Cycloheximide, protein synthesis inhibition

Use of the general protein synthesis inhibitor, cycloheximide, led to the hypothesis that de novo VSV protein synthesis is required for inhibiting cellular protein synthesis because, when cycloheximide was removed from infected cells, viral protein synthesis preceded suppression of cellular protein synthesis inhibition (Wertz and Young-ner, 1972). In our experience, experiments such as these are difficult to interpret because cycloheximide has many side effects, such as inhibition of cellular RNA synthesis (Week and Wagner, unpublished data). 

Those herbicides that block mitotic entry decrease or prevent the formation of mitotic figures in meristems. Amino acid, protein, RNA, DNA, and ATP synthesis and/or utilization can all attest cell growth (163,166). Although not registered as herbicides, cycloheximide [66-81-9] inhibits mitotic entry by inhibiting protein synthesis (167) hydroxyurea/727-(97-/7 inhibits DNA synthesis (168) and actinomycin D [50-76-0] nh2oix.s RNA synthesis (167). 

Other antibiotics inhibit protein synthesis on all ribosomes (puromycin) or only on those of eukaryotic cells (cycloheximide). Puromycin (Figure 38—11) is a structural analog of tyrosinyl-tRNA. Puromycin is incorporated via the A site on the ribosome into the carboxyl terminal position of a peptide but causes the premature release of the polypeptide. Puromycin, as a tyrosinyl-tRNA analog, effectively inhibits protein synthesis in both prokaryotes and eukaryotes. Cycloheximide inhibits peptidyltransferase in the 60S ribosomal subunit in eukaryotes, presumably by binding to an rRNA component. 

Feedback inhibition of amino acid transporters by amino acids synthesized by the cells might be responsible for the well known fact that blocking protein synthesis by cycloheximide in Saccharomyces cerevisiae inhibits the uptake of most amino acids [56]. Indeed, under these conditions, endogenous amino acids continue to accumulate. This situation, which precludes studying amino acid transport in yeast in the presence of inhibitors of protein synthesis, is very different from that observed in bacteria, where amino acid uptake is commonly measured in the presence of chloramphenicol in order to isolate the uptake process from further metabolism of accumulated substances. In yeast, when nitrogen starvation rather than cycloheximide is used to block protein synthesis, this leads to very high uptake activity. This fact supports the feedback inhibition interpretation of the observed cycloheximide effect. 

The use of antibiotics for the control of plant virus diseases( ) is of interest. Several antibiotics have been tested for inhibition of replication of viral nucleic acid and/or protein synthesis within the host cell. Chloramphenicol, cycloheximide, actinomycin D and others are the most used antibiotics and the disease caused by tobacco mosaic... 

Alice et al studied the turnover kinetics of Listeria OTonocytogenex-secreted p60 protein (a murein hydrolase) by host cell cytosolic proteasomes. J774 cells, seeded in flasks and incubated overnight in culture medium, were infected with log-phase cultures of E. monocytogenes for 30 min, washed, and incubated in culture medium for 3 h, with gentamicin (50 tg/ml) added after the first 30 min to inhibit extracellular bacterial growth. Cells then were washed and placed in methionine-free medium with spectinomycin, gentamicin, the eukaryotic protein synthesis inhibitors [cycloheximide (50 tg/mL) and anisomycin (30 tg/ml),] and 25 dVI calpain inhibitor I. After 30 min, [ S]methionine was added, and the cells were pulse-labeled for periods of 20 to 60 min. Cells... 

Kinase inhibitors such as H7, which inhibits serine threonine kinases, resulted in sustained JAK2/STAT activation, suggesting that besides protein dephosphorylation, protein phosphorylation is also required. Furthermore, both inhibition of protein synthesis with cycloheximide and inhibition of... 

Obviously, there is some kind of cross-talk between PKC and cAMP in the modulation of intercellular coupling, since cAMP can inhibit the uncoupling effect of phorbol esters if cells are exposed to both agents from the start of the experiment [Kanno et al., 1984], but this protective effect can be abolished by the protein synthesis inhibitor cycloheximide [Enomoto et al., 1984] in Balb/ cells. 

The induction of the CYPs has been demonstrated in many different species including humans, and in various different tissues as well as the liver. Induction usually results from repeated or chronic exposure, although the extent of exposure is variable. The result of induction is an increase in the amount of an enzyme induction requires de novo protein synthesis, and therefore an increase in the apparent metabolic activity of a tissue in vitro or animal in vivo. Consequently, inhibitors of protein synthesis, such as cycloheximide, inhibit induction. It is a reversible cellular response to exposure to a substance. Thus, it can be shown in isolated cells, such as hamster fetal cells in culture, that exposure to benzo[a]anthracene induces aryl hydrocarbon hydroxylase (AHH) activity (CYP1A1). 

The antibiotic cycloheximide, isolated from Strepomyces griseus, is used as an antifungal agent and for inhibition of protein synthesis. The biosynthesis of cycloheximide via the polyacetate pathway is easily demonstrated by culturing S. griseus in the presence of [l-l3C]- and [2-13C]-acetate and, after the isolation of the antibiotic, by comparing the natural abundance 13CNMR spectrum with those of labeled cycloheximide [1006]. Table 5.40 demonstrates that six carbons (C-4, C-6, C-8, C-10, C-12 and C-14) are incorporated from the carbonyl carbon and six from the methyl carbon (C-3, C-5, C-7, C-9, C-ll and C-13) of acetate. 

