The effects of actinomycin D,cycloheximide and puromycin on the 20-hydroxyecdysone induced autophagocytosis in larval fat body cells of Pieris brassicae

The effects of 20-hydroxyecdysone (5 μg/g body weight), cycloheximide (5 μg/g body weight), puromycin (30 μg/g body weight) and actinomycin D (30 μg/g body weight) were investigated on the larval fat body cells of 48–70-hours-old last instar larvae of Pieris brassicae.20-Hydroxyecdysone was found to induce the formation of autophagic vacuoles 3 hr after its administration, but this was, however, prevented by simultaneous cycloheximide treatment in a parallel experiment. On the contrary, puromycin proved to induce autophagocytosis. These diverse effects of the two translational inhibitors on hormone-induced autophagocytosis may be explained by differences in their modes of action.Actinomycin D, when administered 21 hr prior to the hormone, exerted a preventive effect on induced autophagocytosis, but was ineffective when applied 3 hr before the injection of 20-hydroxyecdysone.It was concluded that the presence of RNA synthesized several hours prior to the hormone treatment was a prerequisite for induction of autophagocytosis by 20-hydroxyecdysone.     

Autophagocytosis in mouse seminal vesicle cells in vitro. Temperature dependence and effects of vinblastine and inhibitors of protein synthesis

Spontaneous autophagocytosis was observed in mouse seminal vesicle cells during incubation for 2 h in vitro. The number of autophagic vacuoles formed was greatest at 37 degrees C and decreased when the temperature was lowered. At 22 degrees C it reached the near-zero value characteristic of non-incubated control cells. Incubation of the cells at 37 degrees C in the presence of 0.1 mg/ml vinblastine sulfate resulted in a marked increase in the number of autophagic vacuoles, but the drug was ineffective at 22 degrees C. Puromycin (10(-3) M) exerted no influence on spontaneous autophagocytosis, but cycloheximide in concentrations from 10(-7) M to 10(-3) M inhibited both spontaneous and vinblastine-induced autophagocytosis. The formation of tubulin paracrystals in vinblastine treated cells was not prevented either by low (22 degrees C) temperature or in the presence of cycloheximide.     

Effect of different protein synthesis inhibitors on the exocrine pancreatocytic autophagocytosis in vivo.

The translational inhibitor cycloheximide is also used as an inhibitor of cellular autophagy and intracellular degradation of endogenous cellular proteins. Some evidence for a similar effect of other inhibitors of protein biosynthesis is also available (largely from in vitro systems). In the present study, the in vivo effects of cycloheximide, emetine and puromycin on autophagy in murine exocrine pancreatic and liver cells were tested using electron microscopic morphometry. The experiments were based on the fact that when the formation of autophagosomes is inhibited, a regression of the autophagolysosomal compartment can be measured, provided intralysosomal degradation in the pre-existing autophagic vacuoles continues at an unchanged rate. To make the measurements easier, autophagolysosomal compartment of the cells was enlarged by administering vinblastine (10 mg/kg b.wt.) for 2 h when the inhibitors were given for an additional 30 min. During this time cycloheximide (0.2 mg/g b.wt.), emetine (0.12 mg/g b.wt.) and puromycin (0.2 mg/g b.wt.), respectively caused 35, 25 and 52% regression of the pancreatocytic autophagolysosomal compartment. Since all the above translational inhibitors inhibited autophagocytosis as well, the possibility of a coupling between the regulation of synthesis and inhibition of proteins arises.     

The effects of vinblastine on acinar cells of the exorbital lacrimal gland of the rat

