DNA POLYMERASES IN FRACTIONATED RAT BRAIN NUCLEI

Abstract— —Adult rat brain nuclei were separated by discontinuous sucrose gradient centrifugation into astrocyte enriched, neuron enriched, and oligodendrocyte/microglia fractions. Nuclear fractions were subjected to velocity sucrose gradient centrifugation and gradient fractions assayed using relatively specific reaction mixtures for DNA polymerase-α, -β and TdT. NEM resistant DNA polymerase activity (DNA polymerase-β) was detected in equivalent amounts in all nuclear fractions. High molecular weight NEM sensitive activity (DNA polymerase-α) was found primarily in the neuron enriched fraction. The significance of the presence of DNA polymerase-α, an enzyme thought to be involved in DNA replication, in a cell incapable of cell division is unknown. TdT was detected in all fractions with increased activity in the neuron enriched fraction. The finding of TdT in thymocytes and neurons further supports the hypothesis that this enzyme is involved in the storage of noninherited information.     

ACIDIC PROTEINS IN RAT BRAIN NUCLEI: DISC ELECTROPHORESIS

—Disc electrophoretic patterns of soluble, acidic proteins in brain and liver nuclei and in the respective tissue homogenates were studied. In brain nuclei, a fast component moving with the electrophoretic front constitutes a relatively large amount of the acidic proteins which migrate toward the anode. Electrophoretic patterns obtained with fractionated brain nuclei (neuronal, astrocytic and glial) were similar to those obtained with total brain nuclei. The S-100 protein could not be detected in any of the nuclei examined. Protein patterns in brain and liver nuclei markedly differed both quantitatively and qualitatively.     

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.     

THE LOCALIZATION OF ENZYME ACTIVITIES IN THE RAT BRAIN

Studies with rat brain illustrate the usefulness of formol-calcium-fixed tissue for studying both enzymatic "chemoarchitectonics" and intracellular organelles. Unembedded frozen sections and polyvinyl alcohol-embedded sections may be used to demonstrate the activities of DPNH-tetrazolium reductase localized in mitochondria and ergastoplasm, TPNH-tetrazolium reductase localized in mitochondria, ATPase (and/or apyrase or ADPase) in cell membranes, and acid phosphatase in lysosomes.¹ Among the observations recorded are: (1) the presence of lysosomes in all cells of the brain; (2) the presence of numerous large lysosomes near the nuclei of capillary endothelial cells; (3) a polarized arrangement of large lysosomes in epithelial cells of the ependyma and choroid plexus; (4) the presence of ATPase activity in the cell membranes of some neurons; (5) the presence of either an apyrase or combination of ATPase and ADPase in the cell membranes of neuroglia and capillaries; (6) the presence of both DPNH- and TPNH-tetrazolium reductase activities in neuroglia; (7) the presence of DPNH- and TPNH-tetrazolium reductase activities in mitochondria and of DPNH-tetrazolium reductase activity in Nissl substance. The possible functional significance of these localizations is briefly discussed, as is their relation to "quantitative histochemistry" data available in the literature.     

RIBOSOMAL RNA PRECURSORS IN NEURONAL AND GLIAL RAT BRAIN NUCLEI

Abstract– The method of T hompson (1973) for isolation and fractionation of brain nuclei was modified by the introduction of 12mM-Mg²⁺ in the isolating media. This technique gives a good yield of pure (85-90%) neuronal and glial rat brain nuclei, with minimal disruption of nuclei and degradation or processing of nuclear RNA. The RNA/DNA ratio of neuronal nuclei is about 3-fold higher than that of glial nuclei. Analysis of nucleolar RNA fractions by urea-agar gel electrophoresis allows the identification of 45S, 41S, 39S, 36S, 32S and 21S pre-rRNA components. The pattern of nucleolar pre-rRNA and rRNA species in neuronal and glial nuclei is identical. These results demonstrate the existence in brain nuclei of multiple pre-rRNA processing pathways qualitatively similar to those observed in other animal tissues.     

