Studies on the amoebo-flagellate transformation inPhysarum polycephalum

Flagellar formation in the true slime mold,Physarum polycephalum, involves a sequence of events during which amoebae are changed into flagellate cells. In the present study a series of inhibitors thought to inhibit RNA and protein synthesis and microtubule assembly were added in an attempt to characterize the metabolic processes associated with this amoebo-flagellate transformation. Proflavin (inhibitor of cellular RNA synthesis), puromycin, cycloheximide and streptomycin (inhibitors of protein synthesis), blocked the transformation; however, actinomycin D (inhibitor of DNA-dependent RNA synthesis) did not block this transformation. On the other hand, 2-mercaptoethanol and dithiothreitol did block flagella formation, but even high concentrations of colchicine failed to have such an effect. Flagellate formation was more strongly inhibited by inhibitors of oxidative phosphorylation than by other respiratory inhibitors; this suggests that oxidative phosphorylation takes part in the energy metabolism of this transformation.     

Cytoplasmic membrane changes in conjugating Tetrahymena

Summary The formation of stacks of smooth-surfaced cisternae was studied in samples of Tetrahymena pyriformis that were prepared for electron microscopy after starvation for 2 days in buffer, and in samples of organisms of different mating type that were starved for 2 days and then mixed to induce conjugation. The number of stacks of cisternae was greater in starved ciliates than in those from stock cultures, and the size and number of stacks increased further after mixing animals of different mating type. When cells were either starved or mixed in buffer to which the protein synthesis inhibitor cycloheximide had been added, the formation of the membranous stacks was almost completely abolished. Addition of the RNA synthesis inhibitor actinomycin D, however, did not result in a significant decrease in the size and number of the stacks of saccules. Conjugation did not occur in the presence of either cycloheximide or actinomycin. The results suggest that protein synthesis is required for the formation of the stacks, but RNA synthesis is no longer necessary for their formation at this stage. The previous identification of the stacks as a Golgi apparatus and the possible functions of these membranes are considered.Supported by grants from NSF (GB-32285) and the American Cancer Society (E-500).The authors acknowledge the technical assistance of Mrs. Sue Thompson.     

Selective inhibition of calcium-stimulated cation-induced pinocytosis by starvation and inhibitors of protein synthesis in Amoeba proteus

The capacity of Amoeba proteus to form pinocytotic channels after pretreatment with either puromycin, cycloheximide, emetine or a long period of starvation was studied. The effect on pinocytosis of the three inhibitors of protein synthesis was similar. They preferentially affected pinocytosis induced by Na+ with little effect on K+-induced pinocytosis. In Ca2+-deficient media, Na+-induced pinocytosis was inhibited, while the addition of Ca2+ restored channel formation. The degree of inhibition of Na+-induced pinocytosis was influenced by the concentration of Ca2+ in the inducing solution. Selective Ca2+-reversible inhibition of Na+-induced pinocytosis also occurred after starvation or treatment with a proteolytic enzyme, subtilisin. The membrane potential in starved or emetine-treated cells in culture medium was normal and their depolarising response to inducers was not diminished in solutions containing Na+. The resting input resistance of these cells was higher than in normal amoebae, but no significant difference in electrical parameters was observed after pinocytosis was induced. It is suggested that starvation, inhibition of protein synthesis, and enzyme digestion deplete the membrane of structures which are necessary for normal Ca2+ functions during induction of pinocytosis by Na+-like inducers.     

The breakdown of Tetrahymena ribosomes in vivo. The effects of inhibitors.

1. When Tetrahymena were deprived of nutrients 50% of the polysomes disaggregated within 20 min and 20% of the total RNA broke down in 2 h. Ribosomal RNA accounted for 75% of the RNA breakdown. 2. RNA labelled by a long incubation with [14C]uridine was stable in growing cells and in the presence of actinomycin D, but broke down at the same rate as bulk RNA in starved cells. 3. The following substances inhibited the loss of RNA during starvation: cycloheximide (which inhibited both polysome disaggregation and protein synthesis), inhibitors of energy metabolism and puromycin (all of which caused polysome disaggregation and inhibited protein synthesis), and chloroquine and 7-amino-1-chloro-3-L-tosylamidoheptan-2-one ('TLCK') (neither of which affected polysomes or protein synthesis). 4. Starvation appears to activate a ribosome degradation mechanism that may involve lysosomal and non-lysosomal enzymes.     