Summary The effects of vinblastine treatment on acinar cells of the rat exorbital lacrimal gland were studied by electron microscopy. Experimental animals of both sexes were given single intraperitoneal injections of (1) vinblastine (4mg/kg body weight) at 1 to 24 h before sacrifice; (2) pilocarpine (20 mg/kg b.w.) for 1 h; or (3) vinblastine for l h followed by pilocarpine for 1 h.Vinblastine treatment caused a number of changes including autophagocytosis, formation of intracisternal granules, and alteration of secretory granules. These changes varied in extent and onset between male and female rats. In addition, the Golgi apparatus was reduced in size and dispersed throughout the cytoplasm. Mitotic figures were commonly observed. Moreover, vinblastine inhibited the pilocarpine-stimulated degranulation of the acinar cells.In view of the known anti-microtubular action of vinblastine, these results suggest that microtubules are involved in various aspects of the transport, packaging, and secretion of exportable proteins in the lacrimal gland. Additionally, autophagocytosis and alteration of secretory granules may partially result from the interaction of vinblastine with membranes.The authors thank Mr. Steve Coriell and Mr. Steve Floyd for preparing the micrographs. Robert Kelly also thanks Dr. George Chapman for his support during the initial phase of this project.     

Regression of autophagic vacuoles in pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells: a comparative morphometric study of the effect of vinblastine and leupeptin followed by cycloheximide treatment

Treatment of mice with both leupeptin (0.06 mg/g body wt) and vinblastine (0.05 mg/g body wt) for 2 h caused a many-fold enlargement of the autophagic-lysosomal compartment of pancreatic acinar, seminal vesicle epithelial, and liver parenchymal cells. In all three types of cells a predominance of large, dense bodies was seen after leupeptin treatment and that of typical autophagic vacuoles were seen after vinblastine treatment. An exponential decrease of the volume fraction of autophagic vacuoles was observed in leupeptin-treated cells after the administration of cycloheximide (0.2 mg/g body wt). The half-life of autophagic vacuoles estimated from the decay curve was 5.3, 5.7, and 6.6 min for pancreatic, seminal vesicle, and liver cells, respectively. Our data suggest that sequestered cytoplasmic material rapidly enters the lysosomes in leupeptin-treated cells and accumulates in this compartment. In contrast, no regression of the autophagic vacuole compartment of pancreatic and seminal vesicle cells was observed after the administration of cycloheximide to animals pretreated with vinblastine, and only a slight decrease was seen in liver cells. These observations show that the lifetime of autophagic vacuoles is prolonged by vinblastine resulting in their accumulation in the cells. However, our measurements also lend support to the view that in addition to the accumulatory effect on undegraded cytoplasmic material, stimulation of sequestration may play a role in the enlargement of the autophagic lysosomal compartment after treatment with leupeptin as well as with vinblastine in all three types of cells investigated.     

RIBOSOME CRYSTALLIZATION IN CHICKEN EMBRYOS : II. Conditions for the Formation of Ribosome Tetramers In Vivo

Slow cooling of fertilized chicken eggs permits the elongation and termination of nascen^t polypeptides in the polysomes but prevents the initiation of new protein chains. This leads to polysome disaggregation during the first 30 min of cooling, and to the formation, of a pool of inactive ribosomes prone to crystallization. After 2 hr these ribosomes began to form tetramers, which do not contain any labeled proteins synthesized during cooling. If protein synthesis is inhibited by cycloheximide, added to eggs before cooling, tetramer formation in the embryos is prevented. Puromycin, on the other hand, leads to polysome disassembly and does not prevent tetramer formation. Rapid cooling of explanted embryos after short incubation at 37°C, with or without cycloheximide, largely prevents polysome disaggregation and the formation of tetramers. On the other hand, the addition of puromycin to explanted embryos promotes tetramer formation after rapid cooling. When cooled eggs are rewarmed, tetramers are disassembled into monomers, even if protein synthesis is inhibited. When those embryos were rapidly recooled tetramers reformed spontaneously from tetramer-derived monomers, even in the presence of cycloheximide. We conclude that the formation of tetramers at low temperature is an inherent property of the normal ribosomes.     

Biochemical research on oogenesis: protein synthesis in whole cells and in cell-free extracts of Xenopus laevis immature ovaries

Nearly all tRNA molecules in previtellogenic oocytes of Xenopus laevis are included in nucleoprotein particles sedimenting at 42S. The tRNA-binding sites of these particles have several properties in common with those of the ribosomes. This suggests that the 42S particles might behave like unprogrammed ribosomes and be the site of a template-independent polymerization of amino acids. We expected this reaction to be insensitive to protein synthesis inhibitors, such as cycloheximide and puromycin. We found that these antibiotics almost completely inhibit the incorporation of labeled amino acids into protein, when added to the incubation medium of whole ovaries or free oocytes. In cell-free extracts of ovaries, the incorporation of amino acids is partially insensitive to cycloheximide and puromycin. When such extracts are fractionated by sucrose density centrifugation and incubated with ATP, a major peak of amino acid incorporation can be detected, which nearly coincides with the 42S particle peak.     

