Biomedical subjects

A K Yeung

Publications and source records attributed to A K Yeung.

6 recordsLinked to original sources

PubMed1999

Biopharmaceutics of transmucosal peptide and protein drug administration: role of transport mechanisms with a focus on the involvement of PepT1.

Non-invasive delivery of peptide and protein drugs will soon become a reality. This is due partly to a better understanding of the endogenous transport mechanisms, including paracellular transport, endocytosis, and carrier-mediated transport of mucosal routes of peptide and protein drug administration. This paper focuses on work related to the elucidation of structure-function, intracellular trafficking, and regulation of the intestinal dipeptide transporter, PepT1.

Biological Transport↗

PubMed1998

Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton- coupled dipeptide transporter (hPepT1).

hPepT1 is a proton-coupled peptide transporter that mediates the absorption of di- and tripeptides. Here we show that tyrosine 167 (Y167) in transmembrane domain 5 (TMD5) of this 12-transmembrane spanning protein contributes to its transport function. We identified this particular amino acid by a computer model of the arrangement of the TMDs of hPepT1 and investigated its role by site-directed mutagenesis and dipeptide uptake studies. [3H]Gly-sar uptake in cells transiently transfected with Y167A-hPepT1 was abolished completely, even though the level of Y167A-hPepT1 expression by Western blot analysis and cell surface expression by immunofluorescence microscopy was similar to those of the wild type. Therefore, mutation affected transport function, but apparently not the steady-state protein level or trafficking of the transporter to the plasma membrane. Moreover, mutation of Y167 into phenylalanine, serine, or histidine all abolished gly-sar uptake in transfected HEK 293 cells. Taken together, these findings suggest that Y167 plays an essential role in hPepT1 function, perhaps due to the unique chemistry of its phenolic side chain.

Blotting, Western↗

PubMed1998

Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1.

The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.

Amino Acid Sequence↗

PubMed1981

Semiautomated assay for indapamide in biological fluids.

A sensitive fluorescence procedure for the determination of indapamide in plasma and whole blood was developed. The procedure requires preextraction of the biological sample followed by continuous-flow analysis. The assay is sensitive to indapamide levels of 25 ng/ml in plasma and blood. A linear response from 25 to 200 ng/ml is observed. The procedure also can be used to measure urinary levels of indapamide. The assay has been used to obtain whole blood and plasma level curves from subjects receiving 2.5 mg of indapamide.

Autoanalysis↗

PubMed1980

Automated analysis of indapamide in drug-rodent food mixtures.

Indapamide, an antihypertensive agent, is an aryl sulfonamide that inhibits carbonic anhydrase in vitro but not in vivo. An assay was developed for indapamide in drug-rodent food mixtures that utilizes this inhibitory effect. Indapamide was extracted from the mixtures with methanol, and an aqueous dilution of the extract was sampled by a continuous-flow system. In the system, the drug was extracted with butanol and then back-extracted into alkali. This solution was neutralized, buffered, and mixed with bovine erythrocyte carbonic anhydrase. The substrate, p-nitrophenyl acetate, was added, the solution was incubated, and the amount of p-nitrophenol formed was measured. The assay was sensitive to 20 micrograms of indapamide/g of food, and 20 unknown samples could be analyzed per hour on the continuous-flow system. It is possible that the method could be extended to the analysis of other toxicological test substances that inhibit carbonic anhydrase in vitro.

Animal Feed↗

PubMed1978

Fluorometric assay for urinary indapamide.

A sensitive fluorescence method for the determination of indapamide was developed. Reaction of indapamide with sodium hydroxide at 100 degrees yielded a fluorescent product, and addition of formaldehyde to the fluorescent product increased its fluorescence intensity by a factor of three. The assay is sensitive to levels of indapamide of 0.025 microgram/ml in an aqueous solution, and a linear response between 0.025 and 2.0 microgram/ml was observed. The procedure was adapted to the analysis of intact indapamide in urine. Concentrations of indapamide of 0.05 microgram/ml can be detected in dogs given 20 mg of the drug.

Animals↗