DISAGGREGATION OF BRAIN POLYSOMES AFTER d-LYSERGIC ACID DIETHYLAMIDE ADMINISTRATION IN VIVO: MECHANISM AND EFFECT OF AGE AND ENVIRONMENT1

Abstract— The cell-free protein synthesis activity and tRNA content of the increased pool of brain monosomes produced after d -lysergic acid diethylamide (LSD) administration were analyzed. Decreased reinitiation of protein synthesis rather than RNase activation or premature termination was shown to be the mechanism which results in brain polysome disaggregation after administration of the drug in vivo. At a constant dosage of 50 μg/kg the degree of polysome shift increases with age from 3-week-old rabbits to adults. There is also an extensive disaggregation of fetal brain polysomes when LSD is administered maternally. The LSD-induced polysome shift was shown to be altered by holding cage environment, pre-LSD sedation and post-LSD handling with brief restraint. It was apparent that elements of environment and physiological arousal were involved in the macromolecular effect of the drug on the protein synthesis apparatus of the brain.     

IN VlTRO BINDING OF PHENYLALANYL-tRNA TO NEONATAL AND ADULT MOUSE BRAIN RIBOSOMES

The ability of brain ribosomes, isolated from mice of various ages, to bind phenylalanyl-tRNA was measured under various reaction conditions. In the presence of template RNA (polyuridylic acid) the binding could be measured by both enzymic and non-enzymic assays. In general, the binding requirements for the brain system were similar to those previously described for microbial and eukaryotic systems. Although previous studies have shown that ribosomes obtained from increasingly older mow brain tissue were less active in polyphenylalanine synthesis, no significant differences in phenylalanyl-tRNA binding to polysome complexes could be detected. The binding of phenylalanyl-tRNA by ribosomes isolated from both neonatal and mature mouse brain tissue was similar with regard to GTP and polyuridylic acid dependence, magnesium ion concentration and reaction kinetics. Similar binding of phenylalanyl-tRNA by young and mature brain ribosomes was also measured with ribonucleoprotein particles previously stripped with puromycin. The results are discussed in light of the rapid alteration of macromolecular synthesis during postnatal brain development and the possible role of the interaction between ribosomes and tRNA.     

TRANSFER RNA AND THE REGULATION OF PROTEIN SYNTHESIS IN RAT CEREBRAL CORTEX DURING NEURAL DEVELOPMENT

Protein synthesis was measured in ribosomal systems derived from the cerebral cortex of 5-and 35-day-old rats. Under optimal conditions incorporation of radioactive leucine per mg ribosomal protein was four times higher with ribosomes from the younger animals than with ribosomes from the 35-day-old rats. This suggests that a decrease in the rate of protein synthesis occurs during neural development. Both ribosomes and the pH enzyme fraction from the cerebral cortex of 35-day-old rats had lower activities than preparations from the younger rats. Cerebral cortical ribosomes from 35-day-old animals had a lower polyribosome content than similar preparations from 5-day-old rats. A three-fold higher requirement for the pH 5 enzyme fraction was observed with the ribosomal system from 5-day-old rats, an observation which correlated with the yields of pH 5 enzyme and ribosomal protein from the younger tissue. The nature of the changes in the composition of the pH 5 enzyme fraction was investigated. Methylated albumin kiesselguhr (MAK) and Sephadex G-75 column chromatography showed that RNA from the pH 5 enzyme fraction was heterogeneous, containing tRNA, rRNA, and a small molecular weight RNA. This latter RNA, perhaps a degradation product of rRNA, comprised the greatest portion of RNA from the pH 5 enzyme fraction of cerebral cortex. The data obtained with MAK chromatography were used to estimate the total tRNA content of the cerebral cortex, with no age-related differences being observed. Since evidence of RNA degradation was seen, tRNA was also isolated by phenol extraction of whole cerebral cortex in the presence of bentonite. Purification of tRNA by NaCl and isopropanol fractionation gave preparations with no detectable rRNA or small molecular weight RNA. With this purification method, the tRNA yield was greater than estimated by the MAK method, demonstrating that losses of tRNA occurred during the cell fractionation steps. With the purification method 1.6 times more tRNA was obtained from the cerebral cortex of 5-day-old animals than from the older tissue. This higher level of tRNA in the younger, more active tissue appeared to involve all tRNA species, since in vitro aminoacyiation studies revealed nearly identical acceptance values for 18 individual amino acids. These results suggest that the rate of protein synthesis in cerebral cortex is regulated in part by the total amount of tRNA present to translate the higher level of polysome-bound mRNA.     

