Altered phospholipid metabolism in a temperature-sensitive mutant of a thermophilic bacillus

The phospholipid metabolism of a temperature-sensitive mutant of a thermophilic bacillus was studied after the shift from a permissive (58°C) to a restrictive (65°C) growth temperature. During the short period of growth of the mutant at 65°C, the proportions of cardiolipin and its 3-acyl derivative (lyso-cardiolipin) increased, and the proportions of phosphatidylglycerol and phosphatidylethanolamine decreased on cell dry weight basis. In ³²P incorporation and turnover experiments, phosphatidylglycerol showed the most rapid uptake and loss of the label. Turnover of cardiolipin, limited to a short period, ceased 18 min after the shift, as did the turnover of phosphatidylethanolamine. In the absence of net phospholipid synthesis, there was a quantitative conversion of phosphatidylglycerol to cardiolipin and an increase in the proportion of lyso-cardiolipin. Chloramphenicol, added to the medium at the time of the shift, reduced the rate of phospholipid synthesis, prevented the increase in the proportions of cardiolipin and lyso-cardiolipin, and slowed the decrease in the proportions of the other two phospholipids. The results indicated a defect in the regulatory mechanism(s) of phospholipid metabolism in the mutant at the restrictive temperature.Nonstandard Abbreviations WT parental strain, thermophilic bacillus - TS-13 temperature-sensitive mutant of a thermophilic bacillus - CL cardiolipin - PG phosphatidylglycerol - PE phosphatidylethanolamine - l-CL lyso-cardiolipin     

Changes in fatty acid distribution and thermotropic properties of phospholipids following phosphatidylcholine depletion in a choline-requiring mutant of Neurospora crassa

Growth of a choline requiring auxotroph of Neurospora crassa on medium lacking exogenous choline produces large changes in the levels of phosphatidylethanolamine and phosphatidylcholine. Whole cell fatty acid distributions were found to vary widely between different phospholipid species of normally growing, choline-supplemented cultures with phosphatidylcholine showing the highest levels of unsaturation and anionic phospholipids and cardiolipin having the lowest. In these lipids, choline deprivation produced little change in fatty acid profiles of phosphatidylethanolamine, whereas changes in fatty acids of phosphatidylcholine and acidic phospholipids resulted in increased levels of unsaturation at both growth temperatures. Microsomal phospholipids also showed fatty acid variability with sharp decreases in phosphatidylcholine unsaturates and increases in acidic phospholipid unsaturated fatty acids at low growth temperatures. Fluorescence polarization of 1,6-diphenylhexatriene in vesicles formed from total cellular and microsomal lipids showed that choline deprivation produces changes in thermotropic properties in the lipids in deprived cultures at either growth temperature. The effective differences in fluorescence polarization between choline-deprived and supplemented cultures grown at a given temperature were found to be comparable to those produced by temperature acclimation in normally growing cultures over a temperature range of 22 K.     

Phylogeny and lipid composition of Thermonema lapsum, a thermophilic gliding bacterium

Abstract 1490 nucleotides of the 16S rRNA gene of a Gram-negative, thermophilic and gliding bacterium, Thermonema lapsum , have been sequenced. Phylogenetic analysis indicates that T. lapsum is related to cytophaga-flavobacteria-bacteriodes (CFB) and is confirmed by the identification signature nucleotides that define this group. Further phylogenetic analysis indicates that T. lapsum forms the deepest branch in the CFB group; this observation was confirmed by the identification of unique nucleotide and nucleotide pairs which separate T. lapsum from all other members of this group. The phospholipid fatty acid (PLFA) profile also confirmed that T. lapsum is related to the cytophaga-flavobacteria-bacteroides group and also to selected members of the genus Flexibacter ; the PLFA profile is unique to T. lapsum .     

