GLYCOSPHINGOLIPIDS IN FETAL TAY-SACHS DISEASE BRAIN AND LUNG CULTURES

Abstract— A study was undertaken of the glycosphingolipids in cell cultures derived from cerebellum of Tay-Sachs disease fetal brain in order to determine the suitability of such cell strains as a model for Tay-Sachs disease. The glycosphingolipids in the Tay-Sachs disease cultured cerebellar cells were compared with those found in normal cultured cerebellar cells, normal and Tay-Sachs cultured lung cells, and normal and Tay-Sachs fetal brain. The glycolipids were separated by TLC, then analyzed by GLC of the trimethylsilyi derivatives of the methylglycosides of the sugar moieties. In the cultured cerebellar lines, the predominant gangliosides were G_M2, G_M3, and G_D3. There was a 4-fold increase of G_M2 in the Tay-Sachs as compared with the normal line. Only G_M3 and G_D3 gangliosides were found in the Tay-Sachs and the normal fetal lung cell cultures. The major neutral glycosphingolipids in all of the cultured cells which were analyzed were glucosylceramide, lactosylceramide, digalactosyl-glucosylceramide, and globoside. When the Tay-Sachs cerebellar cells were labelled with [1-¹⁴C]gluco-samine, some radioactivity was observed in the trihexosylceramide band, indicating the presence of a small amount of a galactosamine-containing trihexosylceramide which may be asialo-G_M2 (G_A2). The trihexosylceramide in Tay-Sachs fetal brain was identified as G_A2 by GLC. Both Tay-Sachs and normal fetal brain gangliosides were more complex than those found in the cultured cells. Long chain fatty acids (C_24:0 and C_24;1) predominated in all of the glycosphingolipids of the Tay-Sachs and the normal cultured cerebellar cells. In contrast, the glycosphingolipids of Tay-Sachs and normal fetal brain contained mainly the shorter chain fatty acids (C₁₆:₀, C₁₈:₀, and C_18:1). The cerebrosides in both the Tay-Sachs and normal fetal brains were mainly glucosylceramide with only small amounts of the galactosylceramide which predominates in infant brain. Cultured cells from the fetal Tay-Sachs disease     

ANOXEMIA AND BRAIN DISEASE

The author advances the concept that anoxemia, either in its general or restricted form, or both, is probably responsible for a considerable portion of “degenerative diseases,” whose etiologic delineation has not yet been traced. It is necessary, he believes, to enlarge greatly the comprehension of the disordered circulatory states to include oxygen want and thereby account for a number of conditions hitherto considered to be of unknown cause. More than this, he finds in oxygen want an explanation of the mechanism of a number of individual lesions or details of lesions otherwise not well understood. The author believes it is very likely that an understanding of cerebral anoxia in its ultimate ramifications will open still wider doors to the understanding of certain clinical syndromes the cause of which remains obscure.     

REGIONAL BRAIN LEVELS OF γ-HYDROXYBUTYRATE IN HUNTINGTON''S DISEASE

A highly sensitive electron capture gas chromatographic method was developed for quantitation of γ-hydroxybutyrate (GHB) in tissue. This method involves an improved, extraction and purification procedure and a one-step derivatization of GHB to the methyl ester-O-heptafluorobutyrate. As low as 5 ng of GHB in tissue was accurately quantitated by this method. By means of this improved method, endogenous levels of GHB in several regions of brains obtained post-mortem from patients with Huntington's disease were determined, and compared with brain samples obtained post-mortem from non-neurological controls. The levels of GHB found in the caudate and substantia nigra obtained from Huntington's patients were significantly higher than the GHB levels found in similar regions of brain obtained from a non-neurological control group. The content of GABA in the same choreic and control brain samples was also determined. No significant correlation between changes in GHB and GABA levels was observed although there was a trend towards an inverse relationship. The high level of GHR in Huntington's disease may be related to the decrease in succinate:oxidoreductase (EC 1.3.99.1) activity reported by Stahl & Swanson (1974). In two subjects (one control and one Huntington patient) the zonal distribution of GHB in substantia nigra was also determined. The zona reticulata from choreic brain contained a substantially higher level of GHB, whereas the zona compacta contained an amount similar to the level found in control brain.     

COPPER DEFICIENCY IN THE DEVELOPING RAT BRAIN: A POSSIBLE MODEL FOR MENKES'' STEELY-HAIR DISEASE

Abstract— In comparison to controls, copper-deficient suckling rats showed an appreciable decrease in body growth, a slight decrease in whole brain and cerebellar growth, and a highly significant decrease in myelination based on the activity of cerebellar 2',3'-cyclic nucleotide 3'-phosphohydrolase—a myelin enriched protein. Specific effects of a fivefold reduction in the copper content of brain were seen in a drastic decrease in cerebellar cytochrome c oxidase and smaller but significant drops in cerebellar super-oxide dismutase and brain norepinephrine concn. These observations are discussed with respect to the neuropathology and biochemistry of Menkes' steely-hair disease, a sex-linked recessive disorder in humans characterized by copper deficiency.     

