Conservation of the alternative oxidase and enhancement of cyanide-resistant respiration in suspension-cultured pear fruit cells by inhibitors of macromolecular synthesis

The contribution of the alternative pathway to the respiration of suspension-cultured pear ( Pyrus communis cv. Passa Crasanne) cells was enhanced, often severalfold, within 2 to 4 days following the addition of cycloheximide, actinomycin D, or 2-(4-methyl-2,6-dinitroanalino)- N -methyl propionamide (D-MDMP). Concomitant inhibition of cellular protein synthesis by cycloheximide and actinomycin D was transient and incomplete. However, inhibition by D-MDMP was virtually complete (>97%) and persisted over several days. [³⁵S]-labelling and polyacrylamide gel separation indicated that cycloheximide precluded the appearance of discernable new proteins in mitochondria. Probes with monoclonal antibodies revealed a conservation of alternative oxidase protein levels in the mitochondria of inhibitor-treated cells. The data, appraised within the complexities of cell-culture dynamics, lead to the conclusion that the observed increases in capacity for cyanide-resistant respiration are the consequence, likely indirect, of inhibited protein synthesis with resultant retention and activation of constitutive alternative oxidase.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were most dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachement completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

Diamine oxidase activity induction in regenerating rat liver

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.     

Effect of hydrophobic compounds on rRNA synthesis in intact cells and isolated nuclei

Papaverine, cycloheximide, 2,4-dinitrophenol (DNP) and actinomycin D at low concentration have been shown to suppress selectively rRNA synthesis in Ehrlich ascite carcinoma cells. rRNA synthesis in isolated nuclei is not sensible to wide range of concentration of papaverine (0,005-0,1 mM), cycloheximide (0,5-100 micrograms/ml) and DNP (5-500 microM). Actinomycin D at low concentration does not act on the rRNA synthesis in vitro either. To suppress rRNA synthesis in this system much higher concentration of this agent (10 micrograms/ml) producing inhibition of all classes of rRNA synthesis in intact cells is required. Selective sensitivity of rRNA synthesis in the cells to papaverine, cycloheximide, DNP and low concentration of actinomycin D does not connect with their direct action on the apparatus of rRNA synthesis.     

Ongoing protein synthesis needed for 1,25-(OH)2D3-mediated rapid increase of cyclic GMP in human skin fibroblasts

Recently we reported that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) through interaction with its specific receptor rapidly (within 1 min) stimulated intracellular cGMP production in cultured human skin fibroblasts. Here we show that this effect of 100 nM 1,25-(OH)2D3 is prevented by brief (30 min) inhibition of RNA synthesis (with actinomycin D or alpha-amanitin) or by brief inhibition of protein synthesis (with cycloheximide or diphtheria toxin). The protein synthesis inhibitors also blocked stimulation of cGMP by other steroids (testosterone or dexamethasone at 100 nM) but did not block cGMP stimulation by sodium nitroprusside. Since the time for the 1,25-(OH)2D3 receptor to increase cGMP seems too short to require de novo protein synthesis, we conclude that the 1,25-(OH)2D3 receptor acts together with rapidly turning over protein(s) to stimulate cGMP synthesis.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors.

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were not dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachment completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

The effect of actinomycin D on the synthesis of ribonucleic acid and protein in rat liver parenchymal cells in suspension and liver slices

1. Rat liver parenchymal cells in suspension are shown to require a higher concentration of actinomycin D than liver slices for equivalent inhibition of the incorporation of [(14)C]adenine, [(14)C]uracil and [(32)P]phosphate into RNA, and of (14)C-labelled amino acids into protein; protein synthesis is much less susceptible to actinomycin D inhibition than RNA synthesis in both the tissue preparations. Possible causes for these differences are discussed. 2. The uptake of [(3)H]actinomycin D in the first few minutes was much greater in the cell suspensions than in the tissue slices; that in the next 1-4hr. was about the same in both the cases. The uptake by both the tissue preparations was at all times proportional to the concentration of the drug within the range 0.5-2.0mug./ml. 3. In the slices actinomycin D taken up initially was concentrated almost exclusively in the nuclei; with time the concentration of the drug in the mitochondria and the supernatant increased more rapidly than in the nuclei though at no stage did it exceed that in the nuclei. In the cell suspension the largest concentration of the drug taken up initially was found in the supernatant; most of the drug taken up subsequently also stayed in the supernatant. 4. When the drug concentration in the incubation medium was 1mug./ml., its concentration within the parenchymal cells in suspension and the parenchymal cells in the slices reached 2.2 and 1.6mug./cm.(3) of cellular volume respectively. On average, 7% of the drug was removed from the medium by the cells in suspension and 23% by the cells in the slices; the average ratio of intracellular to extracellular concentration was 2.4 in the former and 2.1 in the latter case.     