Dietary fatty acid composition differently influences retinoylation reaction in rat testes mitochondria

All-trans-retinoic acid (atRA) is incorporated covalently into proteins of rat testes mitochondria. In this study, the effect of three diets with different fatty acid composition on the retinoylation of proteins of rat testes mitochondria has been investigated. Different groups of rats were fed on a basal diet supplemented with 15% of either coconut oil (CO), olive oil (OO) or fish oil (FO). We found that, when compared with CO, the binding of retinoic acid was decreased in FO- and OO-fed rats. Mitochondrial phospholipids composition was differently influenced by dietary treatments; minor changes were observed in fatty acid composition of phospholipids. Few differences were observed in the Arrhenius plots among the three groups of rats. Kinetic analysis revealed a decrease in the V _max value in FO- and OO- as compared with CO-fed rats. No difference among the three groups were observed in the K _M value. The retinoylation reaction was inhibited by 13-cis-RA and 9-cis-RA.     

Alterations of the composition and metabolism of pulmonary surfactant phospholipids induced by experimental peritonitis in rats

Pulmonary complications often accompany the development of acute peritonitis. In this study, we analyzed the alterations of alveolar surfactant phospholipids in rats with experimentally induced peritonitis. The results showed a reduction of almost all phospholipid fractions in pulmonary surfactant of experimental animals. The most abundant alveolar phospholipids-phosphatidylcholine and phosphatidylglycerol were reduced significantly in surfactant of rats with experimental peritonitis. In addition, analysis of the fatty acid composition of these two phospholipids revealed marked differences between experimental and control animals. The activity of phospholipase A2, which is localized in the hydrophyllic phase of alveolar surfactant, was higher in rats with experimental peritonitis compared to sham-operated ones. Also, a weak acyl-CoA:lysophospholipid acyltransferase activity was detected in alveolar surfactant of rats with experimental peritonitis, whereas in control animals this activity was not detectable. The lipid-transfer activity was quite similar in pulmonary surfactant of control and experimental rats. The total number of cells and the percentage of neutrophils were strongly increased in broncho-alveolar lavage fluid from rats with peritonitis. Thus, our results showed that the development of peritonitis was accompanied by pulmonary pathophysiological processes that involved alterations of the phospholipid and fatty acid composition of alveolar surfactant. We suggest that the increased populations of inflammatory cells, which basically participate in internalization and secretion of surfactant components, contributed to the observed alterations of alveolar phospholipids. These studies would be useful for clarification of the pathogenic mechanisms underlying the occurrence of pulmonary disorders that accompany acute inflammatory conditions, such as peritonitis and sepsis.     

Phospholipid composition and fatty acid desaturation in the roots of rye during acclimatization of low temperature

When the roots of rye plants grown at 20°C were cooled to 8°C the concentration of phospholipid in them more than doubled over a 7 d period in comparison with that in roots remaining at 20°C. The relative abundance of lecithin (PC) declined while that of phosphatidyl ethanolamine (PE) increased; this change was completed after 2 d cooling. Labelling with ³²P suggested that turnover of phospholipids may be inhibited by low temperature. Acyl lipids contained an increased proportion of linolenic acid (18:3) and reduced proportion of linoleic acid (18:2) when roots were cooled at 8°C for 7 d. The ratio of these acids is a relatively more sensitive indicator of desaturation than is the double bond index. Cooling brought about no change in the abundance of the principal saturated acid, palmitic (16:0). In the first 3 days of cooling PC and PE desaturated markedly while there was no change in galactosyl and neutral lipids. Desaturation did not appear to be greatly sensitive to the concentration of dissolved O₂ and was only partly inhibited in 8°C solutions where the oxygen concentration was lowered to 0.5–2.0%. Positional analysis of acyl chains in PC and PE showed that more than 90% of all 16:0 is associated with position I while 65% of the 18:2+18:3 is associated with position II. When roots are cooled the abundance of 18:3 increases in both chains but the relative distribution of saturated and unsaturated fatty acids remains constant in positions I and II. At both 20°C and 8°C there is a high probability that a saturated chain in position I will be paired with the polyunsaturated one in position II.Abbreviations PC Lecithin - PE phosphatidyl ethanolamine - TLC thin layerchromatography - BHT butylatedhydroxytoluene     

Temperature effect on a high stearic acid sunflower mutant.

