The effects of inhibitors of protein synthesis on incorporation of lipids into myelin

Abstract— Following intracranial injections of puromycin, the incorporation of [³H]leucine into brain protein was inhibited by 80 per cent. Conversely, incorporation of [³⁵S]sulphate into sulphatide or [2-³H]glycerol into phosphatidyl choline was not inhibited. Under these conditions, appearance of labelled protein in myelin was inhibited by 90 per cent, while the appearance of newly labelled sulphatide and phosphatidyl choline in myelin membrane was not greatly affected. Experiments with cycloheximide gave similar results with phosphatidyl choline, but incorporation of [³⁵S]sulphate into total sulphatide was decreased by about 30 per cent in animals given cycloheximide. Neither puromycin nor cycloheximide had any inhibitory effect on galactocerebroside sulphotransferase.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

THE PREVENTION BY INHIBITORS OF BRAIN PROTEIN SYNTHESIS OF THE HYPERACTIVITY AND HYPERPYREXIA PRODUCED IN RATS BY MONOAMINE OXIDASE INHIBITION AND THE ADMINISTRATION OF L-TRYPTOPHAN OR 5-METHOXY-N,N-DIMETHYLTRYPTAMINE

Abstract— In rats treated with a monoamine oxidase inhibitor, (tranylcypromine), L- tryptophan produces a stereotyped syndrome of hyperactivity and hyperpyrexia due to an increased rate of brain serotonin (5-hydroxytryptamine) synthesis. Pretreatment of rats with intraperitoneal injections of cycloheximide, acetoxycycloheximide, emetine and dehydroemetine and of mice with puromycin inhibited this syndrome. Cycloheximide also inhibited the hyperactivity caused by tranylcypromine and DL-15-hydroxtryptophan and did not affect the increased rate of brain serotonin ‘synthes’ is produced by tryptophan thus excluding a primary effect on tryptophan-5-hydroxylase. Inhibition of hyperactivity did not occur until brain protein synthesis was inhibited by greater than 65 per cent as measured by the incorporation of L-[U-¹⁴C]tyrosine into brain protein in vivo. Emetine, which has been shown to inhibit brain protein synthesis inhibited hyperactivity whereas isoemetine which did not inhibit brain protein synthesis, did not inhibit hyperactivity. Under conditions where cycloheximide inhibited hyperactivity produced by tranylcypromine and L-tryptophan, a large dose of 5-methoxy-N,N-dimethyltryptamine(5-MeODMT) still produced hyperactivity showing that the rats were still capable of the same pattern of hyperactivity. However, cycloheximide did inhibit hyperactivity due to 5-MeODMT, the degree of this inhibition being dependent upon a balance between the doses of cycloheximide and 5-MeODMT. 5-MeODMT probably acts directly within the brain to cause behavioural excitation and it seems likely that the inhibitors of brain protein synthesis interfere with the mechanism of action of brain 5HT and administered 5-MeODMT rather than upon any aspect of synthesis, storage or release of brain 5HT. It is suggested that the behaviourally excitant effects of brain 5HT and 5-MeODMT are mediated in some way by a brain protein with a short biological half-life. Such a protein may act either as a factor specifically mediating the central effects of brain 5HT or as a factor regulating the neuronal response to excitation by 5HT and 5-MeODMT.     

PROTEIN SYNTHESIS IN ISOLATED NUCLEI FROM ADULT RAT BRAIN

Nuclei from adult rat brains isolated with isotonic sucrose were incubated with [³H]leucine and later purified by centrifugation through hypertonic sucrose solutions. It was found that under these conditions, tritiated leucine was incorporated into TCA precipitable material. Protein synthesis was impaired if the nuclei were treated with the nonionic detergent Triton X-100 or hypertonic sucrose. The presence of puromycin or cycloheximide markedly inhibited the incorporation of the radioactive amino acid. Actinomycin D and RNase did not have any effect on the incorporation. Autoradiography indicated the presence of labelled material within the nuclei and not in cytoplasmic contaminants. Glial nuclei were more actively involved in protein synthesis than neuronal nuclei.     

