Eric G. Brunngraber

Changes in the Concentration and Composition of Human Brain Gangliosides with Aging

Ganglioside content and composition were studied in whole brains from 9 neurologically normal male individuals ranging from 25 to 85 years in age. The content of ganglioside-bound sialic acid decreased from 1,070 to 380 micrograms/g fresh tissue at 85 years. Ten individual ganglioside fractions were identified on high-performance thin-layer chromatography, seven of which were quantified. With age, ganglioside composition shifted to a more polar pattern due to an increase in the relative concentration of the more polar fractions GQ1b, GT1b and GD1b and a decrease in GD1a and GM1. Except for GQ1b, the absolute concentration of all gangliosides decreased with age. All changes were more pronounced in younger ages. Results are discussed in relation to structural changes occurring in the aging brain, and the involvement of gangliosides is suggested.

Absence of ganglioside GM1 in astroglial cells from 21-day old rat brain: Immunohistochemical, histochemical, and biochemical studies

A procedure was developed for the cultivation of cells derived from the cerebral hemispheres of the 21-day old rat. Approximately 98 percent of the cells in a 10 day culture are astrocytes that contain glial fibrillary acidic protein. Analysis of the extracted gangliosides by thin layer chromatography revealed that ganglioside GM1 was absent and that the predominant ganglioside was GM3. Very small amounts of the polysialogangliosides GD1a, GD1b, and GT1b were detected. The concentration of gangliosidic NeuNAc per mg protein in these astrocytes was only 3 percent that observed in the 5 day culture of a mixed cell preparation from newborn rat brain. Immunohistochemical and histochemical studies were performed on the mixed cell population of the minced tissue of 21-day old rat brain prior to cultivation. Astrocytes did not stain for hyaluronectin. These cells also did not provide a positive staining reaction for ganglioside GM1 utilizing the antiganglioside GM1 peroxidase-antiperoxidase procedure and the biotinylated choleragen-avidin-peroxidase procedure. These two histochemical methods for ganglioside GM1 also did not stain astrocytes that had been cultured for 5 days. Oligodendroglial cells, which were also present in the uncultured 21-day-old minced brain tissue, stasined positively for ganglioside GM1 and hyaluronectin. Hyaluronectin had previously been shown to be a marker for oligodendroglia. Oligodendroglial cells which were present in the 5 day cultures of 21-day old brain tissue also provided a positive reaction for ganglioside GM1. It is concluded that ganglioside GM1 is absent in astroglia. The presence of small amounts of polysialogangliosides in the “pure” astrocyte preparation is discussed.

Stereo- and regio-selectivity of diethylaminosulfur trifluoride as a fluorinating reagent for methyl glycosides

Methyl glycopyranosides reacted with diethylaminosulfur trifluoride (DAST) in the absence of solvent to yield methyl dideoxy-difluoro and deoxy-fluoro glycopyranosides. Methyl alpha-D-glycopyranosides produced 6-deoxy-6-fluoro- and 4,6-dideoxy-4,6-difluoro derivatives with Walden inversion at C-4. Methyl beta-D-glucopyranoside also produced a 3,6-dideoxy-3,6-difluoro derivative, with Walden inversion at C-3. Methyl 6-O-trityl-alpha-D-glucopyranoside, reacted with DAST to yield the corresponding 4-deoxy-4-fluorogalactopyranoside derivative.

Distribution of endogenously phosphorylated proteins in subcellular fractions of rat cerebral cortex

The cerebral cortex from adult rats was separated into several subcellular fractions by using established methods of differential and sucrose density gradient centrifugation. Aliquots from each fraction were incubated with γ-32P-ATP, in the presence and absence of adenosine 3′,5′-monophosphate (cyclic AMP), and its protein constituents were separated by means of SDS-slab gel electrophoresis. Fractions containing nuclei, synaptosomes, myelin, microsomes, and soluble proteins each showed a characteristic pattern of protein staining and of endogenously phosphorylated proteins detected by autoradiography of the gels. Cyclic AMP-stimulated phosphorylation of proteins with MW 78K and 84K can serve as markers for membranes of synaptic origin, while cyclic AMP-independent phosphorylation of low-molecular-weight proteins (15K–20K) is characteristic of myelin. The finding of different phosphoproteins in various subcellular fractions may be related to the diversity of cellular functions known to be regulated by phosphorylative activity.

Localization of Hyaluronectin in Oligodendroglial Cells

Abstract: Astroglia and oligodendroglia in primary cell cultures were identified by immunohistochemical staining with antiglial fibrillary acidic protein and anticerebroside antisera, respectively. The antiserum to hyaluronectin (HN) was utilized to show that this hyaluronic acid‐binding glycoprotein was localized in the Oligodendroglial cell. This is consistent with the recent finding that HN is localized in the node of Ranvier. Astroglia did not react with antihyaluronectin.