Vegetable oil with elevated saturated fatty acid content may be useful for producing solid fat without hydrogenation or transesterification. Under the nutritional point of view stearic acid is preferred to other saturated fatty acids because of its neutral effect on serum cholesterol lipoproteins. Selection of a very high stearic acid sunflower (Helianthus annuus L.) line (CAS-14), with up to a 37.3% of stearic acid in the seed oil, and the relationship between the expression of this character and the growth temperature are presented. The mutant was selected from the M(2) progeny of 3000 mutagenized seeds (4 mM sodium azide mutagenesis treatment) by analysing the fatty acid composition of half-seed by gas liquid chromatography. In order to genetically fix the mutant character, plants were grown at high day/night temperatures during seed formation. We found that temperatures higher than 30/20 degrees C are required for good expression of the phenotype, the maximum stearic acid content being obtained at 39/24 degrees C. This behaviour is totally opposed to that observed in normal and previously isolated high-stearic acid sunflower lines that contain more stearic acid at low temperature. Thus, a new type of temperature regulation on the stearate desaturation must occur. This line is the sunflower mutant with the highest stearic acid content reported so far.     

Alterations in growth temperature range and fatty acid composition of Thermus as a result of plasmid elimination

Elimination of plasmids from Thermus flavus, T. thermophilus and three wild Thermus strains caused alterations in growth temperature range, pigmentation and membrane fatty acids without affecting viability. Following plasmid elimination all Thermus strains lost their ability to grow above 70°C. In addition, the minimum growth temperature was lowered by 5–10°C. Fatty acids were reduced by an average of approximately 35%. In addition, the contribution of iso- and anteisobranched fatty acids were altered in four of the five strains. The iso C_15:0/iso C_17:0 ratio approached 1.0 in all strains, whereas the anteiso C_15:0/anteiso C_17:0 was reduced to 0.2. The iso C_16:0/normal-C_16:0 ratio increased in all strains due to an increase in iso C_16:0 in four strains and a reduction in normal-C_16:0 relative to iso C_16:0 in one strain. However, it was evident that the plasmid-free strains were able to compensate for these alterations in membrane fluidity to a certain extent by reducing the average chain length of isobranched acids. Altered fatty acid metabolism at the level of precursors may have influenced membrane composition and consequently growth temperature range.     

Glucose stimulates a decrease of the fatty acid saturation degree in Acinetobacter calcoaceticus

The fatty acid composition of Acinetobacter calcoaceticus 69-V was determined under various growth conditions. Saturated, unsaturated, and hydroxy fatty acids with chain lengths of 12–18 carbon atoms predominated in the fatty acid profile. With acetate or propanol as growth substrates, the ratio of saturated to unsaturated fatty acids varied with changes in the temperature. This was the only adaptive mechanism detected that compensated for the physical effects of temperature alterations on the cell membranes. The fatty acid composition of A. calcoaceticus grown at 40 °C had a saturation degree of approximately 50%; after growth at 20 °C it was approximately 35%. In the presence of a carbon and energy source, A. calcoaceticus was able to respond to temperature reductions under oxic conditions regardless of whether fatty acid biosynthesis was inhibited or not. This suggests an aerobic mechanism of fatty acid biosynthesis and the involvement of a fatty acid desaturase system. Addition of the non-growth substrate, glucose, helped the organism to adapt to lower temperature. The molecular mechanism of the aid is not really understood. The oxidation of glucose could provide the desaturase either with electrons directly via a pyrrolo-quinoline-quinone-linked glucose dehydrogenase or with NADH after fatty acid degradation has been initiated by ATP generated by the oxidation of glucose. Received: 19 June 1998 / Accepted: 28 December 1998     

Main phospholipids and their fatty acid composition in muscle and cephalothorax of the edible Mediterranean crustacean Palinurus vulgaris (spiny lobster)

