Lipid alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

The lipid composition of cells of Pseudomonas aeruginosa strains resistant to polymyxin was compared with the lipid composition of cells of polymyxin-sensitive strains as to their content of readily extractable lipids (RELs), acid-extractable lipids, the fatty acid composition of RELs, and the contents of various phospholipids in the REL fraction. The polymyxin-resistant strains had an increased content of RELs, but a decreased phospholipid content. The REL fraction from the polymyxin-resistant strains had an increased content of unsaturated fatty acids accompanied by a decreased content of cyclopropane fatty acids as compared with the fatty acid composition of RELs from polymyxin-sensitive strains. The phosphatidylethanolamine content was greatly reduced in the polymyxin-resistant strains, whereas the content of an unidentified lipid, thought to be a neutral lipid lacking either a phosphate, free amino, or choline moiety, was greatly increased. Cell envelopes of the polymyxin-resistant strains contained reduced concentrations of Mg2+ and Ca2+ as compared with the cell envelopes of polymyxin-sensitive strains. It appears that polymyxin resistance in these strains is associated with a significant alteration in the lipid composition and divalent cation content of the cell envelope.     

Correlation of Bacterial Lipid Composition with Antibiotic Resistance

The correlation of bacterial lipid composition with antibiotic resistance was investigated with particular emphasis on those organisms in which resistance may be related to membrane or envelope structure or function, as in resistance to tetracyclines and polymyxin. Chloroform-methanol-extractable lipids, phosphatidyl ethanolamine fractions, and both fatty acids of these lipid fractions and total fatty acids were studied by using thin-layer chromatography, gas chromatography, and infrared spectroscopy. Consistent quantitative differences were found between the fatty acid compositions of sensitive and resistant strains. Most notable was the fact that, in gram-negative organisms, resistant strains showed decreases in cyclopropane acids as compared with sensitive strains. These changes were found to be inherent in the strains and not due to growth stage or culture age. No significant qualitative differences were noted. In contrast, no such variation in fatty acid content was observed in penicillin-sensitive and resistant strains of gram-positive cocci. As significant alterations of fatty acid composition were noted in gram-negative strains resistant to antibiotics, we suggest that resistance is correlated to membrane or envelope lipid composition.     

Changes in cellular composition during magnesium deficiency.

1. Concentrations and compositions of liver, serum and milk lipids of cows were measured during 6 days' starvation and serum lipids during 60 days' re-feeding. 2. The concentration of free fatty acid in serum increased fivefold during starvation. 3. The content of total lipid in liver (g/100g of liver dry matter) doubled owing to a 20-fold increase in triglyceride, an eightfold increase in cholesterol ester, a three fold increase in free fatty acid and a 20% increase in cholesterol. There were no changes in the content or composition of liver phospholipids. 4. Starvation lowered the concentrations of total lipid, phospholipid and cholesterol ester of dextran sulphate-precipitable serum lipoproteins. Total lipid and cholesterol ester concentrations in lipoproteins of d greater than 1.055 and in lipoproteins not precipitable by dextran sulphate decreased from day 4 of the starvation period and during the first 20 days' re-feeding. 5. During starvation there were decreases in percentages of stearic acid and increases in oleic acid in serum free fatty acids and triglycerides and in liver neutral lipid. 6. Throughout starvation total milk lipid yield decreased, yields and percentages of C4-14 fatty acids decreased and percentages of C18 fatty acids increased. 7. It is suggested that accumulation of triglyceride in liver may be caused by increased uptake of plasma free fatty acids without corresponding increase in lipoprotein secretion.     

Effect of dieldrin toxicity on acetate and palmitate metabolism in rat liver.

The effect of a single oral dose of dieldrin (30 mg/kg body weight) on lipid metabolism in rats was studied. Liver lipids content increased and this increase was mainly in the triglyceride fraction. The incorporation of acetate-14C into fatty acids was decreased indicating an inhibition of lipogenesis. Fatty acid oxidation was increased. Palmitate-14C incorporation into the triglyceride fraction was enhanced pointing to an overall increased utilization of fatty acids.     

