A new kinetic assay method for quinone-reducing enzymes

A new kinetic method is described for the assay of quinone-reducing enzymes in various biological materials. It is based on polarographic determination of oxygen uptake in spontaneous oxidation of the diphenol formed as a result of 4-anilino-5-methoxybenzoquinone-1,2 (AMOBQ) enzymic reduction. The stoichiometry of the reducing equivalent transfer in the reaction sequence from NAD(P)H to oxygen has been analyzed. Data are presented on quinone-reducing activity distributions in different tissues.     

A new fluorometric assay method for quinolinic acid

A new fluorometric assay method for quinolinic acid is introduced in this study. Quinolinic acid-hydrazine complex, a stable fluorescent compound, is formed after heating quinolinic acid with hydrazine at 215–220°C for 2 min. Fluorescence excitation and emission maxima of the complex are at 285 and 380 nm, respectively. This assay method is rapid and rather sensitive. It takes about 30 min to ascertain the amount of quinolinic acid as low as 50 ng. Specificity of this method is high among biological compounds. An ultrasensitive assay method for uinolinic acid (as low as 20 pg) with diphenylhydrazine instead of hydrazine is also found. After separating the quinolinic acid-diphenylhydrazine complex from residual diphenylhydrazine, this ultrasensitive assay method may be practically applicable.     

A high-performance liquid chromatography method for the simultaneous assay of diaminopimelate epimerase and decarboxylase

A sensitive and comparatively simple method for the assay of diaminopimelate (DAP) decarboxylase, which simultaneously monitors DAP epimerase activity, in the reverse of the biosynthetic direction, is described. The substrate, meso-DAP and products LL-DAP and L-lysine are derivatized with o-phthaldialdehyde and resolved by reversed-phase high-performance liquid chromatography. Separation is achieved on a Spherisorb C18 column using a gradient elution system. This technique offers a high degree of sensitivity as the detection method described can measure picomole quantities of substrate and products.     

A small-scale,inexpensive method for detecting formaldehyde or methanol in biochemical reactions containing interfering substances

A simple, inexpensive microdistillation device is described for capturing methanol or formaldehyde as end products of biochemical reactions or in environmental samples. We demonstrate that the microdistillation protocol, coupled with the use of alcohol oxidase and the formaldehyde-sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole), serves as a quick and inexpensive alternative to chromatographic and mass spectrometer analyses for determining if formaldehyde or methanol is a product of reactions that contain substances that interfere with the Purpald reaction. These techniques were used to affirm formaldehyde as the end product of the dicamba monooxygenase-catalyzed O-demethylation of the herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid).     

A new Multi-PCR-SSCP assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis

Prompt detection of drug resistance in Mycobacterium tuberculosis is essential for effective control of tuberculosis (TB). We developed a Multi-PCR-SSCP method that detects more than 80% commonly observed isoniazid (INH) and rifampin (RIF) resistance M. tuberculosis in a single assay. The usefulness of the newly developed method was evaluated with 116 clinical isolates of M. tuberculosis. Distinct SSCP patterns were observed for different mutations and the correlation between Multi-PCR-SSCP results and DNA sequencing data was strong. Using the culture-based phenotypic drug susceptibility testing as a reference, the sensitivity of the newly developed Multi-PCR-SSCP assay was determined to be 80% and 81.8% for INH and RIF, respectively. The specificity of the assay was 100% and 92%, for INH and RIF, respectively. Multi-PCR-SSCP provides a rapid and potentially more cost-effective method of detecting multidrug-resistant TB.     

A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood

Many bird species host several lineages of apicomplexan blood parasites (Protista spp., Haemosporida spp.), some of which are shared across different host species. To understand such complex systems, it is essential to consider the fact that different lineages, species, and families of parasites can occur in the same population, as well as in the same individual bird, and that these parasites may compete or interact with each other. In this study, we present a new polymerase chain reaction (PCR) protocol that, for the first time, enables simultaneous typing of species from the 3 most common avian blood parasite genera (Haemoproteus, Plasmodium, and Leucocytozoon). By combining the high detection rate of a nested PCR with another PCR step to separate species of Plasmodium and Haemoproteus from Leucocytozoon, this procedure provides an easy, rapid, and accurate method to separate and investigate these parasites within a blood sample. We have applied this method to bird species with known infections of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. To obtain a higher number of parasite lineages and to test the repeatability of the method, we also applied it to blood samples from bluethroats (Luscinia svecica), for which we had no prior knowledge regarding the blood parasite infections. Although only a small number of different bird species were investigated (6 passerine species), we found 22 different parasite species lineages (4 Haemoproteus, 8 Plasmodium, and 10 Leucocytozoon).