Fast and easy in vitro screening assay for cholesterol biosynthesis inhibitors in the post-squalene pathway

A whole-cell assay for screening cholesterol biosynthesis inhibitors in the post-squalene pathway has been developed. HL 60 cells were incubated for 24h with test substances. The nonsaponifiable lipids were extracted by means of liquid-liquid extraction using tert-butylmethylether. The raw extracts were purified by dispersive solid phase extraction using a primary-secondary amine material (PSA) and dried using sodium sulphate. The sterols were derivatized using N-trimethylsilylimidazole. GLC/MS analysis was carried out in less than 12.5 min using fast GLC mode. The obtained sterol patterns indicated which enzyme had been inhibited. Specific sterol patterns which reflect the different enzyme inhibitions were obtained using established inhibitors of cholesterol biosynthesis like AY 9944, NB 598, clotrimazole, aminotriazole and DR 258, a Delta24-reductase inhibitor prepared in our working group. For characterizing IC(50) values we used sodium 2-(13)C-acetate and quantified the incorporation of it into cholesterol relative to control levels after the samples had been normalized to their protein content.     

An easy colorimetric assay for glycosyltransferases

Glycosyltransferases are involved in biosynthesis of both protein-bound and non-bound glycans that have multiple and important biological functions in all species. A variety of methods for assaying glycosyltransferase activity have been developed driven by the specific interests and type of information required by researchers. In this work, a novel colorimetric assay for the glycosyltransferase-catalyzed reaction was established. Compared with measuring the newly formed product, which might not exhibit visible absorption, the unreacted acceptor could be readily detected by measuring the visible absorption of the hydrolysis product. In the assay, 4-nitrophenyl-β-D-glycoside (glycosyl-β-pNP) is used as the glycosyl acceptor, which can be hydrolyzed by a special exoglycosidase to release the p-nitrophenol before glycosylation reactions. Absorbance change of the p-nitrophenolate corresponds to unreacted glycosyl acceptor that accompanied the glycosyl transfer. The assay is demonstrated to be useful in the initial characterization of recombinant glycosyltransferases for their kinetic parameters, optimal metal cofactor, and pH value. It provides a simple, sensitive, and quantitative method for assessing glycosyltransferase activity and is thus expected to have broad applications including automated high-throughput screening.     

An easy colorimetric assay for screening and qualitative assessment of deiodination and dehalogenation by bacterial cultures

Halogenated organic substances are among the main environmental concerns. A number of micro-organisms are able to dehalogenate these compounds. However, the methods for the assessment of micro-organismal ability to dehalogenate are expensive and require complex instrumentation. Here, an easy colorimetric assay for the screening and assessment of the ability of bacterial cultures to deiodinate, and potentially dehalogenate, chemical substances is proposed. The method is based on the oxidation of iodide, released due to biotransformation, to iodine followed by a subsequent detection of iodine by a classical reaction with starch.     

The occurrence of the Entner-Doudoroff pathway in bacteria

The occurrence of the two key enzymes of the Entner-Doudoroff pathway, gluconate-6-phosphate dehydrase and 2-keto-3-deoxygluconate-6-phosphate aldolase, was determined in approximately 150 strains belonging to 37 different bacterial genera. The following results were obtained:

1. 24 out of 37 genera have at least one representative with the Entner-Doudoroff mechanism. It is thus more widespread than previously thought.
2. The Entner-Doudoroff mechanism occurs mainly in gram-negative bacteria with a DNA base composition in the range 52–70% GC. Eighty-five per cent of these organisms contain the system, while only 20% (6 strains) of the gram-negative organisms with less than 52% GC possess both enzymes.
3. This pathway is absent in all gram-positive organisms investigated except in 5 out of 12Nocardia strains.
4. Erwinia and some strains of theAchromobacter-Alcaligenes group are exceptional, since they possess only 2-keto-3-deoxygluconate-6-phosphate aldolase.

     

Pseudomonas cepacia mutants blocked in the Entner-Doudoroff pathway.

