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. 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 RARbeta 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 RARbeta. However, Cys228 lies closer to the opening of the RARbeta ligand binding pocket whereas Cys267 lies more deeply buried.

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. Cotreatment of cells with DDG and glutamate failed to provide significant neuronal protection against glutamate in the three brain regions. Pretreatment of cells with DDG for 4 or 24 h prior to glutamate treatment provided significant neuroprotection as judged by morphological changes and a decrease in LDH activity. Neuroprotection of approximately 15-35% was observed following 4 h of DDG pretreatment, increasing to 60-85% protection after 24 h of DDG pretreatment. Although the mechanism of DDG's neuroprotective action remains to be elucidated, these results demonstrate that both glutamate and DDG have differential specificity for anatomical regions of the brain.

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. The initial decrease in fluorescence intensity results from energy transfer between AmB and TMA-DPH when AmB binds to the fungal cell membrane. The second phase of increasing fluorescence intensity is interpreted in terms of a combination of probe repartitioning and probe segregation as a result of the formation of membrane pores via the aggregation of AmB-ergosterol complexes. An AmB-resistant strain of C. neoformans, containing 94% of aberrant delta-8 double-bonded ergosterol precursors and only 4% of ergosterol (74% ergosterol in wild-type cells), exhibited the first phase of AmB binding but not the second phase of increasing fluorescence intensity. This result suggests that AmB's antifungal activity lies in its ability to form membrane pores due to aggregation of AmB-ergosterol complexes. The AmB-Induced biphasic fluorescence intensity profile may lead to further elucidation of the molecular action of AmB on fungal cells and may provide a sensitive method for screening the development of drug resistance in fungal cells.

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. All fractional inhibitory concentrations (for all strains and species) were calculated to be less than 1, and most were less than 0.6, again demonstrating strong synergism. Other alkyl glycerol ethers with alkyl chain lengths ranging from 8 to 18 carbon atoms were also found to be synergistic with amphotericin B against C. neoformans and C. albicans. Electron microscopy experiments showed that C. neoformans grown in the presence of dodecylglycerol had severely abnormal, deformed capsules. Although the mechanism of action of dodecylglycerol is not known, dodecylglycerol was not simply acting as a detergent. The natural detergent sodium deoxycholate could not substitute for dodecylglycerol. At comparable and higher concentrations, sodium deoxycholate had no fungicidal effect on its own, nor did it potentiate the activity of amphotericin B. Dodecylglycerol did not interact synergistically with the water-soluble antifungal agent fluconazole. The lipid-soluble hydrophobic properties of amphotericin B appear to be important for this synergistic effect, in that alkyl glycerol ethers could promote synergism with amphotericin B by potentially increasing the interaction between membrane-bound ergosterol and amphotericin B.

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. The regulation of TR alpha 1 mRNA by HC appears to be mainly limited to neurons. This conclusion is based on two observations. First, stimulation by HC occurs in cultures highly enriched in neurons at approximately the same time and extent as that seen in mixed-cell cultures (containing neurons, oligodendroglia and astroglia). Second, the stimulation reaches a peak at 13 days in mixed-cell cultures, an age at which neurons but not glia differentiate. In most cases, neither T3 (20 nM) nor RA (10(-7) M) stimulated an increase in TR alpha 1 mRNA steady-state levels. The exception was that RA increased TR alpha 1 mRNA levels at 13 days in culture, but at no other stage of development. In both types of cultures, RA and T3 separately and together produced as much as a complete inhibition of the stimulation by HC. Regulation by T3, when it occurred, was always negative. The fact that T3 can strongly repress the induction of TR alpha 1 mRNA by HC demonstrates that T3 can function through negative cooperativity as a potent regulator of its own receptor in brain.

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. Neither platelet activating factor nor dodecylglycerol at the concentrations used to achieve stimulation of neuronal differentiation compromised the plasma membrane, as indicated by the lack of leakage of cytoplasmic lactic acid dehydrogenase.

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. This accumulation caused by hydrocortisone was shown to be due to a decrease of sulfolipid degradation by arylsulfatase A (ASA) and not due to a stimulation of its synthesis by a sulfotransferase. Both ASA activity and the turnover of sulfolipid were decreased by hydrocortisone to 60-62% of untreated cells. In current work the same decrease in enzyme activity was obtained and enzyme linked immunosorbent assays demonstrate that hydrocortisone decreased the number of ASA protein molecules to 61% of untreated cells [(-)hydrocortisone: 0.31 +/- 0.06 ng ASA/microgram protein; (+)hydrocortisone: 0.18 +/- 0.04 ng ASA/microgram protein]. This decrease in the number of ASA molecules correlates well with the decrease in both the enzyme activity and the sulfolipid turnover, which suggests that the major mode of inhibition of ASA activity by hydrocortisone involves a decrease in the concentration of ASA in the cells rather than some other mechanism of inhibition.

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 7.5-fold. The critical micelle concentration of DDG was determined to be 7.93 mg/ml (0.0305 mM), which is approximately two-fold greater than the minimum inhibitory concentration of DDG determined in the presence of a protein-free chemically defined medium. This finding suggests that DDG is not killing bacteria through its detergent action. Pen G also did not alter the critical micelle concentration of DDG, which indicates that the synergism between these two agents is not related to micelle formation.

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 (0.3 microM)-treated as compared to untreated cultures after 15 days in vitro. Further, this effect of HC on MBP appears to be T3-dependent. Since HC stimulates oligodendroglia to produce MBP, the effect of HC on the activities of the enzymes, glutamine synthetase which is primarily associated with astrocytes, and acetylcholinesterase, which is primarily associated with neurons was was determined. HC stimulated both enzymes, suggesting that all 3 cell types may be regulated by HC.

