The role of Delta(9)-desaturase in the production of cis-9, trans-11 CLA

Biomedical studies with animal models have demonstrated that cis-9, trans-11 conjugated linoleic acid (CLA), the predominant isomer found in milk fat from dairy cows, has anticarcinogenic effects. We recently demonstrated endogenous synthesis of cis-9, trans-11 CLA from ruminally derived trans-11 C18:1 by Delta(9)-desaturase in lactating dairy cows. The present study further examined endogenous synthesis of cis-9, trans-11 CLA and quantified its importance by increasing substrate supply using partially hydrogenated vegetable oil (PHVO) as a source of trans-11 C18:1 and blocking endogenous synthesis using sterculic oil (SO) as a source of cyclopropene fatty acids which specifically inhibit Delta(9)-desaturase. Four cows were abomasally infused with 1) control, 2) PHVO, 3) SO, and 4) PHVO+SO in a 4 x 4 Latin square design. With infusion of PHVO, cis-9, trans-11 CLA was increased by 17% in milk fat. Consistent with inhibition of desaturase, SO treatments increased milk fat ratios for the fatty acid pairs effected by Delta(9)-desaturase, C14:0/cis-9 C14:1, C16:0/cis-9 C16:1, and C18:0/cis-9 C18:1. The role of endogenous synthesis of CLA was evident from the 60-65% reduction in cis-9, trans-11 CLA which occurred in milk fat with SO treatments. cis-9 C14:1 originates from desaturation of C14:0 by Delta(9)-desaturase and can be used to estimate the extent of SO inhibition of Delta(9)-desaturase. When this correction factor was applied, endogenous synthesis was estimated to account for 78% of the total cis-9, trans-11 CLA in milk fat. Thus, endogenous synthesis was the major source of cis-9, trans-11 CLA in milk fat of lactating cows.     

Fatty acid profiles and their chemotaxonomy in planktonic species of Anabaena (Cyanobacteria) with straight trichomes

Twenty-four axenic strains of planktonic Anabaena with straight trichomes, assigned to 7 species, were investigated by analyzing the pattern and content of their fatty acid composition and comparing their fatty acid composition with their morphological properties. In general, the fatty acids in planktonic Anabaena contained 14:0, 16:0, 16:1(cis-), 18:0, 18:1, 18:2, and 18:3(alpha) as their major components, and were classified as Type 2 according to the Kenyon-Murata system. These strains were further divided into 2 subtypes: 18 strains belonging to Type 2A, which contains 16:2 and 16:3, and 6 strains to Type 2B, which lacks 16:2 and 16:3. Fatty acid compositions of strains of A. solitaria, A. smithii, and A. kisseleviana closely corresponded to morphological properties; however, 10 strains of A. planctonica were divided into 4 clusters, and 3 strains of A. affinis into 2 clusters. These clusters should be taxonomically evaluated based on other aspects such as genetic characteristics.     

Acyl-CoA Synthetase in Oilseeds: Fatty Acid Structural Requirements for Activity and Selectivity

The substrate specificities and selectivities of acyl-CoA synthetasesfrom maturing oilseeds were investigated to reveal fatty acidstructures that the enzymes recognize. The synthetases fromrapeseed (Brassica nap us) and castor bean (Ricinus communis)activated palmitic acid 16:0 most rapidly among the saturatedfatty acids tested. Native unsat-urated fatty acids, oleic 18:1cis-9, linoleic 18:2 cis-9,12 and linolenic acid 18:3 m-9,12,15,were all effectively utilized. Palmitoleic acid 16:1 cis-9 wasalso a good substrate, while myristoleic acid 14:1 cis-9 wasa poor substrate. The activation of erucic acid 22:1 cis-13was very slow. Elaidic acid 18:1 trans-9 was utilized at ratessimilar to those of the cis isomer. The efficiencies of petroselinicacid 18:1 cis-6 were half the efficiencies of oleic acid, whilethe rates of activation of m-vaccenic acid 18:1 cw-11 were comparableto those for oleic acid. These findings suggest that acyl-CoAsynthetases of oilseeds producing long-chain fatty acids strictlyrecognize the molecular structures of fatty acids, i.e., thecarbon-chain length between C¹⁶-C¹⁸ and the position of thefirst double bond (     

