Oxidation of Fatty Acids by Leishmania braziliensis panamensis1

Heating cultures of Leishmania braziliensis panamensis (grown at 26°C) to 34°C for 1.5–12 h transformed the cells to an ellipsoidally shaped form. The heat treatment caused an increase in the rate of oxidation of both medium and long chain fatty acids but decreased the rate of oxidation of [1-¹⁴C]glucose. The rate of fatty acid oxidation continued to increase for times as long as 20 h after returning the cultures to 26°C. In both the promastigote and heat-induced ellipsoidal forms, the ratio of ¹⁴CO₂ release from [1-¹⁴C]laurate to that from [12-¹⁴C]laurate was generally larger than four, whereas this ratio from [1-¹⁴C]oleate relative to [10-¹⁴C]oleate was approximately two. These data show that metabolic and morphological differentiation begin after a short heat treatment and that some metabolic changes may continue even after the reverse transformation is initiated. The data also suggest that either the ω-terminal portion of the fatty acids is not completely oxidized to acetyl CoA and/or that there are two functional fatty acid oxidation pathways in Leishmania.     

Circadian Rhythms in Neurospora crassa: Effects of Saturated Fatty Acids

To assess their effects on the conidiation rhythm in Neurospora, 14 saturated fatty acids from 6 to 24 carbons long were used to supplement the bd csp and bd csp cel strains. Both strains express a circadian spore-forming rhythm when grown on solid media; the cel mutation confers a partial fatty acid requirement. Fatty acid supplements from 8 to 13 carbons long lengthened the free-running period of bd csp cel compared with the control value of 21 h; the maximal effect (33 h) was obtained with nonanoic acid (9:0) at a concentration of 5 x 10(-4) M. In contrast, the period of bd csp remained unchanged under all experimental conditions. The short-chain fatty acids (<14 carbons) reduced the rate of advance of the growth front in both strains, compared with unsupplemented controls. However, this inhibition did not appear to be responsible for the lengthened periods in bd csp cel. Nor was direct incorporation of the short-chain (period-lengthening) fatty acids into mycelial total lipids responsible, since such incorporation was not observed. In fact, extensive metabolic conversion of these supplements by both strains was indicated by the disappearance of short-chain fatty acids from the agar media coupled with their absence in mycelial lipids, and by the liberation of (14)CO(2) from cultures supplemented with [1-(14)C]lauric acid (12:0).     

Pyrolytic Methylation-Gas Chromatography of Whole Bacterial Cells for Rapid Profiling of Cellular Fatty Acids

A novel, on-line derivatization technique has been developed which enables generation of fatty acid methyl ester (FAME) profiles from microorganisms by gas chromatography-mass spectrometry without the need for laborious and time-consuming sample preparation. Microgram amounts of bacterial cells are directly applied to a thin ferromagnetic filament and covered with a single drop of methanolic solution of tetramethylammonium hydroxide. After air drying, the filament is inserted into a special gas chromatograph inlet equipped with a high-frequency coil, thus enabling rapid inductive heating of the ferromagnetic filament. This so-called Curie-point heating technique is shown to produce patterns of bacterial FAMEs which are qualitatively and quantitatively nearly identical to those obtained from extracts of methylated lipids prepared by conventional sample pretreatment methods. Relatively minor differences involve the loss of hydroxy-substituted fatty acids by the pyrolytic approach as well as strongly enhanced signals of FAMEs derived from mycolic acids. This type of pyrolysis enables on-line derivatization and thermal extraction of volatile derivatives for analysis, whereas the residual components remain on a disposable probe (ferromagnetic wire) of a pyrolytic device. The reduced sample size (micrograms instead of milligrams) and the lack of sample preparation requirements open up the possibility of rapid microbiological identification of single colonies (thus overcoming the need for time-consuming subculturing) as well as analysis of FAME profiles directly from complex environmental samples.     