Pseudomonas cepacia mutants deficient in either 6-phosphogluconate (6PGA) dehydratase (Edd-) or 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (Eda-) failed to utilize glucose or gluconate despite the prominence of of 6-phosphogluconate dehydrogenase (6PGAD) ii this bacterium and the potential for utilizing the pentose shunt suggested by its growth on ribitol and xylose. The Eda- strains grew normally on glucuronic acid, indicating that in P. cepacia its degradation does not depend upon KDPG aldolase as it does in Escherichia coli. Both 6PGA dehydratase and KDPG aldolase were inducible enzymes, with 6PGA rather than gluconate the apparent inducer. Edd- as well as Eda- strains were sensitive to growth inhibition by glucose, gluconate, fructose, and related carbohydrates when these substrates were present in combination with alternate carbon sources such as citrate or phthalate, presumably as a consequence of accumulation and toxicity of 6PGA, KDPG, or both. Edd- mutants were somewhat less sensitive to such inhibition than were Eda- strains. Certain derivatives of the Edd- strains we examined were able to utilize gluconate despite their deficiency of 6PGA dehydratase. Such mutants formed higher levels of 6PGAD than did the wild type. It is likely that the elevated levels of 6PGAD in these strains prevents accumulation of toxic levels of 6PGA that would otherwise result from a block in he Entner-Doudoroff pathway. The results suggest that P. cepacia can mutate to grow slowly on gluconate utilizing only the pentose shunt.     

An easy assay for histone acetyltransferase activity using a PhosphorImager

A simple radiometric assay for histone acetyltransferase (HAT) activity employing a PhosphorImager is described. In the proposed procedure, following incubation of [1-¹⁴C]acetyl coenzyme A (CoA), histones, and HAT enzyme, radiolabeled histones are fixed on GF/F glass microfiber filter while the excess of acetyl CoA is washed out. Afterward, the filter is exposed to a phosphor-screen and the resulting spot signals are quantified with a PhosphorImager. Given the small volumes required, the new assay reduces reagent consumption and contaminated waste. Moreover, the assay can be performed with a large number of samples simultaneously, is applicable on different protein substrates, and is adaptable to the analysis of other protein modifications.     

Identification of the Entner-Doudoroff pathway in an antibiotic-producing actinomycete species

The metabolic network of the central carbon metabolism represents the backbone of cellular metabolism and provides the precursors and cofactors required for synthesis of secondary metabolites. It is therefore pivotal to map the operating metabolic network in the central carbon metabolism in order to design metabolic engineering strategies towards construction of more efficient producers of specific metabolites. In this context, methods that allow rapid and reliable mapping of the central carbon metabolism are valuable. In the present study, a (13)C labelling-based method was used to identify the primary metabolic pathways of the poorly characterized antibiotic-producing actinomycete Nonomuraea sp. ATCC 39727. Surprisingly, it was found that Nonomuraea sp. ATCC 39272 predominantly metabolizes glucose via the Entner-Doudoroff (ED) pathway. This represents the first time that the ED pathway has been recognized as the main catabolic pathway in an actinomycete. The Nonomuraea genes encoding the key enzymes of the ED pathway were subsequently identified, sequenced and functionally described.     

Promiscuity in the part-phosphorylative Entner-Doudoroff pathway of the archaeon Sulfolobus solfataricus

The hyperthermophilic archaeon Sulfolobus solfataricus metabolises glucose and galactose by a 'promiscuous' non-phosphorylative variant of the Entner-Doudoroff pathway, in which a series of enzymes have sufficient substrate promiscuity to permit the metabolism of both sugars. Recently, it has been proposed that the part-phosphorylative Entner-Doudoroff pathway occurs in parallel in S. solfataricus as an alternative route for glucose metabolism. In this report we demonstrate, by in vitro kinetic studies of D-2-keto-3-deoxygluconate (KDG) kinase and KDG aldolase, that the part-phosphorylative pathway in S. solfataricus is also promiscuous for the metabolism of both glucose and galactose.     

Gluconate dehydratase from the promiscuous Entner-Doudoroff pathway in Sulfolobus solfataricus

An investigation has been carried out into gluconate dehydratase from the hyperthermophilic Archaeon Sulfolobus solfataricus. The enzyme has been purified from cell extracts of the organism and found to be responsible for both gluconate and galactonate dehydratase activities. It was shown to be a 45 kDa monomer with a half-life of 41 min at 95 degrees C and it exhibited similar catalytic efficiency with both substrates. Taken alongside the recent work on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase, this report clearly demonstrates that the entire non-phosphorylative Entner-Doudoroff pathway of S. solfataricus is promiscuous for the metabolism of both glucose and galactose.     

