microsomal assay, cosubstrate NADPH was not required in the cell-based assay, indicating that viable cells might provide a sufficient amount of endogenous NADPH to catalyze the enzymatic conversion of inactive cortisone to active cortisol. Treatment of C2C12 myotubes with cortisone concentration dependently transactivated and transrepressed glutamine synthase and interleukin-6, respectively, which were abrogated by carbenoxolone or RU-486 (mifepristone), a glucocorticoid receptor antagonist. Accordingly, a newly designed cell-based assay using differentiated skeletal muscle cells would be useful for high-throughput screening of 11β-HSD1 inhibitors as well as for understanding the molecular mechanisms of glucocorticoid action.     

AlphaScreen selectivity assay for β-catenin/B-cell lymphoma 9 inhibitors

The aberrant formation of the β-catenin/B-cell lymphoma 9 (BCL9) protein–protein complex is the driving force for many diseases, including cancer. Crystallographic analyses demonstrate that the surface area in β-catenin for interacting with BCL9 is overlapped with that for the β-catenin/E-cadherin interaction. In this study, a robust AlphaScreen selectivity assay was developed to quantify inhibitor potency for the β-catenin/BCL9 interaction and selectivity for β-catenin/BCL9 over β-catenin/E-cadherin interactions. A pilot screen was performed to demonstrat the feasibility of this assay. This selectivity assay is highly sensitive and suitable for adaptation to high-throughput screening. The establishment of this assay lays the foundation for the discovery of selective inhibitors specific for β-catenin/BCL9 interactions.     

A high-throughput apple SNP genotyping platform using the GoldenGate™ assay

EST data generated from 14 apple genotypes were downloaded from NCBI and mapped against a reference EST assembly to identify Single Nucleotide Polymorphisms (SNPs). Mapping of these SNPs was undertaken using 90% of sequence similarity and minimum coverage of four reads at each SNP position. In total, 37,807 SNPs were identified with an average of one SNP every 187 bp from a total of 6888 unique EST contigs. Identified SNPs were checked for flanking sequences of ≥ 60 bp along both sides of SNP alleles for reliable design of a custom high-throughput genotyping assay. A total of 12,299 SNPs, representing 6525 contigs, fit the selected criterion of ≥ 60 bp sequences flanking a SNP position. Of these, 1411 SNPs were validated using four apple genotypes. Based on genotyping assays, it was estimated that 60% of SNPs were valid SNPs, while 26% of SNPs might be derived from paralogous regions.     

Toxicity of ochratoxin A in aBrevibacillus brevis — Growth inhibition assay

Ochratoxin A (OTA) is a nephrotoxic, carcinogenic and immunosuppressive mycotoxin. It can be detoxified by various microorganisms, e.g. different yeast strains, via metabolisation into ochratoxin α (OTα). Within this study a growth inhibition assay was developed to compare the toxicity of OTA and its degradation product OTα. As an indicator organismBrevibacillus brevis was used. The assay was performed in microtiterplates. Growth inhibition was determined by comparing the optical density values ofBrevibacillus brevis cultures grown in medium supplemented with OTA/OTα and OTA/OTα-free medium, respectively. It could be shown thatB. brevis is sensitive to OTA (EC₁₀₀=0.5 mg/L±0.03 mg/L), which is not the case for its metabolite OTα. Therefore this bioassay is a useful tool to show the detoxification of OTA to OTα by microbial degradation. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

A continuous assay for α-methylacyl-coenzyme A racemase using circular dichroism

α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (K_m = 48 ± 5 μM, k_cat = 291 ± 30 s⁻¹), but turned over (2S)-ibuprofenoyl-CoA (K_m = 86 ± 6 μM, k_cat = 450 ± 14 s⁻¹) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His₆-tag had a catalytic efficiency (k_cat/K_m) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.     

Time-resolved Förster-resonance-energy-transfer DNA assay on an active CMOS microarray

We present an active oligonucleotide microarray platform for time-resolved F?rster-resonance-energy-transfer (TR-FRET) assays. In these assays, immobilized probe is labeled with a donor fluorophore and analyte target is labeled with a fluorescence quencher. Changes in the fluorescence decay lifetime of the donor are measured to determine the extent of hybridization. In this work, we demonstrate that TR-FRET assays have reduced sensitivity to variances in probe surface density compared with standard fluorescence-based microarray assays. Use of an active array substrate, fabricated in a standard complementary metal-oxide-semiconductor (CMOS) process, provides the additional benefits of reduced system complexity and cost. The array consists of 4096 independent single-photon avalanche diode (SPAD) pixel sites and features on-chip time-to-digital conversion. We demonstrate the functionality of our system by measuring a DNA target concentration series using TR-FRET with semiconductor quantum dot donors.     

Allelic discrimination using fluorogenic probes and the 5′ nuclease assay

Large-scale screening for known polymorphisms will require techniques with few steps and the ability to automate each of these steps. In this regard, the 5′ nuclease, or TaqMan, PCR assay is especially attractive. A fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye, is included in a typical PCR. Amplification of the probe-specific product causes cleavage of the probe, generating an increase in reporter fluorescence. By using different reporter dyes, cleavage of allele-specific probes can be detected in a single PCR. The 5′ nuclease assay has been successfully used to discriminate alleles that differ by a single base substitution. Guidelines have been developed so that an assay for any single nucleotide polymorphism (SNP) can be quickly designed and implemented. All assays are performed using a single reaction buffer and single thermocycling protocol. Furthermore, a standard method of analysis has been developed that enables automated genotype determination. Applications of this assay have included typing a number of polymorphisms in human drug metabolism genes.     

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.)

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

A carbapenem-based fluorescence assay for the screening of metallo-β-lactamase inhibitors

Reported herein is a fluorescence assay for the rapid screening of metallo-β-lactamase (MBL) inhibitors. This assay employs a fluorogenic carbapenem CPC-1 as substrate and is compatible with all MBLs, including B1, B2 and B3 subclass MBLs. The efficiency of this assay was demonstrated by the rapid inhibition screening of a number of molecules against B2 MBL CphA and 2,3-dimercaprol was identified as a potent CphA inhibitor.