DNA and protein synthesis in cultured lens epithelial cells. II. Activity of synthetic effectors]

Cultivated bovine lens epithelium cells are highly susceptible to inhibitors of DNA-, RNA- and protein synthesis. The strict correlation between inhibition by puromycin of protein and DNA synthesis suggests that, in the cell system investigated, protein synthesis is essential for DNA synthesis to occur. Studies with actinomycin D have shown that in cultivated lens epithelium cells, part of protein synthesis is accomplished through a relatively long-lived mRNA. In long-term cultivation experiments, no further stabilization of mRNA, which is typical of lens fibre cells, could be demonstrated. There are indications that high doses of actinomycin D produce direct inhibition of DNA synthesis. By means of cytosine arabinoside a linear relationship was established between concentration of the effector and inhibition of DNA synthesis. Protein synthesis remains virtually unaffected even after high doses. The strong inhibition of DNA synthesis with protein synthesis continuing ("unbalanced growth") could not be utilized for the synchronization of lens epithelium cells, because it was only partly reversible after changing the medium and applying deoxycytidine.     

Ribosomal RNA synthesis in newly sliced discs of potato tuber

A burst of ribosomal RNA synthesis is induced in potato tissue by slicing, and continues at a decreasing rate for about 12 hours. Ribosomal RNA synthesis in potato discs is sensitive to puromycin, in contrast to non-ribosomal RNA synthesis. Thus, the influence of puromycin on total RNA synthesis is significant only during the first 12 hours following slicing. The function of RNA made after 12 hours in a puromycin-insensitive manner is unknown. However, it is apparently unrelated to protein synthesis, since it has been shown that total inhibition of RNA synthesis by addition of actinomycin D to potato tissue after 12 hours of aging has no effect upon protein synthesis during the ensuing 12 hours.     

Protein synthesis during embryogenesis of Sabellaria alveolata L. (Polychaete annelid)

Protein synthesis before and after fertilization in Sabellaria alveolata has been investigated. Two major increases in protein synthesis were seen: one at fertilization and one after the third cleavage. These are independent of uptake rate modifications and are poorly affected by actinomycin D treatment. No strict correlation was found between cleavage and proteosynthesis inhibition by puromycin.     

The effect of inhibitors in vivo on protein synthesis and the amino acid pool in the sheep blowfly, Lucilia cuprina.

1. The rates of detoxification of cycloheximide (33 mug/g fresh wt.), puromycin (167 mug/g fresh wt.) and actinomycin D (1 mug/g fresh wt.) were assessed in vivo on the basis of acid-insoluble [14C]leucine incorporation in the sheep blowfly, Lucilla cuprina; these were compared with quantitative estimates which took account not only of incorporation data but also of leucine pool size and turnover. Quantitatively, cycloheximide and puromycin were still at least 50% effective in inhibiting protein synthesis after 6.5 and 24.5h of exposure respectively, whereas values based only on incorporation data suggested that cycloheximide was 83% effective and puromycin completely ineffective after the respective periods. Quantitative estimates also showed that actinomycin D effectiveness increased with increasing exposure over 24.5h, in contrast with values based only on incorporation data, which suggested that it was completely ineffective after 24h.2. All inhibitors affected the dynamic state of the amino acid pool; there was a marked decrease in the rate of leucine-pool turnover as well as an increase in the half-life of leucine in the pool. 3. Inhibition of protein synthesis resulted in changes in leucine-pool size; the most pronounced increase occurred with cycloheximide and puromycin and the most pronounced decreases with actinomycin D. 4. Evidence is presented which suggests that proteolysis is functionally linked to protein synthesis, which determines its rate indirectly.     

Is protein synthesis necessary for prostaglandin production by guinea-pig endometrium?

