Effects of bacteriophage M13 infection upon phospholipid and fatty acid compositions ofEscherichia coli

Escherichia coli K38 were grown and infected with wild type and amber mutants of bacteriophage M13 in the early log phase. Lipid compositions of the infected and healthy cultures, grown under identical conditions, were determined 2 hr after infection. From the results, it was observed that total lipid and total phospholipid content remained nearly constant, suggesting that the cell membrane which contained the maximum phospholipids was not damaged by the infection. Moreover, the percentage of diphosphatidylglycerol and lyso-compounds corresponding to phosphatidylethanolamine and phosphatidylglycerol increased, while phosphatidylethanolamine and phosphatidylglycerol decreased. The increase in lyso-compounds may be due to the release of phospholipase A₂ (a periplasmic enzyme) from the cell wall after damage by the infection. Bacteriophage M13 infection had no effect on the fatty acid composition of the phospholipids.     

Metabolism of 12-ketooctadecanoic acid byEscherichia coli K-12 andCandida tropicalis

The mode of degradation of long chain keto acids by two microorganisms was investigated.Escherichia coli K-12 converted 12-ketooctadecanoic acid to 4-ketodecanoic acid, accumulating some amounts of intermediates, 10-ketohexadecanoic, 8-ketotetradecanoic and 6-ketododecanoic acids. In contrast,Candida tropicalis completely metabolized the keto acid with transient accumulation of the metabolites mentioned above. The difference between the metabolism byE. coli of hydroxy acid and keto acid is that 12-hydroxyoctadecanoic acid is degraded as far as 6-hydroxydodecanoic acid, while 12-ketooctadecanoic acid can be degraded as far as 4-ketodecanoic acid.     

Influence of ionic liquids on the growth ofEscherichia coli

Ionic liquids are compounds that composed only of ions and are liquid at room temperature. Thus, it is normally named room temperature ionic liquid (RTIL). In this study, the application of RTILs to the extractive fermentation of biomaterials was investigated as a substitute of organic solvents. The relative toxicity of the RTILs on the growth ofE. coli was tested. The inhibition of cell growth in the presence of various ionic liquids was measured using solid and liquid culture, and EC₅₀ of each RTILs was calculated. The number of viable and total cells was measured by the number of colonies and optical density, respectively. Effective concentrations of toxicity (EC₅₀) in these tested systems were similar with conventional solvents, such as acetone, acetonitrile, and ethanol. The viability ofE. coli was affected by the polarity and ionic properties of ionic liquids. The resistance of the microorganisms against ionic liquids was different with the cations and anions composing ionic liquids. No general influence of the anionic compound of the ionic liquids was found on toxicity comparing with distinctive influence of cationic moiety.     

Lipids of cultured hepatoma cells: IV. Effect of serum and lipid upon cellular and media neutral lipids

Minimal deviation hepatoma 7288C cells were cultured in a modified Swim's medium supplemented with decreasing levels of serum, lipid-free serum, lipid-free serum plus fatty acids, and other additives. Cellular and media neutral lipid classes were quantitated, the fatty acids of triglycerides and sterol esters analyzed, and the carbon number distribution of triglycerides determined. Cellular triglyceride biosynthesis virtually was inhibited when the medium was supplemented with bovine serum alone. This inhibition was not observed when the medium was supplemented with fetal calf serum alone or mixtures of fetal calf serum and bovine serum. Cells cultivated on medium supplemented with lipid-free serum plus palmitic or linoleic acids had much lower levels of free and esterified cholesterol. The fatty acid composition of cellular triglycerides and cholesterol esters differed dramatically from the corresponding media lipid classes. Except when linoleic acid was added to the medium, changes in the media serum and lipid levels had only marginal effects upon the fatty acid composition of cellular triglycerides and cholesterol esters. These data, in conjunction with earlier data that showed the media neutral lipid levels did not decrease during cell growth, indicate that these hepatoma cells utilize little or no serum triglycerides and cholesterol esters. Linoleic acid added to the medium dramatically reduced the level of 18∶1 acids in cellular triglycerides and cholesterol esters. Palmitic acid added to the medium did not change the fatty acid compositions significantly. Comparison of experimentally determined and calculated triglyceride carbon number percentages indicated a random distribution of fatty acids in this glyceride. The fatty acid composition of cellular triglycerides was similar to the composition of the cholesterol esters. The lack of characteristic and distinguishable compositions of these two classes that occur in most normal tissues suggests a loss of specificity in the lipid metabolism of this neoplasm at the class level.     

