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 Effects of Inhibitors of Macromolecular Biosynthesis upon the Persistent Rhythm of Luminescence in Gonyaulax

Certain inhibitors of nucleic acid and protein synthesis, namely actinomycin D, mitomycin C, and puromycin, have been found to block the expression of a persistent daily rhythm of bioluminescence. The action does not inhibit luminescence per se but rather the rhythmicity. Exposure of the cells to these inhibitors for only a few hours, which might be expected to thereby delay the rhythm by a few hours, does not in fact have this effect. Chloramphenicol and amethopterin do not inhibit the rhythm. It is proposed that the functioning of the clock-like rhythmic mechanism depends upon the cell's normal ability to synthesize RNA.     

DIFFERENT LETHAL EFFECTS OF MITOMYCIN C AND ACTINOMYCIN D DURING THE DIVISION CYCLE OF HELA CELLS

The lethal actions of mitomycin C and actinomycin D were followed during the division cycle of HeLa cells. The cells were most susceptible to a 2 hr pulse of mitomycin C during the G₁ phase, whereas their sensitivity to actinomycin D was most pronounced in the S phase. Posttreatment of the cells with acetoxycycloheximide, a potent inhibitor of protein synthesis, increased the survival (colony-forming ability) of cells treated with mitomycin C but had very little effect on the survival of cells treated with actinomycin D. The significance of these findings is discussed.     

Control of normal differentiation of myeloid leukemic cells. VI. Inhibition of cell multiplication and the formation of macrophages.

D+ but not D- myeloid leukemic cells can be induced by the appropriate conditioned medium or by serum from endotoxin treated mice, to undergo cell migration in agar, cell attachment to the surface of a Petri dish and differentiation to mature macrophages and granulocytes. Inhibition of cell multiplication by cytosine arabinoside, hydroxyurea, mitomycin C, thymidine, 5-bromodeoxyuridine, 5-iododeoxyuridine, 5-fluorodeoxyuridine or actinomycin D, but not by vinblastine or cycloheximide, induced cell migration, cell attachment to the Petri dish and the formation of macrophages in D+ cells. There was no induction of cell migration or formation of macrophages and a much lower induction of cell attachment in D- cells. The induction of these changes in D+ cells required protein synthesis and the inhibitors showed the same toxicity for D+ and D- cells. The results indicate, that the inhibitors induced specific surface membrane changes in D+ but not in D- cells.     

Expression of herpes simplex virus type-common surface antigens in clonal cells of an HSV type 2-transformed line: effect of adriamycin

The expression of herpes simplex virus (HSV) type-common surface antigens (CSA) in a representative cell clone (155-4-03) of hamster cell line 155-4 transformed by HSV type 2 was enhanced by treatment with inhibitors of RNA synthesis [adriamycin (ADM) and daunomycin] but not with inhibitors of DNA synthesis (2-iododeoxyuridine, bleomycin, mitomycin C and cytosine arabinoside), although all these drugs decreased the number of viable cells to a similar extent. ADM-enhanced CSA expression in the clone was inhibited by puromycin and 2-deoxy-d-glucose, suggesting that the enhanced expression required both protein synthesis and glycosylation. This enhanced expression was sensitive to protease inhibitors (antipain and p-nitrophenyl-p'-guanidinobenzoate) and procaine, which is known to inhibit trypsin action and the organization of cell membrane-associated cytoskeletal elements (microfilaments and microtubules). Furthermore, low concentrations of ADM (0.1 microgram/ml) and actinomycin D (0.5 microgram/ml) enhanced CSA expression additively, but the most effective concentrations of ADM (0.25 microgram/ml) and actinomycin D (2 microgram/ml) did not. These findings indicated that the two drugs enhance CSA expression in the clone by a common mechanism.     

