Sosamma J. Berger

An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases☆

Abstract A method for measuring nicotinamide-adenine dinucleotide by enzymatic cycling is described which uses malic and alcohol dehydrogenases (EC 1.1.1.37, and EC 1.1.1.1) for the enzyme couple. After cycling, malate is measured with either malic dehydrogenase or malic enzyme (EC 1.1.1.40). The method has a number of advantages compared to those previously described. The cycling rate is high (greater than 30 000/hr); blank values are low; the reaction is linear over a wide range of NAD concentrations; and the terminal indicator reaction requires only one step. In addition the system is well suited for double cycling. This was shown by measurements of NAD in nuclei and cytoplasm from single dorsal root ganglion cells (rabbit). The overall amplification in this case was about 1 000 000.

THE DISTRIBUTION OF GLYCINE, GABA, GLUTAMATE AND ASPARTATE IN RABBIT SPINAL CORD, CEREBELLUM AND HIPPOCAMPUS

The distribution of glycine, GABA, glutamate and aspartate was measured among about 60 subdivisions of rabbit spinal cord, and among the discrete layers of cerebellum, hippocampus and area dentata. A more detailed mapping for GABA was made within the tip of the dorsal horn of the spinal cord. Spinal ventral horn and dorsal root ganglion cell bodies were analyzed for the amino acids and for total lipid. The distribution of lipid and lipid‐free dry weight per unit volume was also determined in spinal cord. Calculated on the basis of tissue water, glycine in the cord is highest in lateral and ventral white matter immediately adjacent to the ventral grey. The distribution of GABA is almost the inverse of that of glycine with highest level in the tip of dorsal horn. It is most highly concentrated in the central 75% of Rexed layers III and IV. Aspartate in the tip of ventral horn is 4‐fold higher than in the tip of the dorsal horn and 3 times the average concentration in brain. Glutamate was much more evenly distributed and is relatively low in concentration with slightly higher levels in dorsal than in ventral grey matter. Large cell bodies in both ventral horn and dorsal root ganglion contained high levels of glycine. As reported by others, GABA was found to be high in cerebellar grey layers, area dentata, and regio inferior of hippocampus. Glycine was moderately high in cerebellar layers but moderate to low in hippocampus and area dentata.

Quantitative histochemical mapping of candidate transmitter amino acids in cat cochlear nucleus.

Levelsofy-amino-butyricacid,glycine,glutamateand aspartatewere measured insamples dissected from freeze-dried sections of cat cochlear nuclei and the immediate surroundings. Refined methods of sampling and record-keeping were systematically applied at the histologic level. A detailed three-dimensional map of the distribution of the amino acids was obtained for one cochlear nucleus, and the results were compared with selected samples from five other nuclei. On a dry weight basis, y-amino-butyric acid showed a 40-fold range, with highest levels in the molecular and fusiform cell layers of the dorsal cochlear nucleus. Glycine had a similar distribution as y-amino-butyric acid although less striking. The aspartate maps differed from those of the other amino acids in that levels were lower in granular regions than in the ventral cochlear nucleus. The distribution of glutamate was least impressive and, on a volume basis, was almost uniform. The auditory nerve lacked high levels of any of the amino acids, as did the olivocochlear bundle, but some unusually high glycine levels were found in parts of the vestibularnerve.The highest y-amino-butyricacid levelswere in the lateralvestibular nucleus,where glutamate levelswere remarkably low. The study of transmitter systems in the cochlear nucleus isin a beginning stage. Some information has been gathered to suggest that acetylcholine is a transmitter at certain synapses (see reference 18). Gamma-amino butync

Diversity of Metabolic Patterns in Human Brain Tumors: Enzymes of Energy Metabolism and Related Metabolites and Cofactors

Abstract: Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01–0.25 μg dry weight, were dissected from freeze‐dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose‐6‐phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6‐phosphogluconate dehydrogenase, and β‐hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose‐6‐phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6‐phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol‐1‐phosphate, and malate averages ranged from 104% to 127% of brain; and fructose‐1,6‐bisphosphate and glucose‐1,6‐bis‐phosphate levels were on the average 50% and 70% those of brain, respectively.