Phospholipid Synthesis in Sindbis Virus-Infected Cells

We investigated the metabolic requirements for the decrease in phospholipid synthesis previously observed by Pfefferkorn and Hunter in primary cultures of chick embryo fibroblasts infected with Sindbis virus. The incorporation of (32)PO(4) into all classes of phospholipids was found to decline at the same rate and to the same extent; thus, incorporation of (14)C-choline into acid-precipitable form provided a convenient measure of phospholipid synthesis that was used in subsequent experiments. Experiments with temperature-sensitive mutants suggested that some viral ribonucleic acid (RNA) synthesis was essential for the inhibition of choline incorporation, but that functional viral structural proteins were not required. The reduction in phospholipid synthesis was probably a secondary effect of infection resulting from viral inhibition of the cellular RNA and protein synthesis. All three inhibitory effects required about the same amount of viral RNA synthesis; the inhibition of host RNA and protein synthesis began sooner than the decline in phospholipid synthesis; and both actinomycin D and cycloheximide inhibited (14)C-choline incorporation in uninfected cells. In contrast, incorporation of (14)C-choline into BHK-21 cells was not decreased by 10 hr of exposure to actinomycin D and declined only slowly after cycloheximide treatment. Growth of Sindbis virus in BHK cells did not cause the marked stimulation of phospholipid synthesis seen in picornavirus infections of other mammalian cells; however, inhibition was seen only late in infection.     

Activation of poly(ADP-ribose)-polymerase inhibits apoptosis of thymocytes, caused by short-wave ultraviolet radiation

The effect of (i) aphidicolin, a specific inhibitor of delta- and epsilon-polymerases, and nucleotide excision repair; (ii) 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase and base excision repair; and (iii) actinomycin D and cycloheximide, inhibitors of protein and RNA synthesis, respectively, on the induction of suppression of apoptosis of rat thymocytes by different doses of short-wavelength ultraviolet radiation was studied by flow cytometry. 3-Aminobenzamide suppressed the inhibition of apoptosis induced by the doses of short-wavelength ultraviolet radiation higher than 20 J/m2, increasing the cell death to a maximum. Thus, the inhibition of apoptosis by high short-wavelength ultraviolet radiation doses depends on the status of poly(ADP-ribose) polymerase and is prevented by 3-aminobenzamide. As opposed to 3-aminobenzamide, aphidicolin did not affect the cell death at short-wavelength radiation doses higher than 10 J/m2 but induced the apoptosis of unirradiated cells and cells irradiated with short-wavelength ultraviolet radiation doses lower than 10 J/m2. The inhibitors of protein and RNA synthesis cycloheximide and actinomycin D prevented the induction of apoptosis caused by low and medium doses but did not abolish the apoptosis-inhibiting activity of high doses of short-wavelength ultraviolet radiation.     

Intracellular Localization of Three l-Glutamate Dehydrogenase Isozymes from Chlamydomonas reinhardtii

The intracellular localization of the activity and synthesis of three isozymes of NAD(P)⁺-glutamate dehydrogenase from the unicellular green alga Chlamydomonas reinhardtii cw-92 has been established. Isozyme activities have been located within mitochondria by using differential centrifugation techniques and discontinuous Percoll gradient separations. Experiments with protein synthesis inhibitors cycloheximide, rifampicin, chloramphenicol, and actinomycin D, under dark and carbon starvation conditions, revealed that synthesis of the three isozymes was likely to occur in cytosol as precursor proteins that are then transported and processed inside the mitochondria.     

Potentiation of cholera toxin-stimulated cyclic AMP production in cultured cells by inhibitors of RNA and protein synthesis

Cyclic AMP increased 8- to 10-fold after a 3-h treatment with 6 nM cholera toxin in rat C6-2B astrocytoma cells. In the presence of cycloheximide, cholera toxin increased intracellular cyclic AMP about 50-fold. Qualitatively similar potentiation of cholera toxin action by cycloheximide was observed in isolated swine aortic vascular smooth muscle cells. Cycloheximide, by itself, had no effect upon cyclic AMP levels and did not alter the apparent Ka for cyclic AMP generation by cholera toxin in the cells. Also, cycloheximide did not appear to augment cholera toxin action via inhibition of cyclic nucleotide phosphodiesterase. Puromycin and actinomycin D also augmented cholera toxin action in C6-2B cells. Potentiation of cholera toxin-increased cyclic AMP formation by cycloheximide was correlated with the inhibition of [14C]leucine incorporation into protein. These results indicate that the ability of cholera toxin to stimulate cyclic AMP production in C6-2B astrocytoma and swine vascular smooth muscle cells is enhanced by inhibition of de novo protein synthesis.     