Using pathway modules as targets for assay development in xenobiotic screening

Toxicology and pharmaceutical research is increasingly making use of high throughout-screening (HTS) methods to assess the effects of chemicals on molecular pathways, cells and tissues. Whole-genome microarray analysis provides broad information on the response of biological systems to chemical exposure, but is not practical to use when thousands of chemicals need to be evaluated at multiple doses and time points, as well as across different tissues, species and life-stages. A useful alternative approach is to identify a focused set of genes that can give a coarse picture of systems-level responses and that can be scaled to the evaluation of thousands of chemicals and diverse biological contexts. We demonstrate a computational approach to select in vitro expression assay targets that are informative and broadly distributed in biological pathway space, using the concept of pathway modularity. Canonical pathways are decomposed into subnetworks (modules) of functionally-related genes based on rules such as co-regulated expression, protein-protein interactions, and coordinated physiological activity. Pathway modules are constructed using these rules but are then restricted by the bounds of canonical pathways. We demonstrate this approach using a subset of genes associated with tumor development and cancer progression. Target genes were identified for assay development, and then validated by using independent, published microarray data. The result is a targeted set of genes that are sensitive predictors of whether a chemical will perturb each pathway module. These selected genes could then form the basis for a battery to test for pathway-chemical interactions under many biological contexts using throughput expression-based assays.     

An ultrahigh-throughput screening assay for estrogen receptor ligands.

Members of the steroid and thyroid hormone receptor superfamily (nuclear receptors) play diverse roles in mammalian physiology, in both normal and pathological states. For this reason, and because nuclear receptors are natural receptors for lipophilic small molecules, they are important therapeutic targets for the pharmaceutical industry. Here we describe a method for screening for ligands for one of these receptors, the estrogen receptor. The method is rapid, robust, and reliable, and has been used in an ultrahigh-throughput robotic screen. The receptor is crosslinked to a scintillant-containing solid support (FlashPlate) via a receptor-specific antibody. Test compounds are assayed for competition with a radiolabeled estrogen for binding to the immobilized receptor. Receptor-ligand complexes are allowed to form and receptor-bound radioactivity is detected in a scintillation counter. The assay has been designed for both isoforms of the estrogen receptor, alpha and beta, using separate antibodies for each. Given a radioactive tracer and an appropriate antibody, many of which are now commercially available, the assay could be established for any nuclear receptor.     

An easy and accurate agarose gel assay for quantitation of bacterial plasmid copy numbers

An assay for quantitation of plasmid copy numbers in bacterial cell cultures has been developed and validated. The method combines isolation of total bacterial DNA (including both plasmid and genomic DNA), running a series of twofold dilutions of total DNA in an agarose gel followed by ethidium bromide staining, and subsequent scanning of the gel picture negatives. We have developed a novel set of rules for integration of the scan data that allows us to achieve high assay precision, accuracy, and sensitivity. The assay validation results were as follows: intra- and interassay precision with %CV of 8.2-9.9 and 7.1-9.8%, respectively; ruggedness with %CV of 9.3-17.5%; spike recovery of 80-102%; and sensitivity of 1 plasmid copy per genome.     

An intact assay for enzymes that labilize C--H bonds.

The activity of enzymes that labilize known CH bonds can be estimated in intact tissue by following the loss to water of tritium from specifically labeled substrates. The accumulated tritium oxide (³HHO) in the medium is recovered by direct sublimation of aliquots of the medium into scintillation vials and the radio-activity measured by liquid scintillation spectrometry.     

Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay

Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified.     

An image-based assay for high throughput screening of Giardia lamblia

Giardia lamblia is a protozoan parasite that causes widespread gastrointestinal illness. Drugs to treat giardiasis are limited, but efforts to discover new anti-giardial compounds are constrained by the lack of a facile system for cell culture and inhibitor testing. We achieved robust and reproducible growth of G. lamblia in 384-well tissue culture plates in a modified TYI-S-33 medium. A high throughput assay for the screening of potential anti-giardial compounds was developed utilizing the WB strain of G. lamblia and automated optical detection of parasites after growth with tested inhibitors. We screened a library of 1600 known bioactive molecules and identified 12 compounds that inhibited growth of G. lamblia at low- or sub-micromolar concentrations. Our high throughput assay should facilitate evaluation of available chemical libraries for novel drugs to treat giardiasis.