The outputs of prostaglandin (PG) F-2 alpha, PGE-2 and 6-keto-PGF-1 alpha from Day-7 and Day-15 guinea-pig endometrium in culture were reduced by the inclusion of actinomycin D, cycloheximide and puromycin in the culture medium, with the output of PGF-2 alpha from Day-15 endometrium being particularly affected during the first 6 h of culture. The intrauterine administration of actinomycin D on Day 10 decreased the outputs of PGF-2 alpha and PGE-2, but not of 6-keto-PGF-1 alpha, from Day-15 endometrium in culture without affecting PG output from Day-15 myometrium in culture. Actinomycin D, cycloheximide and puromycin did not reduce PG output when superfused over the Day-7 and Day-15 guinea-pig uterus in vitro for 20 min, indicating that these compounds do not have a rapid inhibitory effect on endometrial PG synthesis. In fact, they tended to stimulate PG output during this 20-min period, with cycloheximide having a pronounced effect on PGE-2 output. The synthesis of secreted proteins, but not of cellular proteins, was greater by Day-15 than by Day-7 endometrium in culture. Actinomycin D, cycloheximide and puromycin inhibited the synthesis of secreted and cellular proteins by Day-7 and Day-15 endometrium in culture. Protein synthesis and PG synthesis in the endometrium were both inhibited to a greater extent by cycloheximide and puromycin than by actinomycin D. The intrauterine administration of actinomycin D on Day 10 reduced the syntheses of secreted and cellular proteins by Day-15 endometrium in culture. These findings indicate that the endometrial synthesis of PGs, particularly of PGF-2 alpha towards the end of the oestrous cycle, is dependent upon endometrial protein synthesis.     

Inhibition of two-dimensional growth and suppression of ribonucleic acid and protein synthesis in the gametophytes of the fern, Asplenium nidus, by chloramphenicol, puromycin and actinomycin D

Chloramphenicol and puromycin at appropriate concentrations inhibited the induction of two-dimensional growth in the gametophytes of the fern Asplenium nidus without drastically inhibiting germination and continued filamentous growth. Similar responses to actinomycin D were reported earlier. Radioautographic techniques were employed to study the pattern of ribonucleic acid and protein synthesis in gametophytes which were treated with chloramphenicol, puromycin and actinomycin D. Uptake of H³-uridine into ribonucleic acid was strongly inhibited by all three antibiotics. Chloramphenicol and puromycin were not as effective as actinomycin D in inhibiting H³-leucine incorporation. The results are discussed in relation to the quality of light and antibiotics on two-dimensional growth in the gametophytes.     

The light induction of maize phosphoenolpyruvate carboxylase kinase translatable mRNA requires transcription but not translation

We have previously demonstrated that the level of translatable mRNA for phosphoenolpyruvate carboxylase kinase in maize leaves is increased in response to light ( Hartwell et al. 1996 ; Plant Journal 10 , 1071–1078). To identify the steps required for this increase, we have examined the effects of protein and RNA synthesis inhibitors. The RNA synthesis inhibitors actinomycin D and cordycepin (500 μ M ) strongly inhibited the light-induced increases in kinase translatable mRNA and the apparent phosphorylation state of phosphoenolpyruvate carboxylase, as judged by its sensitivity to inhibition by L -malate. The protein synthesis inhibitors cycloheximide and puromycin blocked the light-induced increase in the apparent phosphorylation state of phosphoenolpyruvate carboxylase but not the increase in kinase translatable mRNA. Indeed, the amount of phosphoenolpyruvate carboxylase kinase translatable mRNA after 3 h of illumination of leaves treated with either 1 m M puromycin or 100 μ M cycloheximide was double that in illuminated control leaves. Each inhibitor reduced the light-induction of two control genes, malic enzyme and pyruvate, phosphate dikinase. Thus the light induction of phosphoenolpyruvate carboxylase kinase translatable mRNA requires RNA synthesis, but not protein synthesis.     

Phytochrome destruction: an apparent requirement for protein synthesis in the induction of the destruction mechanism

Examination of the phytochrome destruction reaction as a function of age in etiolated oat (Avena sativa L. cv. Garry) seedlings demonstrates that following illumination of 3-day-old shoots there is a lag, not observed in 4- or 5-day-old oats, prior to the onset of destruction. This light-mediated induction of the phytochrome destruction mechanism in 3-day-old shoots is inhibited by chloramphenicol, actinomycin D, and puromycin suggesting that protein synthesis is required. In 4-day-old shoots, actinomycin D and puromycin do not alter the kinetics of destruction while chloramphenicol partially inhibits the process. Thus, the inhibitors have a specific effect on the induction of the destruction mechanism but not its subsequent operation.     

