Identification of sulfhydryl-modified cysteine residues in the ligand binding pocket of retinoic acid receptor beta.

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 RARbeta mutants: C228A, C258A, and C267A.

Dodecylglycerol provides partial protection against glutamate toxicity in neuronal cultures derived from different regions of embryonic rat brain.

Primary cultures enriched in neurons dissociated from embryonic rat cerebral cortex, cerebellum, or hippocampus were treated in a chemically defined serum-free media with either vehicle, dodecylglycerol (DDG, 3 microM), or glutamate (75 microM), or preincubated with DDG for 4 or 24 h, and further incubated with glutamate. Their morphological and biochemical assessments (lactate dehydrogenase [LDH] release in the culture media, neuronal viability and intracellular Ca2+ mobilization) were made. Neurotoxic effects of glutamate and glutamate-mediated increases in intracellular Ca2+ were maximal in neurons from cerebellum and minimal in neurons from cortex.

Fluorescence studies on the molecular action of amphotericin B on susceptible and resistant fungal cells.

The molecular action of amphotericin B (AmB) on the cell membranes of both AmB-susceptible and AmB-resistant fungal cells was investigated through the use of the fluorescent membrane probe trimethylammonium diphenylhexatriene (TMA-DPH). AmB, the most effective drug for the treatment of systemic fungal infections, is known to interact specifically with membrane sterols, especially ergosterol (the major sterol in fungal cells). Treatment of AmB-susceptible Candida albicans and Cryptococcus neoformans cells with AmB induced a novel biphasic change in TMA-DPH fluorescence intensity over time.

Synergism between the antifungal agents amphotericin B and alkyl glycerol ethers.

The alkyl glycerol ether rac-1-O-dodecylglycerol inhibited the growth of members of two genera of yeasts, Candida and Cryptococcus, and was strongly synergistic with amphotericin B. At one-half its MIC, dodecylglycerol decreased the MIC of amphotericin B by as much as 80-fold. This high degree of synergism between dodecylglycerol and amphotericin B was demonstrated against a number of species of yeasts including Candida albicans, Candida tropicalis, Candida parapsilosis, Cryptococcus neoformans, Cryptococcus albidus, and Cryptococcus laurentii.

Regulation of neuronal thyroid hormone receptor alpha 1 mRNA by hydrocortisone, thyroid hormone and retinoic acid.

The regulation of thyroid hormone receptor alpha 1 (TR alpha 1) mRNA by hydrocortisone (HC), thyroid hormone (T3) and retinoic acid (RA) has been studied in mixed and neuronal primary cultures of cells dissociated from fetal rat cerebra. Steady-state levels of TR alpha 1 mRNA were increased as much as 5-fold at 13 days of development by 0.3 microM HC in both mixed and neuronal-enriched 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.

Regulation of neuronal differentiation in neuron-enriched primary cultures from embryonic rat cerebra by platelet activating factor and the structurally related glycerol ether lipid, dodecylglycerol.

Primary cultures enriched in neurons dissociated from embryonic rat cerebra were used to demonstrate that platelet activating factor and the structurally related ether glycerolipid, dodecylglycerol, are readily taken up in small amounts by neurons and that they stimulate the differentiation of neurons. The stimulation of neuronal differentiation was observed as a precocious development of axon-like extensions which correlated with a concentration-dependent increase in neuronal-specific enzyme activities. This stimulation of morphological and neurochemical factors by either platelet activating factor or dodecylglycerol was almost completely abolished by triazolam, a known inhibitor of platelet activating factor function.

Degrees of cooperativity between triiodothyronine and hydrocortisone in their regulation of the expression of myelin basic protein and proteolipid protein during brain development.

Cultures of cells dissociated from embryonic mouse cerebra were used to demonstrate: (1) that the developmental expression of the mRNA of proteolipid protein is dependent on thyroid hormone; (2) that the expression of the mRNA of proteolipid protein is stimulated not only by triiodothyronine but also by hydrocortisone, which achieve their respective stimulations by an additive and uncompetitive mechanism; (3) the stimulation of the net accumulation of the mRNA of myelin basic protein by hydrocortisone and triiodothyronine is also cooperative, additive, and uncompetitive, and (4) the stimulation of the net accumulation of myelin basic protein, during development by hydrocortisone, is completely dependent on the presence of thyroid hormone. These results suggest that the regulation of the synthesis of myelin basic protein by hydrocortisone requires the presence of triiodothyronine at a posttranscriptional event, but not for transcription itself.