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.) The addition of an equivalent amount of nonradioactive sn-3-DDG to radioactive sn-1-DDG causes more of the radioactivity to accumulate at phosphatidic acid. These results indicate that the monoglyceride kinase (EC 2.7.1.94), lysophosphatidic acid acyltransferase (EC 2.3.1.40) and the monoglyceride acyltransferase (EC 2.3.1.22) enzymatic reactions are not stereospecific, and that the CDP-diglyceride synthase (EC 2.7.7.41) and phosphatidic acid phosphatase (EC 3.1.3.4) metabolic steps are stereospecific in S. mutans BHT. The synthesis of phosphatidic acid and lysophosphatidic acid from sn-3-DDG provides a unique method for synthesizing these glycerol lipids with the uncommon stereochemical configuration in which the phosphate moiety is in the sn-1 position.

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. One type of receptor was the high affinity type (Kd = 7.77 x 10(-9) M; number of receptor sites, Bmax = 350 fmol/mg protein) while the other type was of low affinity type (Kd = 5.75 X 10(-8)M; Bmax = 1150 fmol/mg protein). The specificity of receptors for insulin was also confirmed by showing that [125I]insulin was displaced by non-radioactive insulin but not by glucagon or growth hormone. Insulin displayed a clear dose-dependent stimulation of thymidine incorporation. It also stimulated the activity of the enzyme 2', 3'-cyclic nucleotide phosphohydrolase (CNPase), which is specifically associated with myelin produced from oligodendroglia. Thus insulin has a positive influence on the proliferation and differentiation of brain cells.

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. Cultures grown in hypothyroid calf-serum containing medium had less cAMP dependent and independent protein kinase activity than control cultures grown in normal calf-serum containing medium. However, this activity was restorable to a considerable degree if the cultures grown in hypothyroid calf serum containing medium were supplemented with L-3,5,3'-triiodothyronine (T3). The presence of calcium/calmodulin stimulated protein kinase was also distinctly observed. In comparison, the activity of calcium/phosphatidyl serine stimulated protein kinase was less than the other protein kinases.

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. There was very little cyclic AMP dependent or cyclic GMP dependent protein kinase activities in this myelin-like fraction. Although the myelin basic proteins were the only endogenous proteins phosphorylated, protein kinase of the myelin-like membrane was capable of catalyzing the phosphorylation of exogenous substrates, such as histones.

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. 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, alkyl-glycerols, 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, alkyl-glycerols 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: 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. The concentration of mRNA specific for MBP in control cells grown on a medium containing normal (euthyroid) calf serum increased with increasing age of culture. The greatest increase occurred between 15 and 35 days in culture (5.25-fold increase); whereas between 35 and 50 days in culture, the rate of accumulation slowed to yield a net increase of MBP mRNA of only 10%. The quantity of MBP mRNA was drastically diminished at all ages studied when the cells were grown from the sixth day onward on a medium containing hypothyroid calf serum. Although the amount of MBP mRNA in hypothyroid-treated cells did increase, the change in concentration was less (3.43-fold), and it peaked earlier (at 30 days). Unlike the euthyroid cells, after 30 days the MBP mRNA actually fell in the hypothyroid-treated cells. If hypothyroid media were supplemented with triiodothyronine (T3) on the eighth day in culture, the quantity of MBP mRNA in the cells was restored almost completely to the levels found in the control euthyroid cells at all ages. Therefore, the regulation of the synthesis of MBP by thyroid hormone is at least in part a pretranslational event; that is, thyroid hormone adjusts the concentration of the mRNA specific 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. Radioactive racemic sn-1(3)-dodecylglycerol is readily taken up by the cell and is metabolized primarily to lysophosphatidic acid and phosphatidic acid with smaller amounts converted to phosphatidylglycerol and diacylglycerol. The accumulation of phosphatidic acid and the loss of viability respond in parallel to different concentrations of dodecylglycerol. An increase in CTP is also observed which together with the increase in phosphatidic acid suggests a possible impairment in the synthesis of CDP-diacylglycerol.

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. The studies suggest that beta-adrenergic agonists stimulate adenylate cyclase thereby causing an increased production of cyclic AMP which stimulates the methyltransferases. Cycloheximide addition to the reaction mixture did not affect the stimulation due to cyclic AMP, indicating that new protein synthesis is not involved in the cyclic AMP stimulation of the methyltransferases. Thyroid hormone (T3) has been shown to stimulate MBP methyltransferase [Amur et al, 1984] and could exert its stimulatory effect through beta-adrenergic-dependent systems. But the beta-adrenergic antagonist, propranolol, did not block the stimulation by T3, suggesting that the effect of T3 is not mediated through beta-adrenergic-dependent systems. Thus, the methylation of MBP seems to be regulated both by T3 and by neurotransmitters and/or hormones mediating their effects through cyclic AMP production, whereas the methylation of histones seems to be regulated only by the latter.

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. Cells treated with 30 microM sinefungin, which inhibits myelin basic protein methyltransferase in broken cell preparations about 60%, produced ringlike structures that were devoid of multilamellar periodicity and compactness reminiscent of the vacuolated myelin observed in subacute combined degeneration and in nitrous-oxide- or cycloleucine-treated animals in which methyltransferase activity is also inhibited. The sinefungin-induced change in multilamellar periodicity cannot be attributed to a lack of myelin basic protein, since the ratio of myelin basic protein to total protein did not decrease in sinefungin-treated cells. This primary culture system should be useful for further evaluating the hypothesis that the methylation of myelin basic protein is related to the formation of compact myelin.