Synthesis of Bacterial Flagella I. Requirement for Protein and Ribonucleic Acid Synthesis During Flagellar Regeneration in Bacillus subtilis

A relatively simple immunochemical procedure for estimating flagellar protein was developed. This procedure involved measuring the binding of purified, radioactively labeled, antiflagellar antibodies to bacteria. The assay was used to determine the requirements for ribonucleic acid (RNA) and protein synthesis during flagellar regeneration in Bacillus subtilis. Immediate inhibition of flagella development was observed when chloramphenical or puromycin was added to cells. This inhibition indicated the absence of a large pool of flagella precursors that could be assembled in the absence of protein synthesis. When the cells were starved for uracil or treated with actinomycin D to inhibit RNA synthesis, the ability of the cells to regenerate flagella decayed with a half-life of 5.5 min. When B. subtilis auxotrophs were starved for tryptophan, they continued to synthesize flagella, although this process was also inhibited by actinomycin D. On the basis of these results, we concluded that (i) the system involved in flagellar regeneration does not have unusual metabolic stability, (ii) regeneration requires both concomitant protein and RNA syntheses, and (iii) B. subtilis continues to synthesize messenger RNA during tryptophan starvation.     

Massive synthesis of ribonucleic Acid and cellulase in the pea epicotyl in response to indoleacetic Acid, with and without concurrent cell division

Measurements were made over a 4-day period of the effect of added indoleacetic acid (IAA), puromycin, actinomycin D and 5-fluorodeoxyuridine (FUdR) on growth and the levels of total DNA, RNA, protein and cellulase in segments of tissue at the apex of decapitated etiolated epicotyls of Pisum sativum, L. var. Alaska.

The hormone induced swelling of parenchyma cells and cell division. By 3 days after IAA application, the amounts of DNA and protein were approximately double, RNA triple and cellulase 12 to 16 times the levels in controls. All of these changes were prevented by both puromycin and actinomycin D. FUdR prevented DNA synthesis and cell division but not swelling or synthesis of RNA, protein and cellulase.

It is concluded that IAA-induced RNA synthesis is required for cellulase synthesis and lateral cell expansion, whether or not cell division takes place.

     

Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Biosynthesis of Macromolecules and Wall During Successive Steps

Protoplasts of Bacillus subtilis plated on SDG medium formed L colonies in quantative yield and propagated in the L-form indefinitely. Protoplasts or L bodies placed in 25% gelatin medium formed bacillary colonies. Details of the reversion of these naked bodies to the walled form are reported here. Protoplasts prepared in minimal medium reverted fairly synchronously 3 to 4 hr after inoculation into gelatin, but protoplasts preincubated in casein hydrolysate (CH)-enriched minimal medium were primed to revert within 1 hr in the gelatin. Preincubation for 1.5 hr in 0.44% CH was required for good priming. Cells must be subjected to this preincubation (step 1) in the naked state; it is effective for L bodies as well as protoplasts. Priming was blocked by chloramphenicol, puromycin, and actinomycin D but was not affected by penicillin, lysozyme, or inhibition of deoxyribonucleic acid (DNA) synthesis. It is concluded that protein and ribonucleic acid (RNA) synthesis are required during step 1, that DNA synthesis is not required, and that wall mucopeptide is not made. The reversion of well-primed protoplasts in the gelatin (step 2) proceeded undisturbed in thymine-starved cells with chromosomes arrested at the terminus. It was scarcely slowed by chloramphenicol in the gelatin but was delayed about 3 hr by both puromycin and actinomycin D. Escape from inhibition occurred while the inhibitors were still actively blocking growth. Penicillin and cycloserine inhibited and lysozyme reversed reversion. Momentary melting of the gelatin delayed reversion. It is concluded that mucopeptide synthesis occurs in step 2, that concomitant RNA, DNA, or protein synthesis is not essential, but that physical immobilization of excreted cell products at the protoplast surface is necessary early in step 2. Newly reverted cells were misshapen and osmotically sensitive. Processes which confer osmotic stability after reversion (step 3) did not occur in the presence of chloramphenicol or actinomycin D.     

Microexudation phenomenon in the testing of antibiotics]

Changes in formation of the surface protein-polysaccharide layer (microexudate) on the cell surface under the action of inhibitor antibiotics, such as puromycin, actinomycin D and mitomycin C, as well as protein substances with adhesive action, such as horse serum and triprotamine in low doses were characterized quantitatively on a model of HeLa cells ellipsometrically. Under the action of puromycin, actinomycin D and mitomycin C formation of the microexudate ceased, which was in full accordance with the data on ceasation of the intracellular synthesis of protein, RNA and DNA under the action of the above antibiotics respectively. Inhibition of the microexudate formation was reversible. Still, the time of the inhibitory effect of puromycin and actinomycin D was longer than that of mitomycin C. Under the action of horse serum and triprotamine production of the microexudate by the cells was increased and accelerated. Accounting with the relative simplicity of the ellipsometric method and possibility of rapid estimation of the results, the data substantiate the expediency of using the phenomenon of microexudation as a cytopharmacological test.     