Starvation survival of Salmonella enteritidis.

Washed cells of Salmonella enteritidis harvested from a defined medium during logarithmic growth were subjected to starvation in pH 7 phosphate buffer at 37 C. Viability was measured by slide cultures and plate counts. The survival of cell suspensions equivalent to 1 to 10 mg (dry wt)/ml was influenced by cryptic growth. The rate of cryptic growth, assessed by plate counts, increased with cell density and could not be alleviated by starvation with dialysis. Dialysis of the starving culture did retard the onset of cryptic growth but did not eliminate it, indicating that the major substrates for regrowth were relatively large cellular components. In phosphate buffer, 6.7 homologous heat-killed cells allowed for the doubling of one S. enteritidis cell. Cryptic growth was not observed when cells were starved on the surface of membrane filters or in suspensions equivalent to 20 mug (dry wt)/ml (105 cells/ml). Similar half-life survival times were calculated for both these populations, but the shape of their survival curves differed significantly. These differences were attributed to stress factors encountered during cell preparation and during starvation. The half-life survival time of S. enteritidis starved at 20 mug (dry wt)/ml was 140 h in phosphate buffer, 82 h in 3,6-endomethylene-1,2,3,-6-tetrahydrophthalic acid buffer, and 77 h in tris(hydroxymethyl)aminomethane buffer.     

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.     

Synchronization of the Cell Cycle of Tetrahymena by Starvation and Refeeding*

SYNOPSIS The present report describes a simple and useful method for synchronizing mass cultures of the ciliate Tetrahymena pyriformis. The method employs a nutritional approach which involves starvation of the cells in a non-nutrient phosphate buffer followed by refeeding with an enriched nutritional growth medium. It takes 240 minutes after refeeding before the first cells start to divide. Radioautographic and DNA determinations taken together show that starved cells are stalled in the GI nuclear DNA condition and that essentially all of the cells replicate their DNA prior to their first cell division.     

Changes in the Activity of Certain Enzymes of Hartmannella (Culbertson strain A-1) During Encystment*

SYNOPSIS. Hartmannella (Culbertson strain A-1) was found to undergo encystment (80–90% in 72 hr) on a non-nutrient agar containing 0.015 M MgCl₂ and 0.02 M taurine. Encystment was completely inhibited by 1 × 10^?5 M Mitomycin C, or 1 × 10^?7 M cycloheximide or 1 × 10^?6 M Actinomycin D. The ability of the amoebae to consume glucose increased fourfold within 24 hr incubation in this medium. The specific activities of cellulose synthetase, hexosephosphate transaminase and uridine diphosphosphoglucose pyrophosphorylase were also stimulated. Dehydrogenases mediating electron transfer from pyruvate, malate, succinate, α-ketoglutarate and α-glycerophosphate to triphenyltetrazolium and from glucose-6-phosphate to nicotinamide-adenine dinucleotide phosphate were, however, repressed during this period of incubation in the encystment medium. The results suggested that, during encystment of Hartmannella A-1, there was a metabolic switchover and the enzyme machinery of the amoeba was oriented more towards biosynthesis of cyst wall constituents than towards the aerobic breakdown of carbohydrates.     

Stimulation of lactase synthesis induced by starvation in the jejunum of adult rat

The effects of actinomycin D and of cycloheximide administration have been investigated on the enzyme activities of the jejunal brush border membrane in adult rats after a 48-hour period of starvation. The modifications in the protein and enzyme patterns of the brush border membrane and the incorporation of radiolabelled amino acid in the protein band corresponding to lactase have been studied in the nourished and in the starved animal. The results show that actinomycin D administration did not modify the stimulation of lactase activity caused by starvation whereas cycloheximide completely inhibited this process. The stimulation of lactase activity, in the starved animal, is related to a quantitative increase of the corresponding protein band and with enhanced incorporation of L-[3H]valine in this protein band after separation of brush border proteins by gel electrophoresis. It is concluded that the stimulation of lactase activity observed during starvation is the consequence of de novo synthesis of lactase molecules and that this process is regulated at a translational level. A general hypothesis is proposed in order to clear up partly the mechanism involved in the stimulation of lactase activity by food deprivation in the adult rat.     