Characterization of the hydroxy fatty acid content ofBasidiomycotina

Fatty acids (FA) of nine fungal species belonging to the subphylumBasidiomycotina were identified by using capillary GC-MS, MS and HPLC. The identified fatty acids included 45 saturated (iso-, anteiso-, and 19 hydroxy acids) and 42 monoenoic acids (including 14 hydroxy acids); dienes and polyenes were represented by 13 fatty acids. The proportion of hydroxy acids in the total fatty acids in the fungal species ranged from 4.3 to 10.2%. Very long-chain fatty acids (C₂₄-C₃₀) were also determined. Four fatty acids 16:0 (8.8–14.3%), 18:1(11) (3.9–14.9%), 18:1(9) (7.7–19.0%) and 18:2(6) (7.6–19.4%), were found as major acids. Of the identified acids, 17 were detected inBasidiomycotina for the first time.     

Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis

Conjugated linoleic acids (CLA) are octadecadienoic fatty acids that have profound effects on lipid metabolism. Our previous work showed that CLA (mixture of isomers) markedly reduced milk fat synthesis. In this study, our objective was to evaluate the effects of specific CLA isomers. Multiparous Holstein cows were used in a 3x3 Latin square design, and treatments were 4-day abomasal infusions of 1) skim milk (control), 2) 9,11 CLA supplement, and 3) 10,12 CLA supplement. CLA supplements provided 10 g/day of the specific CLA isomer (cis-9,trans-11 or trans-10,cis-12). Treatments had no effect on intake, milk yield, or milk protein yield. Only the 10,12 CLA supplement affected milk fat, causing a 42 and 44% reduction in milk fat percentage and yield, respectively. Milk fat composition revealed that de novo synthesized fatty acids were extensively reduced. Increases in ratios of C(14:0) to C(14:1) and C(18:0) to C(18:1) indicated the 10,12 CLA supplement also altered Delta(9)-desaturase. Treatments had minimal effects on plasma concentrations of glucose, nonesterified fatty acids, insulin, or insulin-like growth factor-I. Overall, results demonstrate that trans-10,cis-12 CLA is the isomer responsible for inhibition of milk fat synthesis.     

Metabolism of cis-12-octadecenoic acid and trans-9,trans-12-octadecadienoic acid and their influence on lipogenic enzyme activities in mouse liver

High carbohydrate (65% glucose) diets containing cis-12-octadecenoic acid (12c-18:1) or trans-9,trans-12-octadecadienoic acid (9t,12t-18:2) were fed to weanling mice to investigate the influence of fatty acid structure on six hepatic enzyme activities involved in lipid metabolism. Results with these diets were compared to those with diets containing no fatty acids, saturated fatty acids; cis-9-octadecenoic acid (9c-18:1) and cis-9,cis-12-octadecadienoic acid (9c,12c-18:2). These comparisons show saturated fatty acids, 9c-18:1, 12c-18:1, and 9t,12t-18:2, had little or no influence on the activity levels of fatty acid synthetase, malic enzyme (EC 1.1.1.40)citrate cleavage enzyme (EC 4.1.3.8), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and acetyl-CoA carboxylase (EC 6.4.1.2). Neither 12c-18:1 nor 9t,12t-18:2 produced the dramatic enzyme-lowering effect exhibited by the diet containing 9c,12c-18:2 when compared to the diet devoid of fat. Thus, both the 9 and 12 bonds must be present in the same molecule. Also, at least one and probably both bonds must be in the cis configuration to depress liver enzyme activities. Capillary gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were both used for analysis of the methyl esters derived from the hepatic lipids. The GC and GC-MS data provided (a) direct evidence for incorporation of both isomers into hepatic lipids and (b) indirect evidence that 9t,12t-18:2 lowered liver delta 9-desaturase activity. In addition, since these products were found in the complex liver lipids, there is no doubt that the various enzymes concerned with activation and acylation utilize both of these isomeric fatty acids as substrates.     