Pulmonary lipid peroxides and fatty acids of rats fed different lipids and exposed to oxygen at hyperbaric pressure

Semipurified diets containing different lipids were fed to rat dams during lactation and subsequently to their pups for 33 weeks post-weaning. Some rats within each group were exposed to oxygen at hyperbaric pressure (OHP). Lipid peroxide levels were lower in lungs of rats fed 7% hydrogenated coconut oil or 10% butter as compared with their controls, fed 7% corn oil or 10% safflower oil, respectively. Exposure to OHP increased lung peroxide levels. This increase varied with the type of fat in the diet. Studies of the fatty acid composition indicate that lipid peroxide levels generally increased with an increase in the levels of 18∶2 in lung total lipids. The results suggest that the type of dietary lipid may alter the susceptibility of the animal to pulmonary oxygen toxicity.     

Conversion of lipids upon the mechanical degradation of lacustrine sediments

The effect of the mechanical treatment of lacustrine sediments in mechanical activators of various designs on the degree of dispersion, the yields of free and bound lipids, and the carotenoid and chlorophyll contents of lipids was studied. A comparative analysis of changes in the composition of lipids upon the acid and mechanical treatments of lacustrine sediments was performed.     

Effects of polarity and pH on the solubility of acid-treated carbon nanotubes in different media

The aggregation of acid-treated carbon nanotubes (CNTs) as a solid and their solubility in solvents of different polarity and in water of different pH were investigated as a function of acid treatment conditions. The CNTs were found to form solid hydrogen-bonded aggregates, with a higher content of COOH groups resulting in a denser aggregate. The untreated CNTs were non-polar in nature and could dissolve, or be easily dispersed, in non-polar or low polar solvents such as acetone and alcohols (methanol and ethanol), but precipitated from deionized water, a highly polar solvent. In contrast, the acid-treated CNTs dissolved or were well dispersed in the deionized water, but not in acetone or alcohols. The treated CNTs were insoluble in an aqueous solution of pH 0, but soluble in those of pH 4 and higher with their solubility monotonically increasing with pH up to pH 10. The considerable ionic bond strength between carboxylate anions and sodium cations could be a reason for a decrease of their solubility in an aqueous solution of pH 12.     

Effects of dissolved oxygen control on cell growth and exopolysaccharides production in batch culture ofAgaricus blazei

To investigate the effects of DOC on cell growth and EPS production, DOC was controlled at three different levels of 10, 20, and 40% of air-saturation by manipulating agitation speed in a series of batch cultures ofA. blazei. The cellular and EPS productivities increased with the DOC level up to 20%. However, DOC had no significant effects over 20%. When DOC was controlled at 20%, the cellular and EPS productivities were observed to increase 1.6-fold and 2.2-fold, respectively, as compared to the control case with no DOC control in which DOC dropped to and there-after remained at almost zero. Another batch culture was carried out with the DOC controlled at 20% by manipulating the amount of oxygen supply at a rather low agitation speed of 100 rpm. In this case, the cellular productivity was comparable to that of the former case in which DOC was controlled at the same level of 20% by manipulating agitation speed in the range of 100–450 rpm. However, the EPS productivity was about 15% lower than the former case, implying that a sufficient level of agitation is also important for EPS production.     

