A Spectrophotometric Assay of Gamma-glutamylcysteine Synthetase and Glutathione Synthetase in Crude Extracts from Tissues and Cultured Mammalian Cells

Chemico-biological Interactions. Apr, 2002  |  Pubmed ID: 12044560

An assay of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.

Gamma-Glutamyl Transpeptidase and Glutathione Biosynthesis in Non-tumorigenic and Tumorigenic Rat Liver Oval Cell Lines

Carcinogenesis. Apr, 2002  |  Pubmed ID: 12075625

Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.

The Role of Gamma-glutamyl Transpeptidase in the Biosynthesis of Glutathione

BioFactors (Oxford, England). 2003  |  Pubmed ID: 12897436

Detection of Fluorescent-labeled Probes at Subpicomolar Concentrations by Magnetic Modulation

Optics Express. Nov, 2008  |  Pubmed ID: 19582017

A sensitive and rapid method for detecting fluorescent dyes at low concentrations in homogenous solution is experimentally demonstrated. Fluorescent-labeled DNA probes are detected by attaching magnetic beads and applying alternating magnetic field gradient. This condenses the fluorescent probes into a small detection volume and eliminates the scattering noise from solution by synchronous detection. For DNA probes concentration of 1 x 10(-13) M the detection signal was 3.3 times higher than the noise, thereby implying detection sensitivity of 3 x 10(-14) M.

Rapid Homogenous Detection of the Ibaraki Virus NS3 CDNA at Picomolar Concentrations by Magnetic Modulation

Biosensors & Bioelectronics. Dec, 2009  |  Pubmed ID: 19775882

Magnetic modulation biosensing (MMB) system is experimentally demonstrated for rapid and homogeneous detection of the Ibaraki virus NS3 cDNA. A novel fluorescent resonance energy transfer (FRET)-based probe discriminates the target DNA from the control. When detection is made, the FRET-based probe is cleaved using Taq-polymerase activity and fluorescent light is produced. The biotinylated probes are attached to streptavidin-coupled superparamagnetic beads and are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The beads are condensed into the detection area and their movement in and out the orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. 1.9pM of the Ibaraki virus NS3 cDNA was detected in homogeneous solution within 18min without separation or washing steps.

Magnetic Modulation Biosensing for Rapid and Homogeneous Detection of Biological Targets at Low Concentrations

Current Pharmaceutical Biotechnology. Jan, 2010  |  Pubmed ID: 20214612

This paper reviews the development of a magnetic modulation biosensing (MMB) system for rapid, simple and sensitive detection of biological targets in homogeneous solution at low concentrations. It relies on condensation and modulation of the fluorescent-labeled probes attached to magnetic beads using an alternating magnetic field gradient. Condensation of the beads from the entire volume increases the signal while modulation separates the signal from the background noise of the non-magnetized solution. We first discuss the motivation and challenges in specific DNA sequences detection as well as current approaches to overcome some of these challenges. We then present the MMB system, DNA detection schemes and magnetic beads manipulation in solution. Rapid detection at sub-picomolar concentrations of fluorescent-labeled probes as well as of coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) without any washing or separation step is also reviewed. Finally, we show preliminary results of protein detection using a 'sandwich'-based assay.

A Prospective Follow-up of Two 21/7 Cycles Followed by Two Extended Regimen 84/7 Cycles with Contraceptive Pills Containing Ethinyl Estradiol and Drospirenone

The Israel Medical Association Journal : IMAJ. Jul, 2010  |  Pubmed ID: 20862819

Continuous use of combined oral contraceptives is currently attracting growing interest as a means of improving menstrual related symptoms and reducing the number of bleeding days.

[Women's Health: What Are the Screening Tests That Every Woman Should Undergo in Order to Maintain Her Health?]

Harefuah. Jun, 2011  |  Pubmed ID: 21800491

The literature regarding the nature and the frequency of screening tests that every woman should undergo in order to maintain her health is scant and non-systematic. The purpose of the present review is to systematically advise the community physician about the essential screening tests that are required to maintain woman's health. This review does not refer to screening test that should be performed during pregnancy due to the unique nature of such screening tests.