NEUROCHEMICAL ASPECTS OF NEURONAL ONTOGENESIS IN THE DEVELOPING RAT CEREBELLUM: CHANGES IN NEUROTRANSMITTER AND POLYAMINE SYNTHESIZING ENZYMES

Abstract— Changes in the activities of several specific enzymes were measured in the cerebellum during development. Early transient increases were found in both ornithine decarboxylase and S -adenosylmethionine decarboxylase, enzymes involved in the initial steps of polyamine synthesis. Different patterns of changes were found in neurotransmitter synthesizing enzymes. Tyrosine hydroxylase activity achieved adult levels very early, by 3 days after birth, and remained at this level. Glutamic acid decarboxylase activity, while very low at early stages, increased rapidly before birth and then after a lag period of 10 days started to increase rapidly, directly related to the general growth of cerebellar weight and protein content. Choline acetyltransferase activity started to increase rapidly, reaching a peak of about 100% of adult levels at 3-7 days after birth; the activity then gradually declined and at 20 days, after reaching a low of about 55% of adult values, gradually started to increase, reaching adult levels later than 40 days after birth. The development of protein carboxymethylase activity was similar to that of glutamic acid decarboxylase, directly related to the general growth of the cerebellum. Several interpretations of the results are discussed.     

Acetylcholine Synthesis by Adult Bovine Adrenal Chromaffin Cell Cultures

Adrenal chromaffin cells normally synthesize and release catecholamines. In the present study, [3H]acetylcholine synthesis and another characteristic of cholinergic neurons, [3H]choline uptake, were studied in cultures of adult bovine adrenal chromaffin cells. Chromaffin cell cultures took up [3H]choline from the medium and acetylated the [3H]choline to form [3H]acetylcholine. The rate of [3H]acetylcholine synthesis increased after 19 days in culture and continued to increase up to 28 days in culture. [3H]Acetylcholine synthesis could be increased by stimulating the cells with a depolarizing concentration of K+. The ability for K+ to stimulate synthesis of [3H]acetylcholine developed only after 28 days in culture. [3H]Choline was taken up by the cultures through a single mechanism with a high (to intermediate) affinity for choline. [3H]Choline uptake was enhanced by Na+ omission in day-14 cultures, but was at least partially Na+-dependent in day-29 cultures. Hemicholinium-3 (IC50 less than 10 muM) inhibited [3H]choline uptake into chromaffin cell cultures. It is concluded that bovine adrenal chromaffin cells, maintained in culture, are able to exhibit cholinergic properties and this capacity is retained even by the mature adult cell.     

Metabolism of acetylcholine in the nervous system of Aplysia californica. IV. Studies of an identified cholinergic axon

[3H]Choline, injected directly into the major axon of the identified cholinergic neuron R2, was readily incorporated into [3H]acetylcholine. Its metabolic fate was similar to that of [3H]choline injected into the cell body of R2. Over the range injected, we found that the amounts of acetylcholine formed were proportional to the amounts injected; the synthetic capability was not exceeded even when 88 pmol of [3H]choline were injected into the axon. Newly synthesized acetylcholine moved within the axon with the kinetics expected of diffusion. We could not detect any selective orthograde or retrograde transport from the site of the injection. In contrast, as indicated by experiments with colchicine, 30% of the [3H]acetylcholine formed after intrasomatic injection was selectively exported from the cell body and transported along the axon. Most of the [3H]acetylcholine was recovered in the soluble fraction after both intra-axonal and intrasomatic injection of [3H]choline; only a small fraction was particulate. The significance of large amounts of soluble acetylcholine in R2 is uncertain, and some may occur physiologically. The concentrations of choline introduced by intraneuronal injection into both cell body and axon were, however, greater than those normally available to choline acetyltransferase in the cholinergic neuron; nevertheless, these large concentrations were efficiently converted into the transmitter. The synthetic capacity of the neuron supplied with injected choline may exceed the capacity of storage vesicles and of the axonal transport process.     