Formulation and optimization of two culture media for the production of tumour necrosis factor-β in Escherichia coli

Two culture media were designed and optimized by statistical techniques for the growth of Escherichia coli K12 HB101 (pCG402) which expressed human tumour necrosis factor-β (TNF-β). Common compounds, such as MgSO₄ and KH₂PO₄, were used to provide the bacteria with the necessary elements for biomass synthesis. For compounds not required for biomass synthesis, such as thiamine, or compounds used for both biomass synthesis and as an energy source, such as glucose, yield coefficients were used. In formulating the composition of the nutrients, carbon was chosen as the limiting substrate in both media. In the complex medium (CM), casein hydrolysate was selected to supply the two auxotrophic amino acids, proline and leucine. The optimal ratio of glucose to casein hydrolysate was determined to be 1:0·6 by using a centre composite design experiment. In the defined medium (DM), the concentrations of the three carbon sources, glucose, proline and leucine were based on their respective yield coefficients (Y_{biomass/glucose}: 0·5, Y_{biomass/proline}: 7, Y_{biomass/leucine}: 13·5). Shake flask experiments based on a fractional design were used to confirm that the two media were glucose limiting. Bacterial growth was improved in CM whereas DM gave higher TNF-β expression. A 2-dm³ fed-batch fermentation using CM was performed and a dry biomass concentration of 20 g dm^?3 was obtained with the expression of soluble TNF-β being 20 mg g^?1 dry biomass. With this fed-batch system, a high biomass concentration and high expression of TNF-β were achieved concomitantly.     

Intake of different eicosapentaenoic acid-containing lipids and fatty acid pattern of plasma lipids in the rats

The ethyl ester of eicosapentaenoic acid (EPA) is the only pure EPA-containing lipid available in bulk for oral administration. However, there is doubt as to whether EPA ethyl ester can efficiently increase the plasma levels of EPA in comparison with the ability of other kinds of EPA-containing lipids to do so. Therefore, two other kinds of EPA-containing lipids were prepared to study the efficiency of oral administration of those lipids for increasing the EPA content in plasma phospholipids and cholesteryl esters. EPA-containing lipids which were investigated were [A], 1,2,3-trieicosapentaenoyl-glycerol, [B] 2-eicosapentaenoyl-phosphatidylcholine and [C] ethyl ester of EPA. An adjusted amount of lipids [A], [B] and [C] was administered to rats through a gastric tube for 4 days (the first experiment) or for 10 days (the second experiment), and the fatty acid composition of plasma phospholipids and cholesteryl esters was determined. In the first experiment, there were no significant differences in the efficiency for increasing EPA levels in either phospholipids or cholesteryl esters among the lipids. In the second experiment, the EPA levels of both plasma phospholipids and cholesteryl esters of rats administered ethyl ester of EPA were significantly higher than those of rats administered 2-eicosapentaenoyl-phosphatidylcholine. The EPA levels of the rats administered 1,2,3-trieicosapentaenoylglycerol were between the levels of the two groups mentioned above, but the differences in the EPA levels were not significant. Although an ethyl ester-type molecule is not a naturally occurring lipid, ethyl ester of EPA is equal to 1,2,3-trieicosapentaenoyl-glycerol and appears to be superior to 2-eicosapentaenoyl-phosphatidylcholine as to the efficiency for increasing EPA levels in total plasma phospholipids and plasma cholesteryl esters.     

Effect of oxygen levels on the fatty acids and lipids ofMucor rouxii

The effect of aerobic and oxygen limiting (anaerobic) growth conditions upon the fatty acid and lipid composition ofMucor rouxii has been examined. The aerobic cells contained a range of fatty acids typical of phycomycetes, i.e. γ-linolenic acid, with an unsaturation index of 1.20, whereas the anerobic cells contained relatively high levels of shorter chained fatty acids and very low concentrations of unsaturated acids (unsaturation index=0.025). The unsaturated compounds were monoolefinic tetra-, hexa-, and octadecenoic acids; and closer examination of their di-trimethylsilyl derivatives by gas chromatography and mass spectrometry showed that all three acids contained the double bond in the Δ⁹ position. These results were consistent with a microaerobic biosynthetic pathway. In addition, there were major quantitative differences in the lipid composition of the two types of cells; and it was evident that the differences in growth environment markedly affected the cellular lipid and fatty acid compositions.     