Conjugated linolenic acid (CLnA), conjugated linoleic acid (CLA) and other biohydrogenation intermediates in plasma and milk fat of cows fed raw or extruded linseed

Thirty lactating dairy cows were used in a 3 × 3 Latin-square design to investigate the effects of a raw or extruded blend of linseed and wheat bran (70:30) on plasma and milk fatty-acids (FA). Linseed diets, containing 16.6% linseed blend on a dry-matter basis, decreased milk yield and protein percentage. They decreased the proportions of FA with less than 18 carbons in plasma and milk and resulted in cis-9, cis-12, cis-15 18:3 proportions that were more than three and four times higher in plasma and milk, respectively, whereas cis-9, cis-12 18:2 proportions were decreased by 10-15%. The cis-9, trans-11, cis-15 18:3 isomer of conjugated linolenic acid was not detected in the milk of control cows, but was over 0.15% of total FA in the milk fat of linseed-supplemented cows. Similarly, linseed increased plasma and milk proportions of all biohydrogenation (BH) intermediates in plasma and milk, including the main isomer of conjugated linoleic acid cis-9, trans-11 18:2, except trans-4 18:1 and cis-11, trans-15 18:2 in plasma lipids. In milk fat, compared with raw linseed, extruded linseed further reduced 6:0-16:0 even-chain FA, did not significantly affect the proportions of 18:0, cis-9 18:1 and cis-9, cis-12 18:2, tended to increase cis-9, cis-12, cis-15 18:3, and resulted in an additional increase in the proportions of most BH intermediates. It was concluded that linseed addition can improve the proportion of conjugated linoleic and linolenic acids, and that extrusion further increases the proportions of intermediates of ruminal BH in milk fat.     

Analysis of fatty acid composition of anaerobic rumen fungi

The fatty acid (FA) composition of fresh mycelia of anaerobic rumen fungi was determined. The fatty acids methyl esters (FAME) of six strains belonging to four genera (Neocallimastix, Caecomyces, Orpinomyces, Anaeromyces) and one unknown strain were analyzed by gas chromatography. All studied fungi possess the same FAs but differences were found in their relative concentrations. The FA profile of anaerobic fungi comprises carbon chains of length ranging from 12 to 24; the most common fatty acids were stearic (C(18:0)), arachidic (C(20:0)), heneicosanoic (C(21:0)), behenic (C(22:0)), tricosanoic (C(23:0)) and lignoceric (C(24:0)) with relative amount representing >4% of total FA. Significant differences were determined for heptadecanoic, oleic, behenic and tricosanoic acids. Rumen anaerobic fungi can contain very long chain fatty acids; we found unsaturated fatty acids including cis-11-eicosenoic (C(20:1)), cis-11,14-eicosadienoic (C(20:2)), erucic (C(22:1n9)), cis-13,16-docosadienoic (C(22:2)) and nervonic (C(24:1)) acids in very small amounts but their presence seems to be unique for anaerobic fungi.     

发状念珠藻(发菜)细胞质膜的分离与性质分析

使用一种新方法首次从野生发菜(Nostoc flagelliforme Born.et Flah.)中分离得到细胞质膜并对其性质进行了分析,该方法的主要特点为联合使用细胞破碎仪和毛地黄皂甙对发菜细胞进行破碎.经过细胞破碎仪处理两次(80MPa)后,样品(20mg干重/mL)中的细胞可被毛地黄皂甙(3mg/mL)有效破碎,细胞质膜即可通过蔗糖密度梯度离心得以分离.纯化后的质膜,其吸收光谱中类胡萝卜素的3个吸收峰分别位于458、487和524 nm,另外一种叶绿素前体在673 nm处有少量吸收,质膜的荧光发射来自该叶绿素前体.通过变性电泳对其进行多肽组成分析,可分辨出30多条多肽,其中分子量为80、28、19和17 kD的多肽含量最高.其膜脂主要包含4种成分:单半乳糖甘油二酯(62.4%)、双半乳糖甘油二酯(18.9%)、硫代异鼠李糖甘油二酯(16.7%)和磷酯酰甘油(2.0%).膜脂酯酰基连接有棕榈酸(16:0)、十六碳烯酸(16:1[9])、硬脂酸(18:0)、油酸(18:1[9])、亚油酸(18:2[9,12])和亚麻酸(18:3[9,12,15])等六种脂肪酸,其中十六碳烯酸和亚麻酸为主要成分,分别占总脂肪酸含量的32.3%和34.4%.质膜中高含量的亚麻酸可能是发菜具有极强抗旱能力的一个重要因素.     