The viscosity and lipid composition of the plasma membrane of multiple drug resistant and sensitive yeast strains.

Four different plasma membrane preparations were isolated from multiple drug resistant and sensitive isolates of two isogenic groups of Saccharomyces cerevisiae strains: zymolyase ghosts, concanavalin A ghosts, pH 4 nonaggregated vesicles, and sucrose-gradient purified vesicles. The viscosities of these preparations were determined by the use of a fluorescence polarization technique with 1,6-diphenyl-1,3,5-hexatriene. The viscosities of all four membrane preparations within an isogenic set were the same for resistant and sensitive strains. A comparison of the viscosity of zymolyase ghost liposomes showed that zymolyase ghost (glyco) proteins of resistant and sensitive strains had the same effect on viscosity. There was no difference between resistant and sensitive isolates in the mole concentration of the following lipid classes extracted from zymolyase ghosts: phospholipid, sterol, sterol ester, triglyceride, diglyceride, and free fatty acid. The fatty acid distribution of esterified and free fatty acids and the distribution of nine phospholipids was the same in zymolyase ghosts from sensitive and resistant strains. It was concluded that multiple drug resistance does not result from an alteration in plasma membrane viscosity or lipid composition.     

Interrelation of aerobic glycolysis and lipogenesis in isolated perfused liver of well-fed rats

The rates of glycolysis and lipogenesis in isolated perfused liver of well-fed rats were studied. When liver was allowed to synthesize [14C]glycogen prior to perfusion, no more than 9% of the degraded [14C]glycogen was recovered in lactate and 6% in lipid. Addition of glucose, fructose and sorbitol enhanced concomitantly the formation of lactate and pyruvate and the rate of release of triglyceride and free fatty acid. Glucose was less efficient than fructose or sorbitol. The incorporation of 14C from these 14C-labelled substrates into lactate, pyruvate and lipids confirmed their role as carbon sources. Incorporation of 14C into the glycerol moiety of neutral lipid exceeded that found in the fatty acids, suggesting that these substrates contributed largely to the esterification of fatty acids. The total rate of de novo fatty acid synthesis was correlated with the formation of lactate and pyruvate. It is concluded that increased rates of aerobic glycolysis are related to increased rates of lipogenesis.     

Characteristic lipids of Bordetella pertussis: simple fatty acid composition, hydroxy fatty acids, and an ornithine-containing lipid.

The lipids and fatty acids of Bordetella pertussis (phases I to IV) were analyzed by thin-layer chromatography, gas-liquid chromatography, and mass spectrometry and compared with those of B. parapertussis and B. bronchiseptica. The major lipid components of the three species were phosphatidylethanolamine, cardiolipin, phosphatidylglycerol, lysophosphatidylethanolamine, and an ornithine-containing lipid. The ornithine-containing lipid was characteristic of the genus Bordetella. The fatty acid composition of the total extractable cellular lipids of B. pertussis was mostly hexadecanoic and hexadecenoic acids (90%) in a ratio of about 1:1. The hexadecenoic acid of B. pertussis was in the cis-9 form. The fatty acid composition of the residual bound lipids was distinctly different from that of the extractable lipids, and residual bound lipids being mainly 3-hydroxytetradecanoic, tetradecanoic, and 3-hydroxydecanoic acids, with 3-hydroxydodecanoic acid occurring in some strains. It was determined that the 3-hydroxy fatty acids were derived from lipid A. The fatty acid composition of the total extractable cellular lipids of B. parapertussis and B. bronchiseptica, mainly composed of hexadecanoic and heptadecacyclopropanoic acid, differed from that of B. pertussis. Although the fatty acid composition of the residual bound lipids of B. parapertussis was similar to that of the residual bound lipids of B. pertussis, 2-hydroxydodecanoic acid was detected only in the bound lipids of B. bronchiseptica.     