THE TURNOVER AND SUBCELLULAR LOCALIZATION OF THE CHICK FOREBRAIN SMALL NUCLEAR RNAs

Analysis of 2 day old chick forebrain RNA on polyacrylamide gels revealed the presence of the 29S, 18S, 5.5s and 5s rRNAs, 4S tRNA and the small mol wt nuclear RNA (snRNA) species K, L, C, DD and G. The turnover of these molecules was studied following the intracerebral injection of [³H]uridine into 2 day old animals. The specific activities of these molecules declined over a 23 day time course and all displayed first-order decay kinetics. Apparent half-lives (in days) for 29S (7). 18S (S), L (24), C (6.5), DD′ (8.5), 5.5S (7), 5S (13), G (6) and 4S (7) were obtained. The subcellular localization of the snRNAs was studied in 5 day old chick forebrain. The snRNAs accounted for almost 17% of nuclear RNA, in contrast to their low levels in whole forebrain (1.2%). This was due to an approx 20-fold nuclear enrichment of C, DD′, G′ and H. Relatively low levels of K and L were present within the nuclear fraction. However, K and L, and to a lesser extent C and DD, were readily detected within the post-microsomal supernatant. Production of a pH 5 enzyme fraction from this supernatant resulted in a 2-3-fold enrichment of K, L, C and DD′ with respect to tRNA. The polysome containing fractions also displayed significant levels of L with about 1 mol of L per 30 of 5S rRNA. A new method for the determination of the subcellular distribution of the snRNAs is described. It depends upon the purity of the nuclear, microsomal and cell sap subfractions rather than upon total recoveries from such fractions. Utilizing the relative amounts of these RNA species within whole forebrain and the major subfractions, distributions (as a percentage within each fraction) were obtained. The observation of significant levels of K, L, C and DD within the cytoplasmic fractions raises some doubts about previous suggestions of an exclusively nuclear role for these molecules. It is proposed that the snRNAs may be involved in a multistage interaction within the process of eukaryotic gene expression.     

tRNA METHYLTRANSFERASE ACTIVITY IN NEONATAL AND MATURE MAMMALIAN NEURAL TISSUE

Abstract— The activity and kinetic characteristics of tRNA methyltransferases were measured with enzyme preparations obtained from neonatal and adult mouse brain tissue. Both neonatal and mature brain enzyme preparations were shown to contain a considerable amount of protein methylase activity which could interfere with the measurement of the tRNA methyltransferases. When increasing amounts of the unfractionated enzymes from young and adult neural tissue were added to reaction mixtures, the saturation kinetics were found to be considerably different. However, fractionation of the samples by precipitation at pH 5 resulted in an increase in the enzyme activity of preparations obtained from adult brain. Although the precipitation at pH 5 allowed a quantitative recovery of the enzyme activity of immature brain samples, this partial purification step led to an apparent activation of the tRNA methyltransferases in adult preparations. This activation was shown to be independent of differential changes in the thermolability of the enzymes but rather to be associated with an increase in the sites methylated and the measured affinity of the adult enzyme preparations with the tRNA substrate. Nicotinamide, a potent inhibitor of tRNA methyltransferase activity in other tissues, was shown to be ineffective in modulating brain tRNA methyltransferase activity. The results are discussed in light of the possible modulation of the activity of specific enzyme species and the alterations in the synthesis of nucleic acid precursors during neural development.     