DNA SYNTHESIS AND MITOSIS IN MERISTEMS: REQUIREMENTS FOR RNA AND PROTEIN SYNTHESIS

Following provision of sucrose to starved, stationary phase pea root meristems, G₁ and G₂ cells enter DNA synthesis and mitosis, respectively. Puromycin (450 μg/ml) and cycloheximide (5 μg/ml) completely prevent this initiation of progression through the cell cycle. Actinomycin D (10 μg/ml) has no effect on the initial entry of G₁ and G₂ cells into S and mitosis, although later entry is prevented. The resistance of the cells to actinomycin D is lost slowly with time in medium without sucrose, suggesting that an RNA required for the resumption of proliferative activity is being gradually lost. The effects of the inhibitors on transitional and proliferative phase meristem cells indicate that such dividing cells do indeed have sufficient of the requisite RNA for 8-12 hr progression through the cycle, but that protein synthesis is required continuously. It is suggested that this RNA is the one lost slowly during starvation, allowing starved cells to reinitiate progression through the cycle in the presence of actinomycin D.     

Studies on cyst formation inPhysarum polycephalum myxamoebae

Encystment of myxamoebae ofPhysarum polycephalum was induced by transferring the amoebae to a high salt medium of 1/60 M Sørensen buffer (pH 6.0) containing 0.125 M NaCl, 1.6 mM MgCl₂ and 0.18 mM CaCl₂. The induction of cysts was blocked by inhibitors of protein synthesis, such as puromycin, cycloheximide and streptomycin. However, inhibitors of RNA synthesis, such as actinomycin D, proflavin and 8-azaguanine did not block the transformation. These results suggest that in the cyst formation,de novo RNA synthesis is not involved, whereas protein synthesis is required. Cyst formation was more strongly inhibited by inhibitors of oxidative phosphorylation than by other respiratory poisons. It seems that oxidative phosphorylation takes part in the energy supply of this differentiation.     

Purification and characterization of a DNA synthesis inhibitor protein from mouse embryo fibroblasts

A DNA synthesis inhibitor protein was purified from the conditioned medium of cycloheximide treated mouse embryo fibroblasts. This protein has a molecular weight of 45,000 as determined by gel filtration and Polyacrylamide gel electrophoresis. The levels of the [³⁵S] methionine la belled 45 kDa protein in the medium and matrix were monitored across two cell cycles in synchronized cultures. The 45 kDa protein was present in higher levels in the medium of non-S-phase cells depicting a peak between the two S-phases. The DNA synthesis inhibitor protein was immunologically related to a chicken DNA-binding protein which showed similar cell cycle specific variations at the intracellular level. The purified 45 kDa protein inhibited DNA synthesis in murine and human cells. In mouse embryo fibroblasts, the DNA synthesis was inhibited to an extent of 86% by 0.25 μg/ml of the inhibitor, while higher amounts of the inhibitor were required to arrest DNA synthesis in human skin fibroblasts: in these cells, 4 μg/ml of the inhibitor inhibited DNA synthesis to an extent of 50%. The high levels of the 45 kDa protein in the medium of non-S phase cells and its DNA synthesis inhibitory potential suggest that this protein may be involved in the regulation of DNA synthesis during the cell cycle.     

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.     

Inhibition by antibiotics of the bacterial response to long-term starvation of Salmonella typhimurium and the colon microbiota of mice

The number of viable cells of two strains of Salmonella typhimurium and the number of viable cells and the cell size of the colon microbiota of mice were examined during non-growing conditions after exposure to antibiotics with known modes of action. Salmonella typhimurium starved for 1, 2, 4, 5, 12 and 20 d in a phosphate buffer saline solution and subsequently exposed for 2 and 6 h showed the following characteristics. The protein synthesis inhibitors gentamicin and tetracycline, the RNA synthesis inhibitor rifampicin and the membrane potential inhibitor polymyxin all impaired survival of starved cells. The reduction in the number of viable cells caused by the addition of gentamicin, rifampicin and polymyxin was generally more pronounced with extended exposure to energy and nutrient deprivation. Both 2- and 6-h exposure of tetracycline, however, had diminishing inhibitory effects after 20 d compared with 5 d of starvation. Control experiments to verify non-growing conditions in the starvation regime showed that DNA and cell wall synthesis inhibitors had no inhibitory effect after 24-h starvation. The rough mutant strain displayed a lower sensitivity to a hydrophobic rather than a hydrophilic inhibitor as compared to the smooth wild-type strain. The cell size reduction but not viability was partly prevented by protein synthesis inhibitors as seen for both in vivo and in vitro colon microbiota studies.     