Several Cell Responses to Insulin of Cultured Cells Derived from Micromeres, Isolated from Sea Urchin Embryos at the 16 Cell Stage

In micromere-derived cells of sea urchin embryos, treatment with insulin started for up to 24 h during culture at 20°C resulted in augmentation of ³²P incorporation into protein (protein phosphorylation) followed by activation of ³²P incorporation into RNA (RNA synthesis) and then induced pseudopodial cable growth, accompanied by considerable decreases in the rates of protein phosphorylation and RNA synthesis. This augmentation of RNA synthesis and cable growth induced by insulin were blocked by H-7, which inhibited protein phosphorylation, and were also inhibited by actinomycin D without any inhibition of protein phosphorylation. Similar results were obtained on treatment with horse serum, found to contain insulin-like compounds. In cells treated with horse serum treated cells, high rates of protein phosphorylation and RNA synthesis were maintained even after the initiation of cable growth and about 5 h later, spicule rods were produced. Insulin treatment did not induce spicule rod formation. In cells treated with horse serum, actinomycin D treatment started at the time of initiation of cable growth, cables were formed but formation of spicule rods was blocked. These results suggest that horse serum contains some other substance besides insulin-like ones, which induces expression of genes that are indispensable for spicule rod formation.     

Relationship between Increased NADP-linked Glyceraldehyde-3-phosphate Dehydrogenase Activity and Protein Synthesis during the Greening of Etiolated Pea Seedlings

Photoinduction of NADP-linked glyceraldehyde-3-phosphate dehydrogenase activity in etiolated pea seedlings was investigated in the presence of various concentrations of four inhibitors of protein synthesis (cycloheximide, actinomycin D, chloramphenicol and puromycin) and one photosynthesis inhibitor (DCMU), and compared with increase in chlorophyll and total protein contents. The enzymatic activity and chlorophyll showed similar responses to the action of the antibiotics, whereas they were not significantly affected by the presence of DCMU.     

Antibiotic Sensitivity of Micrococcus radiodurans

A wild-type strain of Micrococcus radiodurans and its nonpigmented mutant W(1) were tested for sensitivity to 10 antibiotics selected from the standpoint of their mechanism of action. Representatives of groups of antibiotics inhibiting deoxyribonucleic acid (DNA) synthesis, DNA-dependent ribonucleic acid synthesis, protein synthesis, and cell wall synthesis were selected. M. radiodurans and its mutant exhibited full susceptibility to all antibiotics tested (mitomycin C, actinomycin D, chloramphenicol, dihydrostreptomycin, erythromycin, neomycin, kanamycin, benzylpenicillin, bacitracin, and vancomycin), the degree of susceptibility being of the same order as that of a standard strain of Staphylococcus aureus 209 P, with the exception of dihydrostreptomycin.     

Actinomycin V as a Potent Differentiation Inducer of F5-5 Friend Leukemia Cells

The cell differentiation system of Friend leukemia cells was applied to screening for new types of antitumor antibiotics. F5-5, Friend leukemia cells, were the most suitable for the assay system due to the stability of their response on repeated culture passages. Antibiotics like mitomycin C, adriamycin and actinomycin D, but not cycloheximide, did not induce detectable benzidine-positive cells among the F5-5 cells in the concentration ranges tested. Among the culture fluids of one thousand and fifty-one streptomycete strains subjected to the assay system, actinomycin V, FL-518 and FL-657 were found to be the most active as inducers. Actinomycin V possessing l-4-ketoproline as a substitute for l-proline of actinomycin D at a concentration of 1.0ng/ml caused 39.7% of the F5-5 cells to become benzidine-positive. Furthermore, actinomycin V inhibited the colony formation of F5-5 cells in the soft agar medium at a concentration of 0.004 ng/ml.     

A comparison of the effects of metabolic inhibitors upon direct and antibody-dependent lymphocyte mediated cytotoxicity

The effect of metabolic inhibitors upon the lysis of allogeneic lymphocyte target cells by cytotoxic T lymphocytes and nonimmune lymphocyte K cells were studied by using identical culture conditions. Inhibition of lysis in both systems was induced by cycloheximide, puromycin, actinomycin D, diisopropylfluorophosphate, 2-deoxyglucose, antimycin A, oligomycin, and cytochalasins A and B, whereas ouabain and mitomycin C did not diminish lysis in either system. The strikingly parallel responses to a variety of agents that alter cellular processes suggest that both forms of lysis utilize similar mechanisms.     