COMPARATIVE EFFECTS OF HYPOTHYROIDISM, HYPERTHYROIDISM AND UNDERNUTRITION ON THE PROTEIN AND NUCLEIC ACID CONTENTS OF THE CEREBELLUM IN THE YOUNG RAT

Abstract— The effects of hypothyroidism, hyperthyroidism and undernutrition on the development of the protein and nucleic acid contents of the cerebellum were studied in young rats ranging in age from 6 to 35 days. Foetal and neonatal propylthiouracil treatment caused a delayed cell multiplication in the organ but this delay had disappeared at 35 days. As early as the age of 10 days, the total cerebellar RNA and protein contents in these hypothyroid animals were lower than in the normal animals but the mean cellular contents of RNA and protein were not lowered before the age of 21 days. Slight neonatal hyperthyroidism, induced by the daily injection of a relatively small dose of thyroxine, produced an accelerated cell multiplication during the first postnatal week and afterwards a delay in cell proliferation. In the hyperthyroid animals, the mean cellular protein content, initially higher than normal, tended to be lower at 35 days. At this age, the cell number had nearly returned to normal. Undernutrition directly led to a reduced cell proliferation and the reduced cell number still persisted at 35 days. At first, the cells were on the average larger than normal, but the effect on the mean RNA and protein contents per cell was not persistent in the underfed animals.
This study correlates the degree of neonatal hyperthyroidism with the further development of the cell population of the cerebellum and emphasizes the importance of the events which take place just after birth in the cerebellum submitted to an excess or a deficiency of thyroid hormone. In addition, the results are discussed in relation to data concerning the morphogenetic action of thyroid and of underfeeding on the cerebellum.     

Axoplasmic Transport of Transfer RNA in the Chick Optic System

It has previously been shown that 4S RNA is transported in the optic nerve of the chick, but that no movement of rRNA can be detected. The 4S component behaved as though it were composed mainly of transfer RNA (tRNA), but the possibility remained that it could contain significant amounts of material resulting from RNA degradation. The transport of this 4S component has been examined in more detail to determine its nature. In addition, the transported material was examined to establish whether the transport of tRNA is a general phenomenon or that there are only a limited number of species involved. This was done using the same principles applied in the previous study; i.e., the specific activities of separated 4S RNA species appearing in the optic tectum 4 days after intraocular injection of [3H]uridine were compared with that of 5S RNA, a nontransported species. The separation was accomplished using 2.8-5-10-17% slab polyacrylamide gels, and 18 separate regions of 4S species could be identified. The results show that at least most, if not all 4S RNA species are transported. In a separate series of experiments the 4S RNA was aminoacylated and again separated on slab gels. In this instance, the RNA was labelled with [3H]uridine and the aminoacyl component with [14C]amino acids. Gel profiles of these dual-labelled components showed excellent correspondence between the two labels, demonstrating that 4S RNA species could be aminoacylated and were therefore tRNA species.(ABSTRACT TRUNCATED AT 250 WORDS)     

ENZYMES OF NUCLEIC ACID METABOLISM IN THE BRAINS OF YOUNG AND ADULT RATS

A number of precursors of RNA are incorporated several-fold more readily into the RNA of brain slices from 10-day-old rats than into RNA of slices from adult animals. The brains of the young animals show moderately higher levels of some of the anabolic enzymes of RNA metabolism including RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase; EC 2.7.7.6) and substantially lower levels of the degradative enzymes, the nucleoside phosphorylases. The data suggest that all the enzymes work in a concerted fashion to produce an increased rate of synthesis in young animals rather than that any single controlling enzymic event is responsible.     

