Analytical Determination of Aflatoxin in Ground Corn Check Samples Completed by Multiple Laboratories over Several Years.

Mycotoxins are toxic molecules produced by multiple fungal species, including and . Fungal infection of crops can result in mycotoxins entering the animal and human food supply. Enzyme-linked immunosorbent assays and other immunological assays have been developed to detect mycotoxins in foods.

Overview of Portable Assays for the Detection of Mycotoxins, Allergens, and Sanitation Monitoring.

Background: Many food recalls are related to the presence of undeclared allergens and microorganisms in food products. To reduce these occurrences, portable diagnostic assay kits are available to quantitate mycotoxins, to detect allergens and gluten in foods and on environmental surfaces, and for sanitation monitoring.

Objective: This article reviews diagnostic kits that can detect sources of contamination in food and ingredients as well as on surfaces and clean-in-place rinses.

Development of a rapid multiplexed assay for the direct screening of antimicrobial residues in raw milk.

Antimicrobial residues found to be present in milk can have both health and economic impacts. For these reasons, the widespread routine testing of milk is required. Due to delays with sample handling and test scheduling, laboratory-based tests are not always suited for making decisions about raw material intake and product release, especially when samples require shipping to a central testing facility.

The use of biochemical and biophysical tools for triage of high-throughput screening hits - A case study with Escherichia coli phosphopantetheine adenylyltransferase.

High-throughput screening is utilized by pharmaceutical researchers and, increasingly, academic investigators to identify agents that act upon enzymes, receptors, and cellular processes. Screening hits include molecules that specifically bind the target and a greater number of non-specific compounds. It is necessary to 'triage' these hits to identify the subset worthy of further exploration.

Comparing 2-nt 3' overhangs against blunt-ended siRNAs: a systems biology based study.

In this study, we formulate a computational reaction model following a chemical kinetic theory approach to predict the binding rate constant for the siRNA-RISC complex formation reaction. The model allowed us to study the potency difference between 2-nt 3' overhangs against blunt-ended siRNA molecules in an RNA interference (RNAi) system. The rate constant predicted by this model was fed into a stochastic simulation of the RNAi system (using the Gillespie stochastic simulator) to study the overall potency effect.

Thermodynamic and structure guided design of statin based inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Clinical studies have demonstrated that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) inhibitors, are effective at lowering mortality levels associated with cardiovascular disease; however, 2-7% of patients may experience statin-induced myalgia that limits compliance with a treatment regimen. High resolution crystal structures, thermodynamic binding parameters, and biochemical data were used to design statin inhibitors with improved HMGR affinity and therapeutic index relative to statin-induced myalgia. These studies facilitated the identification of imidazole 1 as a potent (IC 50 = 7.

Phosphopantetheine adenylyltransferase from Escherichia coli: investigation of the kinetic mechanism and role in regulation of coenzyme A biosynthesis.

Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E.

Correlation of carboxylic acid pKa to protein binding and antibacterial activity of a novel class of bacterial translation inhibitors.

Previously we reported the discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding. This report describes efforts directed toward understanding the relationship of the acidity of the carboxylic acid with the extent of protein binding. The pK(a) of the acid was modified via the synthesis of a number of anthranilic acid analogs which vary the aromatic ring substituent at the 4-position.

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA.

Preparation of novel anthranilic acids as antibacterial agents: extensive evaluation of structural and physical properties on antibacterial activity and human serum albumin affinity.

In the past few years a significant effort has been devoted by Pharmacia toward the discovery of novel antibiotics. We have recently described the identification of an anthranilic acid lead 1 and the optimization resulting in the advanced lead 2. In this report, we describe the preparation of several selected analogs to probe the dependency of this template for antibacterial activity and the affinity these compounds have for human serum albumin (HSA).

Binding thermodynamics of substituted diaminopyrimidine renin inhibitors.

Renin is an aspartyl protease involved in the production of angiotensin II, a potent vasoconstrictor. Renin inhibitors can prevent blood vessel constriction and therefore could be useful for the treatment of hypertension. High-throughput screening efforts identified a small molecule renin inhibitor with a core substituted diaminopyrimidine ring.

