Studies on the antigenic S-type lipopolysaccharides of Brucella abortus strains 7 and Mustapha

Antigenic phenol phase S-type lipopolysaccharides (LPS) isolated from Brucella abortus (B. abortus) strains 7 and Mustapha were observed to have 13C n.m.r. spectra which were almost identical to the one reported for the Brucella abortus 1119-3. The glycosyl content of the lipid A obtained from the LPS of strain 7 was found to be 2-acetamido-2-deoxyglucose only while strain Mustapha was found to contain both 2-acetamido-2-deoxyglucose and 2-acetamido-2-deoxygalactose. The fatty acid present in the lipid A of both strains was mainly n-hexadecanoic acid. Octadecanoic acid, 3-hydroxytetradecanoic acid as well as small quantities of 3-hydroxydodecanoic acid were also identified. This contrasts with the earlier reports of the absence of 3-OH-14:0 in the LPS of Brucella abortus.     

Lipopolysaccharides of the cyanobacterium Microcystis aeruginosa

Lipopolysaccharides (LPS) of two isolates of Microcystis aeruginosa were extracted with phenol/water and purified. Cesium chloride gradient ultracentrifugation of these preparations yielded only one fraction. The LPS contained significant amounts of 3-deoxy-D-manno-octulosonic acid, glucose, 3-deoxy sugars, glucosamine, fatty acids, fatty acid esters, hexoses, and phosphate. Heptose, a characteristic sugar component of the polysaccharide moiety of LPS of most gram-negative bacteria was absent. Lipopolysaccharides and lipid A hydrolysate of LPS preparations were active in mouse lethality and Limulus lysate gelation. The lipid A moiety was slightly less active in toxicity and Limulus lysate gelation assays than the intact LPS. The LPS and lipid A moiety of the two isolates of M. aeruginosa were less active in toxicity in mice and Limulus test than LPS of Salmonella abortus equi.     

Purification and fractionation of lipopolysaccharide from gram-negative bacteria by hydrophobic interaction chromatography

By hydrophobic interaction chromatography on octyl-Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild-type smooth-form (S-form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids. Thereby a fractionation of S-form LPS according to the length of the O-polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O-polysaccharide chain from approximately 30 to 4 repeating units. Molecular species of Re-mutant LPS contain four 3-hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence. Among the molecular species of S-form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re-mutant LPS a significant fraction of S-form LPS contains less than four 3-hydroxytetradecanoyl residues. Hydrophobic interaction chromatography also proved an effective one-step purification procedure of LPS as was shown with a crude preparation from S-form S. typhimurium.     

Effect of alcohol on the metabolic conversion of 14C erucic acid in the liver of rats receiving rapeseed or peanut oil]

The effects of addition of ethanol to diets containing rapeseed or ground nut oil on the metabolic conversions of 14 14C erucic and 9-10 3H oleic acid were studied in the rat liver. 1. Whatever the diet more 14C than 3H radioactivity was recovered in liver lipids 2 and 19 hours after injection of labelled fatty acids. Ethanol has little effect on this incorporation. 2. Only small amounts of 3H oleic acid were converted. 3. In all cases, the metabolic conversion of erucic acid was identical: the main part of 14C was not recovered as erucic acid but was present in other monounsaturated fatty acids n-9: oleic acid (18 : 1), which was the most labelled acid, 16 : 1, 20 : 1 and nervonic acid (24 : 1). 4. The amount of erucic acid converted to shorter chain fatty acids was unchanged by addition of ethanol but the alcohol increased the proportion of 14C radioactivity recovered as nervonic acid. This latter effect was opposite to the effect of rapeseed oil diet, which consisted in a decrease in the conversion of erucic to nervonic acid. 5. A high amount of 14C radioactivity was recovered in the F.F.A. fraction of the liver as an unknown compound (13 and 80% of 14C radioactivity respectively after 2 and 19 h.) Its identification is presently under investigation.     

