Biomedical subjects

Stephen K-F Wong

Publications and source records attributed to Stephen K-F Wong.

4 recordsLinked to original sources

PubMed2005

Fully automated solid weighing workstation.

A fully automated, solid-to-solid weighing workstation (patent pending) is described in this article. The core of this automated process is the use of an electrostatically charged pipette tip to attract solid particles on its outside surface. The particles were then dislodged into a 1.2-mL destination vial in a microbalance by spinning the pipette tip. Textures of solid that could be weighed included powder, crystalline, liquid, and semi-solid substances. The workstation can pick up submilligram quantities of sample (=0.3mg) from source vials containing as little as 1mg. The destination vials containing the samples were stored in a 96-well rack to enable subsequent automated liquid handling. Using bovine serum albumin as test solid, the coefficient of variation of the protein concentration for 48 samples is less than 6%. The workstation was used successfully to weigh out 48 different synthetic compounds. Time required for automated weighing was similar to manual weighing. The use of this workstation reduced 90% hands-on time and thus exposure to potentially toxic compounds. In addition, it minimized sample waste and reduced artifacts due to the poor solubility of compound in solvents. Moreover, it enabled compounds synthesized in milligram quantities to be weighed out and tested in biological assays.

Animals↗

PubMed2004

A 384-well cell-based phospho-ERK assay for dopamine D2 and D3 receptors.

Phosphorylation of extracellular signal-regulated kinase (ERK) is linked to activation of many cell surface receptors and kinases. However, phosphorylated ERK has not been used as a biochemical marker to monitor pharmacology of these biomolecules, largely because commonly used methods to detect the phosphoprotein are not quantitative and do not have sufficient throughput. In this article, a high-throughput, 384-well, cell-based functional assay to quantify dopamine agonist-induced ERK phosphorylation in D2- and D3-overexpressed cell lines is described. The assay uses infrared-labeled secondary antibodies to detect phospho-ERK, and the signals in the wells of the microtiter plate are quantified by a LI-COR infrared scanner. V(max), EC(50), and functional K(i) values of various D2 and D3 agonists and antagonists determined in this assay are similar to those in the literature. The assay is nonradioactive, is quantitative, and has a good signal-to-noise ratio. In addition, the signal is stable. This assay can be used to monitor the activities of many G protein-coupled receptors and other signaling biomolecules that are linked to phosphorylation of ERK. The methodology can potentially be used to detect the change in level of any cellular protein in which highly selective antibodies are available.

Biomarkers↗

PubMed2003

Pramipexole inhibits MPTP toxicity in mice by dopamine D3 receptor dependent and independent mechanisms.

The role of dopamine D3 receptors was investigated in mediating the neuroprotective effect of the dopamine D2/D3 receptor agonist (S)-2-amino-4,5,6,7-tetrahydro-6-propylamine-benzothiazole (pramipexole) in vivo. Pramipexole retained the ability to inhibit 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine depletion in mice in which the dopamine D3 receptor had been deleted. However, the neuroprotective efficacy was reduced in the dopamine D3 receptor-deleted mice compared to that in littermates expressing the wildtype receptor. Furthermore, the dopamine D3 receptor selective antagonist 2-(3-[4-(2-tert-butyl-6-trifluoromethyl-4-pyrimidinyl)-1-piperazinyl]propylthio)-4-pyrimidinol (A-437203) partially inhibited the neuroprotective effect of pramipexole in dopamine D3 receptor expressing mice but not in receptor-deleted mice. These results indicate that pramipexole protects dopamine neurons from MPTP-induced toxicity by mechanisms that are both dependent and independent of an interaction with dopamine D3 receptors.

Animals↗

PubMedDate not supplied

G protein selectivity is regulated by multiple intracellular regions of GPCRs.

GTP-binding protein coupled receptors (GPCRs) bind to a vast diversity of extracellular ligands to regulate a wide variety of physiological responses. Upon binding of extracellular ligands, these seven-transmembrane-spanning receptor molecules couple to one or several subtypes of G protein which reside at the intracellular side of the plasma membrane to trigger intracellular signaling events. Amid the large structural diversity at the intracellular regions of GPCRs, there are only 18 different subtypes of G protein belonging to four subfamilies. The question of how GPCRs select and activate a single or multiple G protein subtype(s) has been the topic of intense investigations. This review will attempt to summarize the available data on the structural determinants in GPCRs that regulate the selectivity of G protein activation. The available data suggest that G protein can be activated by structurally diverse cationic alpha-helical structures with no obvious homology in primary sequence. The selectivity of receptor-G protein coupling is maintained by a combination of two functional domains at the intracellular region. One is the 'activation domain' which can activate multiple G protein subtypes, while the other is the 'selectivity domain' which restricts the coupling to the desired signaling pathway(s). A slight change in the conformation at these two functional domains can affect the fidelity of G protein selectivity. This hypothesis can account for the vast structural diversity of GPCRs which link a fascinating variety of extracellular inputs, yet couple to a limited number of intracellular signaling pathways.

Animals↗