METABOLIC PARAMETERS OF ONTOGENESIS OF ELECTRICAL ACTIVITY IN THE BRAIN. SODIUM-POTASSIUM ACTIVATED ADENOSINE TRIPHOSPHATASE IN DEVELOPING CHICK EMBRYO BRAIN

—(1) The activity of the Na-K ATPase in the particulate fraction of the chick embryo brain has been assayed at different stages of development with the objective of finding whether or not changes in the activity of this enzyme bear any relation to the maturation of spontaneous and evoked electrical activity of the growing chick brain. (2) The specific activity of the enzyme is low on day 6 and it rises rapidly between days 10 and 12, at which time it attains a plateau and remains essentially unchanged from day 12 until day 20. Experimental evidence rules out the possible presence of an inhibitor of the enzyme in 8-day-old brain homogenates, suggesting that these developmental changes in the activity of the enzyme may represent new synthesis of enzyme rather than its activation. The period between days 10 and 12 does not represent a unique stage of general protein synthesis. (3) The chick brain particulate enzyme has an optimum activity at pH 7·4 and at 37°. It is optimally activated by a Na⁺ concentration of 100mm and K⁺ concentration of 20 mm . The enzyme is inhibited by ouabain and Ca²⁺. (4) The results have been discussed.     

CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN CULTURED BRAIN CELLS FROM CHICK EMBRYOS

—Dissociated cells from brains of 7-day chick embryos were grown in primary culture for as long as 20 days. Many of the plated cells grew out long processes. Others, which proliferated rapidly, formed a confluent layer of flat cells after 4-6 days. Total DNA and protein increased five-fold, and activity of choline acetyltransferase (EC2.3.1.6) increased about 40-fold in 20 days. Acetylcholinesterase (EC3.1.1.7) increased three-fold by the fourth day of culture and then declined. The pattern of increase for choline acetyltransferase was similar to that for the in vivo development of the enzyme. l -Thyroxine, cyclic AMP (adenosine-3′,5′-monophosphate) or theophylline promoted increased levels of both enzymes by 30-200 per cent. l -Thyroxine also increased the activity of acetylcholinesterase in vivo by 40 per cent. When overgrowth by flat cells was prevented by the addition of 10^-3m -5-flourouracil, there was a decrease in the activity of choline acetyltransferase and an increase in the activity of acetylcholinesterase in comparison to control activities. The addition of 10^-3m -morphine or cocaine produced a 30 per cent elevation in the activity of choline acetyltransferase, but this effect could be mimicked with equimolar concentrations of ammonium ion.     

GUANYL CYCLASE IN CHICK EMBRYO BRAIN CELL CULTURES: EVIDENCE OF NEURONAL LOCALIZATION

Abstract— Guanyl cyclase activity was studied in dissociated chick embryo brain cell cultures presenting different ratios of neuronal to glial elements. The cultures containing neurons in substantial numbers always had higher guanyl cyclase activities than those consisting mainly of glial cells. No guanyl cyclase activity could be found in cultures made up of pure glial or meningeal cells. These results provide further evidence for our conclusion based on subcellular fractionation studies (G oridis & M organ , 1973), that brain guanyl cyclase might be overwhelmingly concentrated in neurons. Guanyl cyclase activity of chick embryo cerebral hemispheres increased sixfold between day 12 and day 16 after fertilization; an increase, though of much smaller magnitude, was also seen in cultured cells of the same age.     

POLYAMINES and NUCLEIC ACID METABOLISM DURING DEVELOPMENT OF CHICK EMBRYO BRAIN

Abstract— Spermine and spermidine reach maximum concentrations in the chick embryo brain between the 12th and 14th day of incubation. Sucrose-density-gradient analysis of polyribosome distribution in the developing chick embryo brain, showed the presence of polyribosomal aggregates in the regions of 147 S and 206 S between the sixth and eighth day of incubation. After the 16th day of incubation the presence of heavier polyribosomal aggregates in the region of 259 S and 280 S was found. The injection of spermine or spermidine into the air space of embryos on the tenth day of incubation leads to a remarkable increase in the incorporation rate of [³H]formate into the ribosomes. Studies under similar experimental conditions, showed an increased radioactivity in the region of 147 S, 206 S, 259 S and 280 S in embryos injected with spermine or spermidine.     