Influence of Macromolecular Biosynthesis on Cellular Autolysis in Streptococcus faecalis

The addition of several different antibiotics to growing cultures of Streptococcus faecalis, ATCC 9790, was found to inhibit autolysis of cells in sodium phosphate buffer. When added to exponential-phase cultures, mitomycin C (0.4 mug/ml) or phenethyl alcohol (3 mg/ml) inhibited deoxyribonucleic acid synthesis, but did not appreciably affect the rate of cellular autolysis. Addition of chloramphenicol (10 mug/ml), tetracycline (0.5 mug/ml), puromycin (25 mug/ml), or 5-azacytidine (5 mug/ml) to exponential-phase cultures inhibited protein synthesis and profoundly decreased the rate of cellular autolysis. Actinomycin D (0.075 mug/ml) and rifampin (0.01 mug/ml), both inhibitors of ribonucleic acid (RNA) synthesis, also reduced the rate of cellular autolysis. However, the inhibitory effect of actinomycin D and rifampin on cellular autolysis was more closely correlated with their concomitant secondary inhibition of protein synthesis than with the more severe inhibition of RNA synthesis. The dose-dependent inhibition of protein synthesis by 5-azacytidine was quickly diluted out of a growing culture. Reversal of inhibition was accompanied by a disproportionately rapid increase in the ability of cells to autolyze. Thus, inhibition of the ability of cells to autolyze can be most closely related to inhibition of protein synthesis. Furthermore, the rapidity of the response of cellular autolysis to inhibitors of protein synthesis suggests that regulation is exerted at the level of autolytic enzyme activity and not enzyme synthesis.     

The mechanism of cell surface changes of guinea pig macrophages activated with purified migration inhibitory factor/macrophage activation factor

Guinea pig MIF (MIF/MAF), which was purified by immunoadsorbent column chromatography using an antibody against MIF/MAF, was observed to induce characteristic cell surface changes in macrophages under scanning electron microscopy (SEM). MIF/MAF induced enlarged petal-like ruffles in both rounded and spreading macrophages. The changes were observed as early as 2 hr after stimulation with MIF/MAF and continued for 24 hr. These morphological changes appeared to be a good indicator of macrophage activation and migration inhibition in the early phase. The mechanism of the characteristic ruffle formation was studied using metabolic inhibitors and reagents known to affect microfilaments and microtubules. When macrophages were treated with MIF/MAF in the presence of mitomycin C, actinomycin D, or puromycin, formation of the petal-like ruffles was not affected. However, vinblastine and cytochalasin B inhibited the induction of these ruffles. These results indicate that microtubule and microfilament assembly, but not synthesis of DNA, RNA, and protein, are required for the formation of the petal-like ruffles. In addition, treatment with a Ca²⁺ ionophore induced the same petal-like ruffles in macrophages, while treatment with dibutyryl-cyclic AMP or-cyclic GMP did not. These findings suggest that Ca²⁺ plays an important role in macrophage activation by MIF/MAF, especially in the early phase.     

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.     

High temperature induced antibiotic sensitivity changes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, which was resistant to a wide variety of antibiotics, became sensitive to several of these antibiotics when grown and tested at 46 degrees C. Cell wall antibiotics such as penicillin G and ampicillin were only effective when added to cells growing at 46 degrees C prior to a temperature shift to 37 degrees C. Antibiotics which penetrate the cytoplasmic membrane to express their inhibiting action present a pattern different from those which are active against the outer cell wall. In order that these compounds be effective, the permeability of the cytoplasmic membrane must be further altered with agents such as EDTA which allow the penetration of actinomycin D. Inhibitors of protein synthesis, such as streptomycin and chloramphenicol, have increased access to their sites of action in cells grown at 46 degrees C. Cells grown at 46 degrees C have 40% less lipopolysaccharide (LPS) than cells grown at 37 degrees C and the LPS aggregates were of large molecular size in cells grown at 46 degrees C. Growth at 46 degrees C affects the permeability properties of the outer cell wall more than the permeability properties of the cytoplasmic membrane and this was due, in part, to the selective release of LPS of LPS-protein complexes at elevated growth temperatures.     