Automated Systems for Identification of Heterotrophic Marine Bacteria on the Basis of Their Fatty Acid Composition

The fatty acid methyl ester composition of a total of 71 marine strains representing the genera Alteromonas, Deleya, Oceanospirillum, and Vibrio was determined by gas-liquid chromatographic analysis. Over 70 different fatty acids were found. The predominant fatty acids were 16:0, 16:1 cis 9, summed-in-feature (SIF) 4 (15:0 iso 2OH and/or 16:1 trans 9) and SIF 7 (18:1 cis 11, 18:1 trans 9, and/or 18:1 trans 6) for all the strains considered, but minor quantitative variations could be used to distinguish the different genera. In addition to a conventional statistical processing method to analyze the data and draw comparison between species and genera, an approach involving neutral network-based elaboration is applied. The statistical analysis and dendrogram representation gave a comparison of the species considered, while the neural network computation provided a more accurate assignment of species to their genera. Moreover, by using neural networks, it was possible to conclude that only 22 fatty acids were important for the identification of the marine genera considered. A database of Alteromonas, Deleya, Oceanospirillum, and Vibrio fatty acid methyl ester profiles was generated and is now routinely used to identify fresh marine isolates.     

发状念珠藻(发菜)细胞质膜的分离与性质分析(英文)

使用一种新方法首次从野生发菜(Nostoc flagelliforme Born. et Flah. )中分离得到细胞质膜并对其性质进行了分析,该方法的主要特点为联合使用细胞破碎仪和毛地黄皂甙对发菜细胞进行破碎。经过细胞破碎仪处理两次(80MPa)后,样品(20mg干重/mL)中的细胞可被毛地黄皂甙(3mg/mL)有效破碎,细胞质膜即可通过蔗糖密度梯度离心得以分离。纯化后的质膜,其吸收光谱中类胡萝卜素的3个吸收峰分别位于458、487和524nm,另外一种叶绿素前体在673nm处有少量吸收,质膜的荧光发射来自该叶绿素前体。通过变性电泳对其进行多肽组成分析,可分辨出30多条多肽,其中分子量为80、28、19和17kD的多肽含量最高。其膜脂主要包含4种成分:单半乳糖甘油二酯(62.4％)、双半乳糖甘油二酯(18.9％)、硫代异鼠李糖甘油二酯(16.7％)和磷酯酰甘油(2.0％)。膜脂酯酰基连接有棕榈酸(16:0)、十六碳烯酸(16:1[9])、硬脂酸(18:0)、油酸(18:1[9])、亚油酸(18:2[9,12])和亚麻酸(18:3[9,12,15])等六种脂肪酸,其中十六碳烯酸和亚麻酸为主要成分,分别占总脂肪酸含量的32.3％和34.4％。质膜中高含量的亚麻酸可能是发菜具有极强抗旱能力的一个重要因素。     

Adverse effects of conjugated alpha-linolenic acids (CLnA) on lipoprotein profile on experimental atherosclerosis in hamsters