UBIQUINONE REDOX STATUS IN BRAIN IN VITRO

—The redox status of ubiquinone (Q) as an index of mitochondrial respiratory chain activity in chopped telencephalon from the rat was investigated. With 12mm -glucose under aerobic conditions Q was 73% oxidized. Three mm -amytal and 2 μm -rotenone, inhibitors of mitochondrial NADH oxidation, resulted in Q being further oxidized by 14 and 16%, respectively. Thus, amytal and rotenone result in shifts in Q redox status of rat telencephalon slices in accord with the known site of action of these compounds in isolated mitochondria. However, under anaerobic conditions Q redox status shifted to 25% oxidized while incubation with 1 mm -NaCN resulted in a shift to only 58% oxidized. This discrepancy may result from concomitant inhibition in the supply of reducing equivalents to Q from substrate by NaCN or it may reflect a differential sensitivity of neuronal and glial mitochondria to NaCN. Evaluation of Q oxidation-reduction can be used to measure the effects of drugs and physiological perturbations on the redox status of the mitochondrial compartment of intact brain preparations.     

TAURINE DISTRIBUTION IN CAT BRAIN

Abstract— The distribution of taurine was investigated in 16 areas of the brain, in cats transected either at collicular or at midpontine level. A comparison was also made between the content in the same areas of the cerebral cortex of the two groups of cats showing respectively either a synchronized or an activated electrocorticogram. Taurine was determined in picric acid extracts by means of column chromatography followed by thin layer chromatography of the eluates. The levels of taurine were fairly uniform in all areas investigated with the exception of the lateral geniculate bodies, the pituitary gland and the pineal gland where the levels were higher than in all other regions. The taurine content of the cortex of cats showing a synchronized EEG pattern was higher than in the cortex of cats showing an activated pattern. The results are discussed in the light of the limited information available on the possible role of taurine in the CNS.     

NUCLEOTIDE METABOLISM IN RAT BRAIN

Abstract— The uptake, the conversion to nucleotides, and their incorporation into RNA for labelled glycine, aspartate, the free bases and nucleosides of purines and pyrimidines were investigated with cortical slices of rat cerebrum. At the end of a 1-hr incubation time the slice-to-medium ratio of the radioactivities for labelled aspartate, glycine, adenine and adenosine were 34, 26, 20 and 5, respectively, while the slice-to-medium ratios for hypoxanthine, inosine, guanine, guanosine, xanthine, orotate, cytidine, cytosine, uridine, and uracil ranged from 1.3:1 to 2:1. Over 99 per cent of the total radioactivity taken up by the cortical slices was present in the TCA supernatant and 86, 82, 65, 50, 34, 23, 20 and 1.6 per cent of this radioactivity was in the form of nucleotides at the end of a 1-hr incubation with labelled adenine, adenosine, hypoxanthine, inosine, uridine, orotate, cytidine, and glycine, respectively. The incorporation of various radioactive precursors into RNA of cortical slices suggests that nucleotides originating from either de novo synthesis or preformed purine derivatives enter the same nucleotide pool utilized for RNA synthesis. The supernatant fraction from homogenized cerebrum was investigated for the presence of various anabolic and catabolic enzymes associated with nucleotide metabolism. These results were correlated with the data from the RNA incorporation studies, and a possible role for AMP: pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, I.U.B. 2.4.2.7) to achieve intercellular transfer of AMP is discussed.     

GLUTATHIONE PEROXIDASE LEVELS IN BRAIN

Glutathione peroxidase activity in brains of various animals was examined. Enzyme activity was low, approximately 10 nmol of glutathione oxidized min^?1 mg protein^?1 or less. This result suggests that brain tissues contain insufficient glutathione peroxidase activity to provide protection from peroxidative damage and that an alternative mechanism may be present.     

THE MEASUREMENT OF TRIGLYCERIDE IN BRAIN AND THE METABOLISM OF BRAIN TRIGLYCERIDE IN VITRO

Abstract—

• 1 Triglyceride has been isolated from brain by thin-layer chromatography and determined by absorption of the carbonyl group at 1740 cm^?1. The means of yields from whole mouse brain, whole rat brain, rat brain grey matter, rat brain stem, and incubated slices of rat brain cortex were 0.15–0.17 μmole/g tissue.
• 2 The distribution of fatty esters varied from preparation to preparation. Palmitate, stearate and oleate usually occurred in greatest amounts. Hydrolysis of a preparation of triglyceride from whole rat brain with pancreatic lipase indicated that palmitate was equally distributed between the α and β esters.
• 3 [1-¹⁴C]Acetate was rapidly incorporated into triglyceride of slices of incubated rat brain cortex. When the resulting triglyceride was hydrolysed with pancreatic lipase the distribution of radioactivity amongst the hydrolysis products was consistent with both the α and β esters of the triglyceride having been radioactively labelled.