PHOSPHOFRUCTOKINASE AND FUMARATE HYDRATASE IN DEVELOPING RAT BRAIN

The developmental patterns of phosphofructokinase, fumarate hydratase and lactate dehydrogenase were determined and compared using homogenates of rat brain. Phosphofructokinase activity, expressed in terms of tissue wet wt., was relatively constant from 5 days before birth to 8 days postnatal; a 110 per cent increase in activity occurred between 12 and 21 days of age, when adult levels were achieved. The degree of inhibition of phosphofructokinase by 1-0 mM-ATP changed little during development; inhibition by 2-5 mM-citrate was about 50 per cent in both newborn and adult brain. Phosphofructokinase development more closely resembled that of lactate dehydrogenase than that of fumarate hydratase.     

LEVEL AND AMINO ACID ACCEPTOR ACTIVITY OF MOUSE BRAIN tRNA DURING NEURAL DEVELOPMENT

Abstract— The level of tRNA in mouse brain tissue was measured at various stages of postnatal development. The amount of tRNA per unit of brain wet weight was little, if at all, altered during the first 22 days after birth and decreased by 26 and 32 per cent by 56 days and maturity, respectively. On a DNA or cellular basis, there was no maturation-dependent decrease in tRNA content. The total amino acid acceptor activity of tRNA for seven different amino acids was measured during neural development. There were considerable differences in the tRNA acceptor activities of individual amino acids within an age group; however on a DNA basis, there was little difference between tRNA preparations obtained from newborn and adult mouse brain tissue. The in vivo levels of aminoacylated-tRNA for the seven amino acids of interest, were measured in brain tissue of 1–, 9–, 34, 70–day-old and adult (over 9 months old) mice. Alterations in tRNA level, total tRNA acceptor activity, for each amino acid, and the levels of in uivo aminoacylation of tRNA were shown to be independent of developmental alterations in brain amino acid pool sizes. The results are discussed with regard to the availability of cellular amino acids for translational events during early mammalian brain development.     

SUBCELLULAR DISTRIBUTION OF ISOCITRATE DEHYDROGENASES IN NEONATAL AND ADULT MOUSE BRAIN

Abstract— The activity and subcellular distribution of NADP- and NAD-isocitrate de-hydrogenases (ICDH) (EC 1.1.1.42 and 1.1.1.41, respectively) in brains of adult and newborn mice have been determined. In the adult, NAD-ICDH activity in whole brain homogeantes was 1–17 mol/kg wet wt of brain/h (MKH), whereas the NADP-ICDH activity was 0.223 MKH. In the newborn, the activity of the NAD-dependent enzyme was only 0.246 MKH, whereas the NADP-dependent enzyme activity was 1.23 MKH. At both ages, 66 per cent of the NADP-ICDH activity was in the cytosol, less than 10 per cent was in the purified mitochondrial fraction and the remainder was in the crude synaptosomal fraction. Less than 10 per cent of the NAD-ICDH activity was in the cytosol in both the newborn and adult, whereas 50 per cent was in the purified mitochondrial fraction. The crude synaptosomal fraction from the newborn and adult brains contained 28 and 22 per cent, respectively, of the total NAD-ICDH activity. The activities of these enzymes in the cytosol and mitochondria were compared with those of succinate dehydrogenase and with three other enzymes which utilize the product, 2-oxoglutarate, as substrate. The relationship of the isocitrate dehydrogenases to the metabolism of adult and newborn brain is discussed.     

FOLATE LEVELS IN DEVELOPING MOUSE BRAIN1

Abstract— The folate coenzymes of mouse brain underwent quantitative and qualitative changes during the first few weeks after birth. The total folate coenzymes per unit wet weight declined by approximately 60 per cent. In the newborn brains a relatively small proportion of the total folates were poly-γ-glutamyl derivatives, but the percentage of the total folates in these forms increased as the brain matured.     