A NOVEL ENZYME IN RAT BRAIN CONVERTING β-ENDORPHIN INTO METHIONINE ENKEPHALIN: AFFINITY CHROMATOGRAPHY AND SPECIFICITY

Abstract— Methionine enkephalin (met-enk), an endogenous opioid, commonly occurs in sequences of lipotropins (LPH) and endorphins, implying a possible biosynthetic pathway for this pentapeptide. In search of the enzyme which generates met-enk from human β-endorphin [LPH(61-91), β-end], it was found that the soluble fraction of rat brain contained such activity. The enzyme was purified by ammonium sulfate fractionation (50-80% saturation), ion-exchange chromatography on DEAE-Sephadex A-50, and affinity chromatography for which LPH(64-67) functioned as affinity ligand and eluant. The final preparation was essentially free of other peptidases, such as kininase, met-enk degrading and post-proline cleaving activities. Studies on specificity revealed that the enzyme selectively cleaved the Met-Thr bond in both LPH(64-67) and β-end. It is possible that this enzyme participates in the biosynthesis of met-enk in vivo .     

5-HYDROXY-l-TRYPTOPHAN DECARBOXYLASE ACTIVITY: MICROASSAY AND DISTRIBUTION IN DISCRETE RAT BRAIN NUCLEI

Abstract— 5-Hydroxy- l -tryptophan decarboxylase (EC 4.1.1.28) activity was measured in discrete regions of the rat brain by means of a sensitive micromethod lhat allows the quantitation of enzyme activity in μg amounts of brain tissue.
A good correlation was obtained between serotonin levels and 5-hydroxy- l -tryptophan decarboxylase activity in all the brain regions examined. Wide differences in enzyme activity were found in the different brain nuclei, with a 20-fold difference in activity between the most active region (the nucleus raphe dorsalis) and the least active regions (the corpus callosum and the optic tract).     

A CHOLESTEROL-ESTERIFYING ENZYME IN RAT CENTRAL NERVOUS SYSTEM MYELIN1

Aontrary to our earlier finding (Eto & Suzuki , 1971), the myelin fraction purified from young adult rat brain consistently showed cholesterol-esterifying activity. The specific activity in myelin was the highest among subcellular fractions. Extensive washing wiih various aqueous salt solutions failed to remove the activity from myelin. The enzyme was evenly distributed among the arbitrarily defined light, medium and heavy myelin subfractions. The myelin-localized activity showed the pH optimum and heat stability identical to the microsome-bound activity. Although there were minor differences in the effect of detergents or exogenous lipids added to the reaction mixture, no firm evidence was obtained to indicate that the myelin-bound cholesterol-esterifying enzyme is different from that in other subcellular fractions. On the other hand, the distribution among the myelin subfractions and heat stability of the myelin-bound cholesterol-esterifying activity were different from those of the myelin-specific cholesterol ester hydrolase. Therefore, the esterification does not appear to be a mere reverse reaction catalyzed by the previously known myelin-specific hydrolase. The rat brain myelin, therefore, is capable of both synthesizing and hydrolyzing cholesterol esters.     

PARTIAL PURIFICATION AND CHARACTERIZATION OF AN ACID RIBONUCLEASE IN BEEF BRAIN NUCLEI

Abstract— In this report we describe the partial purification and characterization of an acid ribonuclease from beef brain nuclei (RNAase BN2). RNAase BN2 was purified approximately 85-fold. The optimum pH is 6-2 and the optimum temperature 55°C. The effect of ions on the RNAase BN2 and its K_m were determined. RNAase BN2 is an endoribonuclease capable of hydrolysing polyA, polyU and polyC. Oligonucleotides produced by the hydrolysis of polyA by the RNAase BN2 have a monophosphate group at the 3' position.     

延髓中缝核区注射谷氨酸钠降低大鼠动脉血压的作用

本工作研究了大鼠延髓中缝核在心血管活动调整中的作用。通过埋植套管向大鼠延髓中缝核区注射神经元兴奋剂谷氨酸钠5-μg可明显降低血压,抑制交感神经传出活动,心率无显著改变。降血压效应可被脊髓蛛网膜下腔注射5-HT受体阻断剂肉桂硫胺50μg,或腹腔注射赛庚啶15mg/kg所部分对抗;并可被皮下注射阿片受体阻断剂纳洛酮1mc/kg所削弱。结果提示:延髓中缝核-脊髓5-HT下行通路对脊髓的血管调节功能起抑制性影响,内源性阿片样物质可能参与该机制。     

