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.

Effect of growth rate on lipid and lipoteichoic acid composition in Streptococcus faecium.

The lipid composition of Streptococcus faecium (S. faecalis ATCC 9790) was analyzed at various growth rates. Diphosphatidylglycerol and the non-ionic lipid fraction containing diacylglycerols and neutral glycolipids appeared to accumulate relative to cellular mass as the culture mass doubling time increased from 30 to 80 min. Within the same range of doubling times the non-ionic lipid fraction appeared to become substantially enriched with diacylglycerols. All lipid species and cellular lipoteichoic acid accumulated relative to the cellular mass at doubling times exceeding 80 min, although diacylglycerol accumulation exceeded that of all other compounds studied.

Effect of thyroid hormone on the synthesis of sialosyl galactosylceramide (GM4) in myelinogenic cultures of cells dissociated from embryonic mouse brain.

A sialyltransferase, which catalyzes the biosynthesis of the myelin-associated sialosyl galactosylceramide (GM4) from galactocerebroside and cytidine-5'-monophospho-N-acetylneuraminic acid, has been detected in primary reaggregating, surface adhering cultures of cells dissociated from embryonic mouse brain. The ontogenetic profile of this enzyme in culture mimics its in vivo developmental pattern in that its activity could be detected only after 28 days in vitro and reached peak values around 48 days in vitro. Between 48 to 75 days in culture (oldest age studied) only a very slow increase in activity is observed. Unlike other myelin marker enzymes whose activities appear at an earlier time in development, the gene expression of the sialyltransferase responds relatively slowly to stimulation by triiodothyronine. However, if exposed to hypothyroid conditions at an early developmental age before the enzyme activity is expressed, little or no activity appears in latter stages of development.

Investigations on myelination in vitro: biochemical and morphological changes in cultures of dissociated brain cells from embryonic mice.

The incorporation of 35SO4(2-) and [3H]galactose into myelin-associated lipids, the activity of enzymes catalyzing the synthesis of these lipids, and the activity of 2',3'-cyclic nucleotide phosphohydrolase were determined in primary cultures of dissociated cells from brains of 15-day embryonic mice. These biochemical parameters of myelination were barely detectable before about 10 days in culture, but their activity increased in parallel after this time and reached a maximum at about 40 days in culture. The activities of the selected enzymes in homogenates of the cultured cells at their optimum age were of the same order of magnitude as the same enzymes derived from fresh brain. Scanning electron microscopic studies showed that the cells after adhering to the surface by the 4th day form aggregates and extensive membranes; the aggregates increase in size and coalesce to form nests of cells by the 15th day; the surface of the aggregates becomes smoother until by the 43rd day the entire surface is covered by and cells are buried in a membrane-like substance. These biochemical measurements and morphological data suggest that the cultures of dissociated cells from brain of 15 day embryonic mice provide a useful system for studying myelination and its regulation in vitro.

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.

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.

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. 4 and 10 d) but had little stimulatory effect at later ages (i.e. 20 and 33 d). Further, this stimulation at the earlier ages was blocked by the dopaminergic blocker, fluphenazine, but not by alpha and beta-adrenergic antagonists. In contrast to dopamine, isoproterenol (a beta-adrenergic agonist) had little stimulatory effect at earlier ages, but its ability to stimulate cyclase activity increased with age. This increase in all age groups was blocked by propranolol (a beta-adrenergic antagonist). Epinephrine-sensitive enzyme activity showed a steady increase with age, which could be blocked with propranolol except in 4-d-old cultures, where it was blocked instead by fluphenazine. Because the cultures are relatively enriched in neurons at earlier ages and in glia in later ages, the results suggest a predominantly neuronal localization for the dopamine sensitive adenylate cyclases and a glial localization of the isoproterenol and epinephrine sensitive adenylate cyclases. Histamine, serotonin, calcium/calmodulin and chloroadenosine were either only slightly or not at all stimulatory.

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.

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. Biol. Chem. 259, 8115-8121).

Investigations on myelination in vitro: thyroid hormone receptors in cultures of cells dissociated from embryonic mouse brain.