STRUCTURAL VARIATIONS DURING MITOSIS IN THE CHICK EMBRYO

Selected tissues from chick embryos were fixed in 2% glutaraldehyde and 1% OsO₄, both buffered at pH 7.6 with Veronal-acetate, and were embedded in Maraglas or Araldite. Two types of cell division have been noted. Generally, epithelial cells divide predominantly by a shortening of the chromosome-to-pole distance rather than by spindle elongation; mesenchymal cells undergo extensive spindle elongation. The presence of numerous continuous microtubules in cells that undergo extensive spindle elongation functionally implicates these tubules in the elongation process. In most embryonic epithelia, the cleavage furrow converges to a fixed site forming a mid-body near the anchoring desmosomes at the free surface; symmetrical furrow formation is typical of mesenchymal cells which lack desmosomes. The hypothesis of cleavage furrow formation and the fate of the mid-body that is formed during cytokinesis are discussed.     

CHANGES IN THE LEVELS OF DNA, RNA, PROTEIN AND DNases IN DEVELOPING AND OLD CHICK BRAIN

—The levels of DNA, RNA, protein and activities of acid and alkaline DNases in developing and old chicken brain were studied. A rapid increase in DNA content was found in the embryonic brain until just prior to hatching. Thereafter, with a transient plateau around the day of hatching, the DNA continued to increase but at a very slow rate. Two-year-old brain was found to have a markedly higher level of DNA as compared to the 10th day postnatal value. RNA showed a steady increase up to the 20th day of embryonic life. Protein levels showed a gradual increase throughout the period studied. Both acid and alkaline DNases exhibited maximum activity during embryonic life, i.e. at a time when rapid cellular proliferation was occurring. With advancing age, the acid DNase activity showed a marked decline thus exhibiting no correlation to the high level of DNA found in the old brain. The alkaline DNase activity, however, was still at a significant level in the 2-year-old brain.     

THE HALF-LIFE OF ACETYLCHOLINESTERASE IN MATURE RAT BRAIN

—The rate of degradation of acetylcholinesterase [EC 3.1.1.7] in mature rat cerebral cortex was determined from the time course of the label introduced into the protein by one intraventricular injection of l -leucine-l-¹⁴C. The half life of the enzyme was 2.84 ± 013 days.     

ACETYLCHOLINESTERASE IN MOUSE BRAIN, ERYTHROCYTES AND MUSCLE

Abstract— The properties of the enzyme acetylcholinesterase (EC 3.1.1.7) from mouse brain, erythrocytes and muscle were investigated. The enzymes were examined by gel filtration in Sephadex G-200, polyac-rylamide gel electrophoresis, immunological reactions, active-site labelling with tritiated di-isopropyl-phosphorofluoridate and also their kinetic properties were compared. All three enzymes appeared to have a single active small mol. wt. component of 80,000 to 82,000 which produced higher mol. wt. forms by aggregation. The partial purification of the enzyme from brain was achieved by affinity chromatography and this product was used to prepare antibodies. The purified immunoglobulin was shown to react with all three enzymes.     

COMPARATIVE PEPTIDE MAPPING AND ISOELECTRIC FOCUSING OF ISOLATED SUBUNITS FROM CHICK EMBRYO BRAIN TUBULIN

The α- and β-subunits of chick embryo brain tubulin have been isolated under denaturing conditions and compared with respect to their molecular weight, amino acid composition, tryptic peptide maps, amide content and isoelectric focusing properties. An 8 M-Urea-containing polyacrylamide gel system with varying acrylamide concentrations was used for calculation of the retardation coefficients (K_R) of the tubulin subunits. A molecular weight of 53,000 was estimated for each subunit by comparison to K_R values for standard proteins. Amide contents of approx 41% of the carboxyl groups of α-tubulin and 48% of the carboxyl groups of β-tubulin were calculated using the average PI value, the pK_intrinsic for the ionizable side chains of the amino acids and the amino acid composition of each subunit. Comparative peptide maps of trypsin digested α- and β-tubulin demonstrated 16 peptides unique to each subunit and 23 peptides which comigrate. Both subunits give rise to multiple species on electrofocusing gels. The average isoelectric points for the α- and β-subunits are 5.4 and 5.2, respectively.     