Age-related qualitative and quantitative changes in tRNA population of rat skeletal muscle

Total tRNA was purified from skeletal muscle of young, adult and old female albino rats. Age-dependent variation of total tRNA was the same with respect to tRNA content and biological activity as measured by amino acid acceptor capacity. The tRNA content was more in young rats and showed a gradual decrease in the adult and old rats. The relative abundancy of eleven aminoacyl-tRNAs were checked at each age and during aging. Arginyl, glutamyl and tyrosyl-tRNAs do not show any quantitative or qualitative change with age.     

Recessive nonsense-suppression in yeast Saccharomyces cerevisiae

Summary The shift of recessive suppressor mutant of yeast Saccharomyces cerevisiae from permissive to restrictive conditions is accompanied by polysome decay and accumulation of 80 S ribosomes (Smirnov et al., 1976). In this paper some properties of 80 S ribosomes are studied. It is demonstrated that polysome decay under non-permissive conditions is not the consequence of the impairement of RNA synthesis. More than 70% of 80 S ribosomes accumulated under non-permissive conditions contain bound peptidyl-tRNAs localized in P-ribosomal site. tRNA moiety of bound peptidyl-tRNA is able to accept all 20 natural amino acids after chemical deacylation. Therefore it is not a specific isoacceptor species but rather total tRNA that is bound to ribosomes. The polypeptide residues of these peptidyl-tRNAs are heterogeneous in size. Their molecular weights are comparable with the molecular weights of the completed polypeptides. Some of the 80 S ribosomes accumulated under non-permissive conditions contain poly-A RNA. In conclusion, possible mechanism of the impairement of translation under non-permissive conditions in recessive suppressor strain is discussed.     

Age related changes in total DNA and RNA and incorporation of uridine and thymidine in rat brain

1. Total brain DNA and total brain RNA and the incorporation of thymidine[14C] and uridine[3H] were measured in young and aged rats. 2. From 20 days to the time of sexual maturation, both DNA and RNA levels increase. Total RNA exceeds total DNA at all ages. Comparatively, the ratio of total DNA/RNA is higher in young than in aged animals. 3. The incorporation of thymidine[14C]/g of DNA and of uridine[3H]/g of RNA decreases with age. This decrease is rapid in young animals. After 350 days of age, the incorporation becomes very low. The significance of data is discussed.     

Glycosaminoglycans of brain during development.

The concentration of hyaluronic acid, chondroitin sulfate, and heparan sulfate was measured in rat brain at 2-day intervals from birth to 1 month of age, and in 40-day-old and adult animals. The levels of all three glycosaminoglycans increased after birth to reach a peak at 7 days after which they declined steadily, attaining by 30 days concentrations within 10% of those present in adult brain. The greatest change was seen in hyaluronic acid, which decreased by 50% in 3 days, and declined to adult levels (28% of the peak concentration) by 18 days of age. Only heparan sulfate showed a significant change in metabolic activity during development (a fourfold increase in the relative specific activity of glucosamine), most of which occurred after 1 week of age. In 7-day-old rats almost 90% of the hyaluronic acid in brain is extractable by water alone, as compared to only 15% in adult animals, and this large amount of soluble hyaluronic acid in young rat brain is relatively inactive metabolically. On the basis of our data we propose that the higher amounts of hyaluronic acid found in very young brain may be responsible for the higher water content of brain at these ages, and that the hydrated hyaluronic acid serves as a matrix through which neuronal migration and differentiation may take place during early brain development.     

