Ronald A. Pieringer

THE BIOSYNTHESIS AND CONCENTRATION OF GALACTOSYL DIGLYCERIDE IN GLIAL AND NEURONAL ENRICHED FRACTIONS OF ACTIVELY MYELINATING RAT BRAIN

—In continuation of our studies on the association of the galactosyl diglycerides of brain with myelination, we have measured the biosynthesis and concentration of these glyceride glycolipids, in oligodendroglial, astroglial, neuronal, and myelin enriched fractions from brains of rats of postnatal age 16, 19 and 29 days. The relative purity of cell fractions and myelin derived from 50 to 60 brains of each age‐group was checked by phase contrast microscopy and 2′,3′‐cyclic nucleotide‐3′‐phosphohydrolase activity. The relative purity was comparable to that reported by other investigators for cell fractions from bovine brain. Of the three cell types, the oligodendroglia had the highest and the neurons had the lowest capacity to enzymatically synthesize and to accumulate monogalactosyl diglyceride. The amount of monogalactosyl diglyceride found in myelin compared to that found in oligodendroglial fraction greatly increased during development between 16 and 29 days of age. The biosynthesis of galactosyl ceramide but not glucosyl ceramide was highest in oligodendroglial enriched cell fraction. However, ceramide glucosyl‐transferase activity, which was greatly affected by the method used for cellular separation, was highest in a microsomal fraction derived from grey matter. Our results support the contention that the oligodendroglial cells are the site of synthesis of myelin constituents of the central nervous system, and that there is a temporal relationship between this site of synthesis and the site of deposition (myelin).

Investigations on Myelination In Vitro: Regulation of 2′,3′-Cyclic Nucleotide 3′-Phosphohydrolase by Thyroid Hormone in Cultures of Dissociated Brain Cells from Embryonic Mice

Abstract: The direct influence of l‐3,3′,5‐triiodothyronine (T3) on the development of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (EC 3.1.4.37, CNPase) is demonstrated by using an in vitro culture system of dissociated embryonic mouse brain cells. Serum from a thyroidectomized calf, which contained low levels of T3 (31 ng/100 ml), and thyroxine, T4 (<1 μg/ml), was used in the culture medium in place of normal calf serum (T3, 103 ng/100 ml; T4, 5.7 μg/ml) to render the culture responsive to exogenously added T3. The lower levels of enzyme activity observed in the presence of such a deficient medium could be restored to normal values by T3 supplementation. Half‐maximal effect was obtained with 2.5 ± 10−9m‐T3. Three days of hormone treatment resulted in the maximal stimulation of CNPase. T4 was less effective in inducing CNPase activity and the inactive analog of the hormone, reverse T3 (3,3′,5′‐T3) was ineffective. The morphological appearance of the cells was characterized by deformed (smaller size and less in number) reaggregates in the cultures, lacking hormone.

Sulfogalactosyl diacylglycerol: Occurrence and biosynthesis of a novel lipid in rat brain

Summary Intracerebral injection of 35 SO 4 = into rats produced a 35 S-labeled lipid which was characterized by mild alkaline deacylation, acid hydrolysis, dioxane solvolysis, periodate oxidation, chromatography and infrared spectrophotometry to be 3′-sulfogalactosyl diacylglycerol. A detergent-solubilized enzyme from brain microsomes catalyzed the synthesis of this lipid from ( 35 S)-3′-phosphoadenosine-5′-phosphosulfate and monogalactosyl diacylglycerol.

Activation of macrophages by ether analogues of lysophospholipids.

SummaryInflammation processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L-α-lysophosphatidyl-DL-glycerol (lyso-Pg) resulted in an enhanced ingestion activity of peritoneal macrophages as did other lysophospholipids. However, lyso-Pg is rather toxic as indicated by a rapid decrease in macrophage activity 3 days after treatment while macrophage activity of lysophosphatidylcholine-treated mice continued to increase at least up to the 6th day after treatment. Alkyl-lysophospholipid derivatives, racemic 1-0-octadecyl-2-methylglycero-3-phosphocholine and -phosphoethanolamine stimulated mouse macrophages for Fc-mediated ingestion. Decomposed products of alkyl-lysophospholipids, alkylglycerols, were also found to be excellent activators of macrophages not only for ingestion of IgG-coated target cells but also antibody-mediated tumoricidal activity. Macrophages from mice treated with alkylglycerols developed superoxide generating capacity. Furthermore, alkylglycerols were found to be tumoricidal by direct contact with retinoblastoma cells. Therefore, the advantage of the potential application of alkylglycerols as chemotherapeutic agents is that they have dual beneficial effects: potentiation of macrophage activity and cytotoxicity to malignant cells.

Investigations on myelinogenesis in vitro: A study of the critical period at which thyroid hormone exerts its maximum regulatory effect on the developmental experession of two myelin associated markers in cultured brain cells from embryonic mice

Cultures of cells dissociated from embryonic mouse brain were used to assess the period in which thyroid hormone exerts its maximum influence on the regulation of the expression of two myelin associated metabolites, sulfolipids and 2′3′-cyclic nucleotide 3′-phosphohydrolase (CNP-ase). Cultures were grown for a specified number of days on a medium containing normal calf serum and then a portion were switched to a medium containing hypothyroid calf serum for 2 days. One half of these cultures were then supplemented with 50 nM triiodothyronine and growth was continued in all cultures for 3 more days. The cells were then assayed for CNP-ase activity and for their ability to incorporate35SO4 into sulfolipids. Studies with both myelin markers showed that in the earlier culture ages of 5, 8, and 11 days, thyroid hormone was able to fully restore the activities when abbed to cultures grown on hypothyroid calf-serum. In contrast, in the intermediate age range (15, 19, and 22 days) the restoration was partial, while in the higher ages, there was practically negligible restoration with T3. Since the culture system eliminates the possibility of a blood brain barrier and drastically decreases the complicity of other hormones, the lack of a myelinogeni response to thyroid hormone after a certain age must be attributed to the loss of sensitivity of the oligodendroglia to T3 possibly through genetic programming.