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.     

The fate of membrane-bound ribosomes following the termination of protein synthesis

Contemporary models for protein translocation in the mammalian endoplasmic reticulum (ER) identify the termination of protein synthesis as the signal for ribosome release from the ER membrane. We have utilized morphometric and biochemical methods to assess directly the fate of membrane-bound ribosomes following the termination of protein synthesis. In these studies, tissue culture cells were treated with cycloheximide to inhibit elongation, with pactamycin to inhibit initiation, or with puromycin to induce premature chain termination, and ribosome-membrane interactions were subsequently analyzed. It was found that following the termination of protein synthesis, the majority of ribosomal particles remained membrane-associated. Analysis of the subunit structure of the membrane-bound ribosomal particles remaining after termination was conducted by negative stain electron microscopy and sucrose gradient sedimentation. By both methods of analysis, the termination of protein synthesis on membrane-bound ribosomes was accompanied by the release of small ribosomal subunits from the ER membrane; the majority of the large subunits remained membrane-bound. On the basis of these results, we propose that large ribosomal subunit release from the ER membrane is regulated independently of protein translocation.     

Ultrastructural alterations in differentiating choroid plexus cells by puromycin

This study centres on the effect of puromycin, applied in various concentrations, on the differentiation of choroid plexus cells in tissue cultures. Puromycin added to these cultures in doses of 25, 50 and 100 μg/ml medium merely produced reversible cell changes when present for 2 h. After 3 h, choroid plexus cells react to continuous feeding with puromycin in a dose of 100μg/ml with an almost total loss of the membrane system of the endoplasmic reticulum. At this time many of the free ribosomes have disappeared. The results of this study suggest a continuous renewal of the membrane-structure of the endoplasmic reticulum.     

Protein synthesis by membrane-bound and free ribosomes of secretory and non-secretory tissues

1. Methods for the separation of membrane-bound and free ribosomes from rat brain (cortex) and skeletal muscle were described and the preparations characterized by chemical analysis and electron microscopy. The attachment of ribosomes to membranes is not an artifact of the separation procedure. 2. The rate of incorporation of l-[(14)C]leucine into protein in vitro by the membrane-bound and free ribosomes from these two predominantly non-protein-secreting tissues is compared with that by similar preparations from rat liver. With all three tissues the initial rate was higher for the membrane-bound preparations. 3. By using the technique of discharging nascent polypeptide chains by incubation with puromycin followed by treatment with sodium deoxycholate (Redman & Sabatini, 1966), a major difference was observed for the vectorial discharge of nascent protein synthesized both in vivo and in vitro on membrane-bound ribosomes from liver, on the one hand, and brain and muscle, on the other. Whereas a large part of nascent protein synthesized on membrane-bound liver ribosomes was discharged into the membranous vesicles (presumably destined for export from the cell), almost all nascent protein from membrane-bound ribosomes from brain and muscle was released directly into the supernatant. Incorporation of [(3)H]puromycin into peptidyl-[(3)H]puromycin confirmed these findings. There was thus no difference between membrane-bound and free ribosomes from brain on the one hand, and from free polyribosomes from liver on the other, as far as the vectorial release of newly synthesized protein was concerned. 4. Incubation with puromycin also showed that the nascent chains, pre-formed in vivo and in vitro, are not involved in the attachment of ribosomes to membranes of the endoplasmic reticulum. 5. The differences in vectorial discharge from membrane-bound ribosomes from liver as compared with brain and muscle are not due to the different types of messenger RNA in the different tissues. Polyphenylalanine synthesized on incubation with polyuridylic acid was handled in the same way as polypeptides synthesized with endogenous messenger. 6. It is concluded that there is a major difference in the attachment of ribosomes to the membranes of the endoplasmic reticulum of secretory and non-secretory tissues, which results in a tissue-specific difference in the vectorial discharge of nascent proteins.     