Lipid-Induced ER Stress: Synergistic Effects of Sterols and Saturated Fatty Acids

Stress within the endoplasmic reticulum (ER) induces a coordinated response, namely the unfolded protein response (UPR), devoted to helping the ER cope with the accumulation of misfolded proteins. Failure of the UPR plays an important role in several human diseases. Recent studies report that intracellular accumulation of saturated fatty acids (SFAs) and cholesterol, seen in diseases of high incidence, such as obesity or atherosclerosis, results in ER stress. In the present study, we evaluated the effects of perturbations to lipid homeostasis on ER stress/UPR induction in the model eukaryote Saccharomyces cerevisiae . We show that SFA originating from either endogenous (preclusion of fatty acid desaturation) or exogenous (feeding with extracellular SFA) sources trigger ER stress and that ergosterol, the major sterol in yeast, acts synergistically with SFA in this process. This latter effect is connected to ergosterol accumulation within microsomal fractions from SFA-accumulating cells, which display highly saturated phospholipid content. Moreover, treating the cells with the molecular chaperone 4-phenyl butyrate abolishes UPR induction, suggesting that lipid-induced ER stress leads to an overload of misfolded protein that acts, in turn, as the molecular signal for induction of the UPR. The present data are discussed in the context of human diseases that involve lipid deregulation.     

Characterization of Clostridia by Gas Chromatography: I. Differentiation of Species by Cellular Fatty Acids

Fatty acids of 41 strains representing 13 species of Clostridium were extracted directly from whole cells and examined as methyl esters by gas-liquid chromatography. Both visual and quantitative comparisons of the resulting chromatograms for the presence and relative amounts of large major peaks allowed rapid differentiation of C. perfringens, C. sporogenes, and C. bifermentans from each other and from 10 other species. Each of the three former species possessed a different characteristic fatty acid methyl ester profile that was exhibited by all strains tested within the respective species. Culture age and growth media influenced the relative proportions of certain of the acids, but such differences did not limit species differentiation.     

Alteration of the Unsaturated to Saturated Ratio of Fatty Acids in Bacterial Lipids by Alcohols

Silver-coated cloth (SCC) effectively controlled root rot that was caused by Pythium aphanidermatum in hydroponically grown cucumber plants. The presence of SCC in the hydroponic solution reduced the root rot from 100% to 10% 20 days after inoculation with zoospores of P. aphanidermatum. It was suggested that the inhibition of SCC was caused not only by the silver ion dissolved from SCC, but also by the metallic silver and silver compounds formed on the surface of the root.     

Lipid Mixtures Containing a Very High Proportion of Saturated Fatty Acids Only Modestly Impair Insulin Signaling in Cultured Muscle Cells

In vitro examinations of the effect of saturated fatty acids on skeletal muscle insulin action often use only one or two different fatty acid species, which does not resemble the human plasma fatty acid profile. We compared graded concentrations (0.1-0.8mM) of 3 different lipid mixtures: 1) a physiologic fatty acid mixture (NORM; 40% saturated fatty acids), 2) a physiologic mixture high in saturated fatty acids (HSFA; 60% saturated fatty acids), and 3) 100% palmitate (PALM) on insulin signaling and fatty acid partitioning into triacylglycerol (TAG) and diacylglycerol (DAG) in cultured muscle cells. As expected, PALM readily impaired insulin-stimulated pAkt^Thr308/Akt and markedly increased intracellular DAG content. In contrast, the fatty acid mixtures only modestly impaired insulin-stimulated pAkt^Thr308M/Akt, and we found no differences between NORM and HSFA. Importantly, NORM and HSFA did not increase DAG content, but instead dose-dependently increased TAG accumulation. Therefore, the robust impairment in insulin signaling found with palmitate exposure was attenuated with physiologic mixtures of fatty acids, even with a very high proportion of saturated fatty acids. This may be explained in part by selective partitioning of fatty acids into neutral lipid (i.e., TAG) when muscle cells were exposed to physiologic lipid mixtures.     