Fatty acid composition and Shwartzman activity of lipopolysaccharides from oral bacteria

The composition and the nature of the linkage of fatty acids and the Shwartzman activity of lipopolysaccharide (LPS) preparations derived from oral gram-negative bacteria including Bacteroides gingivalis, Bacteroides loesheii, Eikenella corrodens, Fusobacterium nucleatum, and Actinobacillus actinomycetemcomitans were examined. 3-Hydroxylated and nonhydroxy fatty acids of various chain lengths were found in all of the LPS preparations. All nonhydroxy fatty acids were found to be ester-bound, and part of the 3-hydroxy fatty acids in the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans were shown to be involved in ester linkage. It was also suggested that the hydroxy group of the ester-bound 3-hydroxy fatty acid of the LPS of F. nucleatum and A. actinomycetemcomitans is at least partly substituted by another fatty acid, but in the LPS of B. gingivalis and E. corrodens it is not. The main amide-linked fatty acid of the LPS of B. gingivalis, E. corrodens, F. nucleatum, and A. actinomycetemcomitans was 3-hydroxyheptadecanoic, 3-hydroxydodecanoic, 3-hydroxyhexadecanoic, and 3-hydroxytetradecanoic acid, respectively. The results of the Shwartzman assay showed that the E. corrodens LPS was the most active among the preparations tested, and that the Shwartzman toxicity of Bacteroides LPS is extremely low.     

Biochemistry of development in insects. Incorporation of fatty acids into different lipid classes.

1. To study the different metabolic behaviour of various stages of development of the insect Ceratitis capitata, the incorporation of labelled decanoic, lauric, myristic, palmitic, stearic, oleic, and linoleic acids into triacylglycerols by insect homogenates was investigated. The time-course of incorporation of labelled fatty acids was firstly studied by using oleic acid; it showed that after 10 min of incubation the levels of radioactivity incorporated into triacylglycerols and those remaining in the free fatty acids were practically unchanged. 2. All labelled fatty acids were efficiently incorporated by larval homogenates; however, most of the radioactivity remained as free fatty acids in the presence of pharate adult homogenates, palmitic, and stearic acids being the most scarcely incorporated by this stage of development of the insect. 3. Plots of triacylglycerol and free fatty acid radioactivites versus the stage of development defined a crossing-zone in coincidence with the larval-pupal apolysis. This metabolic difference between larval and pharate adult homogenates could not be explained through differences in the acyl-CoA synthetase activity of the insect; this enzyme activity was notably higher in pharate adult homogenates than in the larval homogenates whatever would be the nature of the fatty acid. 4. [14C]Triolein was scarcely hydrolyzed by both larval and pharate adult homogenates. 5. Double-label experiments were carried out by incorporating either [3H]oleic acid or [3H]-palmitic acid and [14C]glycerol 3-phosphate by larval and pharate adult homogenates at different incubation intervals. Diacylglycerols, triacylglycerols, and phosphoglycerides were isolated and the 14C/3H molar ratio calculated. Results suggest the existence of a different acyltransferase activity in the different stages of development of the insect.     

Microbial metabolism of amino alcohols. Biosynthetic utilization of ethanolamine for lipid synthesis by bacteria.

1. Ten bacteria utilizing [2-14C]ethanol-2-amine as the sole or major source of nitrogen for growth on glycerol + salts medium incorporated radioactivity into a variety of bacterial substances. A high proportion was commonly found in lipid fractions, particularly in the case of Erwinia carotovora. 2. Detailed studies of [14C]ethanolamine incorporation into lipids by five bacteria, including E. carotovora, showed that all detectable lipids were labelled. Even where phosphatidylethanolamine was the major lipid labelled, radioactivity was predominantly in the fatty acid rather than the base moiety. The labelled fatty acids were identified in each case. 3. The addition of acetate to growth media decreased the incorporation of radioactivity from ethanolamine into both fatty acid and phosphatidyl-base fragments of lipids from all the bacteria except Mycobacterium smegmatis. Experiments with [3H]ethanolamine and [14C]acetate confirmed that unlabelled acetate decreased the incorporation of both radioactive isotopes into lipids, except in the case of M. smegmatis. 4. Enzyme studies suggested one of two metabolic routes between ethanolamine and acetyl-CoA for each of four bacteria. A role for ethanolamine O-phosphate was not obligatory for the incorporation of [14C]ethanolamine into phospholipids, but correlated with CoA-independent aldehyde dehydrogenase activity.     

