Soll Berl

Acetate and fluoroacetate as possible markers for glial metabolism in vivo.

[3H]Acetate has been shown by light autoradiographic methods to label the neuropil but not the perikarya in brain and retina. [3H]Fluoroacetate behaves similarly. The study provides anatomical data which support the concept of metabolic compartmentation of glutamic acid and associated metabolites previously derived from biochemical studies. It is suggested that these may be markers of non-neuronal metabolism, probably mostly glial, and may be used to develop procedures which will provide complementary data to that obtained with 2-deoxyglucose on regional metabolism in brain.

Purification and Characterization of a Soluble and a Particulate Glutamate Dehydrogenase from Rat Brain

Glutamate dehydrogenase (GDH) activity was determined in high‐speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low‐speed pellet (480 g for 10 min). Approximately 60% of the high‐speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similar Km values for the two GDH fractions for the substrates μ‐ketoglutarate, ammonia, and glutamate; however, there were small but significant differences in Km values for NADH and NADPH. Although the allosteric stimulation by ADP and L‐leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mM ADP, with apparent Ki values of 18.5 and 9.0 μM GTP for the soluble and particulate fractions, respectively. The HIll plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45°C and pH 7.4 in 50 mM phosphate buffer.

Effect of kainate on ATP levels and glutamate metabolism in cerebellar slices

The levels of ATP and amino acids were measured in rat cerebellar slices incubated in the presence of the neurotoxin, kainic acid (KA). 0.1--1 mM KA caused a significant decrease in tissue content of ATP, glutamate, aspartate and glutamine. The levels of glutamate and aspartate, but not glutamine, rose concomitantly in the incubation medium. The results are consistent with a multiaction mechanism for the neurotoxicity of KA.

Alterations in uptake and metabolism of aspartate and glutamate in brain of thiamine deficient animals

The high affinity uptake systems of aspartate, glutamate, glycine and taurine were studied in synaptosomal preparations isolated from the cerebellum, medulla-pons and the telencephalon of rats made thiamine deficient (TD) by diet or pyrithiamine (PT). There was a significant enhancement in the uptake of asparate/glutamate (probably transported by the same carrier) by the synaptosomal preparations of the cerebellum only, in both groups of thiamine-deficient animals as compared to controls. This was due to an increase in the number of uptake sites and not to an alteration of the binding affinity. Aspartate levels decreased significantly in all three brain areas of PT-treated animals and this change was greatest in the medulla-pons and the cerebellum and least in the cortex. Glutamate and serine levels were significantly decreased only in the medulla-pons whilst the concentration of glutamine was significantly increased in the three brain regions studied. The changes in both uptake and levels of amino acids in TD rats were reversed by thiamine therapy. Though the uptake studies do not discriminate between an altered aspartergic or glutamergic system, the changes in the levels of aspartate in the cerebellum suggest that the aspartergic system is involved. Since earlier studies showed a selective impairment in the high affinity uptake of serotonin by cerebellar synaptosomes, thiamine deficiency could impair cerebellar function by inducing an imbalance in its neurotransmitter systems.

Effect of thiamine deficiency on brain serotonin turnover.

Serotonin turnover has been investigated in regional brain areas of rats made thiamine deficient by pyrithiamine (PT). Following intracisternal injection of [14C]5-hydroxytryptamine ([14C]5-HT), a marked increase in the accumulation of [14C]5-hydroxyindoleacetic acid ([14C]5-HIAA) was found in the medulla-pons, hypothalamus and cerebral cortex. [14C]5-HT levels were normal in all of the brain areas except the cerebral cortex which had an increase of 58%. The ratio of [14C]5-HIAA/[14C]5-HT was significantly increased in every brain region of PT-treated rats except the cerebral cortex. Part of this increase in [14C]5-HIAA was shown to be due to impairment of active transport of this 5-HT metabolite out of the brain. However, increased 5-HT synthesis in the cerebellum, hypothalamus, striatum, hippocampus and cerebral cortex was demonstrated by measurement of 5-HT accumulation after inhibition of brain monoamine oxidase. PT-induced increase in endogenous 5-HIAA in the medulla-pons occurred simultaneously with the onset of neurological signs and both parameters were reversible by thiamine administration. These results suggest that acute thiamine deficiency, induced by PT, both increases brain 5-HT synthesis and impairs 5-HIAA efflux from the brain. There is a close correlation between neurological manifestations and changes in brain 5-HT metabolism in acute thiamine deficiency.

