Non-specific Effects Of Inhibitors Research Articles

Determination of ROS generation rates in plant mitochondria in vitro using fluorescent indicators: Non-specific effects of inhibitors of terminal oxidases

A possibility of quantitative analysis of the ROS production by mitochondria isolated from etiolated winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings was studied by following hydrogen peroxide accumulation in medium of organelle incubation using a new highly sensitive test-system including the fluorogenic indicator Amplex Red and horseradish peroxidase. The rates of the process were about 100 pmol H2O2/(mg protein min) under state 3 conditions; they were almost doubled after mitochondria transition into state 4, and further increased by more than 50% after the addition of alamethicin, the poreforming antibiotic inducing the organelle inner membrane permeabilization for low-molecular-weight compounds, H2O2 molecules among them. Experimental evidence is presented indicating that the classical inhibitors of the terminal oxidases of the plant mitochondrial respiratory chain, such as cyanide, salicylhydroxamic acid, and propyl gallate, can inactivate the ROS-detecting test-systems in vitro because of interactions with their functional components. These results are discussed in comparison with literature data obtained by similar test-systems and indicate that there are considerable limitations for obtaining reliable experimental evidence of the antioxidant role of alternative cyanide-resistant oxidase in plant mitochondria.

Simple PCR-based method for synthesis of molecular calibrators and controls.

The use of molecular methods such as PCR to quantify nucleic acids has become widely established for both research and diagnostic applications. Increasingly, such methodologies recognize the need for inclusion of internal standards and controls. These are used to define the efficiencies of nucleic acid extraction, reverse transcription, amplification, and detection and to quantify the effects of nonspecific inhibitors. Examples include the “Qs” controls included in Amplicor PCR assays (Roche Molecular Systems Inc.) (1) and the “Qa, Qb, Qc” controls used in Nuclisens assays (Organon Technica) (2). Control constructs (RNA or DNA) are derived from the nucleic acid sequence that the test method is designed to assay and span the primer binding sequences used in the assay. Thus, when added to the test sample, the control is coamplified in the reaction with a single primer pair. To distinguish the control from the test sequence during the detection phase of the assay, a short sequence between the primer binding sites is replaced by a defined sequence to allow differential probing with labeled oligonucleotides. Nucleic acid controls are usually 1 kb or longer, as shorter lengths may not by efficiently purified by the commonly used silica capture or precipitation methods. Previously, the construction of such controls required the identification or engineering of unique restriction sites between the primer binding sites to allow excision of the wild-type sequence and replacement with the probe sequence (1)(2). The process also required ligation of the probe sequence into the target sequence backbone and cloning into a plasmid containing a transcription promoter in the case of RNA controls. The method described here simplifies the procedure to a two-stage PCR amplification and allows the probe sequence to be placed anywhere between the primer binding sites without the need to identify or engineer restriction sites. Similar approaches …

Some applications of statistical mechanics in enzymology 3. Effects of nonspecific inhibitors and activators on enzyme reaction

Effects of nonspecific inhibitors and activators on enzyme reaction are discussed. It has been shown that using the method proposed in this paper, the number of sites for ligand binding which are essential to enzyme activity can be obtained from the experimental data even without knowing the total number of sites for binding of ligand and the detailed information about ligand binding to the enzyme.