Antifungal 3-hydroxy fatty acids from Lactobacillus plantarum MiLAB 14

We report the identification and chemical characterization of four antifungal substances, 3-(R)-hydroxydecanoic acid, 3-hydroxy-5-cis-dodecenoic acid, 3-(R)-hydroxydodecanoic acid and 3-(R)-hydroxytetradecanoic acid, from Lactobacillus plantarum MiLAB 14. The concentrations of the 3-hydroxy fatty acids in the supernatant followed the bacterial growth. Racemic mixtures of the saturated 3-hydroxy fatty acids showed antifungal activity against different molds and yeasts with MICs between 10 and 100 micrograms ml-1.     

Studies on lipogenesis in vivo. Effect of dietary fat or starvation on conversion of [14C]glucose into fat and turnover of newly synthesized fat

1. Lipogenesis was studied in vivo by giving mice 250mg. meals of [U-(14)C]glucose and measuring the disposition and incorporation of label. About 48% of the (14)C dose was eliminated as (14)CO(2) in the first 2hr. At 60min. after administration, 1.0, 1.9 and 11.9% of the dose was recovered as liver glycogen, liver fatty acid and carcass fatty acid respectively. Of the [(14)C]glucose converted into fat in the epididymal pads about 90% was present as glyceride fatty acid and 10% as glyceride glycerol. 2. Hepatic synthesis of fatty acid was depressed by dietary fat to a much greater extent than was synthesis outside the liver. Both feeding with fat and starvation decreased the proportion of the label taken up by adipose tissue present as fat (triglyceride) and increased the proportion of triglyceride label present as glyceride glycerol. These results are consistent with the hypothesis that the primary action of both these conditions in decreasing fat synthesis is to inhibit synthesis of fatty acids. 3. Turnover of body fat labelled in vivo from [U-(14)C]glucose was estimated from the decline in radioactivity measured over the first 24hr. of the experiment. The half-life of liver and extrahepatic fatty acids (excluding epididymal fat) was 16hr. and 3 days respectively. In contrast, no measurable decrease in radioactivity of the fatty acids of epididymal fat was observed for 7 days after administration of the [U-(14)C]glucose.     

Characterization of the lipopolysaccharide from Budvicia aquatica DLR 20186

A lipopolysaccharide (LPS) from Budvicia aquatica DRL 20186 was isolated, studied, and chemically identified. It was shown to be lowly toxic, but highly pyrogenic. Its fatty acid composition was similar to that of the LPS from other Enterobacteriaceae, with predominance of tetradecanoic (32.7%) and 3-hydroxytetradecanoic acids (23.8%). Hexadecenoic (20.4%), hexadecanoic (11.8%), and dodecanoic acids (8.4%) were also revealed. Double immunodiffusion in agar by the Ouchterlony method revealed antigenic activity of the B. aquatica DLR 20186 LPS in a homologous system. In cross reactions, however, it did not interact with the antisera of other B. aquatica strains.     