Composition and positional distribution of fatty acids in polar lipids from Chlorella ellipsoidea differing in chilling susceptibility and frost hardiness

The composition and positional distribution of fatty acids in the polar lipids from 4 strains of Chlorella differing in chilling susceptibility and frost hardiness were analyzed by enzymatic hydrolysis and gas-liquid chromatography. Analysis of the polar lipids from chilling-sensitive, chilling-resistant and chilling-sensitive revertant strains of Chlorella ellipsoidea IAM C-102 showed that the sum of palmitic and trans -3-hexadecenoic acid in phosphatidylglycerol (PG) is about 60% for the sensitive strains and 53% for the resistant strain. The sum of dipalmitoyl and 1-palmitoyl-2-( trans -3-hexadecenoyl) PG as estimated from the positional distribution of their fatty acids, is about 10% in the case of each of the three strains. The contents of unsaturated fatty acids in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were higher in the resistant than in the sensitive strain. This suggests that unsaturation of fatty acids in not only PG but also PC and PE is involved in chilling sensitivity of Chlorella . On the other hand, lipid changes during the development of frost hardiness of C. ellipsoidea IAM C-27, a frost hardy strain, were examined. The results showed that fatty acids in most lipid classes are unsaturated in the hardening process but their degree of unsaturation is not greatly different from that of the chilling-resistant strain, suggesting that not only unsaturation of fatty acids in lipids but also other factors are necessary for the development of frost hardiness.     

Selective increase in acyl hydrolase activity by graminicides in wheat

Seedlings of wheat were grown for 24 h in control nutrient solution and in solutions containing haloxyfop, alloxydim, diquat or paraquat, and thereafter the roots were used for microsomal preparations. Phosphatidylcholine or diacylglycerol with various 1-(14)C-labelled fatty acids (oleic, linoleic, linolenic or ricinoleic acids) in position sn-2 were added to the prepared microsomes. After incubation for 2 h at 30 degrees C, the lipids were extracted and the distribution of radioactivity among lipid classes was determined. In the microsomal preparations of plants treated with diquat and paraquat, the amounts of fatty acids released were similar to the control, whereas they were 1.4-2 times higher in the microsomal preparation of plants treated with haloxyfop and alloxydim. Thus, the data indicate that graminicides could increase lipid catabolism in sensitive plants and that this is not a general phenomenon connected with inhibition of growth.     

Uptake and distribution of cis-unsaturated fatty acids and their effect on free radical generation in normal and tumor cells in vitro

We have previously shown that cis-unsaturated fatty acids (c-UFAs) possess a selective tumoricidal action that can be blocked by antioxidants. This property of c-UFAs might be due to various factors, including increased uptake, unusual distribution, or an ability to alter free radical generation in tumor but not normal cells. 14C-labelled linoleic acid (LA) uptake was almost the same in normal and tumor cells, whereas that of 14C-labelled arachidonic acid (AA) and 14C-labelled eicosapentaenoic acid (EPA) in tumor cells was substantially less than in normal cells. Tumor cells incorporate major portions of the fatty acids in the ether lipid and phospholipid fractions, whereas normal cells incorporate the fatty acids primarily in the phospholipid fraction. LA, AA, and EPA augmented nitroblue tetrazolium reduction, an indication of free radical generation, selectively in the tumor cells. These results suggest that there are significant differences between normal and tumor cells in fatty acid uptake and distribution, and in the ability of fatty acids to generate free radicals.     

Genesis of fatty liver and hyperlipemia in the fetal guinea pig.

