生物化学技术

Bulk protein degradation in the cell is catalyzed by the ubiquitin-proteasome system (UPS). At the heart of the UPS is the proteasome, a large multisubunit tightly-regulated protease. The UPS performs key functions in protein quality control by monitoring and eliminating potentially t ...

Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is involved in regulation of protein function, nuclear t ...

Inhibitors of the proteasome have long been used in studies of protein turnover, but in a notable example of successful translational research they have made the leap from the laboratory into the clinical arena. The proteasome inhibitor bortezomib (VELCADE�, formerly known as PS-341), has r ...

The identification of monogenic variants of Parkinson’s disease (PD) has provided novel insights into its unknown pathogenesis. As the first protein linked to autosomal-recessive forms of PD, Parkin became a welcome tool to explain biochemical and neuropathological observatio ...

Chapters 10-20 describe assay methods for determining enzyme activities that are frequently employed as markers for specific cytochrome P450 (P450) enzymes in human tissues. However, under certain circumstances, e.g., when the objective is to verify that individual cDNA-express ...

Although much has been learned from the study of relatively abundant cytochromes P450 (e.g., P450cam from Pseudomonas putida cytosol, P4501A2 and P4502B4 from rabbit liver microsomes, and P45Oscc from bovine adrenal cortex mitochondria), the development of cytochrome P450 hetero ...

The baculovirus expression system is a highly effective system for the largescale productron of recombinant proteins in insect cells. The major attraction of baculovn-us as an expression vector system is the virus-encoded polyhedrin and p 10 genes. These genes produce large amounts of po ...

Recent extensive research on cytochrome P450 (P450) has clarified that P450 constitutes a superfamily of hemoproteins, which are believed to have evolved from a common ancestor. The versatility of P450 is mainly owing to the existence of a large number of P450 species, each of which shows a broad but ...

In the past few years, much research effort has been directed toward the expression of human cytochrome P450 cDNAs in mammalian cell Imes. The use of such cell lines allows the investigation of the metabohsm of xenobiotics by mdivtdual cytochromes P450 (1, 2). Stable expression of cytochrome P450 ...

Antibodies against cytochrome P450 enzymes can be produced by immunizing animals with synthetic peptides that mimic small regions of the apoprotein. As the amino-acid sequences of a large number of cytochrome P450 enzymes are known (1), this approach to raising antibodies can be applied to m ...

Monoclonal antibodies (MAbs) are produced by the “hybridoma” technology developed by Kohler and Milstem in 1975 (1), which is a major and defining achievement of modern biology (2). The hybridoma methodology producmg MAbs was derived from the clonal selection theory of Burnet and colleagu ...

According to the current model for the subcellular compartmentalization of proteins, any protein entering the secretory pathway will be transported by bulk flow to the cell surface unless a signal mediating its organelle-specific retention is present. Endoplasmic reticulum (ER) m ...

With genome projects spewing forth DNA sequence at tens of Megabases per year, the problem of genetic nomenclature becomes daunting. In the field of cytochrome P450 (P450), there are more than 750 sequences and they are accumulating rapidly. At the current rate, there will be more than 1000 P450 sequ ...

Many attempts to describe the regulation of mdivldual cytochrome P450 (CYP) gene products in animal tissues have been frustrated by the lack of sensitivity and specificity of the assays used. Most enzymatic assays are hampered by the overlapping substrate specificity of these enzymes, a ...

Collagen-gel cultures are relatively new in vitro models for long-term culture of functional hepatocytes. In these models, the cells are cultured between two layers of the extracellular matrix protein, hydrated collagen type I.

To survive for more than a few hours, hepatocytes must attach to a support When cultured under conventional conditions they form a monolayer that usually survrves for about 1 week. However, the hepatocytes undergo phenotypic changes, including dedifferentiation and the selective loss of ...

Liver parenchyma shows a remarkable heterogeneity of the hepatocytes along the porto-central axis, particularly with respect to the expression of cytochromes P450 and other enzymes involved in the biotransformation and conjugation of xenobiotics (1 and references therein). Th ...

Cultivation of cells in Petri dishes or culture flasks is usually performed by changing the medium after certain periods of time, e.g., 24 h (stationary cultivation). Clearly, the concentration of nutrients and other components as well as of added hormones will decrease over this period becau ...

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 ...