DISTRIBUTION OF POLYSOMES IN MOUSE BRAIN TISSUE

Abstract— Polysomes were isolated from several different fractions of mouse brain tissue. After homogenization, the extract was centrifuged to yield a post-mitochondrial supernatant fraction and a pellet fraction. Sucrose gradient analysis of the material in the post-mitochondrial supernatant fraction indicated that 80 per cent of the ribosomes were present in polysomes and that little, if any, of the pellet fraction was present. Sucrose gradient analysis of the solution obtained after washing the pellet showed that very little polysomal material was present. The remaining pellet fraction was resuspended in a detergent mixture of deoxycholate-Tween 40. Sucrose gradient analysis of the resulting detergent-soluble solution indicated that large amounts of ribosomal material, in which 60–70 per cent of the ribosomes were associated in polysomes, were present.
In brain tissue from young animals, 20 per cent of the polysomes were found in the post-mitochondrial supernatant fraction whereas 80 per cent of the polysomes were released from the pellet fraction by detergent treatment. In contrast, in brain tissue from adult animals, 40 per cent of the polysomes were found in the post-mitochondrial supernatant fraction, whereas 60 per cent of the polysomes were released from the pellet fraction by detergent treatment.     

Decreased weight, DNA, RNA and protein content of the brain after neutron irradiation of the 18-day mouse embryo

Pregnant mice were irradiated with 0.5 Gy fission neutrons on the eighteenth day of their gestation. The average litter size at birth was unchanged but mortality increased 5-6 fold in the first 3 days. The irradiated mice were the same weight as control mice at birth but showed a progressively increasing weight deficiency up to at least 36 days as compared to controls. Brain weight was 37, 45 and 25 per cent less in 2-, 3- and 52-week old irradiated animals, respectively, and the ratio of brain weight to body weight was 25, 27 and 13 per cent less. The concentrations of DNA, RNA and protein (mg/g wet tissue) were the same in irradiated and control mice in both brain and liver at all three ages. Total DNA, RNA and protein contents of whole brain after irradiation were 56-75 per cent of the control levels. No definite decrease was observed in liver. Histological study at 6 hours after irradiation showed nuclear pyknosis in the central nervous system from definite to very severe according to the part examined. It is concluded that damage to the central nervous system of the 18-day mouse foetus after neutron irradiation is mainly due to killing and/or inhibition of the differentiation of neuroblasts.     

Protein metabolism in the small intestine of the ethanol-fed rat

The effects of chronic ethanol feeding on the small intestine were investigated in young rats. Rats were fed a nutritionally-adequate liquid diet, containing 36 per cent of total energy as ethanol (treated, n = 7), or isovolumetric amounts of the same diet in which ethanol was substituted by isocaloric glucose (controls, n = 7). After six weeks the wet weight and total tissue contents of protein, RNA and DNA were significantly reduced by 21 per cent, 23 per cent, 16 per cent and 28 per cent respectively, (p less than 0.014). Rates of protein synthesis were measured with L[4(3H)]phenylalanine and fractional rates (defined as the percentage of constituent tissue protein synthesised each hour, i.e. ks, % h-1) were calculated from the specific radioactivity of free phenylalanine in both tissue homogenates and plasma. Ethanol-feeding reduced ks by approx 10 per cent (p less than 0.181). The amount of protein synthesized unit-1 RNA was also reduced by approx 15 per cent (p less than 0.059) but the amount of protein synthesis unit-1 DNA was unaffected by ethanol-feeding (p less than 1.000). In contrast, the absolute rates of protein synthesis were reduced by approximately 30 per cent (p less than 0.022). It was concluded that, as the small intestine contributes to approx. 20-25 per cent of whole body synthesis these results may have an important effect on whole body nitrogen homeostasis and may have implications for the gastrointestinal effects of ethanol seen during chronic alcoholic abuse.     

ACETYLCHOLINE,CHOLINE AND CHOLINE ACETYLTRANSFERASE ACTIVITY IN THE DEVELOPING BRAIN OF NORMAL AND HYPOTHYROID RATS1

Abstract— Acetylcholine, choline and choline acetyltransferase activity were measured in the whole brains of normal and hypothyroid rats during development. At 1 day postpartum, brain acetylcholine was 73 per cent of adult levels. Propylthiouracil-induced hypothyroidism up to age 20 days did not alter brain acetylcholine concentrations, but at 30 days resulted in significantly decreased levels. At day 1, brain choline was 20 per cent higher than adult levels and decreased between days 8 and 10. In hypothyroid rats this phenomenon did not occur until days 15–20. At day 1 postnatally, choline acetyltransferase activity was only 7 per cent of adult levels, then between days 5 and 20 rose to 77 per cent of adult levels. Beginning at day 8, hypothyroidism resulted in significantly decreased enzyme levels. This effect could be reversed at day 17 by concurrent tri-iodothyronine substitution therapy. In hypothyroid rats, maximum brain choline acetyltransferase activity was 30 per cent less than normal adult levels.     