Effects of In Vitro Anoxia and Low pH on Acetylcholine Release by Rat Brain Synaptosomes

Acetylcholine and choline release from rat brain synaptosomes have been measured using a chemiluminescent technique under a variety of conditions set up to mimic anoxic insult, including conditions of low pH (6.2) and the presence of lactate plus pyruvate as substrate. Lactate plus pyruvate as substrate consistently gave higher respiration rates than glucose alone, but with either substrate (glucose or lactate plus pyruvate) the omission of Ca2+ caused an increase in respiration whereas a low pH caused a decreased respiration. Acetylcholine release under control conditions (glucose, pH 7.4) was Ca2+-dependent, stimulated by high K+ concentrations, and decreased significantly during anoxia but recovered fully after a period of postanoxic oxygenation. Low pH (6.2) suppressed K+ stimulation of acetylcholine release, and after a period of anoxia at low pH the recovery of acetylcholine release was only partial. With lactate plus pyruvate as substrate, the effects of anoxia and/or low pH on acetylcholine release and its subsequent recovery were exacerbated. Choline release from synaptosomes, however, was not affected by anoxic/ionic conditions in the same way as acetylcholine release. At low pH (6.2) there was a marked reduction in choline release both under aerobic and anoxic conditions. These results suggest that acetylcholine release per se from the nerve is very sensitive to anoxic insult and that the low pH occurring during anoxia may be an important contributory factor.     

Crystal structure of thiamindiphosphate-dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid.

The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 A resolution and refined to a crystallographic R-factor of 20.5% (Rfree 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open alpha/beta topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis, but with a relative difference of 20 degrees in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan.     

Effect on prostatic growth of 2-difluoromethylornithine, an effective inhibitor of ornithine decarboxylase.

2-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, causes marked changes in the polyamine metabolism of ventral prostate when given to adult rats in drinking water (20 g/l) for 3 consecutive days. A 90% inhibition of ornithine decarboxylase activity is accompanied by approx. 80% decreases of the concentrations of putrescine and spermidine and by a 36% decrease in spermine. Concomitantly, S-adenosylmethionine decarboxylase activity increases 7-fold and the concentration of decarboxylated S-adenosylmethionine 450-fold. When DFMO is given to immature rats for 12 consecutive days the above described changes are accompanied by a marked reduction in the age-dependent increases of the wet weight and RNA and DNA contents of the ventral prostate. In adult rats DFMO decreases the weight and RNA content of the ventral prostate within 4 days by 32% and 24% respectively and maintains them constant for the next 19 days. After 23 days of treatment, the prostatic weight is 46% of that of control animals of the same age, whereas the weights of other organs are only slightly decreased. Cytological studies carried out at this time show that DFMO reduces the size of both prostatic acini and the epithelial cells lining the acini.     

Regulation of S-adenosylmethionine decarboxylase activity in rat liver and prostate

Two methods were used for the quantitation of S-adenosylmethionine decarboxylase protein. The first involved titrating the active site of the enzyme by reduction of the Schiff base between 3H-decarboxylated S-adenosylmethionine and the pyruvate prosthetic group with sodium cyanoborohydride. The second method was radioimmunoassay with rabbit antiserum which was used to determine the total immunoreactive enzyme protein. It was found that the increased S-adenosylmethionine decarboxylase activity produced in rat prostate by treatment with alpha-difluoromethylornithine and in both prostate and liver by methylglyoxal bis(guanylhydrazone) were due entirely to increases in the amount of enzyme protein. The ratio of enzyme activity to protein (measured by either method) remained constant in rats treated with the drugs. Treatment with 2% alpha-difluoromethylornithine in the drinking water for 3 days increased prostatic S-adenosylmethionine decarboxylase protein by 5-fold. A substantial part, but not all, of this increase could be accounted for by a slowing of the rate of degradation of the enzyme. The half-life for loss of activity and titratable protein after inhibition of protein synthesis by cycloheximide was increased from 35 to 108 min by treatment with alpha-difluoromethylornithine. However, the half-life for loss of immunoreactive protein which was considerably longer was only increased from 139 to 213 min. The molecular weight of the S-adenosylmethionine decarboxylase subunit determined by immunoblotting was 32,000, and no smaller immunoreactive fragments were detected. These results indicate that spermidine depletion produced by alpha-difluoromethylornithine affects the degradation of S-adenosylmethionine decarboxylase at an early step involving the loss of the active site without substantial breakdown of the protein.     

Regulation of kinase reactions in pig heart pyruvate dehydrogenase complex.

1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.     

