Level of expression

The extracellular calcium Ca -sensing receptor plays a central role in maintaining a nearly constant level of extracellular calcium by sensing small changes in Ca and directly and/or indirectly altering the translocation of calcium ions into or out of the extracellular fluid so as to normalize CaQ+. Changes in the level of expression and/or function of the CaR reset the level of CaQ+. Recently developed activators (calcimimetics)... 

Figure 10. Tissue specific expression of p-subunit proteins in floral tissues. Stage 3 (fully opened) flowers were collected, dissected and cell wall proteins (5 pgm) from the indicated organs isolated and analyzed for p-subunit antigen. Note the high level of expression in stigma/style and anthers/pollen and restriction of the larger antigen to stigma/style tissues. PGl lane, 1 gg of purified fruit PGl protein.

B. brevis is not so well studied. It also shows low extracellular protease activity, and a protease-deficient strain is available [43]. High [44] and low [45] copy number plasmids are constructed for different levels of expression. Modification of signal sequences can enhance yields of eukaryotic proteins [46]. The yields can be up to 3 gL-1 media. 

TES-32 is the most abundant single protein product secreted by the parasite. It is also heavily labelled by surface iodination of live larvae (Maizels et al., 1984, 1987), and is known by monoclonal antibody reactivity to be expressed in the cuticular matrix of the larval parasite (Page et al, 1992a). TES-32 was cloned by matching peptide sequence derived from gel-purified protein to an expressed sequence tag (EST) dataset of randomly selected clones from a larval cDNA library (Loukas et al., 1999). Because of the high level of expression of TES-32 mRNA, clones encoding this protein were repeatedly sequenced and deposited in the dataset (Tetteh et al., 1999). Full sequence determination showed a major domain with similarity to mammalian C-type (calcium-dependent) lectins (C-TLs), together with shorter N-terminal tracts rich in cysteine and threonine residues. Native TES-32 was then shown to bind to immobilized monosaccharides in a calcium-dependent manner (Loukas et al., 1999). 

Over the past years it has become apparent that the cell type is an important determinant of the extent of oxidative stress that may occur. Both the latent activities of cytoprotective enzymes in specific cell types, as well as the ability of the cell to respond rapidly to an oxidative insult by the upregulation of such enzymes, will be important predeterminants of the fate of the cell. Table 10.1 shows the concentrations of both antioxidants and cytoprotective enzymes in a variety of tissues. While the liver is well provided with antioxidant protection, the brain has very low levels, so the ability to respond rapidly to an oxidative insult by upregulation of gene transcription and translation will be an important determinant of survival or death. Cells such as hepatocytes have high levels of expression and... 

From the above, it is clear that the gut wall represents more than just a physical barrier to oral drug absorption. In addition to the requirement to permeate the membrane of the enterocyte, the drug must avoid metabolism by the enzymes present in the gut wall cell as well as counter-absorptive efflux by transport proteins in the gut wall cell membrane. Metabolic enzymes expressed by the enterocyte include the cytochrome P450, glucuronyltransferases, sulfotransferases and esterases. The levels of expression of these enzymes in the small intestine can approach that of the liver. The most well-studied efflux transporter expressed by the enterocyte is P-gp. 

The extent to which the cell line supports appropriate expression of the cDNA. The level of expression achieved is determined by interactions of the vector/ expressed protein with the cell. These interactions include the strength of the promoter (weaker promoters can be compensated for by using a vector which is present at high copy number), the adequacy of the selective agent (not all agents are toxic to all cells), the stability of the expressed protein (some proteins may be rapidly degraded in some cells), and whether the expressed protein exerts any deleterious effects on the viability of host cells (some efflux transporters could deplete the cell of essential components). Finally, transporters must be expressed in a polarized manner in the host cell (i.e., preferentially on either the basolateral or apical side of the cell). 

The catalytic activity of CYP enzymes requires functional coupling with its redox partners, cytochrome P450 NADPH oxidoreductase (OR) and cytochrome bs. Measurable levels of these two proteins are natively expressed in most cell lines. Therefore, introduction of only the CYP cDNA is generally needed for detectable catalytic activity. However, the levels of expression of the redox partner proteins may not support maximal CYP catalytic activity, and therefore enhancement of OR levels may be desirable. This approach has been used successfully with an adenovirus expression system in LLC-PKi cells [12],... 

---