EFFECTS OF POTASSIUM ON INDICATOR SPACES AND FLUXES IN SLICES OF BRAIN CORTEX FROM ADULT AND NEW-BORN RATS

—Inulin, sucrose and chloride spaces were measured in slices of brain cortex from adult and from new-born rats incubated in‘balanced', potassium-rich and sodium-rich media. The efflux of the radioactive markers was followed in the two first media and the following results were obtained: (1) In brain slices from new-born rats inulin and sucrose spaces were of identical magnitude (35 per cent). The space magnitude was essentially unaffected by excess potassium. The chloride space was somewhat larger than the inulin (sucrose) space, and the difference increased continuously but relatively slightly with the external potassium concentration. By far the largest amount (i.e. about 90 per cent) of the efflux of radioactive inulin, sucrose and chloride occurred from a rapidly exchanging compartment during incubation in both ‘balanced’ and potassium-rich media. (2) In brain slices from adult rats the inulin space (35 per cent) was significantly smaller than that of sucrose (50 per cent) and of chloride (65 per cent); it seemed to represent the extracellular space relatively well although 10 per cent of the efflux occurred from a slowly exchanging (probably intracellular) compartment. High concentrations of potassium led to a reduction of the inulin space which was probably a result of the concomitant intracellular swelling. The hyperosmolarity per se did not affect the space magnitude, but an increase of the sodium concentration exerted a competitive inhibition of potassium effects on the inulin space. Of the sucrose efflux, 20 per cent occurred from a slowly exchanging compartment in both ‘balanced’ and potassium-rich media, and 30 per cent of the chloride exchanged with this compartment when the tissue was incubated in a ‘balanced’ medium. An increase of the external potassium concentration caused a drastic increase of the chloride space and a reduction of the slowly exhanging fraction of chloride efflux to less than 10 per cent.     

FATTY ACID COMPOSITION OF CEREBROSIDES IN MICROSOMES AND MYELIN OF MOUSE BRAIN

Abstract— The fatty acid composition of cerebrosides isolated from myelin and from light and heavy microsomes of adult mouse brain was determined. 2-Hydroxy fatty acids represented 80 per cent of the fatty acids in myelin cerebrosides and approximately 55 per cent of the fatty acids in both light and heavy microsomes. In myelin, the majority of the fatty acids, both normal and hydroxy, were of chain length > C-20; in microsomes, shorter chain acids (C-16 to C-20) predominated.     

Isolation and partial characterization of a sulfogalactoglycerolipid from rat brain.

The brain of adult rats were analyzed for the presence of 35-SO4-containing glycolipids following intraventricular injection of Na2-35SO4. Radiochromatographic analyses revealed the presence of two minor 35-SO4-containing glycolipids, in addition to sulfogalactosylceramide. One of these two minor sulfolipids was isolated and tentatively identified as a 1-O--alkyl-2-0-acyl-3-(3'-sulfogalactosyl)-glycerol, a compound recently demonstrated to be the major glycolipid of mammalian testis. The alkyl and acyl compositions of the compound from rat brain are more heterogeneous than those from rat testis. The non-sulfated form of the galactoglycerolipid was also detected in rat brain. The amount of the sulfogalactoglycerolipid in rat brain is 0.19 mumol per gram wet weight, approximately one-third of the amount in rat testis (per gram wet weight), and is approximately one-fifteenth that of sulfogalactosylceramide in rat brain. The possible significance of the common occurrence in brain and testis of sulfated and non-sulfated galactolipids is discussed.     

Correlation between [U-14C]glucose metabolism and function in perfused cat brain

In brain perfusion experiments conducted with blood containing [U-14C]glucose the relative specific activity (RSA) of blood glucose carbon incorporated in brain intermediate metabolites was measured. It was demonstrated that the so-called metabolic pattern of Geiger is not constant, but it bears a close relation to the function of the brain. The results of the study may be summarized briefly as follows. (1) In a group (A) of cats with a high level of brain function, the RSA of lactic acid was 75 per cent; that of glutamic acid 80 per cent; aspartic acid 75 per cent; glutamine 61 per cent; GABA 43 per cent; and respiratory CO2 55 per cent. It was observed that the major part of the carbon of amino acids, such as glutamic acid and aspartic acid, which are directly associated with the tricarboxylic acid cycle are derived from blood glucose. (2) In a group (B) showing a low level of brain function, the RSA of each amino acid was considerably lowered. The RSA of glutamic acid and aspartic acid was about 50 per cent and that of respiratory CO2 was 27 per cent. (3) In a group (C) with a still lower level of brain function, each amino acid as well as the respiratory CO2 had still lower RSA values. (4) The metabolic pattern of Geiger corresponds to values obtained during low functional activity of the brain in our experiment.     