Endorphin-regulated protein phosphorylation in brain membranes

Abstract This study was initiated to determine whether opioid peptides exert direct effects on the phosphorylation of specific proteins in membranes from rat neostriatum. It was found that low concentrations of β-endorphin (0.1–10nM) inhibit the phosphorylation of specific proteins designated F and H (M.W. 47,000 and 10–20,000 respectively). In addition, β-endorphin produced an overall stimulation of phosphate incorporation into other membrane proteins, the phosphorylation of which is dependent on calcium ions. The stimulatory effects were blocked by naloxone, but the inhibitory effects were not. The regulation of membrane protein-phosphorylation by endorphins may constitute a biochemical mechanism mediating for some of the physiological affects of these peptides on neuronal function.

Binding of concanavalin A to the brain-specific proteins obtained from human white matter by affinity chromatography.

A PROTEIN fraction was obtained from EDTA-saline extracts from human white matter by affinity chromatography utilizing specific antibodies as immunoadsorbents (WARECKA & BAUER. 1966; WARECKA e t al., 1972). The isolated protein material was shown immunologically to be brain-specific. The material was recently reportcd to stimulate the multiplication of cultured lymphocytes from patients suffering from multiple sclerosis and related diseases; such stimulation was not observed with lymphocytes obtained from norinal individuals (DELMOTTE & WARLCKA, 1973). The active preparation has been shown to contain several glycoprotein and protein bands by means of gel electrophoresis in the presence of sodium dodecylsulphate (BRUNNGRABER er d., 1974). A glycoprotein that showed a strong staining reaction with the periodic acid-Schiff test was found to be a prominent componcnt. It migrated a t a rate that corresponded to an apparent molecular weight of 50,000. The presence of brain-specific glycoproteins in the aqueous extract of white matter had first been shown by immunoelectrophoresis; a strong heterogeneous precipitation line in the alpha2 area stained with the periodic acid-Schiff reagent and was modified by pretreatment with neuraminidase (WAKICKA & BAUER. 1967). In this paper we report that the active fraction contains glycoproteins that bind to concanavalin A (Con A) and that these can be separated from other antigens by adsorption to this lectin. The active preparation obtained bq adsorption to and elution from specific brain antibodies characteristically contained a high concentration of carbohydrate material. Although the ratio of hexoses (determined by the phenolsulphuric method of Dunors rt ul., 1956) to protein (cstimated by the method of LOWKY ef ul.. 1951) ,varied from preparation to preparation, the preparation contained on the average 0.25 mg of hexose per mg of protein. Estimation of the sugar composition (Table I ) indicated the presence of mannose, glucose, galactose, fucose, N-acetylgiucosainine. N-acetygalactosamine. and N-acetylneuraminic acid

Electrophoretic analysis of human brain-specific proteins obtained by affinity chromatography

GLIAL cells and aqueous extracts from human white matter contain a brain-specific glycoprotein that can be isolated by means of Sepharose immunoadsorbents (WARECKA et a/., 1972; TKIPATZIS er af.. 1971). In the final step of this procedure, the brain-specific protein antigens are adsorbed by their antibodies which have been coupled to Sepharose 4B. The protein antigens are subsequently eluted with acidic glycine buffer. The antibodies have been prepared against an aqueous extract from human white matter; antibodies to proteins that are common to liver and brain tissue and serum were previously removed by adsorption. Evidence for the existence in the aqueous extract from human white matter of at least one other brain-specific protein in addition to theplycoprotein has been reported (WAN C K A r r d.. 1972). The purpose of the experiments reported in this communication was to determine the degree of heterogeneity of the brain-specific protein preparation (GSP) obtained by affinity chromatography and the molecular weight of the individual components.

Synthesis and release of sulfated glycoproteins by cultured glial cells

Both primary cultured glial cells and cloned (C-6) glioma cells have been shown to synthesize and release sulfated glycoproteins. It was found that N-linked tri- and tetra-antennary glycopeptides recovered from the glycoproteins contained most of the (35S) sulfate label. C-6 glial cells showed a higher rate of oligosaccharide sulfation than the primary glial cultures. Both cell types exhibited a high rate of release of sulfated glycoproteins into the medium. The ratio of 35S/3H incorporated from (35S) sulfate and (3H) glucosamine in the released material was higher than that of the glycoproteins associated with the cell, indicating an enrichment of sulfated glycoproteins in the secreted materials. Monensin inhibited both the synthesis and the release of sulfated glycoproteins.

Biochemistry, Function, and Neuropathology of the Glycoproteins in Brain Tissue

Two reviews that summarize our knowledge of neural glycoproteins have appeared (12, 14). In this review, we will summarize some of the more recent developments in this field of neurochemistry.