Effect of puromycin and cycloheximide on glycosphingolipid biosynthesis by PHA stimulated human lymphocytes

The effect of puromycin and cycloheximide on the biosynthesis of neutral glycosphingolipids and gangliosides by PHA stimulated lymphocytes is studied. Under conditions of permanent inhibition of protein synthesis, and depending on the time of lymphocyte stimulation, inhibition of the biosynthesis of neutral glycosphingolipids was either restricted only to certain species or was very low for all glycosphingolipid species. The degree of inhibition of the various ganglioside species was affected by the time of incubation. After transient inhibition of protein synthesis and at times when protein biosynthesis had recovered, neutral glycosphingolipid and ganglioside biosynthesis inhibition was very prominent and did not recover. The possibility is discussed that glycosphingolipid biosynthesis does not depend directly on concurrent nascent peptide formation and it is proposed that inhibition of glycosphingolipid biosynthesis is related primarily to impairment of the endoplasmic reticulum and to inhibition of galactose transferases, secondary to the binding of the inhibitors.     

Puromycin reaction of the A-site bound peptidyl-tRNA.

AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.     

Inhibition of the puromycin reaction with benzamidine and related compounds

Incubation of mouse cells with N-methyl-N′-nitro-N-nitrosoguanidine causes a strong inhibition of DNA replication the extent of which varies with the cell line used. Analysis of the products synthesized in drug-treated cells indicates a particularly severe effect on the joining of replicons while other steps in DNA synthesis like initiation and chain elongation are much less affected. The data indicate that replicon fusion may be extremely sensitive to changes in the topology of DNA induced by the introduction of rare single-strand breaks during repair of N-methylated purines produced by incubation of cells with small amounts of the methylating agent     

Effects of puromycin and cycloheximide on properties of cells from Xenopus laevis early embryos

The effects have been studied of puromycin and cycloheximide on the reaggregation of ectoderm cells dissociated from Xenopus laevis blastulae. Puromycin or cycloheximide can inhibit reaggregation, suggesting that cell reassociation is dependent upon protein synthesis. If the cells are allowed a 3 h 'recovery' period in culture medium following dissociation, before being exposed to either puromycin or cycloheximide, higher concentrations of the inhibitors are required to prevent cell aggregation, suggesting that significant synthesis of the proteins required for reaggregation occurs in the 3 h immediately following dissociation. Lower concentrations of puromycin permit cell reaggregation but reduce the normal formation of cilia. The effects have also been observed of puromycin on the scanning electron microscopical appearance of Xenopus blastula ectoderm cells cultured singly in vitro. Puromycin reduces the normal formation of pseudopodia, suggesting that puromycin might inhibit reaggregation partly by inhibiting cell movement. Puromycin also produces some elongated cells, possibly by inhibition of cytokinesis.     

The stimulation of the phosphorylation of ribosomal protein S6 by cycloheximide and puromycin

The administration of cycloheximide or puromycin to rats in amounts that all but completely inhibited hepatic protein synthesis caused an increase in the amount of radioactive phosphate incorporated into the liver ribosomal protein S6; there was also an increase in the prominence of the derivatives of S6 which contain increasing numbers of phosphorylated serine residues.     

Protection of Chinese hamster ovary cells from hyperthermic killing by cycloheximide or puromycin

Cycloheximide (CHM) and puromycin (PUR) were used at various concentrations up to maxima of 10 micrograms/ml and 100 micrograms/ml, respectively, which inhibited protein synthesis by 95% without any cytotoxicity. The drugs were added to the cells for a maximum period of 7 h, with various combinations for treatment before, during, and after heating. Maximum protection, i.e., a 10,000-fold increase in survival from 5 X 10(-6) to 5 X 10(-2) after 4 h at 43 degrees C, required both 1-2 h of treatment before heating and 1-2 h of treatment during heating. For treatments at 45.5 degrees C, the protection was less, i.e., a 100-fold increase in survival from 10(-5) to 10(-3). Little or no protection was observed if after treatment, the drug was removed before heating, or if the drug was added at the start of heating and left on for 5 min to 3 h after heating. For both drugs, the amount of protection increased as inhibition of protein synthesis increased. However, the amount of protection from the drugs was the same only at about 95% inhibition; at 60-85% inhibition, CHM afforded more protection than PUR. Therefore, the modes of action of the drugs might be common at high drug concentrations, but different when intermediate concentrations are used.     

