Lipid variation at different temperatures on two species of Xenorhabdus

This paper describes the lipid variation of Xenorhabdus nematophilus N₂4 and Xenorhabdus luminescens HP88 grown at different temperatures (25 °C, 18 °C and 5 °C). In both strains phosphatidylethanolamine was identified to be the major phospholipid accounting about 80% of lipid phosphorus. In X. luminescens HP88 a significant portion of cardiolipidin was found. The major fatty acid found was palmitic acid (C16:0), palmitoleic acid (C16:1) and oleic acid (C18:1). However, lauric acid (C12:0), myristic acid (C14:0), C17 cyclopropane acid (C17-cy) and stearic acid (C18:0) were also detected. Decreasing growth temperatures from 25 °C to 18 °C was accompanied by increase in percentage of unsaturated fatty acid C16:1 at the expense of saturated fatty acids like C16:0, C18:0 and C17-cy. Further decrease in temperature to 5 °C led to decline in C17-cy in favour of C16:1 in X. nematophilus N₂4 but it could hardly get any change in fatty acid composition of X. luminescens HP88.     

Cellular fatty acids derived from normal alkanes by Candida rugosa

In an attempt to examine the specific metabolic relationship between the alkane substrates and lipid products the pattern of formation of cellular lipid, especially fatty acids, has been studied in the course of incubation of cells of C. rugosa with n-alkanes in mineral medium. C_18:2, C_18:1, C_16:1 and C_16:0 fatty acids were the major products formed, irrespective of the odd or even chain length of the alkane used as substrate. These unsaturated fatty acids reached a maximum in the stationary phase. In the case of cells grown on odd-chain n-alkanes (from C₁₁ to C₁₉), the proportion of odd-chain cellular fatty acids was markedly high, reaching 77–88% in the n-pentadecane and heptadecane-grown cells. The resulting acids are then metabolized by β-oxidation or inserted directly or after elongation with C₂-units into the cellular fatty acids. The total lipid content of n-alkane (n-C₁₀-C₂₀) grown cells in the stationary phase was 16.3–19.0% of the dry cell weight, which was about three times as much as that of glucose-grown cells. The chain length of alkane substrates had a significant effect on the lipid content. Ergosterol production from n-hexadecane was twice higher than that from glucose.     

Acyl Bis(monoacylglycero)phosphate,assumed to be a marker for lysosomes,is a major phospholipid of vaccinia virions

A hitherto unknown major phospholipid of purified vaccinia virions harvested from lysed cells was identified as acyl bis(monoacylglycero)phosphate by chromatographic comparison with synthetic bisphosphatidic acid and various analogs. It accounts for 20–30% of the total viral phospholipids. Digestion with pancreatic phospholipase A₂ liberates fatty acids and results in bis(monoacylglycero)phosphate, which is thought to represent a phospholipid marker for cellular lysosomes. Possible implications for the intracellular virus dissemination are discussed.     

Relationship between Hydroxy Fatty Acids and Prostaglandin E2 in Gingival Tissue

Bacterial hydroxy fatty acids and alpha-hydroxy fatty acids have been demonstrated in complex lipid extracts of subgingival plaque and gingival tissue. However, little is known about the relationship between these hydroxy fatty acids in plaque and gingival tissues or the significance of these complex lipids in promoting inflammatory periodontal disease. The present study determined the percentages of ester-linked and amide-linked hydroxy fatty acids in complex lipids recovered from plaque and gingival tissue samples and the relationship between bacterial hydroxy fatty acids and alpha-hydroxy fatty acids in the lipid extracts. To evaluate a potential role for these hydroxy fatty acids in inflammatory periodontal disease, gingival tissue samples were examined for a relationship between prostaglandin E₂ (PGE₂) and hydroxy fatty acids recovered in gingival lipid. This investigation demonstrated that alpha-hydroxy fatty acids are only ester linked in plaque lipids but are largely amide linked in gingival tissue lipids. Furthermore, the level of alpha-hydroxy fatty acid in gingival lipid is directly related to the level of the bacterial hydroxy fatty acid 3-OH iso-branched C_17:0 (3-OH iC_17:0) in the same lipid extract. However, the relationship between hydroxy fatty acids in gingival lipids does not parallel the fatty acid relationship observed in plaque lipids. Finally, alpha-hydroxy fatty acid levels in gingival tissue lipids correlate directly with the recovery of PGE₂ in the same tissue samples. These results demonstrate that alpha-hydroxy fatty acid levels in gingival lipids are directly related to both 3-OH iC_17:0 bacterial lipid levels and PGE₂ levels. These results indicate that in periodontal tissues there are unusual host-parasite interactions involving penetration of bacterial lipid in association with an altered gingival lipid metabolism and prostaglandin synthesis.     