Effect of protein synthesis inhibitor cycloheximide on starvation,fasting and feeding oxygen consumption in juvenile spiny lobster Sagmariasus verreauxi

Metabolism in aquatic ectotherms evaluated by oxygen consumption rates reflects energetic costs including those associated with protein synthesis. Metabolism is influenced by nutritional status governed by feeding, nutrient intake and quality, and time without food. However, little is understood about contribution of protein synthesis to crustacean energy metabolism. This study is the first using a protein synthesis inhibitor cycloheximide to research contribution of cycloheximide-sensitive protein synthesis to decapod crustacean metabolism. Juvenile Sagmariasus verreauxi were subject to five treatments: 2-day fasted lobsters sham injected with saline; 2-day fasted lobsters injected with cycloheximide; 10-day starved lobsters injected with cycloheximide; post-prandial lobsters fed with squid Nototodarus sloanii with no further treatment; and post-prandial lobsters injected with cycloheximide. Standard and routine metabolic rates in starved lobsters were reduced by 32% and 41%, respectively, compared to fasted lobsters, demonstrating metabolic downregulation with starvation. Oxygen consumption rates of fasted and starved lobsters following cycloheximide injection were reduced by 29% and 13%, respectively, demonstrating protein synthesis represents only a minor component of energy metabolism in unfed lobsters. Oxygen consumption rate of fed lobsters was reduced by 96% following cycloheximide injection, demonstrating protein synthesis in decapods contributes a major proportion of specific dynamic action (SDA). SDA in decapods is predominantly a post-absorptive process likely related to somatic growth. This work extends previously limited knowledge on contribution of protein synthesis to crustacean metabolism, which is crucial to explore the relationship between nutritional status and diet quality and how this will affect growth potential in aquaculture species.

     

Effect of Glucose Starvation on Glucose Transport in Neuronal Cells in Primary Culture from Rat Brain

The regulation of glucose transport into cultured brain cells during glucose starvation was studied. On glucose deprivation for 40 h, 2-deoxy-D-glucose (2-DG) uptake was stimulated twofold in neuronal cells but was not changed significantly in astrocytes. On refeeding, the increased activity of neuronal cells rapidly returned to the basal level, an observation indicating that the effect of glucose starvation was reversible. The increase was due solely to change in the Vmax, a finding suggesting that the number of glucose transporters on the plasma membrane is increased in starved cells. Cycloheximide inhibited this increase. In the presence of cycloheximide, the activity of 2-DG uptake of starved cells remained constant for 12 h and then slowly decreased, whereas that of fed cells decreased rapidly. These findings suggest that glucose starvation regulates glucose transport by changing the rate of net synthesis of the transporter in neuronal cells in culture.     

Inhibition by antibiotics of the bacterial response to long-term starvation of Salmonella typhimurium and the colon microbiota of mice

The number of viable cells of two strains of Salmonella typhimurium and the number of viable cells and the cell size of the colon microbiota of mice were examined during non-growing conditions after exposure to antibiotics with known modes of action. Salmonella typhimurium starved for 1, 2, 4, 5, 12 and 20 d in a phosphate buffer saline solution and subsequently exposed for 2 and 6 h showed the following characteristics. The protein synthesis inhibitors gentamicin and tetracycline, the RNA synthesis inhibitor rifampicin and the membrane potential inhibitor polymyxin all impaired survival of starved cells. The reduction in the number of viable cells caused by the addition of gentamicin, rifampicin and polymyxin was generally more pronounced with extended exposure to energy and nutrient deprivation. Both 2- and 6-h exposure of tetracycline, however, had diminishing inhibitory effects after 20 d compared with 5 d of starvation. Control experiments to verify non-growing conditions in the starvation regime showed that DNA and cell wall synthesis inhibitors had no inhibitory effect after 24-h starvation. The rough mutant strain displayed a lower sensitivity to a hydrophobic rather than a hydrophilic inhibitor as compared to the smooth wild-type strain. The cell size reduction but not viability was partly prevented by protein synthesis inhibitors as seen for both in vivo and in vitro colon microbiota studies.