Further studies of the transport of protein to nerve endings

Mice were injected intracerebrally with [l-¹⁴C]leucine, and the specific activities of subcellular fractions of brain and effractions of isolated nerve endings were determined. There was a progressive increase in the specific activity of protein associated with isolated nerve endings after incorporation of [l-¹⁴C]leucine into whole brain protein had terminated. Although, the incorporation of [¹⁴C]leucine into soluble protein of whole brain did not differ significantly in mice which were 3 months or 1-year old, the subsequent increase in specific activity of soluble protein isolated from nerve endings was significantly greater in the younger animals; 6-month-old mice were intermediate. Therefore, changes in some aspect of the transport of protein to nerve endings is altered even after sexual maturity. Anaesthetization with pentobarbitone during incorporation of [¹⁴C]leucine into protein, and inhibition of protein synthesis with acetoxycycloheximide after incorporation of [¹⁴C]leucine was complete, did not interfere with the subsequent appearance of radioactive protein at the nerve ending. Evidence is presented for the transport, from a proximal site of synthesis, of protein associated with particulate components of the nerve ending, including synaptic vesicles.     

ANISOMYCIN,ACETOXYCYCLOHEXIMIDE, CYCLOHEXIMIDE,AND PUROMYCIN AS INHIBITORS OF RAT BRAIN ACETYLCHOLINESTERASE IN VITRO1

A kinetic analysis of the interaction of anisomycin, acetoxycycloheximide, cycloheximide, and puromycin with acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) in rat brain homogenate shows that all of these protein synthesis inhibitors are also inhibitors or this enzyme. Puromycitl aminonucleoside, a puromycin analog without antibiotic activity, was also found to be an inhibitor of acetylcholinesterase activity much like puromycin. Anisomycin appeared to be a competitive inhibitor whereas all of the other compounds showed mixed inhibition. The apparent 10.5 values for inhibition of rat brain acetylcholinesterase at 50 μM substrate were: anisomycin, 3 mM; acetoxycycloheximide, 1 mM; cycloheximide, 2.2 mM; puromycin, 0.5 mM and puromycin aminonucleoside, 0.6 mM.     

Inhibitors of protein synthesis involved in memory disruption: A study of their effects on sympathetic ganglion isolated in vitro

The effect of puromycin, puromycin-aminonucleoside, cycloheximide and acetoxycycloheximide has been tested on the function of the superior cervical ganglion of rat, by recording the postganglionic response evoked by preganglionic stimulation. All the compounds examined exerted a depressant effect, which was prompt and reversible with puromycin and its aminonucleoside, but slow and irreversible with the glutarimides. A correlation of these effects with inhibition of protein synthesis has been examined. The relevance of these findings for experiments on memory disruption reported in the literature is discussed.     

Differential effects of protein synthesis inhibitors on porcine oocyte activation

This study was designed to evaluate the effects of cycloheximide and puromycin on activation and protein synthesis of porcine oocytes. When matured oocytes were electrostimulated, then cultured in the presence of cycloheximide (5 μ/ml) for 6 or 24 hr, 92% of oocytes were activated as indicated by pronuclear formation, vs. 2.8% for untreated oocytes, 5.3% for oocytes not electrostimulated but cultured with cycloheximide, and 60.0% for those only electrostimulated. When cultured with L-[³⁵S]methionine in the presence of cycloheximide, puromycin (100 μg/ml), or no protein synthesis inhibitor for 24 hr, oocytes had mean radiolabeled incorporation rates of 36.5, 2.21, and 32.0 fmol/4 hr/oocyte, respectively. Thus, cycloheximide had little effect on protein synthesis after 24 hr of culture. A 1D-SDS PAGE showed that oocytes cultured with puromycin or cycloheximide are not activated, while electrostimulated oocytes are activated, as characterized by the conversion of a 25-kDa polypeptide to a 22-kDa polypeptide. The radiolabeling experiment was repeated, except that oocytes were cultured for 4 or 24 hr. At 4 hr, mean incorporation rates were lower in the cycloheximide group (2.34 fmol/4 hr/oocyte), but similar in the puromycin (15.7 fmol/4 hr/oocyte) and control groups (18.9 fmol/4 hr/oocyte). At 24 hr, the puromycin group (5.73 fmol/4 hr/oocyte) had a lower rate of incorporation, while the cycloheximide (22.6 fmol/4 hr/oocyte) and control (26.0 fmol/4 hr/oocyte) groups were similar. Cycloheximide was more effective earlier during culture, while puromycin was more effective later. When combined with ES, puromycin did have a higher rate (P = 0.10) of activation (87.8%) than with electrostimulation alone (73.0%). A final experiment evaluated the development to blastocyst after transfer to a ligated oviduct. Cycloheximide treatment in conjunction with an electric pulse did not increase the rate of compact morula or blastocyst formation. In conclusion, puromycin and cycloheximide have differential effects on protein synthesis, and although cycloheximide alone will not induce activation in porcine oocytes, it is very effective in generating activated oocytes in combination with electrostimulation. © 1995 Wiley-Liss, Inc.     