Observations on the lipids ofOochoristica agamae (Cestoda)

An investigation of the lipids ofOochoristica agamae, an anoplocephalid cestode of theAgama lizard, was undertaken. Total lipids of the parasite accounted for 8.4% of the fresh weight; neutral lipids comprised 82.98% of the total, glycolipids, 5.01%, and phospholipids, 12.03%. The major lipid classes inO. agamae include triglycerides, cholesterol, phosphatidyl choline, and phosphatidyl ethanolamine. The 16-and 18-carbon fatty acids were predominant in the parasite. Hexadecenoic acid, usually found at low concentrations in the lipids of helminth parasites, was the most abundant of the 16-carbon fatty acids ofO. agamae (notably in the neutral lipid fraction). Although octadecatrienoic acid occurred only in trace amounts in the intestinal contents of the host, significant amounts of this fatty acid were detected in the parasite. A lack of 20-carbon fatty acids was determined in the lipids of the host's intestinal contents and the neutral lipid fraction of the parasite.O. agamae is suspected to be capable of modifying fatty acids obtained from dietary sources by chain elongation.     

Lipids of parasitic and saprophytic leptospires

The lipid composition of five parasitic and six saprophytic leptospires was compared. Lipids comprise 18 to 26% of the dry weight of the cells after chloroform-methanol extraction. No residual (bound) lipid was found after acid or alkaline hydrolysis of the extracted residue. The total lipid was composed of 60 to 70% phospholipid, and the remaining lipid was free fatty acids. The phospholipid fraction contained phosphatidylethanolamine as the major component, and phosphatidylglycerol and diphosphatidylglycerol were minor components with traces of lysophatidylethanolamine sometimes found. The major fatty acids of leptospires were hexadecanoic, hexadecenoic, and octadecenoic acids. Both the unusual cis-11-hexadecenoic acid and the more common cis-9-hexadecenoic acid were synthesized by the leptospires. Neither the parasitic nor the saprophytic leptospires can chain elongate fatty acids. However, they were capable of β-oxidation of fatty acids. Both groups of leptospires desaturate fatty acids by an aerobic pathway. When the parasite canicola was cultivated on octadecanoic acid, 87% of the hexadecenoic acid was the 11 isomer, whereas the saprophyte semeranga consisted of 10% of this isomer. In addition, the saprophytic leptospires contained more tetradecanoic acid than the parasites. No differences were observed in the lipid composition of virulent and avirulent strains of canicola.     

Lipid composition of metacestodes of Taenia taeniaeformis and lipid changes during growth

