THE REGULATION OF RNA SYNTHESIS AND PROCESSING IN THE NUCLEOLUS DURING INHIBITION OF PROTEIN SYNTHESIS

The effect of protein synthesis inhibition by cycloheximide on nucleolar RNA synthesis and processing has been studied in HeLa cells. Synthesis of 45S RNA precursor falls rapidly after administration of the drug. However, the nucleolar content of 45S RNA remains relatively constant for at least 1 hr because the time required for cleavage of the precursor molecule into its products is lengthened after treatment with cycloheximide. The efficiency of transformation of 45S RNA to 32S RNA remains constant with approximately one molecule of the 32S RNA produced for each cleavage of a molecule of 45S RNA. However, shortly after the cessation of protein synthesis the formation of 18S RNA becomes abortive. The amount of 32S RNA present in the nucleolus remains relatively constant. After long periods of protein synthesis inhibition the 28S RNA continues to be synthesized and exported to the cytoplasm but at a greatly reduced rate. When the protein synthesis inhibitor is removed, a prompt, although partial, recovery in the synthesis rate of 45S RNA occurs. The various aspects of RNA synthesis regulation and processing are discussed.     

ON THE MECHANISM OF ELECTROSHOCK-INDUCED INHIBITION OF PROTEIN SYNTHESIS IN RABBIT CEREBRAL CORTEX

Abstract— The mechanism of electroshock (ES)-induced inhibition of protein synthesis in rabbit cerebral cortex has been investigated by using a cell-free system. The protein biosynthetic activity of the post-mitochondrial supernatant (PMS) obtained from the cerebral cortex of ES-treated animals was found to be markedly lower than in controls (C). This inhibition was accompanied by a decrease of polysomes and an increase of monomers. In addition, a relative increase in light polysomes was evident at short intervals after ES treatment. No difference was found in the total soluble activity and in the activity of the elongation factors and ribonuclease present in the cell sap of C and ES animals. The biosynthetic activity of ES-total. free and membrane-bound ribosomes was approx 45% lower than that of the corresponding C fractions: polysome/monomer ratios were similarly reduced. The total content of cortical ribosomes was not affected by ES. Following ES treatment there was no change in the ribo-somal ability to elongate, terminate and release polypeptide chains, nor a decrease in the polysomal content of poly(A)-containing mRNA. These data strongly suggest that the ES-induced inhibition of protein synthesis results from a defect in the initiation process. The possible mechanisms mediating this defect have been discussed.     

THE SYNTHESIS OF 5S RNA AND ITS REGULATION DURING EARLY SEA URCHIN DEVELOPMENT

De novo synthesis of 5S RNA and of transfer RNA (tRNA) has been demonstrated previously to occur by mid-cleavage (128-cell stage) in sea urchin embryos (24). The present study focused on determining more precisely the time of onset of activity of the genes for 5S RNA and for tRNA during sea urchin embryogenesis by preloading the GTP precursor pools of unfertilized eggs. The results showed that newly-made 5S RNA and tRNA could be detected as early as the 32-cell stage. In order to determine whether newly-synthesized 5S RNA accumulates coordinately during development with newly-made 26S (34) and 18S ribosomal RNAs (rRNAs), the relative rates of accumulation of these three RNA molecules were measured and compared at each of several stages of sea urchin embryogenesis. In contrast to the coordinated accumulation of newly-synthesized 26S and 18S rRNAs, newly-made 5S RNA accumulated in excess at the mesenchyme blastula (9-fold excess), midgastrula (5-fold excess) and prism (3-fold excess) stages. The 5S RNA/26S RNA molar ratios only approached unity in advanced (48 hr) plutei. The non-coordinated accumulation of newly-made 5S RNA with that of 26S and 18S rRNAs suggests that the accumulation of these newly-synthesized RNAs is differentially regulated during early sea urchin development.     

