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N Chiang

Publications and source records attributed to N Chiang.

25 records · Page 2Linked to original sources
PubMed1996

Site-directed mutagenesis of cysteinyl and serine residues of human thromboxane A2 receptor in insect cells.

A thromboxane A2 receptor cDNA was isolated from a human placenta library by polymerase chain reaction (PCR) and was expressed in insect (Sf21) cells using baculovirus system. The recombinant receptor exhibited [3H]-SQ29548 and [125I]-BOP binding activities with Kd values of 1.01 +/- 0.09 nM and 1.63 +/- 0.23 nM, respectively. The receptor binding activity was inhibited by dithiothreitol in a time- and concentration-dependent manner, indicating the involvement of disulfide linkage in ligand binding. The role of the four conserved cysteinyl residues in ligand binding was further examined by site-directed mutagenesis. Each of the four cysteinyl residues was respectively mutated to a serine residue. C102S, C105S, and C183S mutants exhibited no ligand binding activity although successful expression was achieved as revealed by immunoblot analysis, whereas C257S mutant retained most of the binding activity. Homology analysis of all prostanoid receptors indicates that Cys-105 (first extracellular loop) and Cys-183 (second extracellular loop) are conserved and are presumed to form a disulfide bond for receptor stability as suggested by the inhibition of ligand binding by dithiothreitol reduction. Loss of binding activity by C102S mutant revealed that the sulfhydryl group of Cys-102 must play an essential role in ligand binding. Molecular modeling proposed that the Ser-201 is involved in interacting with TXA2 by forming hydrogen bonding. Point mutations of both Ser-201 and a conserved Ser-255 did not affect the ligand binding specificity and affinity for [3H]-SQ29548, but have significantly altered Kd values for [125I]-BOP. These results indicate that various cysteinyl and serine residues of thromboxane A2 receptor may play different roles in ligand binding.

Amino Acid Sequence↗
PubMed1996

Analysis of heregulin-induced ErbB2 phosphorylation with a high-throughput Kinase receptor activation enzyme-linked immunosorbant assay.

A rapid, sensitive, and high-throughput assay has been developed to quantify ligand-induced receptor tyrosine kinase activation in terms of receptor phosphorylation. The assay, termed a kinase receptor activation enzyme-linked immunosorbant assay (KIRA-ELISA), consists of two separate microtiter plates, one for cell culture, ligand stimulation, and cell lysis/receptor solubilization and the other plate for receptor capture and phosphotyrosine ELISA. The assay was developed for analysis of heregulin-induced ErbB2 activation and utilizes the stimulation of intact receptor on the adherent breast carcinoma cell line, MCF-7. Membrane proteins are solubilized via Triton X-100 lysis and the receptor is captured in ELISA wells coated with ErbB2-specific antibodies with no cross-reaction to ErbB3 or ErbB4. The degree of receptor phosphorylation is then quantified by antiphosphotyrosine ELISA. A reproducible standard curve is generated with a EC(50) of approximately 360 pM for heregulin beta 1(177-244) (HRG beta 1(177-244). When identical samples of HRG beta 1(177-244) are analyzed by both the KIRA-ELISA and quantitative antiphosphotyrosine Western blot analysis, the results correlate very closely with one another. The assay described in this report is able to specifically quantify tyrosine phosphorylation of ErbB2 that results from the interaction of HRG with ErbB3 and/or ErbB4.

Animals↗
PubMed1985

The in vitro development of extended-release solid oral dosage forms.

A system is described for developing extended-release products, based upon predicting or simulating the entire plasma level-time curve to be expected from the administration of a controlled-release oral dosage form. Termed "biorelevant dissolution," it employs the product's in vitro dissolution behavior and the drug's pharmacokinetic parameters in conjunction with a classical pharmacokinetic model. The basic system is described along with some pertinent examples.

Administration, Oral↗