The characteristics of enhanced DNA synthesis in L cells treated with interferon and arginine-deprived

DNA synthesis as measured by incorporation of radioactive thymidine or deoxyadenosine is enhanced up to ten-fold in interferon-treated L929 cells as compared to cells not treated with interferon when both were incubated in arginine-deficient medium. Rates and amounts of RNA and protein synthesis were only modestly increased by comparison. The effect was marked after 2 days of arginine starvation and at an interferon dose of 20 PRD₅₀. The labelled product was DNasesensitive and its synthesis was inhibited by cytosine arabinoside and hydroxyurea, but not by puromycin or actinomycin. Autoradiographic studies indicated that label incorporation was entirely intranuclear in interferon-treated cells and substantially greater than non-treated cells.     

Amino acid uptake in the developing chick embryo heart. The effect of insulin on glycine and leucine accumulation

1. The accumulation of [1-(14)C]glycine and the uptake, accumulation, incorporation (into protein, lipid, glycogen) and oxidation of l-[1-(14)C]leucine in 5-day-old chick embryo hearts were investigated in vitro, and the effects of insulin, puromycin and 4-methyl-2-oxopentanoic acid on these processes were studied. 2. With glycine, the ratio of concentration of the labelled amino acid in the cell water to that in medium markedly exceeded unity. Insulin significantly increased this ratio. Puromycin did not prevent the insulin effect. 3. With leucine, the concentration ratio of the labelled amino acid between intracellular and extracellular water approached unity in the absence of puromycin and was doubled by its presence. In neither case did insulin substantially alter this ratio. The addition of 4-methyl-2-oxopentanoic acid had no effect in the absence of insulin, but produced a significant increase of the concentration ratio in the presence of the hormone. 4. Leucine uptake was increased slightly by insulin in all experimental conditions except in the presence of puromycin, where a more pronounced stimulation was observed. The hormone had no effect on the incorporation of the labelled amino acid into protein, but accelerated its oxidation to carbon dioxide; the latter effect was particularly evident in the presence of puromycin and disappeared after the addition of 4-methyl-2-oxopentanoic acid.     

Protein metabolism in goldfish brain

Abstract— Protein metabolism of goldfish brain was studied in vivo by means of intraperitoneal or intracranial injections of [³H]leucine and compared with concomitant studies in the mouse. Heterogeneity of turnover values was observed. Long turnover times were seen relative to other organs examined. The free amino acid pools of goldfish brain were determined, and the fate of tritium from labelled leucine was followed at various times after injection. Following ‘chasing’ with large amounts of unlabelled leucine or protein inhibitors shortly after isotope injection, further incorporation was arrested, but examination of the labelled protein over a period of 2 weeks indicated a slow decay, similar to that seen without ‘chasing’. Possible use of ‘pulse-chase’ experiments in vivo in animals is discussed in relation to behavioural studies.     

Effects of Cycloheximide upon Formation of Ribonucleic Acid Cytidylic and Uridylic Acids

Concentrations of cycloheximide as low as 3 μg/ml inhibited incorporation of labeled orotic acid or uridine into RNA cytidylic acid of soybean (Glycine max) hypocotyl sections. Even lower concentrations of this well known protein synthesis inhibitor interfered with conversion of labeled cytidine into RNA uridylic acid. Both cycloheximide and puromycin inhibited absorption of ³H-phenylalanine and its incorporation into protein, but puromycin did not significantly affect the labeling patterns of RNA cytidylic and uridylic acids when orotic acid-6-¹⁴C was fed. Results give further support to the hypothesis that cycloheximide inhibits the interconversion of uridine and cytidine nucleotides, presumably by acting as a glutamine antagonist in the glutamine-dependent reaction catalyzed by cytidine triphosphate synthetase.     

Effect of puromycin on cartilage proteoglycan structure and capacity to bind hyaluronic acid

Rat chondrosarcoma chondrocytes were cultured in the presence of puromycin to induce premature termination of core protein precursor. The structure and function of intracellular and extracellular proteoglycans were assessed by molecular sieve chromatography and polyacrylamide gel electrophoresis. [³H]Serine incorporation was maximally inhibited by 3 × 10^?4m puromycin but unaffected by 10 ^?5m puromycin. Proteoglycans synthesized in the presence of puromycin exhibited increased monomer size due to increased chondroitin sulfate chain size, typical of proteoglycans synthesized in the presence of protein synthesis inhibitors, but no loss in ability to bind to hyaluronic acid; and no loss in core protein size was observed after treatment with chondroitinase. These data suggest that chondrocytes select only completed or nearly completed core protein molecules to process into proteoglycans.     