GABA对小鼠大脑皮质中GABA受体胚胎发育的调节

本文用ＧＡＢＡ及其受体激动剂和拮抗剂处理培养的胚胎小鼠大脑皮层神经细胞以及精确计时的妊娠小鼠,用放射配体结合法检测ＧＡＢＡＡ及ＧＡＢＡＢ的结合位点数目,研究了ＧＡＢＡ对小鼠大脑皮层ＧＡＢＡ受体胚胎发育的调节作用,结果表明：①ＧＡＢＡ可使培养１５—１７天妊龄的胚胎小鼠大脑皮层神经细胞及出生第１天的仔鼠大脑皮层中的ＧＡＢＡＡ及ＧＡＢＡＢ受体数目增加,这种作用可被蝇蕈醇（Ｍｕｓ）及巴氯芬（Ｂａｃ）分别模拟,对ＧＡＢＡＡ受体的作用可为荷包牡丹碱（Ｂｉｃ）所阻断;②用ＧＡＢＡ处理妊娠７—１３天的小鼠,仔鼠出生第１天其大脑皮层的ＧＡＢＡＡ及ＧＡＢＡＢ受体数目均无变化;③用ＧＡＢＡ处理妊娠１４—１９天的小鼠,仔鼠出生的第１天其大脑皮层中的ＧＡＢＡＡ受体数目增加而ＧＡＢＡＢ受体数目不变;④用ＧＡＢＡ处理妊娠７－１９天的小鼠,仔鼠出生第１天其大脑皮层中ＧＡＢＡＡ及ＧＡＢＡＢ受体数目增加。这说明在胚胎发育的特定时期内,ＧＡＢＡ可诱导其受体数目的增加,这个作用是由ＧＡＢＡ受体调节的。     

CATIONIC COMPOSITION OF RAT CEREBRAL CORTEX SLICES. COMPARATIVE STUDY DURING DEVELOPMENT

Abstract—

• 1 Slices of cerebral cortex isolated from 20-day-old rats, when incubated in an appropriate oxygenated saline, show only a slight increase in weight (± 0.4 per cent of initial fresh wt.). At 30 days, under the same conditions, this increase in weight is 12.4 per cent. This is similar to the value observed in adult animals (13.6 per cent).
• 2 Modifications of the cationic content of slices in the case of 20-day-old animals are very slight while in 30-day-old animals they are greater and similar to those observed with adults.
• 3 The inulin space of 20- and 30-day-old rats are the same. The swelling exhibited by the slices isolated from 30-day-old animals may reasonably be attributed to a cellular uptake of extracellular fluid. This is accompanied by an increase of the Naf concentration and by a decrease of the K⁺ concentration. The values observed are similar to those observed with adult rats.
• 4 Comparing our results with previous histological and histochemical data, it may be suggested that the modifications of cationic content, as well as the swelling of slices isolated from 30-day-old rats or adult animals, are solely attributable to changes affecting glial cells and more specifically the astrocytes.

     

癫痫大鼠大脑皮质及海马神经元NOS的变化

给大白鼠侧脑室注射马桑内酯（Ｃｏｒｉａｒｉａ Ｌａｃｔｏｎｅ, ＣＬ）（１７５×１０－ ２ｍ ｏｌ／Ｌ２μｌ）后可诱发癫痫,用ＮＡＤＰＨｄ 组织化学方法观察大脑皮质及海马ＮＯＳ阳性神经元的变化, 结果： 大脑皮质ＮＯＳ阳性神经元数目逐渐增加, 至２ｈ 达高峰, 与生理盐水组相比差异具有非常显著性意义（Ｐ＜ ００１）, 随着ＣＬ作用时间延长ＮＯＳ反应由弱变强;海马区ＮＯＳ阳性神经元２ｈ 时才出现染色明显加深。对体外培养的大脑皮质及海马神经元用ＣＬ （２５×１０－ ５ｍ ｏｌ／Ｌ） 作用１／２ｈ、１ｈ、２ｈ、４ｈ 后ＮＯＳ阳性神经元均未见明显增加。     

REGULATION OF PROTEIN SYNTHESIS DURING POSTNATAL MATURATION OF THE BRAIN

—The activities of sRNA-aminoacyl synthetases (EC 6.1.1) were investigated in the cerebral white and grey matter of rabbits subjected during their prenatal life to a single X-ray dose of 150 rad. The results of investigations have shown that ionizing radiation acting during intrauterine development of the experimental animal brings about a distinct depression of all sRNA-aminoacyl synthetase activities in the newborn irradiated litter. During the postnatal development of these animals the activities of some of the synthetases further decreased and even at adulthood, where they are normally very low, their activities were below the control values. The activities of some other synthetases, after the initial depression, showed no further decrease and at adulthood had values comparable to controls. Our results indicate clearly that prenatal exposure to ionizing radiation also affects the step of protein biosynthesis which depends on the activity of sRNA-aminoacyl synthetases.     