Conjugated linoleic acids (CLAs) such as rumenic acid (RA) have the potential to alter blood lipid profiles in animals and in humans. In contrast, physiological effects of conjugated α-linolenic acids (CLnAs), which concomitantly are omega-3 and conjugated fatty acids, are still unknown. The aim of this study was to evaluate the potential of CLnA to interfere in early steps of atherosclerosis by altering lipoprotein profiles and fatty streaks in the aortas. F1B hamsters were fed a control or one of the three hypercholesterolemic (HC) diets: HC-control, HC-RA (18:2 cis-9, trans-11) or HC-CLnA (CLnA: equimolar mixture of 18:3 cis-9, trans-11, cis-15 and cis-9, trans-13, cis-15) diet. In low-cholesterol control-fed hamsters, the proportion of high-density lipoprotein cholesterol (HDL-C) was around 45% while in HC-fed hamsters, HDL-C was around 10% and cholesterol was mostly (80%) carried by triglyceride-rich lipoproteins (TRL). Low-density lipoprotein (LDL) triglycerides (TGs) increased by approximately 60% in hamsters fed either HC-RA or HC-CLnA compared with HC-controls but not compared with the low-cholesterol control diet. HDL cholesterol decreased by 24% and 16% in hamsters fed HC-RA and HC-CLnA, respectively. Small dense LDL-cholesterol increased by approximately 60% in hamsters fed HC-RA and HC-CLnA compared with the HC-control group and by more than a 100% compared with hamsters on the control diet. The relative percentage of liver cholesteryl ester content increased by 88% in hamsters fed HC diets compared with the control diet. Significant differences in fatty streaks were observed between control and HC-diet-fed hamsters. However, no significant difference was observed among the HC-diet-fed hamsters. This study shows that animals fed any one of the HC diets developed an adverse lipoprotein profile compared with a normolipidic diet. Also, HC-RA or HC-CLnA diets altered lipoprotein profile compared with animals fed the HC-control diet but had no beneficial effects on atherosclerosis.     

Incorporation profiles of conjugated linoleic acid isomers in cell membranes and their positional distribution in phospholipids

Although the conjugated linoleic acids (CLA) have several isomer-specific biological effects including anti-carcinogenic and anti-adipogenic effects, their mechanisms of action remain unclear. To determine their potential effects on membrane structure and function, we studied the incorporation profiles of four CLA isomers (trans-10 cis-12 (A), trans-9 trans-11 (B), cis-9 trans-11 (C), and cis-9 cis-11 (D)) in CHO and HepG2 cells. All four isomers were incorporated into cellular lipids as efficiently as linoleic acid (LA), with the majority of the incorporated CLA present in membrane rafts. Of the four isomers, only CLA-A increased the cholesterol content of the raft fraction. Over 50% of the incorporated CLAs were recovered in phosphatidylcholine of CHO cells, but in HepG2 the neutral lipids contained the majority of CLA. The desaturation index (18:1/18:0 and 16:1/16:0) was reduced by CLA-A, but increased by CLA-B, the effects being apparent mostly in raft lipids. The Δ? desaturase activity was inhibited by CLAs A and C. Unlike LA, which was mostly found in the sn-2 position of phospholipids, most CLAs were also incorporated significantly into the sn-1 position in both cell types. These studies show that the incorporation profiles of CLA isomers differ significantly from that of LA, and this could lead to alterations in membrane function, especially in the raft-associated proteins.     

The enrichment of a ruminal bacterium (Megasphaera elsdenii YJ-4) that produces the trans-10, cis-12 isomer of conjugated linoleic acid

AIMS: To isolate predominant ruminal bacteria that produce trans-10, cis-12 conjugated linoleic acid (CLA) from linoleic acid (LA). METHODS AND RESULTS: Mixed bacteria from ruminal contents of a cow fed grain were enriched with DL-lactate and trypticase. They produced more trans-10, cis-12 CLA than those that were not enriched (7 vs 2 microg mg protein(-1), P < 0.05). Enrichments had an abundance of large cocci that produced trans-10, cis-12 CLA from LA. Strain YJ-4 produced the most trans-10, cis-12 CLA (approx. 7 microg mg protein(-1)) and 16S rDNA sequencing indicated that YJ-4 was a strain of Megasphaera elsdenii. Megasphaera elsdenii T81 produced approx. 4 microg trans-10, cis-12 CLA mg protein(-1) while strains B159, AW106 and JL1 produced < 0.5 microg mg protein(-1). The trans-10, cis-12 CLA production of YJ-4 was first order with respect to cell concentration (0-800 microg protein ml(-1)), but kinetics were not first order with respect to substrate concentration. CONCLUSIONS: Some M. elsdenii strains produce significant amounts of trans-10, cis-12 CLA. SIGNIFICANCE AND IMPACT OF THE STUDY: Trans-10, cis-12 CLA appears to cause milk fat depression in cattle fed diets supplemented with grain and polyunsaturated fatty acids, but predominant ruminal bacteria that produced trans-10, cis-12 CLA from LA had not previously been isolated.     