DIFFERENCES IN THE NUCLEAR RIBONUCLEIC ACID CONTENT IN DIFFERENT AREAS OF THE HUMAN ADULT AND FOETAL BRAIN*

Abstract— Four different areas of ten brains from adults in the age group between 44 and 74 years and two areas of six foetal brains were studied. The cellular density was similar in the different areas of the brain of adult man; it averaged 11.9 × 10⁷/g wet tissue. The number of neurons per g wet tissue varied from 2.22 × 10⁷ in the cortex to 0.15 × 10⁷ in the centrum semiovale. In the brain of adults the overall RNA content of nuclei was higher for the cortex than for the corpus callosum or the centrum semiovale. The RNA content of neuronal nuclei was higher than that of non-neuronal nuclei. In the brain of foetuses the nuclear density was higher than in adults. The DNA content of a nucleus in foetal brain was 2–3 times as high as in adult brain and it approached adult values with increasing maturity.     

Regional concentration and chromatographic characterization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the brain of the bullfrog,Rana catesbeiana

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide which functions as a hypothalamic factor for pituitary hormone release, and as a neurotransmitter, neuromodulator and neurotrophic factor in both frogs and mammals. This study examined the quantitative distribution and chromatographic characterization of immunoreactive PACAP in the central nervous system (CNS) of the bullfrog, Rana catesbeiana, using an enzyme immunoassay (EIA), named avidin-biotin complex detectable EIA for PACAP, and high-performance liquid chromatographic (HPLC) analysis. The brain of adult bullfrogs contained relatively high levels of immunoreactive PACAP (344.63 pmol/g wet weight of tissue). The average concentrations of immunoreactive PACAP in the regions of the telencephalon, diencephalon, tectum, cerebellum, rhombencephalon, and spinal cord were 213.84, 767.14, 524.94, 192.71, 237.67, and 362.04 pmol/g wet weight of tissue, respectively. The concentrations of immunoreactive PACAP increased with the brain development during metamorphosis, and the concentration of immunoreactive PACAP in the brain of tadpoles at the end of metamorphosis was approximately 200 pmol/g wet weight of tissue. The predominant form of immunoreactive PACAP in the CNS of adult and tadpole was eluted closely with synthetic PACAP38, but another smaller immunoreactivity also appeared in a the fraction, which corresponded to the retention time of synthetic PACAP27, as analyzed by reverse-phase HPLC.     

Microdensitometric measures of cytoplasmic RNA and total protein in pyramidal neurons of the insular cortex and midfrontal gyrus in patients with Alzheimer's disease

Scanning and integrating microdensitometry of azure B- and Coomassie brilliant blue G-stained tissue sections was used to measure the levels of RNA and protein, respectively, in pyramidal neurons of the insular cortex (INS) and midfrontal gyrus (MFG) in patients with Alzheimer's disease (AD) and age-matched, nondemented control subjects. AD was associated with a decreased neuronal RNA (by 7·4 per cent) and protein (by 28·7 per cent) content in INS. Although the neuronal RNA content was maintained at the control amounts in MFG, the average protein level was lower (14·7 per cent) in AD patients. These results demonstrate a disease-related impairment in metabolic function in two brain regions connected via discrete corticocortical pathways. Such findings support the hypothesis that a primary site of pathology occurs in AD, and specific neural deficits occur secondarily in certain connected brain regions.     