NUCLEOTIDE METABOLISM IN RAT BRAIN

Abstract— The uptake, the conversion to nucleotides, and their incorporation into RNA for labelled glycine, aspartate, the free bases and nucleosides of purines and pyrimidines were investigated with cortical slices of rat cerebrum. At the end of a 1-hr incubation time the slice-to-medium ratio of the radioactivities for labelled aspartate, glycine, adenine and adenosine were 34, 26, 20 and 5, respectively, while the slice-to-medium ratios for hypoxanthine, inosine, guanine, guanosine, xanthine, orotate, cytidine, cytosine, uridine, and uracil ranged from 1.3:1 to 2:1. Over 99 per cent of the total radioactivity taken up by the cortical slices was present in the TCA supernatant and 86, 82, 65, 50, 34, 23, 20 and 1.6 per cent of this radioactivity was in the form of nucleotides at the end of a 1-hr incubation with labelled adenine, adenosine, hypoxanthine, inosine, uridine, orotate, cytidine, and glycine, respectively. The incorporation of various radioactive precursors into RNA of cortical slices suggests that nucleotides originating from either de novo synthesis or preformed purine derivatives enter the same nucleotide pool utilized for RNA synthesis. The supernatant fraction from homogenized cerebrum was investigated for the presence of various anabolic and catabolic enzymes associated with nucleotide metabolism. These results were correlated with the data from the RNA incorporation studies, and a possible role for AMP: pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, I.U.B. 2.4.2.7) to achieve intercellular transfer of AMP is discussed.     

INCORPORATION OF PHOSPHATE INTO RAT BRAIN DURING SLEEP AND WAKEFULNESS

Abstract— Labelled inorganic phosphate (³²P₁) was administered intraventricularly to unrestrained sleeping and waking adult rats. After about 20 min of sleep or a comparable period of wakefulness, as monitored by EEG and EMG, the animals were frozen in liquid nitrogen and the brains were analysed. One group of animals (A) was not previously acclimatized to the apparatus. A second group (B) was acclimatized. The specific radioactivity of a phosphoprotein fraction was elevated during sleep in group A but not in group B. The specific radioactivity of the phosphatides of group B was depressed in sleeping as compared with waking animals. This effect was not observed in group A. No significant difference was detected between the EEG patterns of sleeping animals in groups A and B, as evaluated by standard criteria. These observations suggest that the physiological conditions attributable to environmental, emotional or other determinants can influence shifts in brain metabolism during the sleep-wakefulness cycle.     

EFFECT OF ELECTROSHOCK ON THYMIDINE INCORPORATION INTO RAT BRAIN DNA

Administration of electroconvulsive shocks in rats induces a marked inhibition of the process of thymidine incorporation into cortical DNA. The inhibitory effect reaches its maximum after 10–15 min. Percentage incorporation returns to normal values within one hour of the treatment. These findings may suggest the participation of a metabolically active fraction of DNA in neural functions.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

EXISTENCE OF ASCORBIC ACID AS AN ACTIVATOR OF THIAMINE DlPHOSPHATASE IN RAT BRAIN

The effect of the supernatant fraction (105,000 g for 60 min) of rat brain on the microsomal thiamine diphosphatase activity was examined. The thiamine diphosphatase activity was increased by addition of the supernatant fraction. The factor activating the enzyme was a heat-stable and dialyzable substance. It caused lipid peroxidation in the microsomes and the increase of the enzyme activity was mediated through lipid peroxidation of the preparation. When the supernatant fraction was chromatographed on columns of Sephadex G-25 and Dowex 1 × 2, the activator was eluted in fractions containing ascorbic acid. The inhibitory factor of ATPase present in the supernatant fraction was also eluted with the activator. The u.v.-spectrum of the active fraction obtained by these chromatographies was the same as that of ascorbic acid. These findings indicate the existence of ascorbic acid as an activator of thiamine diphosphatase in rat brain and confirm the previous finding that the soluble factor inhibiting ATPase activity is ascorbic acid.     

RESPIRATION IN IMMATURE RAT BRAIN MITOCHONDRIA

—Respiration was studied polarographically in mitochondria isolated from immature rat cerebral hemispheres. Respiratory rates are compared as a function of age, substrate, and the requirement for a phosphate acceptor. 1. All respiratory rates are low in the first week of life. These rates increase during the first month and then decline to about the newborn rate by 5 weeks of age. 2. With the NAD-linked substrate pair, glutamate and malate, the changes with age are significant only for the rate of ADP-dependent respiration. With succinate as substrate, significant age-dependent changes in respiration occur only in ADP-independent respiration. 3. In mitochondria from animals less than five weeks of age, the ADP-dependent respiratory rate is significantly greater with the NAD-linked substrate pair than with succinate. In mitochondria from older animals, both ADP-dependent and ADP-independent rates are greater with succinate.