Little work has been done on the effect of anabolic inhibitors on cellular heat dissipation probably because there is empirical evidence that anabolic processes do not contribute significantly to it (see p. 312). Loike et al. (1981) found that 0.07 mmol dm-3 cycloheximide, an antibiotic inhibitor of protein synthesis, had no effect in 30 minutes on the heat production of murine macrophages. On the other hand, Krakauer and Krakauer (1976) showed that long-term exposure of lymphocytes from immunized horses to 1 mg dm-3 cycloheximide considerably reduced heat production. This was likely to be due to a secondary effect of the antibiotic arresting catabolism by inhibiting the turnover of short half-life enzymes. 

To stop the translation reaction and further stabilize the ribosomal complexes, cycloheximide can be added in the eukaryotic system (Gersuk et al., 1997). For the same purpose chloramphenicol, an antibiotic that inhibits bacterial protein synthesis by binding to the 23S ribosomal RNA in the peptidyl transferase center, can be used in the E. coli system (Mattheakis et al., 1994). However, chloramphenicol was found to have no influence on the efficiency of E. coli ribosome display (Hanes and Pluckthun, 1997). 

Administration of cycloheximide (60 mg/kg) 1 hr prior to tilorone administration inhibited the interferon response43,44. The inhibition by cycloheximide suggested that protein synthesis was involved in the appearance of interferon in the serum. For a more complete discussion of interferon induction, see the review on synthetic interferon inducers written by DeClercq45. ... 

Induction of de novo synthesis of a-amylase by GA in isolated aleurone layers is evident after a lag period of approximately 8 hr following administration of the hormone. In keeping with hormone responses generally, GA must be present continuously if the de novo synthesis of hydrolases is to be sustained. Synthesis of new RNA is essential to the GA-induction of de novo synthesis of hydrolases. Actinomycin D, an inhibitor of RNA synthesis, inhibits the synthesis and release of a-amylase if the inhibitor is presented during the first 7 to 8 hr after treatment. Inhibitors of protein synthesis, such as cycloheximide, also inhibit GA-induction of hydrolases. And, interestingly, abscisic acid, a growth-inhibiting hormone, inhibits GA-induced a-amylase synthesis as well. 

Although a few subunits of mitochondrial membrane proteins are coded by mitochondrial DNA and synthesized in the mitochondrial matrix, most membrane proteins including the adenine nucleotide carrier are coded by nuclear genes and synthesized on cytoplasmic ribosomes [80,81], Chloramphenicol, an inhibitor of mitochondrial protein synthesis, does not inhibit incorporation of radioactive leucine into the carrier in growing Neurospora crassa, but cycloheximide, an inhibitor of cytoplasmic protein synthesis, does inhibit leucine incorporation [78]. Also, a yeast nuclear respiratory mutant has been shown to cause a defect in adenine nucleotide transport [81], and the nuclear gene responsible for coding the carrier in yeast is currently being cloned for further studies [82]. 

Indeed, inhibitors of protein synthesis have been used in the in vivo rabbit [60] and in the rat LangendorfT models [61]. Cycloheximide or actinomycin D did not reverse the PC effect [60]. This is probably due to non-complete inhibition of protein synthesis. The latter study [61] showed that the PC effect was abolished only with cycloheximide, but not with actinomycin D, indicating that PC protection is regulated at the post-transcriptional level. Recent findings [62] have reconfirmed the translational control of protein synthesis in ischemic (but not pharmacological) PC. These are in accord with our findings concerning de novo synthesis of ferritin. 

The amount of each mRNA is regulated with respect to the time after infection. RNAs 1 and 2 are both formed shortly after the primary transcript is made, although more of mRNA-2 is made. Later in the viral life cycle, mRNA-1 is not made and mRNA-2 is abundant. If cycloheximide, an inhibitor of protein synthesis, is added to the infected cells before the shift in splicing pattern takes place, the shift does not occur. This inhibition implies that a newly synthesized protein is a positive effector of the shift. Cycloheximide has a similar effect on other mRNA species derived from other primary transcripts at late times. 

The answer is c. (Murray, pp 452-467. Scriver, pp 3-45. Sack, pp 1-40. Wilson, pp 101-120.) In a general sense, the mechanism of protein synthesis in eukaryotic cells is similar to that found in prokaryotes however, there are significant differences. Cycloheximide inhibits elongation of proteins in eukaryotes, while erythromycin causes the same effect in prokaryotes. Thus, one is an antibiotic beneficial to humans, while the other is a poison. Cytoplasmic ribosomes of eukaryotes are larger, sedimenting at SOS instead of 70S. While eukaryotic cells utilize a specific tRNA for initiation, it is not formylated as in bacteria. Finally, eukaryotic mRNA always specifies only one polypeptide, as opposed to prokaryotic mRNA, which may specify the synthesis of more than one gene product per mRNA. 

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