发育中小鼠和鸡胚脑微管蛋白的合成与甲状腺功能间的关系

利用 ³H-秋水仙碱与微管蛋白间的特异结合及DEAE纤维素对微管蛋白的离子交换作用,连续测定小鼠、鸡胚脑发育过程中的脑微管蛋白的合成变化。结果表明脑微管蛋白的合成速度均在其脑发育的临界期时达到最高峰。此时恰是甲状腺功能逐渐完善的时期。当小鼠进入育龄期时,雌雄鼠脑微管蛋白含量差异显著。可能说明性激素对微管蛋白的合成有重要影响。     

ACETYLCHOLINESTERASE TURNOVER IN BRAIN, CEREBROSPINAL FLUID AND PLASMA

—By assay of acetylcholine hydrolysis to measure total cholinesterase activity and acetyl-β-methylcholine hydrolysis to measure acetylcholinesterase (E.C 3.1.1.7) activity, patterns of regeneration of enzyme activity were measured in seven areas of brain, cerebrospinal fluid and plasma of cats after administration of an irreversible inhibitor. Halftimes of recovery of total cholinesterase in the brain tissues ranged from 0·9 to 3·8 days (av = 2·5 days) and acetylcholinesterase recovery halftimes ranged from 1·2 to 5·3 days (av = 3·6 days). Regeneration of total cholinesterase was also followed in subcellular fractions of guinea-pig and rat brains after similar inhibition. In both species, the fastest recovery occurred in the soluble fraction with halftimes of 1·8 and 1·6 days, while the synaptosomal fractions exhibited the slowest recoveries with halftimes of 8·3 and 4·1 days. Regeneration of activity in plasma and CSF most nearly resembled that of the soluble brain fraction.     

THE RELATION BETWEEN THE INTRACELLULAR RIBONUCLEIC ACID DISTRIBUTION AND AMINO ACID INCORPORATION IN THE LIVER OF THE DEVELOPING CHICK EMBRYO

The RNA-P and DNA-P content of the nucleus and the RNA-P content of the whole cell of the livers of 8- to 20-day chick embryos and of adult fowls have been determined. The DNA-P content of the liver nuclei was slightly higher in the 8- and 10-day embryo than in all the other stages examined. A significant decrease in the RNA content of the cell occurred during embryonic development. The RNA content of the adult cell was the same as that of the 14- to 16-day embryo. The proportion of the cellular RNA contributed by the nucleus also decreased during development. In respect to both nuclear RNA content and distribution of RNA between nucleus and cytoplasm, the adult resembled the 8- to 12-day embryo. Examination of the fine structure of the cell showed that, as development progressed, free ribosomes decreased in number and the rough membranes increased. Slices of 8-, 14-, and 20-day embryonic livers and of adult livers were incubated with ¹⁴C-leucine, and the amount of labeled amino acid incorporated into whole tissue protein and into the proteins of the subcellular fractions was measured. Embryonic liver incorporated ¹⁴C-leucine 15 to 30 times more rapidly than adult liver. The microsomal protein was always more highly labelled than the protein in any other subcellular fraction; however, in the 8-day embryonic and the adult liver the proportion of total counts found in the nuclear fraction was considerably higher than in the 14- or 20-day embryonic liver. The significance of an apparent correlation between the proportion of the cell's RNA contributed by the nucleus and the proportion of total counts in the nuclear fraction is discussed.     

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.