Effects of metabolic inhibitors on spontaneous and interferon-boosted human natural killer cell activity

Human natural killer (NK) cell activity can be augmented by pretreatment with partially purified preparations of human interferon (IF). Studies have now been performed to determine the metabolic processes required for and involved in spontaneous NK activity and augmentation of cytotoxicity. A 4-hr 51Cr release cellular cytotoxicity assay was used to measure the NK activity, and peripheral blood leukocyte cells (PBL) were treated with: a) x-ray or mitomycin C; b) actinomycin D; or c) emetine, cycloheximide, pactamyhcin, or puromycin to assess the roles of DNA, RNA, and protein synthesis, respectively, in spontaneous NK activity and in boosting by IF. Prolonged incubation (18 hr) of PBL after blockage of synthesis of DNA almost completely abrogated NK activity; however, NK activity could be partially or totally restored to these populations by incubation of the effector cells for 1 hr at 37 degrees C with IF. Blockage of DNA synthesis for 1 hr had no effect on spontaneous NK activity or on boosting by IF. Inhibition of RNA synthesis also had no effect on spontaneous NK activity. Treatment of PBL with actinomycin before exposure to IF prevented boosting, but treatment with the RNA synthesis inhibitor after boosting with IF for 5 to 6 hr no longer had an appreciable effect on cytotoxicity. The effect of protein synthesis inhibitors on spontaneous NK activity was dependent on the inhibitor selected. Emetine and puromycin totally abrogated spontaneous NK activity at concentrations of inhibitor that blocked 3H-leucine incorporation 90% or more. In contrast, cycloheximide and pactamycin had only minimal effects on spontaneous NK activity but totally abrogated the boosting of IF.     

Effect of metabolic inhibitors on pyrogen production by rabbit leukocytes.

The metabolic inhibitors, actinomycin D, cycloheximide, puromycin dihydrochloride, puromycin aminonucleoside, and p-fluorophenylalanine did not inhibit the release of leukocytic pyrogen whether endotoxin was preincubated with cells for 20 min at 37 degrees C before addition of inhibitor or inhibitor was preincubated with cells for 1 hr before addition of endotoxin. On the other hand, cortison inhibited release of pyrogen under both experimental conditions. Poly(I): poly(C) was not effective in inducing rabbit leukocytes to produce an endogenous pyrogen.     

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.     

Effect of Tobacco Mosaic Virus Infection on Amino Acid Incorporation into Isolated Mitochondria from Tobacco Leaves

Mitochondria isolated from tobacco leaves incorporated ¹⁴C-leucine into the protein and the rate was enhanced by tobacco mosaic virus (TMV) infection as compared with noninfected level. In vitro amino acid incorporation by mitochondria required adenosine triphosphate (ATP), Mg²⁺, and KC1 and the energy sources from oxidative phosphorylation as well as from ATP-generating system. This incorporation was inhibited by ribonuclease (RNase), deoxyribonuclease (DNase), actinomycin D, mitomycin C, puromycin, and chloramphenicol added in the reaction medium. The pretreatment of the mitochondria with DNase and actinomycin D reduced the rate of incorporation. The mitochondria incorporated ³H-guanosine triphosphate (GTP) and this activity was blocked by actinomycin D. The presence in this system of 15,000 g supernatant cell sap fraction or bacterial contamination was carefully checked obtaining a negative result. The reaction product into which ^l4C-amino acids incorporated was solubilized by trypsin. The nature of the amino acid incorporating activity of isolated mitochondria obtained from TMV-infected tobacco leaves is discussed.