A sub-population of rat liver membrane-bound ribosomes that are detached in vitro by carcinogens and centrifugation.

The chemical-carcinogen-induced detachment of ribosomes from rat liver endoplasmic reticulum was studied in vitro. Incubation of postmitochondrial supernatant with 0.2 mM-diethylnitrosamine or N-2-acetylaminofluorene removed approx. 16% of membrane-bound ribosomes, measured as differences in RNA/protein values of membrane separated from unbound ribosomes by flotation. These ribosomes are also detached by exposure to high centrifugal forces (160000g) and are among those removed by NADPH-catalysed lipid peroxidation. Extensive lipid peroxidation prohibits any measurement. The ribosomes (polyribosomes) removed are not those detached from the membrane by exposure to high KC1 concentrations (loosely bound) or high KC1 concentrations in the presence of puromycin (tightly bound). It is concluded then that centrifugally labile and carcinogen-sensitive represent a previously unreported sub-population of membrane-bound ribosomes.     

Biosynthesis of cytochrome P-450 on membrane-bound ribosomes and its subsequent incorporation into rough and smooth microsomes in rat hepatocytes

Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis.     

Morphological and biochemical differentiation in HeLa cells. Effects of cycloheximide on butyrate-induced process formation and ganglioside metabolism.

When butyrate-treated HeLa cells are trypsinized and replated in the absence of butyrate, their neurite-like processes re-extend transiently. Process formation after replating is prevented when the cells are exposed to cycloheximide during butyrate treatment, whereas it is not prevented by prior exposure to the calcium ionophore A23187 plus butyrate. These results indicate that butyrate induces protein(s) required for process extension which can accumulate in the absence of processing and promote processing in the absence of inducer. Transient process re-extension is followed by spontaneous retraction of processes and reversion to normal morphology. Reversion is not prevented or delayed by puromycin. Surprisingly, however, cycloheximide completely prevents reversion even at low concentrations (< 0.5 μg/ml). Levels of the ganglioside sialolactosylceramide (G_M3), synthesis of which is induced by butyrate, return to basal levels after removal of the inducer. Cycloheximide at 0.5 μg/ml prevents the decline of G_M3 levels after removal of butyrate although the biosynthetic enzyme sialyltransferase decays at the same rate in the presence or absence of the drug and the activity of the sialidase is not affected. The results further support the hypothesis that the ganglioside G_M3 is necessary for the morphological differentiation induced in HeLa cells by butyrate.     

fMet-tRNA F Met binding and peptidyl transferase function in free and bound ribosomes from normal and puromycin aminonucleoside-treated rats.

Treatment of rats with the aminonucleoside of puromycin, which increases the incorporation of labelled phenylalanyl-tRNA into polypeptide chains in liver ribosome preparations studied in vitro, did not change the factor-dependent binding of fMet-tRNA f Met to ribosomes nor the peptidyl transferase function of the ribosomes. Peptidyl transferase function, as measured by fMet-tRNA f Met-puromycin formation, was comparable in the free and bound ribosome preparations. Similarly, the factor-dependent binding of fMet-tRNA f Met to ribosomes was the same in free ribosome preparations obtained from rat liver as it was in bound ribosome preparations that had been freed of membranes by puromycin incubation and high salt wash.     

Study of the regulation of plastid development in Euglena gracilis. II. Functional localisation and synthesis of ribosomal chloroplast particles (author's transl)]

Chloroplast ribosomes in greening cells of Euglena gracilis are found either in the stroma or bound to thylakoid membranes. The membrane-bound chloroplast ribosomes are of two main types: those which can be released by 0.5 M KCl or by puromycin and 0.5 M KCl, and those which are released by detergent (deoxycholate or Triton X-100) and KCl. The ribosomes which are released by puromycin are presumably bound to chloroplast membrane by nascent peptide chains. Ribosomes released by puromycin are found only during the course of plastidial differentiation at the time of active thylacoid membrane synthesis. Following greening, those ribosomes remain bound to the membranes but can be removed by KCl alone. An analysis of RNA labelling showed that 30-S but not 53-S subunits of membrane-bound ribosomes are of uniform specific activity. This suggests that 30-S subunit exchange in a common pool while 53 S subunits remain membrane bound and do not exchange in a common pool. Membrane-bound chloroplast ribosomes which are released either by puromycin or by detergent are originally derived from loosely bound particles, released by 0.5 M KCl.     