Actin-Stimulated Myosin Mg2+-ATPase Inhibition by Brain Protein

Abstract: A low‐molecular‐weight protein, isolated from bovine brain, inhibits the actin‐stimulated Mg‐ATPase activity of myosin from striated muscle. This inhibition is probably related to its ability to cause actin to revert from its polymerized to its depolymerized state and to prevent the polymerization of actin. Its effect on the polymeric state of the actin has been demonstrated by viscosity studies, DNase inhibition assay, and electron microscopy. Heavy meromyosin can overcome the effect of the brain protein and stimulate the polymerization of actin. The inhibition of ATPase activity is in part dependent upon the relative amounts of brain protein, actin, and myosin. The apparent molecular weight of the brain protein is approximately 20,000 daltons. It appears to be a heat‐labile glycoprotein containing 5–6% carbohydrate.

Spiroperidol Binding in the Human Caudate Nucleus

The displacement of spiroperidol binding by the optical isomers of butaclamol in synaptosomal preparations of the caudate nucleus obtained from parkinsonians and non-parkinsonians at post mortem were investigated. A group of spiroperidol binding sites found in the non-parkinsonians was less strongly expressed or absent in analogous tissue of the parkinsonian. The significance of these findings in relation to Parkinsons disease and its treatment are discussed.

Distribution of actin and myosin in a rat neuronal cell line infected with herpes simplex virus

SummaryThe indirect immunofluorescence technique was used to study alterations in the distribution of actin and myosin filaments in a rat B 103 neuronal cell line infected with herpes simplex virus type 1 (HSV-1). In uninfected cells, actin filaments were arranged in parallel and ran lengthwise from one end of the cell to the other; although myosin filaments were closely associated with actin filaments, additional myosin formed a netlike distribution which did not stain for actin. In infected cells, actin filaments became more randomly aligned and were concentrated along with myosin in close association with rosette-like formations of nuclei in syncytial cells; structural organization of actin and myosin within these intensely staining areas was no longer evident. The possible role of contractile proteins (actin and myosin) in viral infections of neural tissue is raised.

Uptake and Metabolism of Serotonin and Amino Acids in Thiamine Deficiency

Thiamine deficiency (TD) in humans causes a well known clinical syndrome in which cerebellar ataxia, ophthalmoplegia and mental changes prevail (Wernicke’s encephalopathy). Some patients, who survive this acute encephalopathy, develop a characteristic chronic amnestic syndrome (Korsakoff’s psychosis). Experimental thiamine deficiency, produced acutely with thiamine analogs (pyrithiamine) or chronically with deficient diet, can result in neurological manifestations and brain pathologic changes resembling those seen in patients with Wernicke’s encephalopathy (Dreyfus, 1976). Therefore, such animal models have been used extensively to study the effects of TD in the nervous system.

RESOLUTION OF BRAIN TROPONIN COMPLEX

Affinity chromatography was used to partially purify the troponin complex from crude regulatory proteins obtained from bovine brain cortex. Three components were obtained from this partially purified troponin complex by treatment with 6 M‐urea and 1 mM‐EGTA followed by chromatography on DEAE‐Sephadex‐A50. The effects of the three components on skeletal muscle actin activated MgATPase activity of muscle myosin (ATP phosphohydrolase, EC 3.6.1.3.) suggested that they were analogous to that of the skeletal muscle troponins I, C, and T. The apparent molecular weights of the brain troponin subunits (I, C, and T) were 18, 700, 14, 000 and 36, 400, respectively. The molecular weights of the former two proteins were less than those reported for the analogous skeletal muscle troponins. Thus, brain actomyosin complex may be regulated in a manner similar to that of striated muscle actomyosin.