CYCLIC NUCLEOTIDES and PROTEIN KINASE ACTIVITY IN THE RAT BRAIN POSTMORTEM

Abstract— The components of the cyclic nucleotide system were studied in rat brain at various times after death to determine the stability of the system postmortem. The concentration of cyclic AMP in 4 brain regions was highest 10 min after death and declined to stable levels within 6 h after death. At this time concentrations approximated those normally seen in vitro. The concentration of cyclic GMP in brain regions fell markedly postmortem and was undetectable 6 or 16 h after death. Nor-epinephrine-stimulated cyclic AMP accumulation measured in vitro in slices of the cerebral cortex was the same 10 min and 16 h postmortem. In the cerebellum, however, accumulated levels of cyclic AMP were lower 16 h postmortem, although the degree of stimulation elicited by norepinephrine was the same 10 min and 16 h after death. Cyclic AMP-dependent and independent protein kinase activities were detected in the rat brain at all times after death. There was no change in the cyclic AMP-dependent enzyme activity postmortem, but activity in the absence of cyclic AMP was significantly higher immediately after death compared to 6 or 16 h postmortem. These studies indicate that it should be possible to study the cyclic AMP system in human brain tissue obtained at autopsy.     

DOI诱导的大鼠头部抽动模型脑内多巴胺转运体的研究

目的研究DOI诱导的大鼠头部抽动模型脑内多巴胺转运体改变,探讨Tourette综合征发病与突触前多巴胺神经纤维支配的关系。方法将30只Wistar大鼠随机分为模型组和对照组,应用DOI诱导大鼠头部抽动作为Tourette综合征动物模型。两组以99mTc-TRODAT-l作为配体,采用放射自显影和γ测量方法观察大鼠脑内多巴胺转运体分布及其密度。结果放射自显影结果表明模型组大鼠脑内银颗粒密度显著高于对照组,尤其是纹状体。γ测量结果显示,模型组纹状体每克脑组织含放射性占注射剂量的百分比(%ID/g)与对照组相比显著增高(P<0.01),模型组海马和大脑皮层与对照组相比有统计学差异(P<0.05)。结论DOI诱导的大鼠头部抽动模型脑内多巴胺转运体密度较正常大鼠显著增加,Tourette综合征发病与突触前多巴胺神经纤维支配增加密切相关。     

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.     

正常大鼠肾脏细胞溶酶体膜的构成蛋白

溶酶体是细胞内对其吞噬之物质溶解及消化之主要场所,同时也是细胞自噬作用的主要细胞器。为了进一步了解此细胞器的功能与结构,我们采用免疫荧光标记法,通过5种针对大鼠肝细胞溶酶体膜蛋白的特异性单克隆抗体,对大鼠正常肾脏细胞溶酶体膜蛋白进行了标记,并通过NH_4Cl溶液对溶酶体作了膜膨胀处理,结果显示:(1)细胞内溶酶体膜蛋白是由多种蛋白所构成,其各种蛋白的含量是不同的;(2)所有溶酶体膜蛋白均表达于该细胞器之表面;(3)NH_4Cl溶液能有效地使溶酶体扩张,这将有和于进一步研究溶酶体的结构。     

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.     

ADENOSYLMETHIONINE DECARBOXYLASE IN DEVELOPING RAT BRAIN

Adenosylmethionine decarboxylase from rat brain has been found to be similar to the same enzyme isolated from other rat tissues in regard to kinetic parameters, pH optimum, putrescine requirement, and subcellular location. Evidence is presented that pyridoxal phosphate is not the functional cofactor in enzymatic decarboxylation by the rat brain preparation. The capacity for spermidine synthesis in developing rat brain was determined by measurement of the activity of adenosylmethionine decarboxylase. The activity increased dramatically after 10 days of postnatal age. This increase occurred after the period of maximum nucleic acid synthesis, an observation which suggests that spermidine may have a role in the functional development of the brain.     