The total lipids of muscle and cephalothorax of Mediterranean lobster Palinurus vulgaris were found to be 1.0% and 2.4% of the wet tissue of which the phospholipids represented 66.5% and 47.5%, respectively. The main PhL saturated, monounsaturated and polyunsaturated fatty acids in muscle and cephalothorax were C16:0, C18:0, C18:1ω-9, C18:1ω-7, C20:4ω-6, C20:5ω-3 and C22:6ω-3. 2-OH C14:0 and cyclo-17:0 fatty acids were also identified though in low percentages. The main individual PhL in muscle were found to be phosphatidylcholine (53.5%), 72.0% of which corresponded to the structure of 1,2-diacyl-glycerocholine while the rest 28.0% to 1-O-alkyl-2-acyl-glycerocholine or 1-O-(1-alkenyl)-2-acyl-glycerocholine and phosphatidylethanolamine (19.3%), 75.0% of which corresponded to the structure of 1,2-diacyl-glyceroethanolamine and 25% to 1-O-alkyl-2-acyl-glyceroethanolamine or 1-O-(1-alkenyl)-2-acyl-glyceroethanolamine. Cephalothorax main PhL were found to be PC and PE (66.4% and 18.8%, respectively). In muscle and cephalothorax PC ω-3 fatty acids amounted 7.78% and 8.60%, while in PE amounted 30.77% and 23.65% respectively. Furthermore, in both tissues PhL, cardiolipine phosphatidylserine, phosphatidylinositol, sphingomyelin and lysophosphatidylcholine, were also found.     

Lipid Composition and Temperature Adaptation of the Nervous System of the Leech Hirudo medicinalis L.

The lipid composition of the nervous system of the leech Hirudo medicinalis was investigated following acclimatization of animals at 25 degrees C and 5 degrees C. Choline, ethanolamine, and serine plus inositol phosphoglycerides are the major phospholipid classes of the leech ganglionic chain; minor amounts of lysophosphatidylcholine, phosphatidic acid, and sphingomyelin are also present. Neither the phospholipid pattern nor the cholesterol to total phospholipid molar ratio was dependent on the acclimatization temperature, whereas the fatty acid patterns of choline and serine plus inositol phosphoglycerides were significantly affected. Both for choline and serine plus inositol phosphoglycerides, a significant increase of the unsaturation index and a decrease of saturated to unsaturated fatty acid ratio was observed in animals acclimatized at 5 degrees C in comparison with those acclimatized at 25 degrees C. These observations, which point to increased lipid fluidity of the nervous system of cold-adapted leeches, are strengthened by results obtained by the fluorescence polarization method using 1,6-diphenyl-1,3,5-hexatriene as a probe: a decrease of the fluorescence polarization value was observed throughout the temperature range selected (5-40 degrees C).     

Effect of growth temperature on the fatty acid composition of Mycobacterium phlei

In Mycobacterium phlei, fatty acid unsaturation increased with decreasing temperature. The 10-hexadecenoic acid content increased as the temperature was reduced from 35°C to 26–20°C. At lower temperatures tuberculostearic acid decreased while oleic and linoleic acids increased, the latter being found in M. phlei for the first time. Concomitantly palmitic acid content decreased, and the 6- and 9-hexadecenoic acids increased slightly on reducing the temperature from 35 to 10°C. Thus, down to 26–20°C palmitic acid was mainly replaced by 10-hexadecenoic acid. From this range down to 10°C, palmitic and tuberculostearic acids were replaced by oleic and linoleic acids. Consequently, fatty acid branching decreased and mean chain length increased, as the temperature was reduced. These observations support the view that regulation of membrane fatty acid composition is part of microbial temperature adaptation, and that themechanism behind the responses might be more complex than generally believed.Abbreviations ACP acyl carrier protein - FAS I (Type I) fatty acid synthetase I - FAS II (Type II) fatty acid synthetase II - MGLP methylglucose containing lipopolysaccharide - MMP methylmannose containning polysaccharide     

大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间的关系

通过对大肠杆菌生长温度、膜脂肪酸组成和压力抗性之间关系研究发现,10℃培养,对数期细胞有最大的压力抗性,随着培养温度的升高直到4 5℃,压力抗性呈下降的趋势;相反,10℃培养,稳定期的细胞对压力最敏感,随着培养温度的升高,压力抗性呈增加趋势,30～37℃时达到最大,之后到4 5℃有下降。对数期和稳定期细胞膜脂中不饱和脂肪酸的组成随温度的上升而下降,这与从全细胞中抽提的磷脂的熔点密切相关。因此,对数期细胞压力抗性随着膜流动性的增大而升高;但稳定期细胞,膜流动性与压力抗性之间不存在简单的对应变化关系     

Phospholipid characteristics and neutral lipid fatty acid composition related to temperature and nutritional conditions in ecologically important amphipod species from the northern Baltic Sea