Determining molecular binding sites on human serum albumin by displacement of oleic acid.

An NMR method was developed for determining binding sites of small molecules on human serum albumin (HSA) by competitive displacement of (13)C-labeled oleic acid. This method is based on the observation that in the crystal structure of HSA complexed with oleic acid, two principal drug-binding sites, Sudlow's sites I (warfarin) and II (ibuprofen), are also occupied by fatty acids. In two-dimensional [(1)H,(13)C]heteronuclear single quantum coherence NMR spectra, seven distinct resonances were observed for the (13)C-methyl-labeled oleic acid as a result of its binding to HSA.

Multi-selective one dimensional proton NMR experiments for rapid screening and binding affinity measurements.

High-throughput ligand-based proton NMR screening performed in the presence of a spy molecule and a control molecule is a valuable tool for identifying drug leads. A limitation of the technique is represented by the severe overlap encountered in the screening of large chemical mixtures. An approach for overcoming this overlap problem is the use of multi-selective R(1) filtered and COSY or TOCSY experiments.

Fluorine-NMR experiments for high-throughput screening: theoretical aspects, practical considerations, and range of applicability.

Competition ligand-based NMR screening experiments have recently been introduced to overcome most of the problems associated with traditional ligand-based NMR screening. Molecules with marginal solubility and high affinity for a given target can be easily identified in a high-throughput manner by screening chemical mixtures against the target in the presence of a weak- to medium-affinity ligand of known binding constant. While the original competition-based approaches utilized (1)H detection, significant advantages are obtained using (19)F detection.

Spontaneous aggregation and cytotoxicity of the beta-amyloid Abeta1-40: a kinetic model.

The time dependency of the spontaneous aggregation of the fibrillogenic beta-amyloid peptide, Abeta1-40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-Abeta antibody 6E10, raised against residues 1-17, at concentrations of 200-300 nM delayed significantly the aggregation of 50 microM amyloid peptide.

Identification of a mutant amyloid peptide that predominantly forms neurotoxic protofibrillar aggregates.

The amyloid peptide (Abeta), derived from the proteolytic cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases, undergoes multistage assemblies to fibrillar depositions in the Alzheimer's brains. Abeta protofibrils were previously identified as an intermediate preceding insoluble fibrils. While characterizing a synthetic Abeta variant named EV40 that has mutations in the first two amino acids (D1E/A2V), we discerned unusual aggregation profiles of this variant.

Physical methods to determine the binding mode of putative ligands for hepatitis C virus NS3 helicase.

Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K(d)'s) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay.

X-ray crystal structure of Staphylococcus aureus FemA.

The latter stages of peptidoglycan biosynthesis in Staphylococci involve the synthesis of a pentaglycine bridge on the epsilon amino group of the pentapeptide lysine side chain. Genetic and biochemical evidence suggest that sequential addition of these glycines is catalyzed by three homologous enzymes, FemX (FmhB), FemA, and FemB. The first protein structure from this family, Staphylococcus aureus FemA, has been solved at 2.

Determination of ligand-MurB interactions by isothermal denaturation: application as a secondary assay to complement high throughput screening.

We used a temperature-jump isothermal denaturation procedure with various methods of detection to evaluate the quality of putative inhibitors of MurB discovered by high-throughput screening. Three optical methods of detection-ultraviolet hyperchromicity of absorbance, fluorescence of bound dyes, and circular dichroism-as well as differential scanning calorimetry were used to dissect the effects of two chemical compounds and a natural substrate on the enzyme. The kinetics of the denaturation process and binding of the compounds detected by quenching of flavin fluorescence were used to quantitate the dose dependencies of the ligand effects.

Expression, purification, and characterization of peptidyl-tRNA hydrolase from Staphylococcus aureus.

Bacterial peptidyl-tRNA hydrolase (Pth) activity ensures the rapid recycling of peptidyl-tRNAs that result from premature termination of translation. Pth has been shown to be essential for growth in Escherichia coli suggesting that its homologue in Staphylococcus aureus is a potential molecular therapeutic target for the development of antibacterial agents. In this report we describe the cloning of a DNA fragment (573 bp) containing the pth gene from a S.