A lipid analysis was performed on developing metacestodes of Taenia taeniaeformis removed from the livers of rats at times varying from 3 to 35 weeks post infection. Lipid accounted for 7–21% of the dry weight of the parasites. The highest proportions were found at the earlier stages. The distribution was as follows; neutral lipid 27–45%; glycolipid 5–11%; and phospholipid 50–61%. The major neutral lipid was cholesterol, and minor neutral lipids were sterol esters, triglycerides, diglycerides and monoglycerides. Hydrocarbons were present throughout development, but in the highest amounts at the earlier stages. Five different glycolipids were found, all of which were identified as glycosphingolipids. An increase in the proportion of more complex glycolipids was noted as parasites grew older. Ten different phospholipids were identified, with the major components being phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. Other phospholipids were: lysophosphatides, phosphatidylinositol, phosphatidic acid, diphosphatidylglycerol, sphingomyelin, and an unknown phospholipid component. Changes in the relative amounts of the two major phospholipids were found when the early and late stages were compared. Two lipids found throughout development were identified as glycosylated dolichol phosphates, and they comprised between 1 and 3% of the total phospholipid fraction. Nineteen fatty acids were detected, and the fatty acid distribution for each lipid class at each stage was determined. Seven major fatty acids were common to each. These were: hexadecanoic, octadecanoic, oleic, linoleic, arachidonic, docosanoic, and docosahexaenoic.     

Lipids and fatty acids of tachyzoites and purified pellicles ofToxoplasma gondii

Various lipids were extracted from tachyzoites and from purfied pellicles ofToxoplasma gondii. Extracts from both sources were found to have a low cholesterol/phospholipid ratio. The major phospholipid in these fractions was phosphatidylcholine associated with a low amount of sphingomyelin. Oleic acid represented one-third of whole-cell fatty acids and 44% of pellicular fatty acid content. The lipid composition of the pellicle ofT. gondii is consistent with the previously reported high fluidity of this membrane.     

Lipid Composition of the Hemagglutinating Active Fraction Obtained from Chick Embryos Infected with Chlamydia psittaci 6BC

The lipid composition of the concentrated hemagglutininating active fraction (HF) of allantoic fluid from infected eggs, but free from Chlamydia psittaci 6BC, was compared to concentrated normal allantoic fluid (NAF). Phosphatidyl-choline (PC) and phosphatidylethanolamine were the major lipid classes of the total phospholipid fraction. Some quantitative differences were found in the amount of PC and phosphatidylserine present in HF and NAF. Lysophosphatidylcholine was present in HF but absent in NAF. Triglycerides and sterols were the major lipid classes found in neutral lipids of HF and NAF. Quantitative data showed distinct differences in the amount of different neutral lipid classes present between HF and NAF. The fatty acids of various classes of lipids were examined, and differences were noted in a number of phospholipids, sterol esters, and the free fatty acids. Branched-chain saturated fatty acids were found in many lipid classes of the HF, particularly in the phosphatidylethanolamine fraction, but were absent in the NAF.     

Lipid biomarkers of glioma cell growth arrest and cell death detected by 1H magic angle spinning MRS

Biomarkers of early response to treatment have the potential to improve cancer therapy by allowing treatment to be tailored to the individual. Alterations in lipids detected by in vivo MRS have been suggested as noninvasive biomarkers of cell stress and early indicators of cell death. An improved understanding of the relationship between MRS lipids and cell stress in vitro would aid in the translation of this technique into clinical use. Rat BT4C glioma cells were treated with 50 µ m cis‐dichlorodiammineplatinum II (cisplatin), a commonly used chemotherapeutic agent, and harvested at several time points up to 72 h. High‐resolution magic angle spinning ¹H MRS of cells was then performed on a 600‐MHz NMR spectrometer. The metabolites were quantified using a time domain fitting method, TARQUIN. Increases were detected in saturated and polyunsaturated fatty acid resonances early during the exposure to cisplatin. The fatty acid CH₂/CH₃ ratio was unaltered by treatment after allowing for contributions of macromolecules. Polyunsaturated fatty acids increased on treatment, with the group –CH = CH–CH₂–CH = CH– accounting for all the unsaturated fatty acid signals. Transmission electron microscopy, in addition to Nile red and 4',6‐diamino‐2‐phenylindole co‐staining, revealed that the lipid increase was associated with cytoplasmic neutral lipid droplets. Small numbers of apoptotic and necrotic cells were detected by trypan blue, annexin V–fluorescein isothiocyanate‐labelled flow cytometry and DNA laddering after up to 48 h of cisplatin exposure. Propidium iodide flow cytometry revealed that cells accumulated in the G1 stage of the cell growth cycle. In conclusion, an increase in the size of the lipid droplets is detected in morphologically viable cells during cisplatin exposure. ¹H MRS can detect lipid alterations during cell cycle arrest and progression of cell death, and has the potential to provide a noninvasive biomarker of treatment efficacy in vivo. Copyright © 2012 John Wiley & Sons, Ltd.     