Pyruvate decarboxylase inactivation by interaction with substrate and molecular oxygen]

Pyruvate may promote the yeast pyruvate decarboxylase inactivation when affected by molecular oxygen. In the presence of pyruvate and O2 inactivation of enzyme increases with the initial substrate concentration increasing. pH-dependence of pyruvate decarboxylase inactivation under joint action of substrate and O2 has maximum in the region 6.9-7.5. It is suggested that the influence of pyruvate and molecular oxygen is connected with the coenzyme-substrate complex oxidation in an active site of yeast pyruvate decarboxylase on the steps preceding the release of free acetaldehyde.     

Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions.

Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate.     

The Utilization of Choline and Acetyl Coenzyme A for the Synthesis of Acetylcholine

Acetylcholine synthesis in rat brain synaptosomes was investigated with regard to the intracellular sources of its two precursors, acetyl coenzyme A and choline. Investigations with α-cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, indicated that pyruvate must be utilized by pyruvate dehydrogenase located in the mitochondria, rather than in the cytoplasm, as recently proposed. Evidence for a small, intracellular pool of choline available for acetylcholine synthesis was obtained under three experimental conditions. (1) Bromopyruvate competitively inhibited high-affinity choline transport, perhaps because of accumulation of intracellular choline which was not acetylated when acetyl coenzyme A production was blocked. (2) Choline that was accumulated under high-affinity transport conditions while acetyl coenzyme A production was impaired was subsequently acetylated when acetyl coenzyme A production was resumed. (3) Newly synthesized acetylcholine had a lower specific activity than that of choline in the medium. These results indicate that the acetyl coenzyme A that is used for the synthesis of acetylcholine is derived from mitochondrial pyruvate dehydrogenase and that there is a small pool of choline within cholinergic nerve endings available for acetylcholine synthesis, supporting the proposal that the high-affinity transport and acetylation of choline are kinetically coupled.     

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect. this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeasts were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeasts exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.     

Concentrations of putrescine and polyamines and their enzymic synthesis during androgen-induced prostatic growth

1. Castration of adult rats resulted in marked decreases in the amounts of putrescine, spermidine and spermine in the ventral prostate gland. Spermidine concentrations decline rapidly over the first 11 days after androgen withdrawal, reaching a value of only 12% of normal controls. Spermine concentrations diminish more slowly, reaching 24% of normal within 11 days. The spermidine/spermine molar ratio falls from 0.9 to 0.46 under these conditions. Putrescine concentrations decrease by 70% at 7 days after castration and then remain constant for some days. 2. After daily injections of testosterone propionate to rats castrated 7 days previously, prostatic spermidine and putrescine concentrations increase significantly within 24h; normal or even greater values are observed within 8 and 4 days respectively. In contrast, the spermine concentration does not increase until 5 days after commencement of androgen treatment. 3. The activities of two enzymes involved in polyamine biosynthesis (ornithine decarboxylase and a putrescine-activated S-adenosyl-l-methionine decarboxylase system) were greatly decreased soon after castration: after 7 days the respective values were 15% of normal for ornithine decarboxylase and 7% of normal for putrescine-dependent decarboxylation of S-adenosyl-l-methionine. Injection of testosterone propionate into animals castrated 7 days previously induced a rapid increase in both enzymic activities: ornithine decarboxylase was doubled in 6h, and increased three- to four-fold within 48h, whereas the putrescine-dependent decarboxylation of S-adenosyl-l-methionine doubled in 3h and increased tenfold within 48h of commencement of daily androgen treatments. 4. The activity of these enzyme systems was very low in the ventral prostates of hypophysectomized rats and was increased by administration of testosterone in a manner similar to that found in castrated rats. 5. Alterations in the activity of two ventral-prostate enzymes involved in ornithine production (arginase) and utilization (ornithine–2-oxoglutarate transaminase) that result from changes in the androgenic status of rats are described. 6. The findings presented suggest that the activities of ornithine decarboxylase and the putrescine-dependent S-adenosyl-l-methionine decarboxylase system, rather than ornithine concentrations, are rate-limiting for the formation of putrescine and polyamines in rat ventral prostate. 7. The relation of polyamines to androgen-induced prostatic growth is discussed with particular reference to the biosynthesis of proteins and nucleic acids.     

Choline increases serum insulin in rat when injected intraperitoneally and augments basal and stimulated aceylcholine release from the rat minced pancreas in vitro.

