化学生物学实验技术

Bell et al. (1) showed that a dermal equivalent can be obtained by mixing living fibroblasts with a solution of collagen that will gel and further contract into a firm lattice by extrusion of water. Epidermal keratmocytes can be seeded on top of these lattices, giving rise to a simplified skin (2, 3).

DNA transfection has become an important technique in molecular biology. The fusion of putative gene-regulatory sequences to reporter genes and their introduction into a eukaryotic cell can be used for a detailed analysis of cisacting DNA regulatory sequences (1). DNA regulatory elem ...

Functional analysis of the 5′-flanking region of a gene has become a routme procedure for identifying the cis-acting DNA elements involved in the control of the transcriptional activity of the gene (1). This experimental approach requires the transfection of reporter plasmids, harbori ...

The gel-retardation, band-shift, or electromobility shift assay is used to investigate protein—DNA interactions. Nuclear protein from the desired source is incubated with a radiolabeled DNA fragment. The products of the reaction are then analyzed by electrophoresls through a non ...

A number of common functtonally significant genetic polymorphisms in genes encoding certain cytochromes P450 have been described and characterized (for review, see refs. 1 and 2). Individuals may lack certain enzyme activities or have higher or lower than normal activities owing to the p ...

Inducible proteins are attractive candidates for biomarkers-i.e. biological responses that reflect exposure to, or effects of, environmental pollutants in an organism (1). Blomarkers are increasingly regarded as powerful and informative tools in ecotoxicology and environ ...

The ability to manipulate the murine genome via the use of gene targeting in pluripotent embryonic stem cells is arguably one of the most important scientific achievements of the last decade (1–4). This technology holds the potential to produce murine strains and cell-culture systems that ha ...

The ability to purify cytochrome P450 from liver microsomes was the result of the collective efforts of a number of laboratories, borrowing from each others’ efforts and observatrons. Early efforts to solubrlize the hemoprotein from microsomes through the use of proteases were not succe ...

Identtfication of the cytochrome P450 profiles in extrahepatic tissues such as the brain, breast, and prostate is a prerequisite for evaluation of the role of these enzymes in in situ activation of procarcinogens and tissue-specific metabolism of hormones and pharmaceuticals. The main ...

Cytochrome P450 (P450) enzymes catalyze the blotransformatlon of a broad range of structurally diverse foreign chemicals and endogenous substances. Many P450 enzymes have been isolated and identified in liver and other tlssues, including kidney, lung, intestines, and brain. Alth ...

The cytochrome P450 enzyme CYP2B6 plays an important role in the metabolism of structurally diverse drugs, including the anticancer drug cyclophosphamide, and may be an important determinant of clinical responses to these agents. A spectrofluorometric method is described for the d ...

Paclitaxel (Taxol) is a naturally occurring member of the taxane family of antitumor drugs, which act by stabilizing microtubules. Paclitaxel is inactivated in human liver by a cytochrome P450 (P450)-catalyzed 6α-hydroxylation reaction. A reverse-phase, high-performance liqu ...

A reverse-phase, high-performance liquid chromatography method is described for the quantification of 4′-hydroxymephenytoin formed enzymatically from 14C-labeled (S)-mephenytoin following incubation with cDNA-expressed CYP2C19 or human liver microsomes. Analy ...

A reverse-phase, high-performance liquid chromatography method is described for the quantification of 1′-hydroxybufuralol formed enzymatically by the incubation of bufuralol with cDNA-expressed CYP2D6 or human liver microsomes. Analytical separation is achieved usi ...

Testosterone and other steroid hormones have been studied as prototypic examples of endogenous substrates for hepatic cytochrome P450 (P450) enzymes. CYP3A enzymes from various species, including human, metabolize testosterone by a 6β-hydroxylation reaction, which is unique ...

A normal-phase, isocratic high-performance liquid chromatography assay is described for cholesterol 7α-hydroxylation catalyzed by CYP7A1, which corresponds to the first and rate-limiting step in the conversion of cholesterol into bile acids. This method is based on the convers ...

7-Ethoxycoumarin is metabolized by many cytochrome P450 enzymes active in foreign compound metabolism and has been used as a prototypic substrate to monitor P450 (P450) activity in both hepatic and extrahepatic tissues. A spectrofluorometric method is described for determinati ...

Aspects of cytochrome P450 (CYP) nomenclature are addressed. The rules for naming a P450 are outlined, though individuals should not name their own genes. The nomenclature is presented as a unifying principle to enhance communication across disciplines. Because of the historical natu ...

Benzydamine is a nonsteroidal anti-inflammatory drug that undergoes flavin-containing monooxygenase (FMO)-dependent metabolism to a stable N-oxide. This metabolite can be quantified with high specificity and sensitivity by using a simple reverse-phase high-performan ...

Amino acid analysis is an important technique that has many applications in biochemical, pharmaceutical, and biomedical fields. Profiling of plasma amino acids is also of great interest in clinical practice. Many diseases are known to be associated with disorders in amino acid metaboli ...