Preliminary studies of lipids of the trematodes Eurytrema pancreaticum,Calicophoron erschowi and the turbellarian Penecurva sibirica

The amount of total lipids, phospholipids, neutral lipids and fatty acids was determined in cattle parasites, namely the trematodes Eurytrema pancreaticum, Calicophoron erschowi, and in the free-living turbellarian Penecurva sibirica. Neutral lipids of these flatworms contained sterols, sterol esters, triacylglycerols and free fatty acids. P. sibirica also contained diacylglycerols.Parasitic and free-living flatworms differed in phospholipid composition: the turbellarian did not contain phosphatidylserine, and the trematodes had practically no sphingomyelin or lysophosphatidyl choline. E. pancreaticum, C. erschowi and P. sibirica contained high levels of phosphatidylcholine and phosphatidylethanolamine as well as lysophosphatidylethanolamine and cardiolipin.The fatty acid composition of total lipid extracts of flatworms and of the pancreas and rumen of the host animals were determined. The fatty acid composition of the flatworm lipids reflected the fatty acid composition of the host tissue but was not identical to it.     

Analysis of human serum lipoprotein lipid composition using MALDI-TOF mass spectrometry

This study used matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify all lipid classes in human serum lipoproteins. After the major lipoproteins classes were isolated from serum by ultracentrifugation, the lipids were extracted and mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) dissolved in Folch's solution (chloroform/methanol 2:1, v/v). MALDI-TOF MS analysis of the samples identified phospholipids (PLs), lysophospholipids (lysoPLs), sphingolipids (SLs), triglycerides (TGs), cholesteryl esters (CEs), and free cholesterol; it also showed the characteristics of individual fatty acid chains in serum lipids. MALDI-TOF MS allowed analysis of strongly hydrophobic and non-polar molecules such as CEs and TGs as well as hydrophilic molecules such as phospholipids. Direct analysis of fatty acids was not possible. The concentrations of lipids were not consistent with the ion peak intensities, since the extent of polarity affected the ionization characteristics of the molecules. However, lipid molecules with similar molecular structures but various fatty acid chains, such as phosphatidylcholine (PCs), were analyzed quantitatively by MALDI-TOF MS. Quantitative measurement of cholesterol was possible with the use of an internal standard. This study shows that MALDI-TOF MS can be used for direct investigation and quantitative analysis of the phospholipid composition of serum lipoproteins.     

Carbon stripping extracts serum free fatty acids: implications for media supplementation of cultured type II pneumocytes