Hydrocortisone regulates arylsulfatase A (cerebroside-3-sulfate-3-sulfohydrolase) by decreasing the quantity of the enzyme in cultures of cells dissociated from embryonic mouse cerebra.

Previous work from our laboratory (Biochem. J. 219:689-697 (1984] had shown that hydrocortisone stimulated the net accumulation of the myelin-specific sulfolipid in cultures of cells dissociated from embryonic mouse cerebra.

Synergism between penicillin G and the antimicrobial ether lipid, rac-1-dodecylglycerol, acting below its critical micelle concentration.

rac-1-Dodecylglycerol (DDG) and penicillin G (Pen G) act synergistically to dramatically lower the minimum inhibitory concentration (MIC) of each other in four Gram-positive bacteria studied. At one-half its MIC, DDG ether lowered the MIC of Pen G 10- to 80-fold. Under the same conditions, Pen G lowered the MIC of DDG 4- to 7.

Effect of hydrocortisone on myelin basic protein in developing primary brain cultures.

The hormones hydrocortisone (HC) and triiodothyronine (T3) are known to regulate myelinogenic parameters in cultures of brain cells. However, the effect of glucocorticoids on the myelin-specific metabolite, myelin basic protein, has not been previously studied. In the present studies we show that the concentrations of myelin basic protein (MBP) in developing primary cultures from mouse cerebra are significantly higher in HC (0.

In vivo studies on stereospecificity of the monoglyceride kinase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase and CDP-diglyceride synthase of Streptococcus mutans BHT using the stereoisomers of the ether lipid, dodecylglycerol.

Streptococcus mutans BHT metabolizes radioactive 3-dodecyl-sn-glycerol (sn-3-DDG) almost exclusively to lysophosphatidic acid, phosphatidic acid and 1,3-diradyl-sn-glycerol, whereas the cells of this organism metabolize 1-dodecyl-sn-glycerol (sn-1-DDG) to all of the glycerol lipids of S. mutans BHT, with the largest amounts incorporated into phosphatidylglycerol and diradylglycerol (mostly the 1,2- but also the 1,3-isomer). (The common names of lipids, such as phosphatidic acid, are used in the broader sense to mean that the lipid may contain alkyl as well as acyl groups.

Insulin: its binding to specific receptors and its stimulation of DNA synthesis and 2',3'-cyclic nucleotide phosphohydrolase activity in cerebral cells cultured from embryonic mouse brain.

The presence and specificity of insulin receptors was investigated in culture cells obtained from 15-16 days old embryonic mouse cerebra. Developmental studies suggested that the maximum insulin binding occurred at about 11 days in vitro (DIV). Scatchard analysis of binding data revealed two types of binding sites.

Investigations on myelinogenesis in vitro: II. The occurrence and regulation of protein kinases by thyroid hormone in primary cultures of cells dissociated from embryonic mouse brain.

The occurrence and regulation by thyroid hormone of four protein kinases (cyclic AMP independent and dependent, calcium/calmodulin stimulated, and calcium/phosphatidyl serine stimulated protein kinases) was studied in primary cultures of cells dissociated from embryonic mouse brain. Serum from a thyroidectomized calf, which contained low levels of L-3,5,3'-triiodothyronine, T3 (less than 25 ng/100 ml), and thyroxine, T4 (less than 1 microgram/100 ml) was used in the culture medium in place of normal calf-serum (T3, 130 ng/100 ml; T4 5.9 micrograms/100 ml) to render the cultures responsive to exogenously added T3.

Investigations on myelinogenesis in vitro: I. The occurrence of endogenous protein kinase and its role in the phosphorylation of myelin basic proteins in "myelin-like membranes" isolated from cerebral cell cultures.

The presence of a protein kinase capable of phosphorylating endogenous as well as exogenously added myelin basic proteins has been demonstrated in a myelin-like membrane fraction isolated from reaggregating and surface adhering, primary cultures of cells dissociated from embryonic mouse brain. Only the large and small components of myelin basic proteins were found to be phosphorylated when myelin-like membrane fraction was incubated with [gamma-32P]ATP. The protein kinase endogenous to the myelin-like membrane fraction was mainly of the cyclic AMP independent type.