Components of the plasma membrane of growing axons. III. Saxitoxin binding to sodium channels

The density of sodium channels was measured in growing and mature axons of the olfactory nerve of the bullfrog, using as a probe the drug saxitoxin (STX). The toxin binds to control nerves from adult animals in a saturable manner with a dissociation constant of approximately 23 nM at 4 degrees C and a capacity of 72 fmol/mg wet weight, equivalent to about five sites per square micrometer of axolemma. In growing nerves, obtained from adult frogs 4-5 wk following removal of the original nerve, the STX-binding capacity per wet weight of tissue is markedly reduced, to approximately 25% of control values, and appears to decrease in the proximodistal direction. STX-binding data, expressed as STX/mg wet weight, was converted to STX/micron 2 of axolemma using stereologically derived values of membrane area per milligram wet weight of nerve. The axolemmal content (area/mg wet weight) of all regions of growing nerve is substantially decreased compared to controls, but increases in the proximodistal direction by 60%. These changes in axolemmal area result in calculated STX receptor densities (per unit axolemmal area) which, in distal regions, are approximately at the level of the mature nerve and, in proximal regions, are actually increased above controls by 50 to 70%. Upon comparing the axolemmal density of intramembrane particles, reported in the companion paper, with the calculated density of STX receptors in both mature and growing nerves, we find a correlation between STX receptors and intramembrane particles with diameters of 11.5-14.0 nm. The growing axon's gradient of sodium channels and the shift from this gradient to a uniform distribution in the mature axon suggest (a) that sodium channels are inserted into the perikaryal plasmalemma and diffuse from there into the growing axolemma, and (b) that the axolemma undergoes functional maturation during growth.     

Sphingolipids of developing human central nervous tissue: changes in composition of sphingosine bases

—Developmental changes in composition of sphingosine bases of cerebrosides, sulphatides and sphingomyelins were investigated in samples from brain stem or corpus callosum from premature infants and patients aged 14 months, 39 and 59 years who died of non-neurological causes. Since insufficient material was obtainable from the premature brains, sulphatides were studied only in older cases. Individual sphingolipids were isolated by combinations of column chromatography and TLC, and were examined for purity by analytical TLC. Sphingosine bases were released by acid-catalysed methanolysis, and analysed as aldehydes by GLC. Effectiveness and limitations of methods used for analyses of sphingosine bases were evaluated. In contrast to adult sphingolipids in which approximately 95 per cent of the sphingosine bases was 18: sphingosine, as much as 10 per cent of the total was 18: dihydrosphingosine in immature sphingolipids. A compound tentatively identified as 20: sphingosine was present in foetal, infant and adult sphingomyelin at 5, 3 and 1 per cent of the total, respectively. Composition of sphingosine bases of non-ganglioside sphingolipids in human brain varies with age, presumably in a complex manner.     

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of ¹⁴C incorporation into brain DNA at 30 min after administration of [2-¹⁴C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 10⁶ and 18 × 10⁶ cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.     

Effects of nitrogen uptake on rice yield in northern Australia

Summary The yield responses to nitrogen uptake are described for water-sown IR8 rice grown in the wet season in northern Australia. Various patterns of nitrogen uptake were achieved by different times and amounts of nitrogen application in the range 0 to 300 kg per ha. Grain yields increased with nitrogen uptake at flowering stage up to approximately 120 kg per ha but decreased when uptake exceeded this level. The grain yield increase was associated mainly with increased panicle production, while grain yield decrease was associated with a reduction in the weight of ripened grain per panicle. Nitrogen concentration in grain over all experiments increased linearly with nitrogen uptake at flowering, but grain yield was reduced when grain nitrogen exceeded approximately 1.5 per cent. High nitrogen uptake at flowering was presumed to lead to depletion of carbohydrate reserves during the critical grain-ripening phase.     

Changes of sulfated mucopolysaccharides and mucopolysaccharidases during fetal development

The changes of sulfated mucopolysaccharides and mucopolysaccharidases during bovine fetal development were analyzed. It is shown that chondroitin sulfate C increases in concentration up to the 50th day of fetal development and then decreases progressively until its complete disappearance in most adult tissues. Likewise, hyaluronidase also reaches a peak on the 50th day and decreases in activity until its disappearance in adult tissues. On the other hand, heparitin sulfate and chondroitin sulfate B as well as beta-glucuronidase and beta-N-acetylglucosaminidase remain without significant changes during the whole period. The fetal chondroitin sulfate C is tissue specific with different molecular weights depending on the tissue of origin. Some properties of fetal muscle and brain hyaluronidase are also described. The possible role of chondroitin sulfate C and hyaluronidase in the processes of differentiation and division is discussed in view of the present findings.