In vivo and in vitro biosynthesis of free fatty alcohols inEscherichia Coli K-12

In vivo studies have indicated that exogenous free fatty acids may serve as precursors of the free fatty alcohols ofEscherichia coli K-12. Following disruption of the cells, the enzymatic activity capable of catalyzing the reduction of long chain fatty aldehydes to fatty alcohols was localized in the 100,000 x g supernatant fraction. Nicotinamide adenine dinucleotide phosphate, reduced form, was the required cofactor. The product of the reaction was characterized rigorously as 1-hexadecanol when hexadecanal was the substrate. Three independent, but complementary, assay methods were developed to assay the aldehyde reductase activity. By employing these methods, an equivalence between nicotinamide adenine dinucleotide phosphate, reduced form, oxidation and 1-hexadecanol synthesis was established. Two protein fractions catalyzing the reduction of fatty aldehydes to fatty alcohols were detected in the 100,000 x g supernatant fraction following ammonium sulfate fractionation and diethylaminoethyl-cellulose chromatography. Enzymatic activity (70%) applied to the diethylamino-ethyl-cellulose column was eluted at a phosphate concentration of 0.115 M. The remaining 30% was eluted at a concentration of 0.23 M. Following sephadex chromatography, it was observed that the enzyme eluting at 0.115 M phosphate had an apparent mol wt of 250,000 Daltons while that eluting at 0.23 M had an apparent mol wt of 62,000 Daltons. The enzymes were similar with respect to substrate specificity, pH optima, ionic strength optima, and stability with respect to thiol inhibitors, suggesting different sized aggregates of similar subunits.     

Partial purification and conversion of the particulate-bound diglyceride kinase ofEscherichia coli to a water soluble,detergent free state

Alkali (pH 11.5) treatment of a washed, ammonium sulfate-precipitated residue derived from a triton-X-100 extract of a 30,000 Xg particulate diglyceride kinase preparation ofE. coli converts much of the enzyme to a 100,000 Xg soluble, detergent free state. This procedure improves the enzyme as an analytical tool for the structural analysis of triglycerides, and may be applicable for the solubilization of the many other glyceride- and phosphoglyceride-forming enzymes which have all resisted further purification because of insoluble, particulate properties.     

Effect of long and medium chain length lipids upon aqueous solubility of α-tocopherol

The efficiency by which α-tocopherol is solubilized in vitro into mixed bile salt micelles containing different lipids was studied. Alterations in solubility due to addition to the incubation media of triglyceride, free fatty acid, monoglyceride, and lecithin of either long or medium chain length were examined. Results are expressed as a partition ratio between a micellar and an oil phase. The triglyceride of both long and medium chain length fatty acids greatly decreased the solubility of α-tocopherol in bile salt solutions. When added singly, monoglyceride and lecithin of long chain length fatty acids increased the α-tocopherol solubilized four- to fivefold; fatty acids of either chain length and medium chain monoglyceride when added singly had no significant effect upon the tocopherol solubilized. An additive effect was observed when a combination of long chain monoglyceride and lecithin was added. Addition of fatty acid to this combination, however, significantly decreased the α-tocopherol solubility into the micellar phase. Although the solubility of α-tocopherol was increased by all combinations of medium chain length polar lipids, except the fatty acid-monoglyceride pair, the effect was three to seven times less than for the corresponding long chain mixture.     

Distribution of deuterium-labeledcis-andtrans-12-octadecenoic acids in human plasma and lipoprotein lipids

Triglycerides containingcis- andtrans-12-octadecenoic acid (12c-18∶1 and 12t-18∶1) andcis-9-octadecenoic acid (9c-18∶1) labeled with deuterium were fed to 2 young adult male subjects. These fatty isomers each contained a different number of deuterium labels, which allowed mass spectrometric analysis to distinguish among them after they were fed as a mixture. This approach results in a direct comparison of the absorption and distribution of these 3 monoenoic acids into blood plasma and lipoprotein lipids. Plasma lipid data indicated that all phospholipid fractions selectively incorporate 12c-18∶1 and 12t-18∶1 in preference to 9c-18∶1. Discrimination against 12c-18∶1 and 12t-18∶1 compared to 9c-18∶1 was found in the plasma neutral lipids, with a strong discrimination against 12t-18∶1 incorporation into the cholesteryl ester fraction. Considerable reduction in the percentage of linoleic and arachidonic acid was observed when 12–18∶1 isomers were incorporated in plasma triglyceride, phosphatidylcholine and sphingomyelin samples. Chylomicron lipid analyses indicated that all isomers were well absorbed. Variation was observed in the relative distribution of 12c-18∶1, 12t-18∶1 and 9c-18∶1 between the very low density, low density and high density lipoprotein lipid classes. No desaturation of 12c-18∶1 to linoleic acid was detected.     