促甲状腺激素释放激素受体-1(TRH-R1)蛋白在大鼠出生后睾丸不同发育阶段的表达和定位

目的研究促甲状腺激素释放激素受体-1(thyrotrophin-releasing hormone receptor type-1, TRH-R1)在大鼠睾丸出生后不同发育阶段的表达,探讨其在生殖发育调节中的作用.方法应用蛋白质免疫印迹杂交技术以及免疫组织化学ABC法检测TRH-R1在8d、15d、20d、35d、60d和90d大鼠睾丸中的表达和定位,并结合图像分析技术对免疫组化结果进行统计学分析观察其在发育过程中的变化.结果免疫印迹杂交发现TRH-R1蛋白表达于15d以后各阶段的大鼠睾丸;而运用免疫组化在第8d即检测到TRH-R1的表达,以后发育过程中的各个阶段均有阳性反应细胞, TRH-R1定位于大鼠睾丸的间质细胞;免疫反应阳性物均位于胞膜和胞质,胞核区为阴性;图像分析结果表明,随着大鼠睾丸的发育,TRH-R1表达量呈增多趋势,且具有统计学差异(P＜0.01).结论本实验证明TRH-R1在出生后8d大鼠的睾丸内即有表达,并持续表达于其后各个发育阶段;TRH-R1定位于睾丸的间质细胞,其表达量随着增龄变化呈增多趋势,即同发育过程相关.     

PATTERN OF RNA SYNTHESIS IN RABBIT CORTEX DURING SLEEP

The pattern of RNA biosynthesis in rabbit cerebral cortex was examined during periods of physiological sleep and wakefulness monitored with EEG techniques. The types of newly synthesized RNA were analysed by sedimentation on sucrose gradients. In experiments lasting 60 and 105 minutes the labelling ratio of RNA sedimenting in two regions of the gradient (28 S-50 S50 S) increased with decreasing percentages of EEG synchronization. Analysis of the radioactivity distributions of five homogeneous experiments in terms of moments confirmed the dependence of the RNA synthetic pattern on the electric state of the cortex. The possible nature of the RNA species involved in this effect is discussed.     

THE PRESENCE OF BIOLOGICALLY LABILE COMPOUNDS DURING ISCHEMIA AND THEIR RELATIONSHIP TO THE EEG IN RAT CEREBRAL CORTEX AND HYPOTHALAMUS

—Two surgical methods are described in the present paper, allowing for the approximate determination of in vivo levels of ATP, lactate, glucose, pyruvate and glycogen in anatomically uninjured cortex and hypothalamus from unanaesthetized rats. It was not possible to obtain such levels for P-creatine in the 2 mm thick samples used in the present investigation. No fundamental difference was observed between the cortical and the hypo-thalamic levels of these substrates nor in their fluxes. The substrate fluxes during ischemia were correlated with electrical activity in the rat cortex and hypothalamus, recorded by means of telemetrically transmitted electroencephalograms. The electrical activity declined precipitously at 9.6 s after decapitation in the cortex, and after 12.1 s in the hypothalamus. High levels of glycogen, glucose and ATP were present at this moment, while P-creatine had declined sharply.     