Composition of O-antigenic lipopolysaccharides from Enterobacter cloacae

Analyses have been carried out on lipopolysaccharides (LPS) from 14 strains of Enterobacter cloacae representing different O serotypes. All of the products appeared to have a composition and architecture typical of enterobacterial LPS, but points of interest include the absence of phosphate residues from the core oligosaccharide, the presence of both L-glycero-D-mannoheptose and D-glycero-D-mannoheptose (ratio usually about 4:1), and the presence in lipid A of small amounts of fatty acids with odd numbers of carbon atoms (mainly C13) in addition to tetradecanoic acid and 3-hydroxytetradecanoic acid. Monosaccharides identified as components of polymeric fractions from the LPS were glucose, galactose, mannose, rhamnose, glucosamine, galactosamine, fucosamine, and galacturonic acid. Most polymeric fractions also probably contained an O-acetyl substituent. Closely similar chemotypes found for the polymeric fractions from the LPS of cross-reacting serotypes support the view that these fractions contain the O-antigenic determinants and represent the side chains of the LPS.     

Hepatic lipase-mediated hydrolysis versus liver uptake of HDL phospholipids and triacylglycerols by the perfused rat liver

Hepatic triacylglycerol-lipase-mediated hydrolysis and liver uptake of high-density lipoprotein (HDL) lipid components were studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and active at the vascular bed. Human native HDL were labelled with tri-[3H]oleoylglycerol, [N-methyl-3H]dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl,2-[14C]linoleoylphosphatidylcholine (PLPC), 1-palmitoyl,2-[14C]linoleoylphosphatidyl-ethanolamine (PLPE) and 1-palmitoyl,2-[14C]palmitoylphosphatidylethanolamine (DPPE). (1) Relative degradation rates of phosphatidylethanolamine molecular species were 2- to 10-fold higher than those of phosphatidylcholine. Considering [14C] PLPC and [14C] PLPE as representative of HDL phosphatidylcholine and phosphatidylethanolamine, respectively, the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine generated after a 60 min perfusion were comparable. The enzyme showed a clear preference for the molecular species bearing an unsaturated fatty acid at the 2 position of glycerol; this was the most pronounced in the case of phosphatidylethanolamine molecular species. (2) Relative liver uptake of HDL-phosphatidylethanolamine was 4- to 5-fold higher than that of HDL-phosphatidylcholine, irrespective of the constitutive fatty acids. Nevertheless, mass estimation indicated that 3 times more molecules of phosphatidylcholine than of phosphatidylethanolamine were transferred. No correlation could be found between the relative degradation rates of phospholipids and their relative liver uptake, indicating a dissociation between the two processes. (3) Perfusate decay and relative liver uptake of labelled HDL-triacylglycerol were higher than that of any phospholipid class. No circulating radiolabelled free fatty acids accumulated in the perfusate, but they were found acylated into liver cell phospholipids and triacylglycerols. (4) A prior 10-12-min washout of the liver vascular bed with heparin removed over 80% of the hepatic lipase activity, as assessed by specific immunoinhibition. Hepatic lipase-depleted liver displayed impaired phospholipid hydrolysis and triacyglycerol uptake, whereas the transfer of HDL phospholipids to liver tissue was unaffected.     

The transfer of fatty acids across the sheep placenta

Maternal and fetal plasma concentrations of free fatty acids, triacylglycerols and phospholipids and the profile of their fatty acids were measured in three catheterized and unanaesthetized sheep. Fetal concentrations of all three lipid fractions were low and did not correlate with maternal concentrations. There were no measurable umbilical venous-arterial differences. Linoleic acid concentrations were low in both mother and fetus. The fatty acid composition of fetal adipose tissue, liver, lung and cerebellum of five animals was analysed. Again linoleic acid levels were very low, but phospholipids contained 2-8% arachidonic acid. [14C] linoleic acid and [3H] palmitic acid were infused intravenously into three ewes. Only trace amounts of labelled fatty acids were found in fetal plasma and these were confined to the free fatty acids. 14C-label was incorporated into fetal tissue lipids, but most of this probably was due to fetal lipid synthesis from [14C] acetate or other water-soluble products of maternal [14C] linoleic acid catabolism. It is concluded that only trace amounts of fatty acids cross the sheep placenta. They are derived mainly from the maternal plasma free fatty acids and might just be sufficient to be the source of the small amounts of essential fatty acids found in the lamb fetus, but are insignificant in terms of energy supply or lipid storage.     