The effect of puromycin and cycloheximide on vacuole formation and exocytosis in Tetrahymena pyriformis GL-9

It has been shown that both puromycin and cycloheximide, at concentrations of 434 and 100 g/ml respectively, produce a marked inhibition of vacuole formation and exocytosis in Tetrahymena pyriformis GL-9. These effects were analysed in a quantitative manner. At the same time as these inhibitions occurred the incorporation of 1-C¹⁴ leucine into trichloroacetic acid precipitable material was inhibited by 90% and 100% respectively over a 40 min period. This inhibition of protein synthesis by cycloheximide occurred almost immediately, whereas the inhibition of vacuole formation and egestion was delayed. The results suggested that the latter processes were dependent upon a continuing supply of proteinaceous material, of which there was only a small store within the cell. Cycloheximide inhibited exocytosis completely under the conditions employed (with 100% inhibition of protein synthesis) whereas puromycin (with a 90% inhibition of protein synthesis) only inhibited it by about 50%. This suggested that the amount of newly synthesized protein required for the exocytic egestion process was very small in relation to the total cell requirement for protein synthesis. The entry of both inhibitors into the cell was by means other than vacuole formation. Puromycin appeared to have some effect on vacuole formation which was unconnected with protein synthesis. Microscopic observations of living cells indicated that oral apparatus function and endocytic vacuole formation were probably both affected by the inhibitors. Chloramphenicol, at 200 g/ml, had little effect on vacuole formation by starved cells with an exposure of an hour. The uptake of 1-C¹⁴ leucine from the growth medium was found to be a selective process, giving a concentration of about 2000 times into the cells over a 1 hr period. The results are discussed.     

Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins

After sodium arsenite (100 microM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10(-6) to 10(-1) after 4 hr at 43 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43 degrees C with little resistance at 45.5 degrees C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45 degrees C caused by heating at 43 degrees C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.     

Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells

Gilden, R. V. (Wistar Institute, Philadelphia, Pa.), and R. I. Carp. Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells. J. Bacteriol. 91:1295-1297. 1966.-Synthesis of the simian virus 40 (SV40) T antigen in primary African green monkey kidney cells was abolished when cycloheximide was added up to 10 hr postinfection. In contrast, puromycin, another inhibitor of protein synthesis, did not suppress antigen production. The basis of this differential effect was the inability of puromycin to inhibit protein synthesis in the cells used. This was shown by the failure of the drug to depress the incorporation of labeled amino acid into protein and also failure to inhibit poliovirus synthesis. The puromycin preparation used was very effective in inhibiting poliovirus synthesis in HeLa cells. Thus, appearance of the SV40 T antigen is dependent on protein synthesis in infected cells.     

Phase shifting the circadian rhythm of neuronal activity in the isolated Aplysia eye with puromycin and cycloheximide. Electrophysiological and biochemical studies

The effects of pulse application of puromycin (PURO) or cycloheximide (CHX) were tested on the circadian rhythm (CR) of spontaneous compound action potential (CAP) activity in the isolated Aplysia eye. CAP activity was recorded from the optic nerve in constant darkness at 15degreesC. PURO pulses (6, 12 h; 12--134 mug/ml) and CHX pulses (12 h, 500--2,000 mug/ml) caused dose-dependent phase delays in the CR when administered during projected night. PURO pulses (6 h, 125 mug/ml) caused phase advances when given during projected day and caused phase delays when given during projected night. In biochemical experiments PURO (12 h, 20 mug/ml) and CHX (12 h, 500 mug/ml) inhibited leucine incorporation into the eye by about 50%. PURO (12 h; 50, 125 mug/ml) also changed the molecular weight distribution of proteins synthesized by the eye during the pulse. The effect of PURO (12 h, 125 mug/ml) on the level of incorporation was almost completely reversible within the next 12 h but the phase-shifted eye showed an latered spectrum of proteins for up to 28 h after the pulse. In electrophysiological experiments spontaneous CAP activity and responses to light were measured before, during, and after drug treatments. In all, eight parameters in three periods were analyzed quantitatively. Of these 24 indices, only 3 showed significant changes. PURO increased spontaneous CAP frequency by 67% 0-7 h after the drug pulse and increased the CAP amplitude of the tonic light response by 23% greater than 7 h after the pulse. CHX increased the intraburst spontaneous CAP frequency by 33% during the pulse and CAP frequency of the tonic light response by 32% 0- 7 h after the pulse. The above data indicate that phase-shifting doses of PURO and CHX inhibit protein synthesis in the eye without causing adverse electrophysiological effects, and suggest that protein synthesis is involved in the production of the CR of the isolated Aplysia eye.