Intraperitoneal injection of choline (30-90 mg.kg-1) produced a dose-dependent increase in serum insulin, glucose and choline levels in rats. The increase in serum insulin induced by choline (90 mg.kg-1) was blocked by pretreatment with the muscarinic acetylcholine receptor antagonists, atropine (2 mg.kg-1), pirenzepine (2 mg.kg-1) and 4-diphenylacetoxy-N-methylpiperidine (2 mg.kg-1) or the ganglionic nicotinic receptor antagonist, hexamethonium (15 mg.kg-1). The effect of choline on serum insulin and glucose was enhanced by oral glucose administration (3 g.kg-1). Choline administration was associated with a significant (P < 0.001) increase in the acetylcholine content of pancreatic tissue. Choline (10-130 microm) increased basal and stimulated acetylcholine release but failed to evoke insulin release from the minced pancreas at considerably higher concentrations (0.1-10 mm). Hemicholium-3, a choline uptake inhibitor, attenuated the increase in acetylcholine release induced by choline augmentation. Choline (1-32 mm) inhibited [3H]quinuclidinyl benzilate binding to the muscarinic receptors in the pancreatic homogenates. These data show that choline, a precursor of the neurotransmitter acetylcholine, increases serum insulin by indirectly stimulating peripheral acetylcholine receptors through the enhancement of acetylcholine synthesis and release.     

Incorporation of [32P]phosphate into the pyruvate dehydrogenase complex in rat heart mitochondria.

1. Evidence is given for three sites of phosphorylation in the alpha-chains of the decarboxylase component of purified rat heart pyruvate dehydrogenase complex, analogous to those established for procine and bovine complexes. Inactivation of rat heart complex was correlated with phosphorylation of site 1. Relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. 2. Methods are described for measurement of incorporation of 32Pi into the complex in rat heart mitochondria oxidizing 2-oxoglutarate + L-malate (total, sites 1, 2 and 3). Inactivation of the complex was related linearly to phosphorylation of site 1 in mitochondria of normal or diabetic rats. The relative initial rates of phosphorylation were site 1 greater than site 2 greater than site 3. Rates of site-2 and site-3 phosphorylation may have been closer to that of site 1 in mitochondria of diabetic rats than in mitochondria of normal rats. 3. The concentration of inactive (phosphorylated) complex was varied in mitochondria from normal rats by inhibiting the kinase reaction with pyruvate at concentrations ranging from 0.15 to 0.4 mM. The results showed that the concentration of inactive complex is related linearly to incorporation of 32Pi into site 1. Inhibition of 32Pi incorporations with pyruvate at all concentrations over this range was site 3 greater than site 2 greater than site 1. 4. With mitochondria from diabetic rats, pyruvate (0.15-0.4 mM) inhibited incorporation of 32Pi into site 3, but it had no effect on the concentration of inactive complex or on incorporations of 32Pi into site 1 or site 2. It is concluded that site-3 phosphorylation is not required for inactivation of the complex in rat heart mitochondria. 5. Evidence is given that phosphorylation of sites 2 and 3 may inhibit reactivation of the complex by dephosphorylation in rat heart mitochondria.     

High-Affinity Choline Uptake in the Hippocampus: Its Relationship to the Physiological State Produced by Administration of Barbiturates and Other Treatments

Abstract: Choline uptake in hippocampal synaptosomes was not inhibited by pentobarbital administration when rats were decapitated immediately upon loss of the righting reflex (3–4 min) even though it was inhibited at later times post-injection, when the rats were still unable to right themselves. Choline uptake was increased when the animals were decapitated at convulsion after an injection of picrotoxin, high doses of bicuculline, or one of the convulsant barbiturates. However, another convulsant barbiturate, as well as strychnine and lower doses of bicuculline, did not increase choline uptake even though the animals also convulsed. Thus loss of righting reflex or convulsion is not directly correlated with changes in choline uptake. At 7 min after injection, levels of pentobarbital in the hippocampus (and other brain regions) were correlated with the degree of inhibition of choline uptake up to about 50% inhibition; however, greater inhibition could not be achieved with much higher brain levels of the drug. Although hippocampal uptake was partially inhibited at 1 h after septal lesions, 3 h after the lesion the inhibition was no longer apparent. Inhibition was almost complete 10–12 days after the lesion. These results suggest that other factors in addition to impulse flow influence choline uptake.     