Lipid composition of the benthic, stenothermal amphipod Monoporeia affinis collected from constantly cold deep-water (> 80 m) regions of the northern Baltic Sea was analysed in detail to study phospholipid characteristics, its relation to thermal adaptation as well as potential effects of food quality and seasonal variability. Similar measurements were performed on the littoral, eurythermic amphipod Gammarus spp. Fatty acid (FA) composition of storage lipids of Pontoporeia femorata, a sibling species of M. affinis, was also studied. Interannual and interspecies variability in selected FA was observed both in triacylglycerols (TAG) and phospholipids (PL). Differences in monounsaturated vs. polyunsaturated (MUFA/PUFA) FA combinations of phosphatidyl ethanolamine (PE) diacyl and alkylacyl subgroups were also observed. Seasonal variability in M. affinis was considerably lesser compared to Gammarus spp. A literature synopsis shows that in diacyl PE the share of the FA combination MUFA/PUFA increases with lowering body temperature (40-55% in cold-adapted organisms vs. 5-15% in warm adapted ones), signifying that this characteristic is probably in key position concerning regulation of membrane fluidity in temperature adaptation. According to this feature M. affinis belongs to the group of cold-adapted stenothermic species (Group 1) while eurythermic Gammarus spp. settles in the Group 2 (cold-acclimatized) or 3 (warm-acclimatized/tropical) depending on the status of temperature adaptation. Omnivory and/or periodical food item switching is an important strategy in benthic organisms in high-latitude areas characterised by recurring long poor-nutritional periods. Since many benthic species utilise a time-averaged, integrated food source from phytal and animal matter from various sources the FA composition of TAG of the amphipod species measured here do not exclusively point towards any specific feeding mode or food source. In general, using selected FA as food source markers to assess the diet of field collected more-or-less omnivoric species cannot be considered as an optimal approach. The current study gives more insight to the biochemistry of biological membranes of aquatic crustaceans that is essential in estimation of the capacity of the thermal adaptation/acclimatization of organisms as well as the potential effects of food quality on storage lipids.     

Isolation,sequencing, and expression of a cDNA for the HXM-A form of xenobiotic/medium-chain fatty acid:CoA ligase from human liver mitochondria

The purification of xenobiotic/medium-chain fatty acid:CoA ligases (XM-ligases) from human liver mitochondria resulted in the isolation of two chromatographically separable forms (HXM-A and HXM-B). These two forms were purified to near homogeneity, cleaved with cyanogen bromide, the resulting peptides separated, and the N-terminus of two of the peptides partially sequenced. Identical sequences were obtained for HXM-A and HXM-B for the two peptides. These sequences were used to design probes for screening a human liver cDNA library. This resulted in the isolation of two overlapping cDNAs. Using these sequences we were able to design PCR primers that resulted in the isolation of a full-length cDNA from a human cDNA library. The cDNA contained 1731 bp of open reading frame and coded for a 64230-Da protein. This protein bears 56.2% amino acid homology to the MACS1 (medium-chain acyl-CoA synthetase) enzyme, 58.7% homology to the bovine XL-III XM-ligase, and 81.5% homology to the bovine XL-I XM-ligase. The cDNA could be expressed in COS cells, and the expressed enzyme had greater benzoate activity than phenylacetate activity, which is consistent with the known substrate specificity of HXM-A.     

Seasonal changes in the composition of storage and membrane lipids in overwintering larvae of the codling moth,Cydia pomonella

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. It overwinters as a diapausing fifth instar larva. The overwintering is often a critical part of the insect life-cycle in temperate zone. This study brings detailed analysis of seasonal changes in lipid composition and fluidity in overwintering larvae sampled in the field. Fatty acid composition of triacylglycerol (TG) depots in the fat body and relative proportions of phospholipid (PL) molecular species in biological membranes were analyzed. In addition, temperature of melting (T_m) in TG depots was assessed by using differential scanning calorimetry and the conformational order (fluidity) of PL membranes was analyzed by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in membrane vesicles. We observed a significant increase of relative proportion of linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) in TG depots during the larval transition to diapause accompanied with decreasing melting temperature of total lipids, which might increase the accessibility of depot fats for enzymatic breakdown during overwintering. The fluidity of membranes was maintained very high irrespective of developmental mode or seasonally changing acclimation status of larvae. The seasonal changes in PL composition were relatively small. We discuss these results in light of alternative survival strategies of codling moth larvae (supercooling vs. freezing), variability and low predictability of environmental conditions, and other cold tolerance mechanisms such as extending the supercooling capacity and massive accumulation of cryoprotective metabolites.     

