A SIMPLE, SENSITIVE, AND ECONOMIC ASSAY FOR CHOLINE AND ACETYLCHOLINE USING HPLC, AND ENZYME REACTOR, AND AN ELECTROCHEMICAL DETECTOR

A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G-10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine

Numerical simulation of powder compaction for two multilevel ferrous parts, including powder characterisation and experimental validation

The paper presents a summary of two case studies that were carried out by the scientific team in the Thematic Network PM Modnet. During the life of this project, the compaction of complex multilevel ferrous components was investigated. These formed a vehicle to explore methods to characterise the yield and friction properties of the powder, perform simulation,of the compression stage of the forming process, complete experimental trials, and compare experimental and simulated results. Density comparisons were made with results from Archimedes,. quantitative metallography, and computerised tomography and force levels were compared with recordings from the pressing trials. The results highlight differences between equipment and experimental techniques used in characterising powders. They also show that hardness, metallographic analysis, and computerised tomography may be used to measure density variations throughout the compact. The prediction of density variation was reasonably consistent when using different simulations, whereas punch force prediction showed good consistency. It was found that predicted and measured density distributions agree within 0.05 to 0.5 g cm(-3) and that punch force levels may be predicted within 10 to 30%. The study effectively establishes a benchmark with which to compare and improve future simulations

Optimization of Tetrahydroindazoles as Inhibitors of Human Dihydroorotate Dehydrogenase and Evaluation of Their Activity and In Vitro Metabolic Stability

Human dihydroorotate dehydrogenase (DHODH), an enzyme in the de novo pyrimidine synthesis pathway, is a target for the treatment of rheumatoid arthritis and multiple sclerosis and is re-emerging as an attractive target for cancer therapy. Here we describe the optimization of recently identified tetrahydroindazoles (HZ) as DHODH inhibitors. Several of the HZ analogues synthesized in this study are highly potent inhibitors of DHODH in an enzymatic assay, while also inhibiting cancer cell growth and viability and activating p53-dependent transcription factor activity in a reporter cell assay. Furthermore, we demonstrate the specificity of the compounds toward the de novo pyrimidine synthesis pathway through supplementation with an excess of uridine. We also show that induction of the DNA damage marker gamma-H2AX after DHODH inhibition is preventable by cotreatment with the pancaspase inhibitor Z-VAD-FMK. Additional solubility and in vitro m etabolic stability profiling revealed compound 51 as a favorable candidate for preclinical efficacy studies