Selective impairment of acetylcholine release and content in the central nervous system following intracerebroventricular administration of ethylcholine mustard aziridinium ion (AF64A) in the rat

Ethylcholine mustard aziridinium ion (AF64A) was administered via intracerebroventricular injection to rats. Unilateral injection of 40 nmol AF64A resulted in pronounced toxicity with an 80% mortality rate. Administration of 10 nmol unilaterally resulted in a significant reduction in both acetylcholine content and ouabain stimulated acetylcholine release in the hippocampus 2, 4 and 7 days after treatment. Non-specific changes in hippocampal levels of dopamine, noradrenaline and 5-hydroxytryptamine were also observed.Bilateral injection of 5 nmol AF64A was more effective than a unilateral 10 nmol injection in reducing acetylcholine release from hippocampus 4 and 7 days after treatment. Hippocampal acetylcholine content was also reduced (to 35% of control). In contrast, there was less effect on acetylcholine content in striatum and frontal cortices, and acetylcholine release from these areas was not decreased. Although there was a transient reduction in hippocampal 5-hydroxytryptamine content 4 days after treatment, this had recovered to control levels within 7 days. 5-Hydroxytryptamine levels in striatum or cortex were not affected, nor were there any changes in noradrenaline or dopamine contents in the areas studied.This study indicates that, in the correct dose range, AF64A can exert selective effects on cholinergic systems, particularly in the hippocampus. The selective cholinotoxicity of this compound makes it a useful tool in developing animal models of cholinergic dysfunction.     

Stimulation by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine of rat hepatic polyamine biosynthesis in vivo

Intraperitoneal injection of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), resulted in a rapid and transient induction of rat hepatic ornithine decarboxylase (ODC) activity. Maximal activity was found about 5 hr after application. The levels of putrescine and spermidine increased accordingly, reaching a maximum at 7 and 12 hr following injection, respectively, while the concentration of spermine remained almost constant. The implications of these findings are discussed in relation to the mechanism of induction of ornithine decarboxylase and concomitant polyamine biosynthesis.     

A Simple Bioassay for Chemicals Active to the Photosynthetic or Respiratory Systems of Plants

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet with or without 0.5% nicotinamide for 13 days, 180 mg/kg body weight of alloxan was injected in- traperitoneally into the rats. The rats were kept for 18 days with the same diet. The level of blood glucose was increased 6-fold in the group on a 20% casein diet by the injection of alloxan, while there was only a 2-foid increase in the group on a nicotinamide-containing diet and the decreased body weight was also lower in the group on the nicotinamide diet than the group on the casein diet. The body weight was indirectly related to the concentration of blood glucose. A marked increase was observed in the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase upon the injection of alloxan with both diets; on the other hand, the activities of kynureninase and NAD⁺ synthetase were decreased by the injection of alloxan. The activity of kynurenine aminotransferase increased in the group on the 20% casein diet by the injection of alloxan, while in the group on the nicotinamide-containing diet its activity was not increased by the injection. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to niacin is lower in the alloxan diabetic rat than normal rat. It was found that the activities of tryptophan oxygenase, aminocarboxymuconate-semialdehyde decarboxylase, and nicotinamide methyltransferase were directly related to the concentration of blood glucose, and that the activities of kynureninase and NAD⁺ synthetase were inversely related. There was no difference in the activities of 3-hydroxyanthranilic acid oxygenase and nicotinamide mononucleotide adenylyltransferase upon the injection of alloxan with both diets.     

THE EFFECT OF dl-ALLYLGLYCINE ON POLYAMINE AND GABA METABOLISM IN MOUSE BRAIN

Abstract— dl -Allylglycine, a potent inhibitor of glutamate decarboxylase in vivo when given intraperitoneally, causes a marked decrease in brain GABA concentration and at the same time a dramatic increase in l -ornithine decarboxylase activity and a simultaneous decrease in S -adenosyl- l -methionine decarboxylase activity followed by putrescine accumulation. It does not, however, alter the degree of GABA formation from putrescine. The timing of the recovery of glutamate decarboxylase activity after the injection of dl -allylglycine is concomitant with that of the GABA concentration, indicating that it is probably glutamate decarboxylase that is solely responsible for making up the GABA deficit caused by dl -allylglycine, and that the changes in polyamine metabolism are associated in some indirect way with the recovery process.