Diversity of metabolic patterns in human brain tumors--I. High energy phosphate compounds and basic composition.

Abstract— A total of 25 human brain tumors and 4 specimens of human brain were rapidly frozen at the time of operation and analyzed for ATP, ADP, AMP, UTP, total nucleoside triphosphates, P‐creatine, creatine, inorganic P, creatine kinase, lipid and glycogen. Analyses were made on submicrogram samples dissected from frozen dried sections in order to obtain material as free as possible from admixture with brain, necrotic tissue, blood, etc. A method was developed to estimate the original water content of the frozen dried samples. The brain specimens contained five times as much glycogen as small mammal brains, otherwise the values were similar. The tumors were in fair to excellent energy status. Within the areas chosen for assay, most of ATP and total adenylate were substantially higher than in brain in the case of 5 out of 15 gliomas, 3 of 5 meningiomas, and 1 of 4 schwannomas. UTP was almost invariably higher and other nucleotide triphosphates (besides ATP and UTP) lower than in brain. Glycogen was extremely variable, ranging among the gliomas from 0.05% to 6% of dry wt (4 times the level in the human brains). Creatine plus P‐creatine, compared to cerebral cortex levels, ranged from 15 to 85% in gliomas, was about 25% in meningiomas and the only medulloblastoma, and varied between 6 and 8% in the schwannomas. P‐Creatine varied more or less in keeping with the energy status. Creatine kinase was exceedingly variable. It was almost zero in the schwannomas, the medulloblastomas, 3 of 5 meningiomas, and 2 of 15 gliomas, whereas in some of the gliomas the activity approached that found in brain.

Levels of putative transmitter amino acids in the guinea pig cochlea.

Although considerable effort has been directed toward establishing acetyichobine as a chemical transmitter at the obivocochlear synapses, investigation of the robe of other putative chemical transmitters in cochbear function is in a preliminary’ stage (7, 10). In order to provide some basic information relevant to the possible robe of amino acids as transmitters in the cochlea, we have measured levels of ‘y-aminobutyrate (GABA), glycine, glutamate and aspartate in 0.2to 2.ig samples of tissue dissected from a total of six freeze-dried cochbeas, each obtained from a separate guinea pig. The sampling methods have been described previously ( 14). Samples were obtained from the various cochbear regions without attempting to sample systematically the entirety of each region or check the exact contents of each sample. The organ of Corti samples were split into inner and outer hair cell regions by a cut through the tunnel. The basilar tunnel fibers traveling at the floor of the tunnel were not in the organ of Corti samples but rather were likely to be in the basilar membrane samples. The assay procedures, by which all four of these amino acids were measured for each individual freeze-dried sample in oil wells, will be presented elsewhere’ but are based on published methods (1, 2, 4, 17). Low bevels of GABA were found in all cochlear regions examined, whereas levels of glycine were high for all regions (Fig. 1). Glutamate and aspartate levels were higher in the organ of Corti than in any other cochlear region. Additionally, there were gradients of aspartate bevels along the cochbear length of both inner and outer hair cehl regions. It should be mentioned that, since we were measuring all four amino acids in each sample and since the levels of all except GABA were quite high, the samples used did not provide amounts of GABA far above the bower limit of sensitivity of our GABA assay. The GABA bevels measured in individual organ of Corti samples had a considerable range: between -0.52 and +3.52 mmoles/kg dry weight for outer hair cell region and between -0.19 and +4.87 mmoles/kg

Amplification of pyridine nucleotide pools in mitogen-stimulated human lymphocytes.