10 to 20% of [1-14C] palmitate injected into pregnant guinea pigs was recovered in lipids of their fetuses. From these data and the rate of transport of palmitate in maternal blood, it appears that placental transport of free fatty acids can account for the accumulation of lipids in late gestational fetuses. About 80% of the labeled palmitate in the fetus appeared initially in lipids of the liver. 14C appeared in plasma triglyceride fatty acids after a few minutes and subsequently accumulated in lipids of white and brown adipose tissue, suggesting that much of the palmitate deposited in adipose tissue were derived from hepatogenous triglyceride fatty acids. By contrast, 14C was usually maximal in heart and carcass lipids before it appeared in plasma triglyceride fatty acids. Lipoprotein lipase activity in fetal adipose tissue was low, and activity of cofactor protein of lipoprotein lipase in fetal blood plasma was much lower than that observed in other mammalian species. On the basis of these and earlier observations, it is concluded that the accumulation of triglycerides in liver and blood plasma of fetal guinea pigs during late gestation is at least partly the result of the large uptake of maternally derived free fatty acids by the fetal liver accompanied by rapid synthesis and secretion of triglyceride-rich very low density lipoproteins into the blood. However, limited uptake of triglyceride fatty acids in adipose tissue may contribute to the fatty liver and hyperlipemia.     

Incorporation of acetate-1-14C into lipids by the perfused liver of normal, X-irradiated, or partially hepatectomized rats

In order to study lipid metabolism in the liver without interference due to transport from and to the liver the isolated livers of normal, X-irradiated, and partially hepatectomized rats were perfused with acetate-1-(14)C and the distribution of radioactivity in total lipids, total fatty acids, individual lipids, and fatty acids of individual lipids was determined. In X-irradiated animals, an increased incorporation of acetate into many lipids, particularly into cholesterol, was observed. Lipids in the liver of the partially hepatectomized rats exhibited a marked increase in triglyceride content together with a decreased rate of incorporation into all but the phospholipid fractions. It is concluded that the increase usually observed in lipid pontent of the regenerating liver is due to the changes in transcort rather than to changes in synthesis. The changes observed in irradiated liver could be the result of alterations in the metabolism of precursors common to most lipids.     

Lipids of Bacteroides melaninogenicus

The lipids of Bacteroides melaninogenicus were readily extractable with chloroform-methanol. Three per cent of the fatty acids were not extractable. The neutral lipids contained 4% of the extractable fatty acids, the stench characteristic of these organisms, and 0.5 mumole of vitamin K(2) isoprenologues K(2)-35, K(2)-40, and K(2)-45 per g (dry weight). This is one-fifth to one-tenth of the vitamin K(2) level found in other bacteria. Ninety-six per cent of the extractable fatty acids were associated with the phospholipids (60 mumoles of lipid phosphate/g, dry weight), which consisted of the diacyl lipids phosphatidic acid, phosphatidyl serine, and phosphatidyl ethanolamine (with phosphatidyl glycerol and cardiolipin in one strain). The unusual phosphosphingolipids ceramide phosphorylethanolamine, ceramide phosphorylglycerol, and ceramide phosphorylglycerol phosphate accounted for 50 to 70% of the lipid phosphate. In protoheme-requiring strains, the protoheme concentration in the growth medium regulated the growth rate and the amount of enzymatically reducible cytochrome c. There were no gross changes in the lipid composition in cells containing different levels of enzymatically reducible cytochrome c.     

Metabolism of triglyceride fatty acid by the perfused rat heart

1. Chyle lipids, labelled with (14)C, are taken up and oxidized by the isolated perfused rat heart. 2. In recirculatory perfusions, when chyle lipids are the sole exogenous energy source, about 24% of the total oxygen uptake is accounted for by their oxidation. This proportion is not changed by starvation of the rats for 48hr. and falls when an external work load is imposed on the left ventricle. 3. With albumin in the perfusion medium, the rate of (14)CO(2) output is reduced by half and there is a rise in the proportion of (14)C-labelled free fatty acids in the medium. 4. Clearing-factor lipase appears in the perfusion medium when chyle lipids are perfused through the heart. In the absence of albumin, the activity of the medium enzyme is low and only a small proportion of the (14)CO(2) output can be accounted for by the oxidation of free fatty acids released by it. In the presence of albumin, the enzyme is more active in the medium. 5. When a substantial proportion of the total clearing-factor lipase is removed from the heart by a prior perfusion with heparin, (14)C-labelled chyle lipid perfused subsequently is oxidized at only half the normal rate.     