Brain cells dissociated from 15-day-old embryonic mice and grown in culture contain both cytosolic and nuclear receptors for L-3,5,3'-triiodothyronine (L-T3). KD values for L-T3 of 3.05 X 10(-9) M and 4.2 X 10(-9) M were determined with the cytosolic and nuclear receptors respectively. These cultured cells, which are suitable for studying the regulation of myelination by T3 in vitro, display a high specificity for L-T3 in that the receptors for L-T3 do not bind D-T3, D-thyroxine, L-diiodothyronine, or DL-thyronine, and bind only small amounts of L-thyroxine.

Phosphatidylkojibiosyl Diglyceride: metabolism and function as an anchor in bacterial cell membrane.

The recently discovered phosphoglycolipid, phosphatidylkojibiosyl diglyceride (PKD), was first observed as a biosynthetic by-product of glycosyl diglyceride metabolism in Streptococcus faecalis (faecium) ATCC 9790. Its structure is 1, 2-diacyl-3-O-alpha-Dglucopyranosyl-6'-O-phosphoryl- [1'', 2''-diacyl-3''-O-sn-glycerol]-alpha-D-glucopyranosyl)-sn-glycerol. The biosynthesis of phosphatidyl-kojibiosyl diglyceride occurs by a novel transphosphatidylation reaction in which a phosphatidyl 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. Phosphatidyl-kojibiosyl diglyceride is bound covalently through a phosphodiester bond to the polyglycerol phosphate moiety of membrane lipoteichoic acid from S. faecalis. Phosphatidylkojibiosyl 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 enviroment of the membrane of S. faecalis and probably other gram-positive bacteria as well.

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. The radioactive label is incorporated exclusively into the fatty acid moieties of the glycerolipids. The increase in the radioactive content of the extracellular lipids reflects an actual net increase in the total fatty acid content as determined by a chemical assay. During a 4-hr incubation in the presence of penicillin, the extracellular fatty acid ester concentration (per mg cell dry weight) increases 1.5 fold, even though there is no growth or cellular lysis. No change is observed in the intracellular fatty acid ester content. An indication of the relative rate of fatty acid synthesis was most readily obtained by placing S. mutans BHT in a buffer containing 14C-acetate. Under these nongrowing conditions free fatty acids are the only lipids labeled, a factor which simplifies the assay. The addition of glycerol to the buffer causes all of the nonesterified fatty acids to be incorporated into glycerolipid. The cells excrete much of the lipid whether glycerol is present or not. Addition of penicillin to the nongrowth supporting buffer system does not stimulate the incorporation of [14C]-acetate into fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A micromethod for the stereospecific determination of triglyceride structure.

Triglyceride lipase and diglyceride kinase can be used in a sensitive stereospecific analysis of the separate fatty acid compositions at the 1, 2 and 3 positions of a triglyceride.Diglyceride kinase fromEscherichia coli selectively catalyzes the phosphorylation of 1,2-diglycerides but not the 2,3-diglycerides.The composition of the 3-position in rat liver triglycerides is clearly different from that at the 1-position.

Excretion of extracellular lipids by Streptococcus mutans BHT and FA-1.

Streptococcus mutans BHT and FA-1, when grown to log phase on chemically defined medium containing [14C]glycerol, excreted 15% of the total biosynthesized 14C-lipid into the medium. When grown to early stationary phase, 28 to 33% of the 14C-lipid was found in the medium. The radioactive lipids of these varieties of S. mutans were identified as diacylglycerol, diglucosyl diacylglycerol (DGD), monoglucosyl diacylglycerol, diphosphatidylglycerol, phosphatidylglycerol (PG), and smaller amounts of two other lipids tentatively were identified as amino acyl-PG and glycerol phosphoryl-DGD. All lipids were found as extracellular and intracellular components from cells grown to either log or stationary phase. However, there were some shifts in the relative percentage of these lipids as the cells changed from log to stationary phase. For example, the intracellular lipid content of log-phase S. mutans BHT was composed of 49% PG and 19% DGD, but these percents shifted to 18% PG and 57% DGD when the cells were grown to stationary phase. However, the extracellular lipids of this organism contained 50 to 60% PG and 20% DGD in both log and stationary phases.

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.