A classification of biologic lipids based upon their interaction in aqueous systems

A classification of lipids is presented, based upon their physical properties in bulk aqueous systems and at the air-water or oil-water interface. This is supported by binary-phase diagrams of the various classes of lipids in water. The interactions of the lipids of each class with a lipid of another class is illustrated by a series of different ternary-phase diagrams of two lipids in water. The various types of association and the molecular relation of one lipid to another are indicated. The interaction of three classes of lipids with water is illustrated in two examples by quaternary-phase diagrams of the three lipids in water. As an example of the application of these invitro studies, the composition of bile is correlated with a quaternary-phase diagram cholesterollecithin-bile salt-water. The correlation shows that human bile behaves as a biologic four-component system the physical state of which is entirely predictable from the quaternary-phase diagram. Although bile is a special case, it is probable that the physical arrangement of the lipids in membranes, cellular organelles, lipoproteins, and adipose tissue can be suggested by studies of the interaction of lipid classes with themselves in water.     

Gas chromatographic analysis of reactive carbonyl compounds formed from lipids upon UV-irradiation

Peroxidation of lipids produces carbonyl compounds; some of these, e.g., malonaldehyde and 4-hydroxynonenal, are genotoxic because of their reactivity with biological nucleophiles. Analysis of the reactive carbonyl compounds is often difficult. The methylhydrazine method developed for malonaldehyde analysis was applied to simultaneously measure the products formed from linoleic acid, linolenic acid, arachidonic acid, and squalene upon ultraviolet-irradiation (UV-irradiation). The photoreaction products, saturated monocarbonyl, α,β-unsaturated carbonyls, and β-dicarbonyls, were derivatized with methylhydrazine to give hydrazones, pyrazolines, and pyrazoles, respectively. The derivatives were analyzed by gas chromatography and gas chromatography-mass spectrometry. Lipid peroxidation products identified included formaldehyde, acetaldehyde, acrolein, malonaldehyde, n-hexanal, and 4-hydroxy-2-nonenal. Malonaldehyde levels formed upon 4 hr of irradiation were 0.06 μg/mg from squalene, 2.4 μg/mg from linolenic acid, and 5.7 μg/mg from arachidonic acid. Significant levels of acrolein (2.5 μg/mg) and 4-hydroxy-2-nonenal (0.17 μg/mg) were also produced from arachidonic acid upon 4 hr irradiation.     

Effects of hydrogen peroxide on lipoproteins and associated lipids

Lipoproteins isolated from human or chimpanzee serum were treated with H₂O₂ and allowed to stand varying lengths of time before quantitative analysis in the ultracentrifuge. Marked instability of ultracentrifugal boundaries (convection) occurred during the first 24 hr, but diminished thereafter. Simultaneously, the quantity of lipoprotein decreased. The instability of boundaries in H₂O₂-treated samples was presumed to reflect loss of lipid-protein affinity and breakdown of lipoproteins under the force of ultracentrifugation. Analysis of extracted lipids showed that H₂O₂ caused little loss of phospholipid, significant loss of triglyceride, and apparent loss of cholesteryl ester. The latter loss, however, was accompanied by appearance of esterified cholesterol in the free cholesterol eluent. Apparently H₂O₂ converted some cholesteryl esters to a more polar form which was eluted later from the column, with the free cholesterol fraction. Gas chromatographic analysis of the fractions eluted from the column showed that selective degradation of polyunsaturated fatty acids was most marked with cholesteryl esters, somewhat less with triglycerides, and negligible with phospholipids. It was postulated that the loss of lipid-protein affinity caused by H₂O₂ in vitro may reflect a similar process in vivo, i.e., that one process contributing to development of atherosclerosis can be oxidation of lipoprotein, with loss of lipid-protein affinity and accumulation of lipid products in (or on) cells of the vascular system. Presented at the AOCS Meeting, Washington, D. C., April, 1968.