Thin-layer chromatography and high-performance liquid chromatography for the assay of fatty acid compositions of individual phospholipids in platelets from non-insulin-dependent diabetes mellitus patients: effect of eicosapentaenoic acid ethyl ester administration

Eight major phospholipids were separated by a TLC method with a one-dimensional developing system without any pretreatment of the plate and the fatty acids incorporated into each phospholipid class were analysed by an improved HPLC method with a simple elution system, which has advantages with respect to resolution and analysis time. The fatty acid compositions of individual phospholipids in platelets were investigated following administration of ethyl cis-5,8,11,14,17-eicosapentaenoate for more than 13 weeks to patients with non-insulin-dependent diabetes mellitus. The cis-5,8,11,14,17-eicosapentaenoic acid compositions of all phospholipid classes were significantly increased with decreasing platelet aggregation rates after the administration. These results suggested that the present method provides the complete separation of individual phospholipids in sufficient amounts to allow fatty acid analysis on the isolated phospholipid moieties.     

The effects of temperature on the fatty acid and phospholipid composition of four obligately psychrophilicVibrio Spp.

The free fatty acid and phospholipid composition of 4 psychrophilic marineVibrio spp. have been determined in chemostat culture with glucose as the limiting substrate over a temperature range 0–20°C. All the isolates show maximum glucose and lactose uptake at 0°C and this correlates with maximum cell yield. None of the isolates contain fatty acids with a chain length exceeding 17 carbon atoms.Vibrio AF-1 andVibrio AM-1 respond to decreased growth temperatures by synthesizing increased proportions of unsaturated fatty acids (C15:1, C16:1 and C17:1) whereas inVibrio BM-2 the fatty acids undergo chain length shortening. The fourth isolate (Vibrio BM-4) contains high levels (60%) of hexadecenoic acid at all growth temperatures and the fatty acid composition changes little with decreasing temperature. The principal phospholipid components of the four psychrophilic vibrios were phosphatidylserine, phosphatidylglycerol, phosphatidylethanolamine and diphosphatidylglycerol. Lyso-phosphatidylethanolamine and 2 unknown phospholipids were additionally found inVibrio AF-1. The most profound effect of temperature on the phospholipid composition of these organisms was the marked increase in the total quantities synthesized at 0°C. At 15°C phosphatidylglycerol accumulated in the isolates as diphosphatidylglycerol levels decreased. Additionally inVibrio BM-2 andVibro BM-4 phosphatidylserine accumulates as phosphatidylethanolamine biosynthesis was similarly impaired. The observed changes in fatty acid and phospholipid composition in these organisms at 0°C may explain how solute transport is maintained at low temperature.Abbreviations PS Phosphatidylserine - PE phosphatidylethanolamine - PG phosphatidylglycerol - DPG diphosphatidylglycerol - lyso PE lysophosphatidylethanolamine     

13种微藻的脂肪酸组成分析

分析了13种微藻(包括7种绿藻,5种杂色藻和1种红藻)的总脂含量和脂肪酸组成,结果表明,不同门类微藻的脂肪酸组成差异较大:绿藻的脂肪酸组成以C16和C18为主;杂色藻类的脂肪酸组成相近,金藻门含有14:0、16:0、18:1、18:4等特征脂肪酸,三角褐指藻主要的脂肪酸为14:0、16:0、16:1、16:3和20:5,而粉核油球藻的脂肪酸以14:0、16:0、20:5为主;紫球藻的脂肪酸组成以16:0、20:4和20:5为主。在测试的13种微藻中,杜氏盐藻的亚麻酸含量最高,占总脂肪酸的60.9%;等鞭金藻的十八碳四烯酸含量最高,占总脂肪酸的19.6%;紫球藻和粉核油球藻中花生四烯酸与二十碳五烯酸(EPA)含量分别占总脂肪酸的17.1%和20.9%。