Action of long-chain fatty acids in vitro on Ca2+-stimulatable, Mg2+-dependent ATPase activity in human red cell membranes.

Human red cell membrane Ca2+-stimulatable, Mg2+-dependent adenosine triphosphatase (Ca2+-ATPase) activity and its response to thyroid hormone have been studied following exposure of membranes in vitro to specific long-chain fatty acids. Basal enzyme activity (no added thyroid hormone) was significantly decreased by additions of 10(-9)-10(-4) M-stearic (18:0) and oleic (18:1 cis-9) acids. Methyl oleate and elaidic (18:1 trans-9), palmitic (16:0) and lauric (12:0) acids at 10(-6) and 10(-4) M were not inhibitory, nor were arachidonic (20:4) and linolenic (18:3) acids. Myristic acid (14:0) was inhibitory only at 10(-4) M. Thus, chain length of 18 carbon atoms and anionic charge were the principal determinants of inhibitory activity. Introduction of a cis-9 double bond (oleic acid) did not alter the inhibitory activity of the 18-carbon moiety (stearic acid), but the trans-9 elaidic acid did not cause enzyme inhibition. While the predominant effect of fatty acids on erythrocyte Ca2+-ATPase in situ is inhibition of basal activity, elaidic, linoleic (18:2) and palmitoleic (16:1) acids at 10(-6) and 10(-4) M stimulated the enzyme. Methyl elaidate was not stimulatory. These structure-activity relationships differ from those described for fatty acids and purified red cell Ca2+-ATPase reconstituted in liposomes. Thyroid hormone stimulation of Ca2+-ATPase was significantly decreased by stearic and oleic acids (10(-9)-10(-4) M), but also by elaidic, linoleic, palmitoleic and myristic acids. Arachidonic, palmitic and lauric acids were ineffective, as were the methyl esters of oleic and elaidic acids. Thus, inhibition of the iodothyronine effect on Ca2+-ATPase by fatty acids has similar, but not identical, structure-activity relationships to those for basal enzyme activity. To examine mechanisms for these fatty acid effects, we studied the action of oleic and stearic acids on responsiveness of the enzyme to purified calmodulin, the Ca2+-binding activator protein for Ca2+-ATPase. Oleic and stearic acids (10(-9)-10(-4) M) progressively inhibited, but did not abolish, enzyme stimulation by calmodulin (10(-9) M). Double-reciprocal analysis of the effect of oleic acid on calmodulin stimulation indicated noncompetitive inhibition. Addition of calmodulin to membranes in the presence of equimolar oleic acid restored basal enzyme activity. Oleic acid also reduced 125I-calmodulin binding to membranes, but had no effect on the binding of [125I]T4 by ghosts. The mechanism of the decrease by long chain fatty acids of Ca2+-ATPase activity in situ in human red cell ghosts thus is calmodulin-dependent and involves reduction in membrane binding of calmodulin.     

Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Isolation and Characterization of the Cytoplasmic Membrane from the Terrestrial Cyanobacterium -Nostoc flagelliforme

Cytoplasmic membrane of Nostoc flagelliforme Born. et Flah. was isolated for the first time with a new method, the unique feature of which is the combined use of French pressure cell and digitonin to disrupt cells. After passed twice through French pressure cell (at 80 MPa), cells in sample (20 mg of dry weight/mL) were disrupted effectively by digitonin (3 mg/mL), and then the cytoplasmic membrane was isolated by density gradient centrifugation. The membrane contained carotenoids with absorption peaks at 458, 487 and 524 nm and a precursor of chlorophyll a with a minor peak at 673 nm. The fluorescence emission peaks of the membrane were emitted from the precursor of chlorophyll a. More than 30 polypeptides were detected in the membrane, in which the most obvious corresponded to the polypeptides with molecular mass of 80,28,19 and 17 kD. The membrane contained four types of glycerolipids: MGDG(62.4%), DGDG (18.9%), SQDG (16.7%) and PG (2.0%). 16:0, 16:1 [9], 18:0, 18:1 [9], 18:2 [9, 12] and 18:3 [9,12,15] fatty acids were determined in the membrane, in which 16:1 and 18:3 fatty acids were the main components, representing 32.3% and 34.4% of the total fatty acids respectively. High proportion of 18:3 fatty acid in the cytoplasmic membrane may be an important factor of N. flagelliforme in its remarkable drought-tolerant ability.     