Carbon stripping is a process that is widely used to remove hormones from serum. Because addition of serum to culture media also provides exogenous fatty acids that influence lipid metabolism of cultured cells, we investigated the effects of carbon stripping on the composition of the phospholipid and free fatty acid fractions in fetal bovine serum and the effects of these changes on phosphatidylcholine synthesis by cultured adult alveolar type II cells. Carbon stripping resulted in quantitative and qualitative changes in serum free fatty acids. The process effectively extracted greater than or equal to 99% free fatty acids and, to a lesser extent, phospholipids. There were also qualitative changes in the relative composition of the remaining free fatty acids with a selective loss of oleic and linoleic free fatty acids. However, the relative composition of the serum phospholipid fatty acid fraction was unaffected. Type II cells isolated from adult male rat lung and cultured in Dulbecco's modified Eagle's medium supplemented with 10% carbon-stripped fetal bovine serum (FBS-CS) incorporated [3H]choline into phosphatidylcholine at a rate 36% less than the rate of control cells cultured with unstripped FBS. Addition of oleic acid to FBS-CS supplemented media increased total phosphatidylcholine synthesis by adult type II cells by 67-71%. In contrast, addition of palmitic acid inhibited PC synthesis 51-67%. The combination of oleic and palmitic acids resulted in a rate of [3H]choline incorporation into phosphatidylcholine similar to the rate for control cells cultured in FBS-CS-supplemented media alone. Although synthesis of disaturated phosphatidylcholine was unaffected by exogenous fatty acids, addition of fatty acids altered the proportion of disaturated phosphatidylcholine synthesis relative to total phosphatidylcholine synthesis. The presence or absence of the hormones, dexamethasone and triiodothyronine, did not explain the difference in rate of phospholipid synthesis by type II cells cultured in untreated versus carbon-stripped serum supplemented media. These results suggest that the removal of serum free fatty acids by carbon stripping can influence phospholipid metabolism of cultured type II cells. Because serum free fatty acids influence cellular lipid composition and potentially cell metabolic functions, carbon-stripped serum may not be the optimal choice for media supplementation of cultured cells.     

Rapid uptake and esterification of arachidonic acid and other fatty acids by microfilariae of Brugia malayi

We have examined the differential incorporation and esterification of exogenous fatty acids by microfilariae of the human filarial parasite Brugia malayi. Microfilariae incubated with 2 nM [3H]arachidonic acid over 1 h rapidly took up this fatty acid. Palmitic, oleic and linoleic acids were also incorporated by parasites. In contrast to these other fatty acids, little incorporated arachidonic acid remained as free fatty acid within microfilariae. Arachidonate was rapidly esterified into phospholipids, with 66% of incorporated arachidonate esterified into phospholipids at 1 min. Esterification of other fatty acids into phospholipids was quantitatively lesser and occurred into phosphatidylcholine and phosphatidylethanolamine. Arachidonate was preferentially esterified into phosphatidylinositol, which constituted only 10% of the total parasite phospholipid pool, and into phosphatidylcholine. By 1 min these two phospholipid classes, respectively, comprised 53% and 43% of [3H]arachidonyl-phospholipids. Neither the microfilarial incorporation of arachidonate nor its esterification into parasite phospholipids could be saturated by noncytotoxic concentrations of up to 600 microM. Microfilariae, which in vivo are exposed to arachidonate in blood, can rapidly, avidly and with high capacity incorporate exogenous arachidonate and esterify it preferentially into specific classes of phospholipids, including phosphatidylinositol. Like many mammalian cells, these phylogenetically distinct metazoan parasites possess efficient means for utilizing host-derived arachidonic acid.     

Control of Clostridium perfringens-induced necrotic enteritis in broilers by target-released butyric acid,fatty acids and essential oils

The efficacy of target-released butyric acid, medium-chain fatty acids (C₆ to C₁₂ but mainly lauric acid) and essential oils (thymol, cinnamaldehyde, essential oil of eucalyptus) micro-encapsulated in a poly-sugar matrix to control necrotic enteritis was investigated. The minimal inhibitory concentrations of the different additives were determined in vitro, showing that lauric acid, thymol, and cinnamaldehyde are very effective in inhibiting the growth of Clostridium perfringens. The in vivo effects were studied in two trials in an experimental necrotic enteritis model in broiler chickens. In the first trial, four groups of chickens were fed a diet supplemented with butyric acid, with essential oils, with butyric acid in combination with medium-chain fatty acids, or with butyric acid in combination with medium-chain fatty acids and essential oils. In all groups except for the group receiving only butyric acid, a significant decrease in the number of birds with necrotic lesions was found compared with the infected, untreated control group. In the second trial the same products were tested but at a higher concentration. An additional group was fed a diet supplemented with only medium-chain fatty acids. In all groups except for that receiving butyric acid in combination with medium-chain fatty acids and essential oils, a significant decrease in the number of birds with necrotic lesions was found compared with the infected, untreated control group. These results suggest that butyric acid, medium-chain fatty acids and/or essential oils may contribute to the prevention of necrotic enteritis in broilers.     