Cell-to-cell adhesion of dissociated embryonic brain cells; the effects of metabolic inhibitors and temperature

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and rotated for 120 min. The area and density of of the adhesive complexes formed were registered using the method described previously. The adhesiveness of dissociated embryonic brain cells (measured during the 120 min of rotation) was diminished in the presence of inhibitors of protein synthesis (puromycin, cycloheximide and inhibition of mRNA synthesis actinomycin D). The inhibition was, however, not distinct, because 1 microgram/ml of cycloheximide and actinomycin was without any significant effect, and the degree of inhibition evoked by 10 micrograms/ml and 25 micrograms/ml of puromycin bordered on significance. However, protein synthesis inhibitors in long-term aggregation experiments had a pronounced inhibitory effect and/or induced destruction of the aggregates. Metabolic inhibitors (KCN and NaN3) caused an inhibition at the lowest level of significance (p less than 0.05) 10(-3) mol/l KCN reduced the final adhesive product significantly. Cells rotated at room temperature and at +5 degrees C adhere to the same extent as in control experiments (37 degrees C). The adhesion was significantly inhibited at +60 degrees C and also after freezing at -80 degrees C with subsequent thawing. The adhesion of cells exposed for 30 min to between +80 degrees C and 100 degrees C was completely abolished. The process of embryonic brain cell adhesion requires a low energy supply, and is relatively independent of biosynthetic processes and of temperature changes between +5 degrees C and +50 degrees C.     

PUROMYCIN-INDUCED NECROSIS OF CRYPT CELLS OF THE SMALL INTESTINE OF MOUSE

Chemical and radioautographic analysis of the small intestine of mice injected intraperitoneally with puromycin revealed an immediate decrease of precursor incorporation into DNA and protein and a delayed decrease of precursor incorporation into RNA. In addition to this decrease of precursor incorporation, damage to the crypt cells, but not to the cells of the villus of the small intestine, was observed. Further examination of other dividing cells (spleen) and nondividing cells (liver and heart) of these mice showed again that only cells of actively dividing tissues were damaged. The metabolic inhibitors actinomycin D, cytosine arabinoside, actidione, and puromycin aminonucleoside were used in an attempt to clarify the mechanism of cell damage by puromycin. The results showed that there was no clear correlation between cell necrosis and the pattern of inhibition of synthesis of DNA, RNA, or protein.     

Metabolic requirements for the in vitro augmentation of mouse natural killer activity by interferon

The metabolic processes involved in the in vitro augmentation of mouse natural killer (NK) activity by interferon (IF) were studied. Augmentation occurred after a very brief (5–10 min), temperature-independent exposure of spleen cells to IF. This binding of, or triggering by, IF was independent of protein synthesis, since treatment with puromycin before or during contact with IF failed to block augmentation. Spleen cells, however, required new RNA and protein synthesis in the first few hours after contact with IF to develop the boosted reactivity, as shown by their susceptibility to inhibition by actinomycin D, emetine, pactamycin, and puromycin. Mitomycin C did not interfere with the boosting, suggesting that a pool of NK cells exists which rapidly responds to IF without cell proliferation. The need for new RNA and protein synthesis may be for the differentiation of pre-NK cells to functionally active cells or for the increase in lytic efficiency of already active NK cells.     

The circadian rhythm in photosynthesis in Acetabularia in the presence of actinomycin D, puromycin, and chloramphenicol

Anucleate Acetabularia crenulata shows a circadian rhythm in photosynthesis. In this study, an oxygen electrode was employed to measure this photosynthetic rhythm in the presence and absence of the inhibitors, actinomycin D, chloramphenicol, and puromycin. High concentrations of the inhibitors were used: actinomycin D, 20-40 micrograms ml-1; puromycin, 30 and 100 micrograms ml-1; and chloramphenicol, 250 micrograms ml-1. The effectiveness of these inhibitors on protein synthesis was also measured under the same conditions used for the determination of rhythmicity. In spite of large effects of all three inhibitors on the incorporation of 14C leucine, no effect on the period or the phase of the photosynthetic rhythm was observed. The higher concentration of puromycin and chloramphenicol produced toxic effects which were expressed as a reduction in the amount of photosynthesis, but rhythmicity was still apparent. After 3 or 4 days' exposure to actinomycin, Acetabularia became resistant to its effect. Recovery was also observed in the ability to incorporate leucine. The implications of these results for theories of the basic oscillator responsible for circadian rhythmicity are discussed.