Acetylcholine formation from glucose following acute choline supplementation

The effects of choline administration on acetylcholine metabolism in the central nervous system are controversial. Although choline supplementation may elevate acetylcholine (ACh) content in brain, turnover studies with labelled choline precursors suggest that systemic choline administration either has no effect or actually diminishes brain ACh synthesis. Since choline supplementation elevates brain choline levels, the apparent decreases in previous turnover studies may reflect dilution of the labelled choline precursor pool rather than altered ACh formation. Therefore, brain ACh formation from [U-¹⁴C]glucose was determined after choline supplementation. A two to three fold elevation of brain choline did not alter ACh levels or [U-¹⁴C]glucose incorporation into ACh in the cortex, hippocampus or striatum. Although atropine stimulated ACh formation from [U-¹⁴C]glucose in hippocampus, two to three fold increases in brain choline did not augment ACh synthesis or content in atropine pretreated animals. Atropine depressed brain regional glucose utilization and this effect was not reversed by choline treatment. These results suggest that shorttern elevation of brain choline does not enhance ACh formation from [U-¹⁴C]glucose, and argue against enhanced presynaptic cholinergic function after acute, systemic choline administration.Special issue dedicated to Dr. Louis Sokoloff.     

The role of phenylalanine in differentiating amphibian melanocytes

To see whether phenylalanine serves as a substrate in melanogenesis, hanging drop explants of neural crest from amphibian (Ambystoma maculatum and A. mexicanum) embryos were subjected on the seventh day in vitro to treatment with phenylalanine-³H and studied by means of light microscopic radioautography. All melanin-containing cells showed label. On the other hand, when puromycin, an inhibitor of protein synthesis, together with the labeled amino acid was administered to the cultures, no radioactivity was incorporated by pigmented cells. Comparable results were obtained when leucine was substituted for phenylalanine. In control experiments, puromycin and labeled tyrosine or 3,4-dihydroxyphenylalanine (DOPA), both known precursors for melanin synthesis, were administered to the neural crest cultures. In these experiments, puromycin had no effect on the incorporation of label by pigmented cells. Our data strongly indicate that in differentiating amphibian melanocytes with functional pigment-forming systems, phenylalanine is used in protein synthesis, but does not serve as a substrate for the tyrosine-tyrosinase system.In another series of experiments, explants of neuroepithelium (neural crest anlage) were grown from the time of explantation to the seventh day in vitro in the presence of phenyllactic acid, an analog of phenylalanine. Pigment cells developed normally.These results suggest that phenylalanine plays little or no role in pigment cell differentiation.     

Stimulatory effect of gamma-aminobutyric acid upon animo acid incorporation into protein by a ribosomal system from immature rat brain

Abstract—

• 1 GABAstimulated the incorporation of L-[U-¹⁴C]leucine, primarily into the particulate protein of a ribosomal system from immature rat brain, but not from immature rat liver.
• 2 The GABA effect required the presence of Na⁺ and occurred at GABA concentrations which are thought to be physiological (1–5 mM).
• 3 Of all other amino acids tested at tissue extract concentrations in the system, only glycine had a similar effect. No analogues of GABA tested had a significant stimulatory effect upon leucine incorporation into protein, with the exception of homocarnosine which was mildly stimulatory.
• 4 The effect of GABA upon the incorporation of L-[U-¹⁴C]leucine was examined in the presence of added amino acid substrates, both individually and as mixtures. Also, the incorporation of L-[U-¹⁴C]leucine was compared with incorporation of L-[U-¹⁴C]Iysine and L-[U-¹⁴C]phenylalanine. The results are discussed in terms of GABA interaction with activating, transfer and transport mechanisms of other amino acids, inhibition of proteinase activity, and the possibility that GABA is stimulating the synthesis or turnover of specific proteins in the brain ribosomal system.
• 5 The results illustrate the fact that studies of ‘protein synthesis’ in immature rat brain ribosomes, as measured by amino acid incorporation, will yield answers which depend heavily upon substrate conditions and upon the labelled amino acid used as the marker for protein synthesis or turnover.