The role of isoacceptor transfer RNAs in regulation of age-related changes in the rate of protein synthesis

In cell-free protein-synthesizing systems containing an S30 extract from liver and brain cortex tissues of 22-day-old fetuses and of male WAG rats (1-900 days old), the minimal rate of protein synthesis was observed in the fetuses, while the maximal one - in 7-day-old animals. The difference in the rates of protein synthesis correlated with the minimal concentration of total tRNA in the former group and with its maximal concentration in the latter. In fetal tissues, an addition to cell-free systems of total tRNA isolated from homologous tissues of 7-day-old animals augmented protein synthesis up to a level observed in 7-day-old animals, whereas in the tissues of animals belonging to other age groups total tRNA had a far less pronounced stimulating effect which decreased with age. Fractionation of total tRNA and analysis of effects of individual tRNAs on protein synthesis demonstrated that the stimulating influence was induced by tRNA(2Arg), tRNA(4Arg) and tRNA(2Val) from brain cortex and by tRNA(2Leu), tRNA(5Leu), tRNA(2Val), tRNA(1Met) and tRNA(2Met) from liver.     

Effects of quantity and quality of dietary protein on the brain polysome profile in aged rats

The purpose of this study was to determine whether the quantity and quality of dietary protein affected the polysome profile of the brain in aged rats. Two experiments were done on three groups of aged rats (30 wk) given the diets containing 20% casein, 5% casein, or 0% casein (experiment 1), and 20% casein, 20% gluten, or 20% gelatin (experiment 2) for 10 d. The aggregation in brain ribosomes declined with a decrease of quantity and quality of dietary protein except in the hippocampus. The RNA concentration (mg RNA/g protein) did not differ among the three groups varying the dietary protein in any brain regions. The results suggest that the higher quantity and quality of dietary protein improves the polysome profile in the brain of aged rats, and that the polysome profile is at least partly related to the mechanism by which the dietary protein affects brain protein synthesis in aged rats.     

Methyldeficient mammalian 4S RNA: evidence for L-ethionine-induced inhibition of N6-dimethyladenosine synthesis in rat liver tRNA

The nucleotide composition of 4s RNA from livers of rats fed with a diet containing 0.3% D-ethionine was found to be identical with that from untreated animals. In contrast, one single modified nucleotide was absent in 4s RNA from livers of rats fed with a 0.3% L-ethionine diet. The minor nucleo=tide was also absent in liver 4s RNA from rats fed with a 0.3% L-ethionine diet followed by ten days of normal food. It was identified after dephosphorylation by ultraviolet absorption spectra, cochromatography with authentic material and mass spectra as N(6)-dimethyladenosine. It is concluded that S-adenosylethionine, the primary product of L-ethionine in the liver, causes strong and selective inhibition of the specific RNA-methylase responsible for adenosine to N(6)-dimethyl=adenosine methylation in rat liver 4s RNA. Compared to the strong inhibition of N(6)-dimethyladenosine formation described here, L-ethionine-dependent ethylation of liver 4s RNA is far less efficient. The quantitation of l-methyladenosine, ribothymidine and 3'-terminal adenosine in this 4s RNA as well as its aminoacid acceptor activity is typical for tRNA; hence it may be concluded that N(6)-dimethyladenosine is a component of rat liver tRNA. This may demonstrate the first evidence for the existence of specifically methyl-deficient mammalian tRNA. A possible correlation between the activity of L-ethionine as a liver carcinogen and its ability to induce the formation of methyl-deficient tRNA by selectively inhibiting the synthesis of N(6)-dimethyladenosine on the tRNA level in the same organ is discussed.     

PARTICIPATION OF DOPAMINE- AND SEROTONIN-RECEPTORS IN THE DISAGGREGATION OF BRAIN POLYSOMES BY l-DOPA AND l-5-HTP

Abstract— It has previously been shown that the disaggregation of brain polysomes and suppression of brain protein synthesis observed in rats given the amino acids l -dopa or l -5-HTP is mediated by the decarboxylation products dopamine and serotonin. Present studies demonstrate that the poly-some disaggregation is caused by the interactions of the monoamines with specific receptor sites. Thus, dopa-induced disaggregation is blocked if rats are pretreated with haloperidol or pimozide (but not methysergide or cyproheptadine), while 5-HTP-induced disaggregation is blocked by methysergide or cyproheptadine (but not by haloperidol or pimozide).
Pretreatment of rats with MK-486, a drug that inhibits dopa decarboxylase in blood vessels and peripheral tissues but not brain, does not block dopa-induced brain polysome disaggregation; hence this disaggregation depends on the interaction of dopamine with receptors in the brain parenchyma. Brain polysomes are not disaggregated in rats given intraperitoneal apomorphine (or intracisternal dopamine). The disaggregation caused by dopa is not reduced in animals pretreated with sufficient intracisternal 6-hydroxydopamine to cause major damage to catecholaminergic nerve terminals.     