Phosphonopyruvic acid: A probable precursor of phosphonic acids in cell-free preparation of Tetrahymena.

1. In cell-free preparations of Tetrahymena, doubly labelled [32P]phosphoenol-[3-14C]pyruvate gives rise to 2-aminoethylphosphonate and 2-amino-3-phosphonopropionate, labelled with the two isotopes in the same ratio as the starting compound. The result is consistent with an intra-molecular rearrangement of phosphoenolpyruvate in the biosynthetic sequence of carbon-phosphorus bond formation. 2. Incubation of [32P]phosphoenolpyruvate with the same preparation, followed by treatment with 2,4-dinitrophenylhydrazine, yielded labelled hydrazones. When these were subjected to hydrogenolysis, the radioactivity was recovered in 2-aminoethylphosphonate and 2-amino-3-phosphonopropionate, suggesting that 2-phosphonoacetaldehyde and 3-phosphonopyruvic acid were probable precursors of the aminoalkylphosphonic acids. 3. Radioactivity from 2-amino-3-phosphono-[3-14C]propionic acid was incorporated into 2-aminoethylphosphonic acid, but incorporation of the radioactivity into lipids was negligible.     

Chemical structure of the lipid A component of lipopolysaccharides from Fusobacterium nucleatum.

The lipid A component of lipopolysaccharides from Fusobacterium nucleatum Fev 1 consists of beta-1',6-linked D-glucosamine disaccharides, which carry two phosphate groups: one in glycosidic and one in ester linkage. The amino groups of the glucosamine disaccharides are substituted by D-3-hydroxyhexadecanoic acid. The hydroxyl groups of the disaccharide backbone are acylated by tetradecanoic, hexadecanoic, and D-3-hydroxytetradecanoic acids. Part of the ester-bound D-3-hydroxytetradecanoic acid is 3-O-substituted by tetradecanoic acid. Whereas a similar pattern of fatty acids was detected in lipopolysaccharides from two other F. nucleatum strains, the amide-bound fatty acid in F. varium and F. mortiferum was D-3-hydroxytetradecanoic acid. The chemical relationships of lipid A from Fusobacteria and other gram-negative bacteria are discussed.     

Conversion of (U-14C)-glycerol, (2-3H)-glycerol and (1-14C)-palmitate into circulating lipoproteins in the rat.

The in vivo formation of labelled very low density lipoproteins (VLDL) from (U-14C)-glycerol, (2-3H)-glycerol and (1-14C)-palmitate was studied in fed female rats. The rate of disappearance of radioactivity from plasma after the i.v. injection with these tracers was similar for (U-14C)-glycerol and (1-14C)-palmitate. With (2-3H)-glycerol, plasma radioactivity at 10 min was lower than with the other substrates although it did not change thereafter. A certain proportion of radioactivity administered as glycerol appeared in plasma lipids, mainly in the VLDL glyceride glycerol fraction, although when (U-14C)-glycerol was the substrate, a considerable portion also appeared in the esterified fatty acids of these lipoproteins. When using (1-14C)-palmitate, practically all the circulating labelled esterified fatty acids appeared in the VLDL fraction, while the labelled free fatty acids appeared in lipoprotein of higher density, presumable free fatty acid-albumin complexes. This data is discussed in terms of the role of the liver in the rapid, continuous cycling of these substrates to yield VLDL-glycerides for their extrahepatic utilization.     

Sphingolipid metabolism in Bacteroideaceae.