Activation of macrophages by ether analogues of lysophospholipids.

Inflammation processes cause activation of phospholipase A in plasma membranes resulting in the production of various lysophospholipids. Treatment of mice with L-alpha-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.

Investigations on myelinogenesis in vitro: developmental expression of myelin basic protein mRNA and its regulation by thyroid hormone in primary cerebral cell cultures from embryonic mice.

The concentration of myelin basic protein (MBP) mRNA in primary cultures of cells dissociated from embryonic mouse cerebra and grown in the presence of varying amounts of thyroid hormone was measured using a 32P-labeled cDNA probe and a dot-blot procedure. The cDNA probe contained 1.85 kilobases of the gene for MBP.

Studies on the antibacterial activity of dodecylglycerol. Its limited metabolism and inhibition of glycerolipid and lipoteichoic acid biosynthesis in Streptococcus mutans BHT.

Growth-inhibitory concentrations of racemic sn-1(3)-dodecylglycerol inhibit the incorporation of [14C] glycerol into lipids and lipoteichoic acid of Streptococcus mutans BHT and alter the per cent composition of the glycerolipids. Increases in phosphatidic acid and diphosphatidylglycerol (at the expense of phosphatidylglycerol) contribute the most to the change in lipid composition. No cellular lysis occurs under these conditions.

Correlation between inhibition of myelin basic protein (arginine) methyltransferase by sinefungin and lack of compact myelin formation in cultures of cerebral cells from embryonic mice.

Sinefungin, a known inhibitor of protein methylation, inhibited the myelin basic protein (arginine) methyltransferase activity in homogenates of cultured cerebral cells from embryonic mice. Fifty percent inhibition was achieved with 25 microM sinefungin. Electron microscopic examination of the myelin fraction, isolated by gradient density centrifugation and obtained from untreated cells, revealed numerous ringlike multilamellar membranous substructures that had a major dense line periodicity, compactness, and the general appearance expected of myelin obtained by the same technique from whole brain.

Beta-adrenergic stimulation of protein (arginine) methyltransferase activity in cultured cerebral cells from embryonic mice.

Several adrenergic effectors and neurotransmitters were tested as potential regulators of myelin basic protein (MBP) and histone methyltransferase activities. Both enzymes were specifically activated by beta-adrenergic agonists in a stereospecific manner. Cyclic AMP (but not AMP) stimulated the enzymes to the same extent as did the beta-adrenergic agonist, (-) isoproterenol.

Developmental study on the regulation of neurotransmitter-sensitive adenylate cyclase systems in primary cerebral cell cultures from embryonic mice.

An ontogenetic study of the effect of various neurohormones and other activators on adenylate cyclase systems was carried out using cultures of cells from 15-d-old embryonic mouse brain. Dopamine stimulated the enzyme activity at earlier culture ages (i.e.

Investigations on myelinogenesis in vitro: a study of the critical period at which thyroid hormone exerts its maximum regulatory effect on the developmental expression 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 effect of penicillin on fatty acid synthesis and excretion in Streptococcus mutans BHT.

Treatment of exponentially growing cultures of Streptococcus mutans BHT with growth-inhibitory concentrations (0.2 microgram/ml) of benzylpenicillin stimulates the incorporation of [2-14C] acetate into lipids excreted by the cells by as much as 69-fold, but does not change the amount of 14C incorporated into intracellular lipids. At this concentration of penicillin cellular lysis does not occur.

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

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 nM L-3,5,3'-triiodothyronine to hypothyroid medium. These data demonstrate that thyroid hormone can regulate a posttranslational event.

Inhibition of peptidoglycan synthesis of Streptococcus faecium ATCC 9790 and Streptococcus mutans BHT by the antibacterial agent dodecyl glycerol.

Dodecyl glycerol inhibits the synthesis of the peptidoglycans of Streptococcus faecium ATCC 9790 and Streptococcus mutans BHT. This metabolic regulation represents the second known mode by which dodecyl glycerol expresses antibacterial activity. The first mode of action of dodecyl glycerol was shown to stimulate autolysin activity which degrades cell-wall peptidoglycan (Ved HS, Gustow E, Mahadevan V and Pieringer RA, 1984, J.