Relationship between resistance to metallic ions and production of beta-lactamase in strains of Staphylococcus epidermidis.

Thirty five strains of Staphylococcus epidermidis were tested for resistance to penicillin G, erythomycin, metallic ions Zn, As3, As5, Cd, Hg, Pb and activity of beta-lactamase. These studies have shown that the majority of tested staphylococci were resistant to penicillin G, erythromycin, and produced beta-lactamase. No correlation between the activity of beta-lactamase and the resistance to metallic ions has been shown.     

The influence of insulin on glucose and fatty acid metabolism in the isolated perfused rat hind quarter.

Glucose and fatty acid metabolism of resting skeletal muscle were studied by perfusion of the isolated rat hind leg with a hemoglobin-free medium. Tissue integrity was demonstrated by normal ATP, ADP and creatine phosphate levels, by a sufficient oxygen supply, and by a normal appearance of perfused muscle specimens under the electron microscope. The rates of glucose and fatty acid uptake, and of lactate, alanine, glycerol and fatty acid release were constant over a perfusion period of 60 min. Insulin (1 unit/l) caused a more than threefold increase in glucose uptake, a stimulation of lactate production, and a 20% increase in the muscular glycogen levels. Fatty acids and alanine release were significantly diminished by insulin, but glycerol release did not change. The uptake of oleate by the rat hind leg was dependent on the medium concentration in a range of 0.7-1.9mM oleate, and was stimulated by insulin. Glucose uptake was not influenced by oleate, whether sodium was present or not. When the leg was perfused with [1-14C]oleate, 75% of the incorporated fatty acids were found in muscle lipids, 10% were oxidized to CO2, and 5% were recovered in bone lipids. The absolute amount of oleate oxidation was not altered by insulin. In all experiments with and without glucose in the medium, 70-80% of the 14C label incorporated into muscle lipids was found in the triglyceride fraction. In the presence of glucose, insulin significantly increased the incorporation of [1-14C]oleate into muscle triglycerides, whereas no insulin effect, either on fatty acid uptake or on triglyceride formation, could be observed when glucose was omitted from the perfusate. The present results indicate that a "glucose-fatty acid cycle" as found in rat heart muscle does not operate in resting peripheral skeletal muscle tissue. They also demonstrate that the stimulating effect of insulin on muscular fatty acid uptake and triglyceride synthesis is dependent on glucose supply. This finding can be intrepreted as a stimulation of fatty acid esterification by sn-glycerol 3-phosphate derived from an increased glucose turnover, which is in turn due to insulin.     

Lipoidal Components of Bacterial Lipopolysaccharides: Nature and Distribution of Fatty Acids in Aerobacter aerogenes

The fatty acid distribution of Aerobacter aerogenes was studied by comparing the fatty acid composition of the lipoidal component of the endotoxin (lipid A) with the fatty acids of the readily extractable native lipids and total cellular fatty acids. The results for total cellular fatty acids and readily extractable native lipids were generally similar, but both quantitative and qualitative differences exist. In addition, profound differences between these two fractions and lipid A were observed. These differences included fewer fatty acids and lower concentrations of unsaturated and cyclopropane fatty acids in the lipid A. Hydroxy fatty acids persisted in the lipid A. The significance of these differences with respect to mammalian toxicity of endotoxins is discussed.     

Contribution of triglyceride-rich lipoproteins to plasma free fatty acids.