     

Nitrate reductase in agrostemma githago comparison of the inductive effects of nitrate and cytokinin

The effect of nitrate and cytokinin on the induction of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in embryos of Agrostemma githago was compared. Increased enzyme levels in response to treatment with the cytokinin benzyladenine were not correlated with a general stimulation of protein synthesis or a general reduction of protein breakdown. Actinomycin D did not inhibit the formation of nitrate reductase in response to nitrate or the cytokinin. Cycloheximide and puromycin inhibited the induction by the hormone to the same extent as, or even more than, the incorporation of [¹⁴C]leucine into protein. Induction of nitrate reductase by nitrate was much less susceptible to inhibition by cycloheximide and puromycin than induction of the enzyme by benzyladenine. When induction of nitrate reductase was carried out in the presence of ²H₂O, isopycnic equilibrium centrifugation in CsCl showed that incorporation of ²H into the enzyme had occured. The increase in the buoyant density of nitrate reductase was the same whether the enzyme was induced by nitrate or by benzyladenine, indicating that at least part of the nitrate reductase molecule was newly synthesized in both instances.     

PROTEIN SYNTHESIS IN THE ISOLATED MAUTHNER NERVE FIBRE OF GOLDFISH

Abstract— Mauthner nerve fibres isolated from the spinal cord of goldfish were incubated, in the presence of radioactive amino acids for varying periods of time. It was found that the Mauthner fibre synthesizes proteins in the absence of cell nuclei. Amino acid incorporation showed sensitivity to puromycin and to acetoxycycloheximide but resistance to chloramphenicol. Only slight inhibition was caused by actinomycin-D. The contribution of the denuded axon to the total protein synthesis was about 30 per cent per unit length Mauthner fibre. The remaining activity was due to the myelin sheath compartment. Fractionation experiments showed that the incorporation in the sheath was due to components other than the myelin lamellae. The subcellular distribution of newly synthesized proteins in the isolated and incubated Mauthner fibre was compared to that found in the incubated spinal cord. The results strongly suggested the existence in the Mauthner fibre of a primary microsomal, rather than a mitochondrial, protein synthesizing system.     

The influence of early protein-calorie malnutrition on neuronal and glial protein synthesis

The effect of pre- and postnatal undernutrition, produced according to the method of Chow and Lee (3), on the rate of protein synthesis in the brains of rats 11, 21, 34 and 90 days of age was studied by measuring the incorporation ofl-[¹⁴C]valine in vivo andl-[³H]lysine in vitro. Both in vivo and in vitro experiments were performed with high concentration of the precursor to decrease the effects of pool variations and protein degradation. Particular interest was given to the effects of this form of early protein-calorie malnutrition (PCM) on neuronal and glial cells which were isolated from the brains by gradient centrifugation. Brain protein synthesis measured in vivo which showed a peak at 21 days in both animal series, was depressed by PCM at 11 days but stimulated at 34 days of animal age. Small effect was observed in the 90-day-old animals. A similar response as in whole brain was seen for neuronal cells, while glial cells showed a different reaction. Studies of in vitro protein synthesis did not reveal appreciable effects of undernutrition in whole brain. Both neuronal and glial cells showed a moderate but not statistically significant elevation of protein synthesis in animals subjected to early PCM.     

THE EFFECT OF EXTRACELLULAR POTASSIUM CONCENTRATION ON PROTEIN SYNTHESIS IN GUINEA-PIG HIPPOCAMPAL SLICES1,2

Abstract— We have measured the effect of small variations in extracellular potassium concentrations ([K⁺]) upon the incorporation of radioactively labelled amino acid into the protein of the isolated guinea-pig hippocampal slice. The slice is super-perfused with glucose fortified buffer and maintains an ATP concentration of 33–36 nmol/mg protein and incorporates lysine into protein at a rate of 0.82 pmol/(ig protein/h. Within the range of extracellular K⁺ from 1.3 to 8.1 mil the change in the rate of lysine incorporation into protein is proportional to the logarithm of the extracellular K⁺ concentration. Incorporation increases by about 100% over this range. Measurements of the specific activity of the presumed intracellular amino acid pool indicate that the effect of changes in extracellular [K⁺] is to alter the rate of protein synthesis rather than alter the availability of radioactively labelled precursor. Altering extracellular [K⁺] does not affect tissue levels of ATP or creatine phosphate, indicating that the effect on amino acid incorporation does not result from an effect upon energy metabolism. It is suggested that this effect of extracellular [K.⁺] may be a means by which changes in cerebral electrical activity lead to changes in the rate of protein synthesis in brain.