Fatty acid analysis of Stenotrophomonas maltophilia clinical strains showing different susceptibility to antibiotics at 30 and 37 degrees C

Isolates of Stenotrophomonas maltophilia species display the feature "temperature-dependent susceptibility" (TDS) to antibiotics. Both 30TDS strains (at least 4 times lower value of minimum inhibitory concentration (MIC) of an antibiotic at 30 than at 37 degrees C) and 37TDS strains (at least 4 times lower value of MIC at 37 than at 30 degrees C) were described. Changes in the distribution of saturated and unsaturated fatty acids (FA) at 30 and 37 degrees C were considered as one of possible causes of the TDS phenomenon. Gas chromatography was used to determine the distribution of individual FA in five 37TDS strains of S. maltophilia (Group I); in five strains with MIC values unaffected by the cultivation temperature (Group II) and in six 30TDS (four strains) or 30/37TDS (two strains) isolates (Group III). At identical temperatures, no statistically significant differences in the distribution of major FA (iso-15:0, anteiso-15:0, 16:0 and 16:1) were registered between individual groups. Statistically significant (p < 0.05) differences between groups were found in minor FA only (iso-16:0, iso-17:0 and iso-17:1). Distribution changes of cellular FA at 30 and 37 degrees C can be considered to play only a minor role in the formation of the TDS phenomenon.     

Fatty acids of pulmonary surfactant phosphatidylcholine from fetal rabbit lung tissue in culture. Biosynthesis of n-10 monoenoic fatty acids

We have previously reported that fetal rabbit lung tissue in organ culture produces a lamellar body material (pulmonary surfactant) with a lower percentage of disaturated phosphatidylcholine than is typically found in rabbit lung in vivo (Longmuir, K.J., C. Resele-Tiden, and L. Sykes. 1985. Biochim. Biophys. Acta. 833: 135-143). This investigation was conducted to identify all fatty acids present in the lamellar body phosphatidylcholine, and to determine whether the low level of disaturated phosphatidylcholine is due to excessive unsaturated fatty acid at position sn-1, sn-2, or both. Fetal rabbit lung tissue, 23 days gestation, was maintained in culture for 7 days in defined (serum-free) medium. Phospholipids were labeled in culture with [1-14C]acetate or [U-14C]glycerol (to follow de novo fatty acid biosynthesis), or with [1-14C]palmitic acid (to follow incorporation of exogenously supplied fatty acid). Radiolabeled fatty acid methyl esters obtained from lamellar body phosphatidylcholine were first separated by reverse-phase thin-layer chromatography (TLC) into two fractions of 1) 14:0 + 16:1 and 2) 16:0 + 18:1. Complete separation of the individual saturated and monoenoic fatty acids was achieved by silver nitrate TLC of the two fractions. Monoenoic fatty acid double bond position was determined by permanganate-periodate oxidation followed by HPLC of the carboxylic acid phenacyl esters. Lamellar body phosphatidylcholine contained four monoenoic fatty acids: 1) palmitoleic acid, 16:1 cis-9; 2) oleic acid, 18:1 cis-9; 3) cis-vaccenic acid, 18:1 cis-11; and 4) 6-hexadecenoic acid, 16:1 cis-6. In addition, 8-octadecenoic acid, 18:1 cis-8, was found in the fatty acids of the tissue homogenate. The abnormally low disaturated phosphatidylcholine content in lamellar body material was the result of abnormally high levels of monoenoic fatty acid (principally 16:1 cis-9) found at position sn-2. Position sn-1 contained normal levels of saturated fatty acid. The biosynthesis of the unusual n-10 fatty acids was observed from the start of culture throughout the entire 7-day culture period, and was observed in incubations of tissue slices of day 23 fetal rabbit lung. This is the first report of the biosynthesis of n-10 fatty acids (16:1 cis-6 and 18:1 cis-8) in a mammalian tissue other than skin, where these fatty acids are found in the secretory product (sebum) of sebaceous glands.