The lipid composition of the anaerobic Bacteroides thetaiotaomikron has been analyzed. Sphingomyelin, ceramide phosphinicoethanolamine, free even-numbered and branched chain sphingosine bases and ceramide represented about 50% of the total lipid extract. The main ester phospholipid was phosphatidylethanolamine. The alkali-stable sphingophospholipids were predominantly N-acylated with 3-hydroxypalmitic acid, whereas the ester phospholipids are preferentially substituted with normal even and odd-numbered and branched-chain fatty acids. When Bacteroides was grown in a medium supplemented with labelled palmitic acid, this fatty acid was utilized for acylation reactions and to a large extent for the de novo synthesis of sphinganine. This long-chain base was incorporated into the sphingolipids and was also present in free form. The 3-hydroxypalmitic acid present in sphingolipids is not derived from palmitic acid, since labelled palmitate did not serve as a precursor. Free sphinganine added to the culture medium was also utilized efficiently for the biosynthesis of the sphingolipids by growing Bacteroides cultures. The 3H/14C ratio in sphingomyelin and ceramide phosphinicoethanolamine is the same, when [1-14C]palmitic acid and [3-3H]sphinganine serve as precursors. Sphingomyelin, which is usually only present in higher animals, is synthesized de novo in this Bacteroides strain.     

Lipid synthesis in the adult dog heartworm, Dirofilaria immitis.

1. Incorporation studies with three labelled substrates--[14C]2-glycerol, [14C]1-acetate and [14C]1-oleic acid--demonstrated that adult dog heartworms can synthesize all classes of complex lipids present, including free cholesterol. 2. Diacylglycerols and phosphoglycerides were most rapidly labelled regardless of the precursor employed. 3. 14C from glycerol was found in the aqueous phase of saponified lipids, whereas that from oleic acid was in the fatty acid portion. 4. Tag from acetate was predominantly in the fatty acid portion of saponified lipids and also occurred in the unesterified fatty acids. 5. Acetate and unesterified fatty acids, as represented by oleic acid, were more readily used for lipid synthesis than was glycerol.     

Elongation of (omega-14C)oleic acid and (omega-14C)nervonic acid.

During feeding experiments with [omega-14C]oleic acid and [omega-14c]nervonic acid to adult rats, 14C-labelled C26, C28 and C30 fatty acids were recovered from the intestinal mucosa, liver, plasma, kidney and stools. The structures of these fatty acids were determined by g.l.c., radio-g.l.c. and mass spectrometry. The Schmidt and Ginger degradation methods indicated that most of the 14C found in these extra-long fatty acids remained in the omega position. These radioactive extra-long fatty acids were found mainly in the polar lipids of rats killed 3 or 15 h after being fed on labelled oleic acid or nervonic acid. Rats killed 63 h later yielded only traces of these extra-long fatty acids. When the rats were given antibiotics or received the same radioactive fatty acids by intravenous injection, the labelled extra-long fatty acids could not be detected in any of the tissues. We conclude that they were probably synthesized by elongation of oleic acid and nervonic acid by intestinal micro-organisms (probably yeasts) and then absorbed by the intestinal mucosa.     

The metabolism of palmitic acid in the fetal lamb

[1-14C]Palmitic and [9,10-3H]palmitic acids were injected into the femoral artery of fetal sheep in utero about one month preparturition. The experiment was terminated after 5, 15 or 30 min when the main tissues were removed for analysis of the lipid components. 5 min after injection of the label, most was recovered in the plasma but increasing amounts were recovered later in the liver and heart. Selective loss of 14C-label occurred such that in the plasma, 30 min after injection, the ratio of 3H:14C had changed from 1:1 to 8.4:1. Increasing amounts of the labelled lipid were recovered in esterified form with time after injection, and the 3H:14C ratio differed markedly in specific lipids and tissues. Most of the label was recovered in palmitic acid, but some was also present in myristic and octadecenoic acids. Some evidence was obtained that the latter may have been the delta 11-isomer, which was found in much greater amounts in fetal than maternal tissues. It appears that partial-oxidation and resynthesis of fatty acids occurs in a concerted manner at a rapid rate in fetal sheep. The phenomenon has important implications for the interpretation of the results of much previous work with fetal animals in vivo.