Abstract NAD+ levels in resting human lymphocytes obtained from 20 donors were found to be 69.9 ± 21.7 pmols/106 cells. After 3 days of phytohemagglutinin (PHA) stimulation the NAD+ levels rose to 452 ± 198 pmols/106 cells. NADH, NADP+ and NADPH also increased in mitogen-stimulated lymphocytes, but the major portion of the increase in total pyridine nucleotide pools was accounted for by the increase in NAD+. When PHA-stimulated lymphocytes were incubated in nicotinamide-deficient growth medium, there was no significant increase in their total pyridine nucleotide pools; however, the ratios of oxidized to reduced pyridine nucleotides changed in a similar fashion to cells grown in medium containing nicotinamide. When lymphocytes in nicotinamide-deficient medium were stimulated with PHA they increased their levels of DNA synthesis and cell replication in a similar fashion to cells growing in nicotinamide-supplemented media. Human lymphocytes were able to synthesize pyridine nucleotides from nicotinamide or nicotinic acid; however, in the absence of a preformed pyridine ring they did not efficiently use tryptophan for the synthesis of NAD. Uptake of [carbonyl-14C]nicotinamide and conversion to NAD was markedly increased in PHA-stimulated lymphocytes; these cells also showed a marked increase in activity of the enzyme adenosine-triphosphate-nicotinamide mononucleotide (ATP-NMN) adenylyl transferase.

Association of poly(ADP-rib) synthesis with cessation of DNA synthesis and DNA fragmentation

CHO cells and cs-4-D3 cells were used to investigate the association between poly(ADP-rib) synthesis and the cessation of DNA synthesis and DNA fragmentation. The cs4-D3 cells are cold-sensitive DNA synthesis arrest mutants of CHO cells. Upon incubation at 33 degrees C, DNA synthesis in the cs4-D3 cells stops and the cells enter a prolonged G1 or G0 phase. The events that occurred when cs4 cells were incubated at 33 degrees C were similar to those that occurred when wild-type CHO cells grew to high density. (1) In both cases, DNA synthesis and cell growth stopped. (2) The NAD+ concentration/cell was 20-25% lower in growth-arrested cells than in logarithmically growing cells. (3) Poly(ADP-rib) synthesis was 3-4 fold higher in growth-arrested cells than in logarithmically growing cells. (4) The growth-inhibited cells developed DNA strand breaks which resulted in large percentages of their DNA appearing in the low molecular weight range of alkaline sucrose gradients. (5) Both the increased rate of poly(ADP-rib) synthesis and the development of DNA strand breaks appears to be characteristic of the G1 phase of the cell cycle. (6) When growth-inhibited cells were restored to conditions favorable for DNA synthesis and cell growth, the DNA strand breaks were repaired. (7) Prolonged incubation under growth-restrictive conditions resulted in the accumulation of more DNA strand breaks than the cells could repair. This was followed by cell death when the cells were restored to conditions favorable for cell growth.

The measurement of cyclic GMP and cyclic AMP phosphodiesterases

Abstract Methods are described for measuring phosphodiesterases for cGMP and cAMP in the range of activity yielding 10 −12 to 10 −8 mol of product. The 5′-GMP formed is measured by conversion to GDP with guanylate kinase. Amounts of GDP greater than 10 −10 mol are measured directly with an enzyme system which results in stoichiometric oxidation of NADH. This is either determined by the decrease in fluorescence or the excess NADH is destroyed with acid and the NAD + measured by its fluorescence in strong NaOH. With smaller amounts of GDP, sensitivity is amplified 1000-fold with the succinic thiokinase-pyruvate kinase cycle. In the case of cAMP diesterase, larger amounts of 5′-AMP are measured in the same way as 5′-GMP, except that adenylate kinase is substituted for guanylate kinase. With smaller amounts, the 5′-AMP is converted to ATP, and sensitivity is amplified with the adenylate kinase-pyruvate kinase cycle. As little as 20 ng dry weight of average brain is sufficient for accurate assay of the diesterase activity toward either cAMP or cGMP. When there is danger of significant destruction of AMP or GMP by tissue 5′-nucleotidase, this is prevented by adding GMP to the cAMP reagent, AMP to the cGMP reagent, or 5′-UMP to either reagent.

Distribution of Guanine Deaminase in Mouse Brain

Guanine deaminase was measured in nearly 100 different areas of mouse brain. The levels are relatively high in all parts of the telencephalon, both gray and white. It is especially active in parts of the olfactory tubercle and amygdala. Levels in the diencephalon range from low to as high as in the telencephalon. Brain areas caudal to the diencephalon, including all parts of the cerebellum, are almost uniformly below the level of detection. The enzyme is also virtually absent from the retina. The extreme range of concentration suggests that guanine deaminase might play a role in the metabolism of a neuroeffector.