Hemolytic factors in Schistosoma japonicum eggs

Extracts of Schistosoma japonicum eggs were found to exhibit hemolytic activity on erythrocytes of various species. The hemolytic reaction took place more rapidly at 37 degrees C than at 4 degrees C and did not require divalent cations. The degree of hemolysis was dependent on the concentration of the egg extracts. The hemolytic factors seemed to be lipid in nature because of being heat-stable and soluble in chloroform solvent. Further analysis by means of thin-layer chromatography showed that hemolytic activity in the extracts was due to free fatty acids. Analysis by gas chromatography revealed that fatty acids in the egg extracts consist of myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, and arachidonic acids. Among them, based on the experiments with commercially available fatty acids, arachidonic acid was the most intensively hemolytic, followed by linoleic, linolenic, oleic, palmitoleic, and myristic acids in that order, whereas palmitic acid showed weak activity only in the presence of divalent cations. From the estimated calculation, it was assumed that 1 mg of protein of the egg extracts contains as much as 0.22 mg of free fatty acids.     

Growth inhibition of human hepatoma cells (HepG2) by polyunsaturated fatty acids. Protection by albumin and vitamin E

Albumin carries fatty acids and has also been suggested to act as an antioxidant. In the present work, polyunsaturated fatty acids (linoleic, arachidonic, eicosapentaenoic and docosahexaenoic acids) - but not palmitic and oleic acid - inhibited growth of human hepatoma cells in low albumin concentration (0.5%). Growth inhibition by polyunsaturated fatty acids was prevented by albumin in a dose-related manner in the range 0.7–5.0%. Albumin also protected against growth inhibition following catabolism (by lipoprotein lipase) of very low density lipoproteins. Vitamin E strongly counteracted the inhibitory effect of polyunsaturated fatty acids. Vitamin E and albumin appeared to have additive effects in protecting against growth inhibition by polyunsaturated fatty acids. Indomethacin did not greatly modify the polyunsaturated fatty acids effect. Growth inhibition by polyunsaturated fatty acids, as well as the level of thiobarbituric acid reacting substances (a measure of lipid peroxidation) in growth media, increased with increasing number of fatty acids double bonds. Vitamin E and albumin prevented both thiobarbituric acid reacting substances formation and growth inhibition by polyunsaturated fatty acids. The results suggest that the concentrations of albumin and vitamin E in the incubation medium are essential when studying polyunsaturated fatty acids effects on cell growth.     

Alterations in arachidonic acid metabolism in mouse mast cells induced to undergo maturation in vitro in response to stem cell factor

