Biomedical subjects

G Brophy

Publications and source records attributed to G Brophy.

8 recordsLinked to original sources

PubMed2000

Detection of p56(lck) kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384- and 1536-well format.

p56(lck) is a lymphocyte-specific tyrosine kinase that plays an important role in both T-cell maturation and activation. We have developed a homogeneous assay in which p56(lck) catalyzes the transfer of the gamma-phosphate group from [gamma-(33)P]ATP to a biotinylated peptide substrate. The labeled peptide is then captured on a streptavidin-coated scintillation proximity assay (SPA) bead or imaging proximity bead. The SPA is counted in a microplate scintillation counter and the imaging proximity assay is counted in a charge-coupled device-based imaging system called LEADseekertrade mark, recently launched as a homogeneous imaging system by Amersham Pharmacia Biotech. We show, via time-dependence assays and inhibitor studies, that this assay can be performed in 1536-well microplate format using imaging proximity as the method of detection. The results compare favorably with the same assay performed in 384-well microplate format using both SPA and imaging proximity as the detection methods. From this study, we conclude that a kinase assay can be performed in 384- and 1536-well format using imaging as the detection method, with significant time savings over standard scintillation counting. In addition, we show cost saving advantages of 1536- over 384-well format in terms of reagent usage, higher throughput, and waste disposal.

Amino Acid Sequence↗

PubMed1997

Noninvasive, real-time method for the examination of thymidine uptake events--application of the method to V-79 cell synchrony studies.

[14C]Thymidine uptake into V-79 hamster lung fibroblasts has been successfully demonstrated using a noninvasive, real-time method utilizing Cytostar-T scintillating microplates. These plates are standard format, tissue culture-treated, 96-well microplates with an integral scintillating base. The microplates permit the culture and observation of adherent cell monolayers. Biological activities of the cells can be studied by the provision of specific radiolabeled compounds. The biological activities of the adherent monolayer bring the specific radiolabel into proximity with the scintillating base and a scintillation signal is thereby generated. [14C]Thymidine incorporation on the microplates can be used to examine cell proliferation and cell cycle events. Using a combined mitotic shake-off/aphidicolin treatment to achieve synchronization, the thymidine incorporation activities of V-79 cells have been examined on Cytostar-T plates and correlated to traditional methods of determining incorporation. The method was further used to examine the effects of colcemid and olomoucine, both chemical inhibitors of cell proliferation, on synchronous populations of cells. The homogeneous detection format and the microplate nature of the method suggest a role for scintillating microplates in cell biology research and drug discovery.

Animals↗

PubMed1992

Assessment of Pediococcus acidilactici as a Potential Silage Inoculant.

Eighteen Pediococcus strains were screened for their potential as silage inoculants. Pediococcus acidilactici G24 was found to be the most suitable, exhibiting a short lag phase on both glucose and fructose, a rapid rate of acid production, a high sugar-to-lactate conversion efficiency, no detectable breakdown of proteins or lactic acid, and the ability to grow within a broad range of pH and temperature. When tested in laboratory silos using grass with a water-soluble carbohydrate content of 24 g/kg of aqueous extract, P. acidilactici G24 stimulated the natural Lactobacillus plantarum population and accelerated the rates of lactic acid production and pH decrease. After 6 days of fermentation, the inoculated silage exhibited a 12% decrease in ammonia nitrogen and an 11% increase in crude protein levels compared with uninoculated controls. The use of an L. plantarum inoculant at a rate of 10 bacteria per g of grass in conjunction with P. acidilactici G24 produced no additional beneficial effect. Inoculation of grass with a water-soluble carbohydrate level of 8 g/kg of aqueous extract with P. acidilactici G24 led to no acceleration in the rate of L. plantarum growth or pH decrease. However, after 7 days of fermentation the inoculated silage had a 14% lower ammonia nitrogen protein content than did uninoculated controls. The results suggest that P. acidilactici G24 may be useful as a silage inoculant for crops with a sufficiently high water-soluble carbohydrate level.

Journal Article↗

PubMed1990