A very poorly expressed tRNA(Ser) is highly concentrated together with replication primer initiator tRNA(Met) in the yeast Ty1 virus-like particles.

The analysis of the tRNAs associated to the virus-like particles produced by the Ty1 element revealed the specific packaging of three major tRNA species, in about equal amounts: the replication primer initiator tRNA(Met), the tRNA(Ser)AGA and a tRNA undetected until now as an expressed species in yeast. The latter tRNA is coded by the already described tDNA(Ser)GCT. This tRNA is enriched more than 150 fold in the particles as compared to its content in total cellular tRNA where it represents less than 0.1% (initiator tRNA(Met) and tRNA(Ser)AGA being 11 and 4 fold enriched respectively). This tRNA is the only species coded by the tDNA(Ser)GCT gene which is found in three copies per genome since no other corresponding expressed tRNA could be detected. This gene is thus very poorly expressed. The high concentration of tRNA(Ser)GCU in the particles compared to its very low cellular content led us to consider its possible implication in Ty specific processes.     

Methionine Sulfoximine In Vivo Modifies the tRNALys Pool of Developing Rat Brain

Abstract: tRNA was extracted from brains of 3-, 8-, and 18-day-old rats that were injected intracerebrally, 45 min before death, with [³H]methyl methionine or [8-³H]guanosine, and intraperitoneally, 3 h before death, with l -methionine-dl-sulfoximine (MSO), a methylation-activating convulsant agent. Although there was no effect of age or of MSO on the per gram yield of tRNA, its specific radioactivity (dpm/A₂₆₀) was highest at 3 days in both the control and the MSO groups. Age- and MSO-related changes in the tRNA^Lys content of the brain tRNA pool were investigated by means of benzoylated DEAE- cellulose (BDC) and reverse-phase chromatography (RPC). BDC chromatography revealed tRNA^Lys species in the brains of the MSO-treated animals that were absent in control brains. Of particular interest was the finding that differences in RPC-5 chromatographic mobility between control and MSO-tRNA^Lys species were abolished by conversion to lysyl-tRNA, suggesting that the MSO-elicited change(s) in tRNAL^Lys structure involved the binding site(s) for lysine. Two additional findings were made: (a) lysine acceptance by the [³H]methyl-labeled tRNA^Lys purified from brains of the MSO-treated animals was higher than that of controls at 18 days; and (b) omission of the BDC chromatographic step accentuated the differences in mobility on RPC-5 columns between tRNA^Lys species of control and MSO-treated brains. Lastly, we found that some tRNA^Lys species present in the MSO-treated brains contained significantly different proportions of N²-methyl guanine and 1-methyl adenine, relative to controls. These MSO-elicited changes in the methyl base content of tRNA^Lys of immature rat brain are the first evidence of an alteration of brain tRNA structure by a centrally acting excitatory agent.     

Macromolecular synthesis in mitochondria isolated from different regions of developing rat brain

DNA, RNA, and protein synthesis in mitochondria isolated from cerebral hemispheres, brain stem, and cerebellum of 10- and 30-day-old rats was measured. Synthesis of different macromolecules was affected by the respective mitochondrial specific inhibitors, showing a good level of purity of mitochondrial preparations. DNA and protein synthesis in 10-day-old rats was about 70% higher than in 30-day-old animals. In contrast, RNA synthesis did not decrease with age in all the regions examined.