We studied arachidonic acid (AA) metabolism during the maturation of bone marrow–derived cultured mast cells (BMCMCs) into mast cells with phenotypic characteristics, which were more similar to those of connective tissue–type mast cells. BMCMCs were maintained in medium containing 100 ng/ml recombinant rat stem cell factor (SCF) for 1 to 6 weeks. After 3 to 4 weeks in SCF, BMCMCs acquired many phenotypic characteristics of maturation, including enlarged size, numerous electron-dense cytoplasmic granules, and a 50-fold elevation in histamine content. Maintenance in SCF for 6 weeks did not significantly alter the amounts or species of eicosanoids that were produced by BMCMCs stimulated with calcium ionophore A23187. However, SCF-treated mast cells released 2.6 ± 0.13 times more free AA and accumulated 6.4 ± 1.0 times higher levels of intracellular free AA than did immature BMCMCs not exposed to SCF. There was no increase in the mobilization of other fatty acids (e.g., linoleic or oleic acid), indicating specificity for AA. Moreover, there were no differences between the 5-lipoxygenase activities of SCF-treated or untreated cells, as assayed in cell homogenates prepared by nitrogen cavitation. Although the total AA content in SCF-treated cells was significantly elevated, the distribution of AA in phospholipid and neutral lipid classes was not altered by SCF treatment. Total phospholipase (PL)A₂ activity increased 85% ± 11.5% in SCF-treated cells. In homogenates of immature BMCMCs, 51.0% ± 13.7% of the PLA₂ activity was inhibited by 0.5 mmol/L dithiothreitol, whereas the same concentration of dithiothreitol caused only a 2.2% ± 10.7% reduction in the PLA₂ activity in homogenates of SCF-treated BMCMCs (p ≤0.05, n = 4). These findings suggest that SCF treatment induces a dithiothreitol-resistant PLA₂ and that this PLA₂ may contribute to the mobilization of AA that is not further metabolized to eicosanoids. (J ALLERGY CLIN IMMUNOL 1996;97:1329-41.)     

Purification and characterization of a highly thermostable esterase from the actinobacterium Geodermatophilus obscurus strain G20

Intracellular thermostable esterase produced by the extremophilic Geodermatophilus obscurus G20 was purified to homogeneity by a heat treatment, followed by an anion‐exchange chromatography, and then characterized. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was shown to be approximatively 55 kDa. The enzyme showed an optimal activity between pH 8.0 and 9.0 and was stable in the pH range 7.0–10.0. Moreover, it is highly thermostable, with a residual activity greater than 90% after incubation at 80 °C for more than 10 h. The enzyme showed preference for esters of p ‐nitrophenol with short chain fatty acid. When the p ‐nitrophenyl acetate (C2) was used as substrate, the Michaelis–Menten constant (K_m) and maximum velocity for the reaction (V_max) of esterase were 400 μM and 2500 U/mg protein, respectively. The effect of phenylmethanesulphonyl fluoride (PMSF), a serine‐specific inhibitor, on the enzyme activity suggested that the thermostable esterase belong to the serine hydrolase group. Because of its high thermostability, activity at alkaline pH, tolerance to methanol and various metal ions and specificity for short chain fatty acids, this enzyme showed high potential for use in biocatalysis. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Early changes in the phospholipid metabolism of lymphocytes following stimulation with phytohemagglutinin and with lysolecithin

Following stimulation of rabbit lymphocytes with phytohemagglutinin (PHA) [¹⁴C]oleic acid incorporation was augmented into phospholipids but not into neutral fats. The main phospholipid into which oleate was incoporated was lecithin. Stimulation of lymphocytes with lysolecithin in short-time experiments also increased the incorporation of oleate into lecithin. The distribution of the labeled fatty acids in the newly formed lecithin molecules was determined using snake venom phospholipase A. Of the incorporated oleate 80 % was found in position 1 of the glycerol moiety both in unstimulated and stimulated lymphocytes. In addition to natural [1-acyl]-lysolecithin a sythetic non-metabolized lysolecithin analogue also effected lymphocyte stimulation, suggesting that exogenous lysolecithin acts not as a substrate for lysolecithin-acyltransferase. Microsomal membranes of normal and stimulated lymphocytes were examined for enzyme ectivities involved in the incorporation of long-chain fatty acids into phospholipids especially (a) fatty acid: CoA-ligase, (b) lysolecithin-acyltransferase, (c) phospholipase A and (d) lysophospholipase. After 3 h of cultivation with PHA, fatty acid: CoA-ligase (which is rate-limiting for the incorporation of oleate into phospholipids) and phospholipase A were activated. Lysolecithin produced only an activation of phospholipase A supporting the idea that oleate is incorporated by reacylation of endogenously formed [2-acyl]-lysolecithin and consistent with our findings that de novo synthesis of phospholipids, as measured by [¹⁴C]choline uptake, is low during the early phase of stimulation.