PERSISTENCE OF ALTERED RNA SYNTHESIS IN RAT CEREBRAL CORTEX 12h AFTER A SINGLE ELECTROCONVULSIVE SHOCK

The effect of electroshock (ECS) on RNA synthesis in nuclei and cytoplasm of rat cerebral cortex was examined using a double label technique by intraventricular injection of [³H] and [¹⁴C]orotate. At t h after ECS, the incorporation into nuclear RNA was 80% of the control rate and the appearance of newly synthesized RNA in the cytoplasm was only 27.6%. Analysis on composite polyacrylamide-agarose gels of purified RNA showed that the ³H/¹⁴C ratio of each gel slice slowly increased with decreasing M.W. of the RNA. This has been interpreted as an inhibition in the rate of processing of nuclear RNA. When the nuclear RNA was subjected to denaturation with 50% dimethyl sulphoxide (DMSO) this effect was enhanced. In a similar experiment, rats were injected, treated to ECS and killed 12 h later. The overall incorporation into nuclear and cytoplasmic RNA was increased to 174%, and 137.5% respectively. Analysis on gels showed very little variation in the ³H/¹⁴C ratio of the steady state levels of nuclear RNA. They compared well with a control experiment where rats were injected with [³H] and [¹⁴C]orotate as described above but no ECS was applied to the [¹⁴C] labelled animals. However a 1 h pulse label given 11 h after ECS treatment revealed that the rate of incorporation into nuclear RNA still showed a decrease of 81% of the control. The nuclear RYA fractionated on gels clearly showed that the inhibition of the processing rate of nuclear RNA was still occurring. This effect was again magnified on denaturation of the RNA with DMSO. This suggests that ECS may disturb RNA metabolism in nervous tissue for much longer periods than previously realised.     

PROTEIN SYNTHESIS AND RNA SYNTHESIS DURING MITOSIS IN ANIMAL CELLS

Protein synthesis and RNA synthesis during mitosis were studied by autoradiography on mammalian tissue culture cells. Protein synthesis was followed by incubating hamster epithelial and human amnion cells for 10 or 15 minutes with phenylalanine-C¹⁴. To study RNA synthesis the hamster cells were incubated for 10 minutes with uridine-C¹⁴. Comparisons of the synthetic capacity of the interphase and mitotic cells were then made using whole cell grain counts. The rate of RNA synthesis decreased during prophase and reached a low of 13 to 16 per cent of the average interphase rate during metaphase-anaphase. Protein synthesis in the hamster cells showed a 42 per cent increase during prophase with a subsequent return to the average interphase value during metaphase-anaphase. The human amnion cells showed no significant change at prophase but there was a 52 to 56 per cent drop in phenylalanine incorporation at metaphase-anaphase as compared to the average interphase rate. Colcemide was used on the hamster cells to study the effect of a prolonged mitotic condition on protein and RNA synthesis. Under this condition, uridine incorporation was extremely low whereas phenylalanine incorporation was still relatively high. The drastic reduction of RNA synthesis observed under mitotic conditions is believed to be due to the coiled condition of the chromosomes. The lack of a comparable reduction in protein synthesis during mitosis is interpreted as evidence for the presence in these cells of a relatively stable messenger RNA.     

PROTEIN PROFILES OF RAT EMBRYO CEREBRAL CELLS DURING DIFFERENTIATION IN CULTURE

The protein composition of the particulate fraction of dissociated foetal rat cerebral cells during maturation in culture was investigated. SDS polyacrylamide gel electrophoresis showed a general decrease in the histonal components and significant changes in composition of a group of polypeptides with molecular weights ranging from 42 to 60 K. Two of these polypeptides coelectrophoresed with tubulin and actin whereas a 48 K polypeptide comigrated with the major component of the Wolfgram myelin protein. Its relative quantity appeared to approach a plateau after 8 days in culture. The myelin basic and proteolipid proteins were below detection levels in cultured cells at any time point investigated. A group of polypeptides with estimated molecular weights of 47, 51 and 52 K possibly representing synaptic proteins increased with time in culture. The appearance of a prominent band (60 K) in brain cultures and in other cells of divergent origin was demonstrated. This protein may be related to the process of cell adaptation to culture conditions. The developmental changes in the protein profile are discussed in the context of an in vitro myelinogenesis and synaptogenesis and compared with whole brain particulate and subcellular fractions.     

STUDIES ON RNA SYNTHESIS IN TWO POPULATIONS OF NUCLEI FROM THE MAMMALIAN CEREBRAL CORTEX

A method is presented for the rapid separation of cell nuclei from the rabbit cerebral cortex into two populations. The first of these consists largely of nuclei with the morphological characteristics of neuronal nuclei, the second almost entirely of nuclei with the morphological characteristics of glial cell nuclei. From studies based upon sensitivity to the toxin α-amanitin, the ratio of incorporation of different bases, ionic requirements and differential sensitivity to actinomycin D, it is concluded that under both the classical low and high salt conditions described by other workers, two enzymes are active in RNA synthesis. The presence of a third enzyme of low activity cannot be excluded. No qualitative difference in the number of enzymes involved in RNA synthesis in neuronal and glial cell nuclei has been found, but there are quantitative differences in activity between the two nuclear populations.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