Free fatty acids are the major lipid fuel of the body. Dysregulation of adipose tissue lipolysis results in increased plasma free fatty acid concentrations, and via that mechanism contributes to insulin resistance in obesity and type 2 diabetes mellitus. Adipose tissue hormone sensitive lipase is thought to be responsible for the production of the majority of free fatty acids. However, a separate contribution comes from the action of endothelial lipases, especially lipoprotein lipase, on triglyceride-rich lipoproteins via a process known as spillover. The primary substrate for spillover appears to be chylomicrons derived from dietary fat. The spillover of fatty acids into the free fatty acid pool varies from one tissue to another. For example, spillover is low ( approximately 14%) in the forearm of healthy volunteers, suggesting that triglyceride fatty acid storage is relatively efficient in skeletal muscle. In contrast, spillover appears to be higher in adipose tissue and may also be higher in the splanchnic bed, based on preliminary data. If systemic spillover is increased in insulin resistant states such as diabetes, this could represent a mechanism contributing to the abnormal increases in plasma concentrations of free fatty acids in that condition.     

Fatty acid biosynthesis by a particulate preparation from germinating pea

1. Fatty acid synthesis was studied in microsomal preparations from germinating pea (Pisum sativum). 2. The preparations synthesized a mixture of saturated fatty acids up to a chain length of C(24) from [(14)C]malonyl-CoA. 3. Whereas hexadecanoic acid was made de novo, octadecanoic acid and icosanoic acid were synthesized by elongation. 4. The products formed during [(14)C]malonyl-CoA incubation were analysed, and unesterified fatty acids and polar lipids were found to be major products. [(14)C]Palmitic acid represented a high percentage of the acyl-carrier protein esters, whereas (14)C-labelled very-long-chain fatty acids were mainly present as unesterified fatty acids. CoA esters were minor products. 5. The addition of exogenous lipids to the incubation system usually resulted in stimulation of [(14)C]malonyl-CoA incorporation into fatty acids. The greatest stimulation was obtained with dipalmitoyl phosphatidylcholine. Both exogenous palmitic acid and dipalmitoyl phosphatidylcholine increased the amount of [(14)C]-stearic acid synthesized, relative to [(14)C]palmitic acid. Addition of stearic acid increased the amount of [(14)C]icosanoic acid formed. 6. [(14)C]Stearic acid was elongated more effectively to icosanoic acid than [(14)C]stearoyl-CoA, and its conversion was not decreased by addition of unlabelled stearoyl-CoA. 7. Incorporation of [(14)C]malonyl-CoA into fatty acids was markedly decreased by iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). Palmitate elongation was sensitive to arsenite addition, and stearate elongation to the presence of Triton X-100 or fluoride. The action of fluoride was not, apparently, due to chelation. 8. The microsomal preparations differed from soluble fractions from germinating pea in (a) synthesizing very-long-chain fatty acids, (b) not utilizing exogenous palmitate-acyl-carrier protein as a substrate for palmitate elongation and (c) having fatty acid synthesis stimulated by the addition of certain complex lipids.     

Myocardial lipid metabolism in cardiac hyper- and hypo-function. Studies on triiodothyronine-treated and transplanted rat hearts.

Lipid composition of the myocardium and in vitro lipid metabolism were studied in hearts from young rats after 30 days of treatment with triiodothyronine (100 microgram/kg per day) and in heterotopically isotransplanted hearts of inbred adult rats 6 days after surgery. The former served as an experimental model of cardiac hyperfunction, while the latter, empty beating hearts, served as a model of cardiac hypofunction. In hearts from hyperthyroid animals the concentration of phosphatidylcholine, phosphatidylethanolamine, cardiolipin, and the incorporation of 14C-labelled palmitic and erucic acid into these phospholipids were increased significantly as compared with controls. In contrast, the triglyceride concentration and the incorporation of palmitate into triglyceride was significantly decreased. In transplanted hearts, the phospholipid concentration and the incorporation of 14C-labelled fatty acids into phospholipids were significantly decreased as compared with the hearts of the inbred host rats of the same age. The results indicate that the mechanical performance of the heart affects the phospholipid composition, which may be a reflection of increased or decreased proliferation of subcellular membranes in sustained cardiac hyper- or hypo-function.