LIPID PEROXIDE FORMATION IN RAT BRAIN

Abstract— Lipid peroxide formation as measured by the thiobarbituric acid reaction was demonstrated in subcellular fractions of rat brain. The ascorbic acid induced nonenzymic lipid peroxidation was distributed in all the subcellular fractions with a maximum in microsomes. The NADPH dependent enzymic lipid peroxidation occurred mainly in microsomes and to a smaller extent in synaptosomes; NADH could replace NADPH for the enzymic lipid peroxidation under the assay conditions employed. Fe²⁺ but not Fe³⁺ stimulated the NADPH or NADH dependent lipid peroxide formation. The optimum conditions with respect to pH, ascorbic acid or NADPH concentration, time of incubation and protein concentration were studied. Heating the microsomes at 100^oCdid not influence the ascorbate-induced lipid peroxidation but completely abolished the NADPH linked peroxidation. Several heavy metal ions, surface active agents and EDTA were inhibitory to lipid peroxidation. The effect of thiol agents indicated that -SH groups were involved in the enzymic lipid peroxidation. Studies on subcellular fractions of developing rat brain showed an increasing trend in lipid peroxidation with the advancing age of the animal. No significant difference in lipid peroxidation was observed between brains from normal rats and those from rats affected by experimental allergic encephalomyelitis.     

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.     

DISTRIBUTION OF N-ACETYL-l-ASPARTIC ACID IN RAT BRAIN

The distribution of N-acetyl-l -aspartic acid in the rat brain has been studied by means of a new gas-chromatographic method. The results obtained concern twelve different brain areas.     

PROPERTIES OF MEMBRANE-BOUND HEXOKINASE IN RAT BRAIN

Abstract— —-(1) The total hexokinase activity present in the mitochondrial fraction can be solubilized completely by incubation with salt and Triton X-100. This activity cannot be entirely released by washing with sucrose or by freezing and thawing.
(2) A part of the particle bound hexokinase exists in a latent form. The latent form is apparent after incubation with high salt concentrations, detergents or by freezing and thawing.
(3) Solubilization of membrane bound hexokinase is pH-dependent. Incubation in salt solutions increases the specific activity ten-fold. The salt concentration and pH are con-current. At pH 7.0 part of the hexokinase is solubilized. The lower the pH the less salt is required to release the same amount of activity.
(4) Triton X-100 solubilizes particle-bound hexokinase, but to a less extent than salts. The activation of hexokinase is greater with Triton X-100 than with salt.
(5) The possible nature of the bonds between hexokinase and mitochondrial membranes is discussed.     

CATECHOLAMINES IN FETAL AND NEWBORN RAT BRAIN

The levels of dopamine and norepinephrine were determined in the brains of fetal and newborn rats by means of a sensitive, radiometric-enzymatic assay. Catecholamines were converted to their 3-O-methylated derivatives in the presence of catechol-O-methyl transferase (EC 2.1.1.1) and [³ H-methyl]S-adenosylmethionine; and the [³H]-derivatives were isolated by selective extraction. The assay had a sensitivity for dopamine and norepinephrine of 100 picograms and was linear to at least 30 nanograms of catecholamines. Both amines were present at 15 days of gestation and increased 15-fold in content during the last week of gestation. The regional distribution of these neurotransmitters in the brain of the newborn rat correlated with the distribution of their biosynthetic enzymes. An investigation of the effects of reserpine, pheniprazine, α-methyl-para-tyrosine, diethyldithiocarbamate and l -DOPA on the levels of dopamine and norepinephrine in the brains of the 18-day gestational fetus indicated that the levels of these neurotransmitters are under controls similar to those known to occur in the brain of the adult rat.     

REGIONAL TRANSPORT OF TRYPTOPHAN IN RAT BRAIN

Abstract— Tryptophan uptake was studied in brain slices and synaptosomes prepared from regions known to vary in the numbers of serotoninergic cell bodies and nerve endings that they contain. The rate of tryptophan uptake was highest in hypothalamus for both types of preparation. Differences among the regions were much more pronounced in isolated nerve endings (synaptosomes). Loading with tryptophan did not affect the uptake into tissue slices. Tryptophan accumulation in hypothalamus synaptosomes was reduced after intraventricular injection of 5,7–dihydroxytryptamine whereas no change was observed in synaptosomes prepared from cerebellum under the same conditions; accumulation by synaptosomes prepared from the hypothalamic and hippocampal regions was reduced after raphe lesions.     

METABOLIC EFFECTS OF AZASERINE IN RAT BRAIN

The de novo biosynthesis of purine nucleotides in rat brain was markedly and rapidly inhibited by intracerebral injections of azaserine. After 48 hr the animals became paraplegic. The biosynthesis of purine and pyrimidine nucleotides, RNA and protein was investigated by radioactive tracer studies over a 4-day period in an attempt to correlate the azaserine-induced, metabolic lesions found in rat brain and the manifested, peripheral, neurological disturbance.     