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of NADPH-adrenodoxin reductase and adrenodoxin of bovine adrenal cortex mitochondria

Cytoplasmic free and membrane-bound ribosomes were isolated from bovine adrenal cortex, and characterized. Contributions of free and bound ribosomes to the synthesis of NADPH-adrenodoxin reductase (AdR) and adrenodoxin (Ad) were determined by examining the presence of their nascent peptides on isolated ribosomes. Nascent peptides were released from the ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of AdR and Ad were separately isolated by immunoprecipitation using antibodies. AdR nascent peptides were associated with free and loosely-bound ribosomes, whereas Ad nascent peptides were associated with free, loosely-bound and tightly-bound ribosomes. Smaller nascent peptides of AdR were carried by free ribosomes, whereas larger nascent peptides were preferentially carried by loosely-bound ribosomes. In the case of Ad, smaller nascent peptides were more abundant in free ribosomes than in bound ribosomes. The nascent peptides of Ad were released from bound ribosomes of rough microsomes to the aqueous milieu by puromycin treatment, suggesting the release of completed Ad peptides into the cytoplasm in cells.     

Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii.

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.     

Effect of protein synthesis inhibitors on xylose uptake and 125I-insulin binding by rat soleus muscle

Prolonged exposure (90–180 min) to cycloheximide (0.2 mg/ml), puromycin (0.2 mg/ml) or chloramphenicol (0.1 mg/ml) did not affect ¹²⁵I-insulin binding by rat soleus muscle. Chloramphenicol (2 mg/ml) depressed insulin binding and insulin-stimulated xylose uptake; these effects were attributed to the “toxic” effect of chloramphenicol on muscle ATP levels. Cycloheximide and puromycin inhibited insulin-stimulated xylose uptake without affecting ATP. Puromycin and chloramphenicol, but not cycloheximide, also inhibited basal sugar transport. This difference, and the rapid onset of all these inhibitory effects, suggest that they are not due to the inhibition of protein synthesis, but rather to some more direct effect on sugar transport itself.     

Tunicamycin,puromycin and brefeldin A influence the subcellular distribution of neuropeptides in hypothalamic magnocellular neurones of rat

Summary Magnocellular neurones in the supraoptic nuclei of normal Long Evans and homozygous Brattleboro rats were examined electron-microscopically after intracisternal injections of tunicamycin, puromycin, or brefeldin A. Moderate (50 g) or high (200 g) doses of tunicamycin caused the formation of electron-dense filamentous accretions in the endoplasmic reticulum (ER) cisterns of vasopressin neurones, but only the high dose of tunicamycin also caused accretions to form in the ER of some oxytocin neurones. Immunogold labelling of ultrathin sections from tunicamycin-treated rats revealed that, in about 5% of vasopressin neurones, the accretions could be immunogold-labelled for vasopressin and its associated neurophysin. However, in the majority of vasopressin neurones, the sections required trypsinisation before immunolabelling of the accretions could be detected. Small accretions in the ER of oxytocin neurones did not label for oxytocin or its neurophysin without prior trypsinisation, whereas larger accretions in other oxytocin cells could be labelled without prior trypsin treatment. Administration of puromycin resulted in the formation of small ER accretions in both vasopressin and oxytocin neurones. These accretions were immunolabelled with antisera, respectively, to vasopressin and oxytocin, but neurophysin-immunoreactivity was in most cases absent and was not revealed by treatment with trypsin, suggesting that neurophysin-immunoreactive epitopes were absent from truncated peptides forming the accretions. Brefeldin A caused dilatation of ER cisterns and disruption of the Golgi apparatus in both oxytocin and vasopressin neurones, but did not cause accretions to form in the ER.