DEVELOPMENT AND CHARACTERISTICS OF THE HISTAMINE-INDUCED ACCUMULATION OF CYCLIC AMP IN THE RABBIT CEREBRAL CORTEX

Abstract— In slices of adult rabbit cerebral cortex histamine at 5 μM produced a detectable rise in adenosine 3',5'-monophosphate (cyclic AMP). A maximum (20-fold) increase was observed in response to 0–5 mM histamine, with higher concentrations being less effective. The antihistaminic agent, tripelennamine, inhibited the response to 50 μM histamine in a dose-related manner. No effect on basal levels of cyclic AMP was noted with the highest dose of tripelennamine. The cyclic AMP response to 50 μM histamine was sustained for up to 1 h of incubation whether the slices and included medium were assayed together or the slices were assayed separately, although after 60 min of incubation cyclic AMP levels were higher when the medium was included in the assay. During development of the rabbit cerebral cortex, the first detectable increase of cyclic AMP in response to histamine occurred at fetal day 25, and from day 28 to birth the response was a 4-to 5-fold increase. A maximal (10-fold) response was observed at 4–8 days postpartum and by 20 days of postnatal age the response had decreased to the adult levels.     

OXIDATIVE METABOLISM OF THE CEREBRAL CORTEX OF THE RAT IN SEVERE INSULIN-INDUCED HYPOGLYCAEMIA

—Measurements were made of organic phosphates, carbohydrate substrates, amino acids and ammonia in the cerebral cortex, as well as of cerebral blood flow and of cerebral metabolic rate for oxygen and glucose in rats that developed an isoelectric EEG pattern (‘coma’) during insulin-induced hypoglycaemia. The results were compared to those obtained in control animals, as well as in hypoglycaemic animals with an EEG pattern of slow waves and polyspikes. In animals with slow waves and polyspikes, there was a decrease in all citric acid cycle intermediates except succinate and oxaloacetate, and a decrease in the pool size of intermediates. In animals that had an isoelectric EEG for 5–15 min, there were further decreases in citrate, isocitrate, α-ketoglutarate, malate and fumarate, but since the concentration of succinate (and oxaloacetate) increased, the pool size remained the same. In isoelectric animals, the results revealed extensive utilization of amino acids by both transamination and deamination reactions. However, since glycogen had disappeared and the amino acid pattern was constant after the first 5 min of isoelectric EEG, further oxidation must have occurred at the expense of non-carbohydrate, non-amino acid substrates. There were two- to three-fold increases in cerebral blood flow in animals with slow waves and polyspikes and in animals with isoelectric EEG, and no decrease in the cerebral metabolic rate for oxygen. Since less than half of the oxygen consumption could be accounted for in terms of glucose extraction, the data indicate that severe hypoglycaemia is associated with extensive oxidation of endogenous substrates other than carbohydrates and free acids.     

THE EFFECT OF ISCHAEMIA AND RECIRCULATION ON PROTEIN SYNTHESIS IN THE RAT BRAIN

Abstract— Rats were subjected to cerebral compression ischaemia for 15min and were subsequently recirculated with blood for periods up to 3 h. In vivo incorporation of intravenously administered L-[1–¹⁴C]valine into total brain proteins was found to be severely inhibited (about 20% of controls) after 45 min of recirculation. After 3 h, protein synthesis had increased, the specific radioactivity of proteins then being about 40% of controls. The post-ischaemic inhibition of protein synthesis was accompanied by a breakdown in polyribosomes and a concomitant increase in ribosomal subunits. In vitro incorporation of L-[1–¹⁴C]phenylalanine by a postmitochondrial supernatant system derived from animals subjected to 15 min ischaemia and 15 min recirculation was also severely reduced and showed, in contrast to control animals, no response to the addition of a specific inhibitor of polypeptide chain initiation (Poly(I)). Together with the in vivo accumulation of ribosomal subunits this indicates a block in peptide chain initiation during the early stages of recirculation.
Polyribosomes from animals subjected to 15 min ischaemia without recirculation showed a normal rate of in vitro protein synthesis which was inhibited by Poly(I) to a similar extent as polyribosomes from control animals. These results suggest that the post-ischaemic inhibition in chain initiation develops during the early stages of recirculation rather than during the ischaemic period itself.