STUDIES ON HAEM BIOSYNTHESIS IN RAT BRAIN

Abstract— Abnormalities involving haem biosynthesis have been postulated as underlying mechanisms in the aetiology of the neural manifestations of acute porphyria and of lead poisoning. This paper reports a study of the enzymes of the haem biosynthetic pathway and their control in mammalian brain. The activity of rat brain 6-aminolaevulinate synthetase (ALA synthetase), 6-aminolaevulinate dehydratase (ALA dehydratase), uroporphyrinogen I synthetase, uroporphyrinogen decarboxylase and ferrochelatase were found to be between 12.5 and 0.002% of the corresponding values for liver. This accords with the lower concentrations of total haem and cytochrome P₄₅₀ found in brain and with the slower rate of incorporation of [4-¹⁴C]ALA into brain haem in vivo . The subcellular distribution of radioactivity following intraventricular injection of [4-¹⁴C]ALA confirmed that the bulk of brain haemoproteins are intramitochondrial in contrast to liver where the major portion is microsomal. Brain haem biosynthesis was apparently unaffected by factors known to influence this pathway in liver, including starvation and treatment with allylisopropylacetamide or phenobarbitone. These findings suggest that brain haem requirements are considerably less than those of liver and are not subject to significant fluctuations under normal circumstances. Apparent non-inducibility of ALA synthetase suggests that deficient haem and consequently haemoprotein production could result where other enzymes in the pathway become rate-limiting due to genetic defects or inhibition by exogenous agents such as lead.     

SYNTHESIS OF RNA IN DEVELOPING RAT BRAIN IN VITRO

—Incorporation of [8-¹⁴C]adenine into a rapidly-labelled fraction of RNA derived from the nucleus, and into a cytoplasmic RNA of high molecular weight was studied in brain slices from new born rats. The kinetic behaviour of the two fractions of RNA was compatible with a precursor-product relationship between them. The change in the specific activity of adenine and the reduction of radioactivity in prelabelled RNA of brain slices in the presence of actinomycin D, suggest that the observed degradation of nuclear RNA is not due to random changes, but is limited to a relatively small fraction, presumably messenger RNA.     

LOCALIZATION, SOLUBILIZATION AND PROPERTIES GANGLIOSIDE TRANSFERASES IN RAT BRAIN OF N-ACETYLGALACTOSAMINYL AND GALACTOSYL GANGLIOSIDE TRANSFERASES IN RAT BRAIN

Abstract— The subcellular distributions of UDP-N-acetylgalactosamine: G_M3 N-acetyl-galactosaminyl transferase and UDP-galactose: G_M2 galactosyl transferase, two enzymes involved in the biosynthesis of gangliosides, were determined in the 7-day-old rat brain by means of synaptosomal fractionation techniques. The enzymes were located on the synaptic membranes and appeared to be closely associated with gangliosides and acetylcholinesterase. Solubilization of the transferase enzymes from the microsomal particles was achieved and differed from the solubilization of acetylcholinesterase and of the total membrane protein. Competition studies suggest that the ^N-acetylgalactosaminyl transferase involved in the formation of G_M2 from G_M3 is different from the N-acetylgalactosaminyl transferase involved in the formation of GalNAoGal-Glc-ceramide from Gal-Glc-ceramide, whereas in contrast, both the formation of G_M1 from G_M2 and of Gal-GalNAc-Gal-Glcceramide from GalNAc-Gal-Glc-ceramide appear to be catalysed by the same galactosyl transferase.     

HE TURNOVER RATE OF TUBULIN IN RAT BRAIN

The turnover rate of tubulin in rat brain was determined from the decay in specific radioactivity of the protein after pulse-labeling. When precursors were administered by a parenteral route, the shortest half-life, 9.8 days, was obtained with [¹⁴C]NaHCO₃; the longer half-lives obtained with [U-¹⁴C]glucose or [4,5-³H]leucine suggest significant reutilization of label. Furthermore, with leucine as precursor maximal specific radioactivity of tubulin was not obtained until eight days after administration of label. Labeling and decay kinetics obtained with [4,5-³H]leucine were markedly different when the isotope was administered directly into the lateral ventricle. The difference between the turnover rates of the -α and β subunits of tubulin purified by means of high resolution polyacrylamide gel electrophoresis was not statistically significant. A half-life for tubulin of 6.2 days was measured by continuous intravenous infusion of [U-¹⁴C]tyrosine.