Regulation of myelin basic protein (arginine) methyltransferase by thyroid hormone in myelinogenic cultures of cells dissociated from embryonic mouse brain.

Abstract: The ontogenetic expression of myelin basic protein (arginine) methyltransferase in myelinogenic cultures of cells dissociated from embryonic mouse brain is highly dependent on the presence of thyroid hormone. Restoration of myelin basic protein methyltransferase to normal activities occurred 16 h after the addition of 100 nMl‐3,5,3′‐triiodothyronine to hypothyroid medium. These data demonstrate that thyroid hormone can regulate a posttranslational event. On the other hand, histone (arginine) methyltransferase has a different temporal activity pattern, which is not coordinated with myelination, and is not influenced by the lack of thyroid hormone. These data, which suggest the existence of two methyltransferases, were substantiated by demonstrating that the total amount of methylation of added myelin basic protein and histone is the same whether they are incubated together or separately. The requirement of thyroid hormone for the expression of the myelin basic protein methyltransferase and not for histone methyltransferase suggests that thyroid hormone preferentially regulates myelin‐associated events in these cultures.

Regulation of Neuronal Differentiation by Retinoic Acid Alone and in Cooperation with Thyroid Hormone or Hydrocortisone

Cultures highly enriched in neurons obtained from embryonic mouse cerebra were used to demonstrate that: (1) at the optimum concentration of 10(-8) M retinoic acid stimulated the neurons to produce axon- and dendrite-like structures as determined by phase contrast and fluorescent microscopy; (2) the same concentration of retinoic acid stimulated acetyl cholinesterase and choline acetyltransferase activities; (3) treatment of neurons of either prenatal or neonatal equivalent age with retinoic acid produced a sustained stimulation of neuronal differentiation, and (4) retinoic acid cooperatively stimulated neuronal differentiation with either thyroid hormone or hydrocortisone.

Biosynthesis of the phosphatidyl diglucosyl diglyceride of Streptococcus,faecalis (ATCC 9790) from diglucosyl diglyceride and phosphatidyl glycerol or diphosphatidyl glycerol

Abstract A phosphatidyl-group from either phosphatidyl glycerol or diphosphatidyl glycerol is covalently bonded to diglucosyl diglyceride to form phosphatidyl diglucosyl diglyceride in a reaction readily catalyzed by a disrupted membrane enzyme preparation from Streptococcus , faecalis .

Phosphatidylkojibiosyl diglyceride: Metabolism and function as an anchor in bacterial cell membranes

The recently discovered phosphoglycolipid, phosphatidylkojibiosyl diglyceride (PKD), was first observed as a biosynthetic by-product of glucosyl diglyceride metabolism inStreptococcus faecalis (faecium) ATCC 9790. Its structure is 1,2-diacyl-3-O(2′-O-α-D-glucopyranosyl-6′-O-phosphoryl-[1″,2″-diacyl-3″-O-sn-glycerol]-α-D-glucopyranosyl)-sn-glycerol. The biosynthesis of phosphatidylkojibiosyl diglyceride occurs by a novel transphosphatidylation reaction in which a phosphatidyl group is transferred from diphosphatidyl glycerol to the primary alcohol function at the 6 position of the internal glucose of kojibiosyl diglyceride. The reaction is catalyzed by a membrane-derived enzyme. Phosphatidylkojibiosyl diglyceride is bound covalently through a phosphodiester bond to the polyglycerol phosphate moiety of membrane lipoteichoic acid fromS. faecalis. Phosphatidylkojibosyl diglyceride has four nonpolar long chain fatty acyl groups and appears to have the necessary physico-chemical properties to anchor the long hydrophilic glycerol phosphate polymer of lipoteichoic acid to the hydrophobic environment of the membrane ofS. faecalis and probably other gram-positive bacteria as well.

Identification of Sulfhydryl-modified Cysteine Residues in the Ligand Binding Pocket of Retinoic Acid Receptor β

The diverse biological functions of retinoic acid (RA) are mediated through retinoic acid receptors (RARs) and retinoid X receptors. RARs contain a high affinity binding site for RA which is sensitive to treatment with sulfhydryl modification reagents. In an attempt to identify which Cys residues are important for this loss of binding, we created three site-specific RARβ mutants: C228A, C258A, and C267A. The affinity for RA of all three mutant receptors was in the range of that of the wild type protein, suggesting that none of these Cys residues are critical for RA binding. Rather, these modified Cys residue(s) function to sterically hinder RA binding; however, the modified Cys residues critical for the inhibition of binding differ depending on the reagent employed. Only modification of Cys228 is necessary to inhibit RA binding when RARβ is modified by reagents which transfer large bulky groups while both Cys228 and Cys267 must be modified when a small functional group is transferred. These data suggest that both Cys228 and Cys267 but not Cys258 lie in the ligand binding pocket of RARβ. However, Cys228 lies closer to the opening of the RARβ ligand binding pocket whereas Cys267 lies more deeply buried.