Cell free systems mammalian cells

Today, there are a wide variety of laboratory protein expression systems available, ranging from cell-free systems over bacterial and yeast cultures to eukaryotic models including the Xenopus oocytes or insect and mammalian cell cultures, some of which even form polarised epithelial-like cells layers. In Table 24.1, an overview of the most important systems, as well as their particular strength and weaknesses in the expression of transmembrane transport proteins is provided. 

Hybrid poIy(U)-programmed cell-free systems containing heterologous combinations of factors and (factor-free) ribosomes have been used to assess the ability of archaeal factors to cooperate with heterologous ribosomes [127,182]. In accordance with the phylogenetic status of the archaea, archaeal EF-2 and EF-la factors T. acidophilum, M. vannielii) do not cooperate with eucaryal (mammalian) and bacterial ( . coli) ribosomes. The reciprocal condition however, in which archaeal ribosomes are tested with either eucaryal or bacterial factors, is less clear-cut in that bacterial T. thermophilus) EF-Tu appears to significantly cooperate with archaeal Thermoplasma) ribosomes [127]. With this limitation, the functional restrictions define a third class of elongation-factor specificity [127]. 

Theaflavins efficiently and specifically inhibited the proteasomal chymotrypsin-like activity of the purified mammalian 20S and 26S proteasomes in cell-free systems (figure 11.3) as well as 26S proteasomes in tumor cell extracts (table 11.1). The purified M. thermophila recombinant 20S proteasome was more sensitive to theaflavins than mammalian 20S or 26S proteasomes, possibly due to prokaryotic recombinant 20S proteasome without postmodification that forms steric hindrance that prevents theaflavins from binding to the chymotrypsin-like activity subunit of the 20S proteasome. When assaying for proteasomal chymotrypsin-like activity in human cancer cell extracts, the IC50 values of tea polyphenols were apparently increased compared... 

In a cell-free system inhibition can be shown to occur on both 70S and 80S ribosomes. However, in a more realistic in vitro setting intact prokaryotic (i.e., bacterial) cells are much more sensitive. The reason for this selectivity is that tetracyclines are actively transported into bacterial but not mammalian cells. In Gm- bacteria, at least, the more water-soluble compounds seem to cross through membrane channels (pores). The more lipid-soluble drugs (particularly MNC, Table 6-9) diffuse more readily through the lipoidal phases of the membranes. This energy-coupled process then leads to intracellular antibiotic accumulations. 

Germination of Sphaerotheca fuliginea conidia was inhibited at rather low concentrations of the compound and when germ tubes were formed they showed spherical or oval shaped alterations [36]. Regarding the mode of action, mildiomycin interferes in protein synthesis at low concentration (0.02 mM) the compound inhibited incorporation of amino acids into polypeptides in a cell-free system of E. coli, whereas synthesis of polypeptides in mammalian cell-free systems from rabbit reticulocytes proved to be less sensitive to mildiomycin [37]. 

The question remains whether CAP under certain circumstances blocks protein synthesis at the 80 S ribosome. In a cell-free system of reticulocyte microsomes (Weisberger et al., 1964 Beard et oL, 1969) an inhibition of the association of mRNA and the 40 S ribosomal subunit was reported. In the reticulocyte system used 70-100% inhibition of [ Cjleucine incorporation was reported at 0.1 pmole/ml of CAP with either endogenous template RNA or small amounts of poly(U). These results could not be confirmed by Zelkowitz et al. (1968). We also found no striking inhibition of poly (U)-directed amino acid incorporation by CAP in messenger-depleted cell-free systems from rat liver or rat embryos (Uehlin et al, 1974). From all this it seems clear that generally CAP has no striking effect on protein synthesis at the 80 S ribosome level. But inhibition of mammalian microsomal protein synthesis under certain conditions cannot be ruled out. 

In cell-free systems the inhibition of the transfer of aminoacyl-transfer RNAs to polypeptide (at the ribosome level) is probably the primary effect The most interesting effect of cycloheximide is that protein synthesis by isolated mitochondria of eukaryotic cells, like bacterial ribosomes, but unlike mammalian and yeast cytoplasmic ribosomes, is not inhibited over a wide range of concentrations. Despite this selective action, cycloheximide is extremely harmful to the biogenesis of mitochondria in vivo, due to a large contribution of the microsomal protein synthesizing system in the formation of mitochondrial proteins. 

The primary translation product of proteins destined to be anchored by GPI contains both an A-terminal secretory signal sequence and a C-terminal signal sequence that specifies GPI addition. The latter is cleaved and replaced by the GPI precursor in the ER (Figure 3A). This transfer reaction is mediated by a transamidase and involves a nucleophilic attack by the EtN amino group of the GPI precursor on a carbonyl of a specific amino acid residue near the C-terminus. This residue, residing at the co site , will become the new C-terminus of the GPI anchored protein (Figure 3B). In a cell free system that reconstitutes anchor attachment, an alternative nucleophile such as hydrazine or hydroxylamine can substitute for a GPI in this reaction [89]. In a T. brucei cell free system, GPI addition to VSG does not require ATP or other cofactors [90], In a mammalian system, the transfer of the GPI precursor to a protein seems to be enhanced by an ATP-dependent chaperone [91,92],... 

This prevents further ineorporation of aminoaeyl-tRNA by bloeking the binding of EF-Tu GTP. Like the tetraeyelines, fusidie aeid owes its seleetive antimierobial aetion to active uptake by bacteria and exclusion fiom manunalian cells. The equivalent elongation factor in mammalian cells, EF-2 is susceptible to fusidie acid in cell-free systems. 

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