Synthesis of protein and mRNA is necessary for transition of suspension-cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G₁ phase by anisoniycio or cycloheximide caused the arrest of cells in the G₁ phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G₁ phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G₁ phase caused the arrest of cells in the G₁ phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G₁ phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G₁ phase, and that the mRNA(s) that encode these proteins are also synthesized at the G₁ phase.     

In vitro DNA and RNA synthesis by human platelets.

In vitro incorporation of [Me-3H] thymidine and [5-3H] uridine into human platelets was demonstrated. Thymidine incorporation was inhibited by three specific inhibitors of DNA synthesis: hydroxyurea, cytosine arabinoside and daunomycin. The effect was dose-dependent. Uridine uptake by platelets was found to be inhibited by specific inhibitors of RNA synthesis such as actinomycin D, rifampicin and vincristine, the effect of actinomycin D being dose dependent. The drug also led to a time-dependent inhibition of protein synthesis when preincubated with platelets. The platelet RNA profile on polyacrylamide gel was demonstrated to be similar to that of embryonic mouse erythroblast RNA. Synthesis of all three fractions, 28 S, 18 S and 4 S, was inhibited by actinomycin D. These findings show that human platelets are capable of DNA and RNA synthesis, and that these activities play a role in controlling protein synthesis in these cells. Detectable amounts of DNA have been found in whole human platelets, and in isolated mitochondria derived from these cells. Isolated platelet mitochondria incorporated [3H] thymidine and [3H] uridine into their macromolecules. These activities were inhibited by daunomycin and by both rifampicin and actinomycin D, respectively. These results support the assumption that DNA and RNA synthesis found in intact cell preparations takes place most probably in platelet mitochondria.     

Effect of tunicamycin on the turnover of epidermal growth factor receptors in cultured calf aorta smooth muscle cells: Comparison with IMR-90 human lung fibroblasts

Treatment of cultured calf aorta smooth muscle cells with tunicamycin, a potent inhibitor of dolichol-mediated glycosylation, resulted in progressive loss of receptors for epidermal growth factor with 50% of receptors lost after 6 h. Receptor half-life was also 6 h with cycloheximide treatment but was 12 h with either actinomycin D or camptothesin treatment. The epidermal growth factor-induced processing (internalization and/or degradation) of residual receptors remaining after tunicamycin treatment appeared to be unaltered.50% decrease in ¹²⁵I-labeled epidermal growth factor binding was observed also with IMR-90 fibroblasts upon 6 h treatment with tunicamycin, although these cells were less sensitive to inhibition by tunicamycin of glycosylation and protein synthesis.     

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.     

Inhibition by corticosteroids of epidermal growth factor-induced recovery of cyclooxygenase after aspirin inactivation

Cultures of vascular smooth muscle cells superfused with [14C]arachidonic acid synthesized the antiplatelet substance prostacyclin as the major cyclooxygenase product. Prostacyclin synthesis was inactivated by aspirin, which irreversibly acetylates cyclooxygenase. Aspirin-treated cells recovered within 2 h by a process that was blocked by cycloheximide but not by actinomycin D, and that required a serum component identified as epidermal growth factor (EGF). EGF-induced recovery of cyclooxygenase was greatly potentiated by type beta transforming growth factor (TGF-beta). Incubation with EGF and TGF-beta in the 0.1-1.0 nanomolar range stimulated cyclooxygenase recovery up to 20-fold without increasing [35S]methionine incorporation into other cell proteins. Induction of cyclooxygenase by EGF and TGF-beta also was prevented by cycloheximide but not by actinomycin D. EGF-dependent recovery was blocked by preincubation with dexamethasone (2 microM), an effect that was duplicated by pure lipocortin (2-4 micrograms/ml). Incubation of membrane preparations from these cells with EGF selectively activated phosphorylation of a 35-kDa cellular protein that comigrated with lipocortin. The results suggest that cyclooxygenase recovery in aspirin-inactivated vascular smooth muscle cells is mediated by an EGF-dependent translational control that is inhibited by corticosteroids. The findings also provide a new mechanism whereby corticosteroids suppress inflammatory prostaglandins.