Herbicide Transformation: I. Studies with Whole Cells of Fusarium solani

A strain of Fusarium solani isolated from soil by enrichment techniques used propanil (3′, 4′-dichloropropionanilide) as a sole source of organic carbon and energy for growth in pure culture. The primary product of the transformation of propanil by F. solani was isolated and identified as 3,4-dichloroaniline (DCA). This compound accumulated in the medium to a level (80 μg/ml) which stopped further herbicide utilization. Herbicide utilization by F. solani was influenced by various environmental and nutritional factors. It was more sensitive to acid than alkaline pH. Added glucose and yeast extract increased the rate of propanil decomposition, and the reduced aeration retarded growth of the fungus and herbicide utilization. The growth of F. solani on propionate was inhibited by added DCA.     

Lysis of Bacillus subtilis Cells by Glycerol and Sucrose Esters of Fatty Acids

The lytic action of glycerol and sucrose esters of fatty acids with different carbon chain lengths on the exponentially growing cells of Bacillus subtilis 168 was investigated. Of each series of esters, glycerol dodecanoate and sucrose hexadecanoate were the most active. Lysis at 1 h after the addition of 0.1 mM glycerol dodecanoate or 20 μg of sucrose hexadecanoate per ml was 81 or 79%, respectively, as evaluated by the reduction in optical density. During this treatment a great loss of viability occurred that preceded lysis. The results that were obtained suggest that autolysis is induced by these esters. The esters caused morphological changes in the cells, but a seeming adaptation of the cells to esters was seen.     

Fat Metabolism in the West African Oil Palm (Elaeis Guineensis): PART I. FATTY ACID FORMATION IN THE MATURING KERNEL

Using a column chromatographic technique for the estimationand identification of fatty acids, a study has been made offat formation in developing Oil Palm kernels from 10 weeks afterpollination to full maturity (20 weeks), during which time thefat content may increase a hundredfold. Nuts from three differenttrees have been analysed and differences between these in ratesof maturation (as indicated by appearance of the endosperm)can be directly correlated with changes in character of theoil present. Amounts of reducing sugars, sucrose, and starch in the developingendosperm have also been followed, but these carbohydrates arepresent throughout in low concentration, and it is assumed thattranslocation from the rest of the plant to the developing kernelmust account for the major part of storage material. The mature kernel contains in its fat an unusual mixture ofeight different saturated fatty acids. The major such componentis lauric acid (46·1 to 49·5 per cent.) and thereare present two common unsaturated acids, oleic (15·7to 16·5 per cent.) and linoleic acid (0·7 to 3·1per cent.). At the earliest stage examined (10 weeks after pollination)all these acids are.present but in altered proportions, unsaturatedacids forming a larger fraction (36·5 to 81·2per cent., according to the tree investigated), and lauric acid(1·4 to 8·5 per cent.) a smaller. The results suggest that young kernels contain a small quantityof a largely unsaturated ‘protoplasmic’ fat, andthat at a certain stage in development some physiological changein the tissue results in the formation, in large quantities,of a new, highly saturated storage fat. No fatty acid interconversionscould be demonstrated although there is some suggestion thatoleic acid behaves anomalously. There is evidence that free fatty acids are not accumulatedprior to esterification.     

Microbial Assimilation of Hydrocarbons I. Fatty Acids Derived from Normal Alkanes

Fatty acids derived from Micrococcus cerificans growing at the expense of odd- and even-carbon normal alkanes were studied. Results demonstrated that cultures grown with a variety of nonhydrocarbon substrates serving as sole carbon and energy source yielded only even-carbon fatty acids. Even-chain alkanes, dodecane through octadecane serving as sole carbon source, resulted in even-carbon fatty acids with direct correlation between carbon number of the major fatty acid species and carbon number of the alkane substrate. Odd-carbon alkanes, undecane through heptadecane serving as sole carbon source, yielded both odd- and even-carbon fatty acids. A transitional shift from even-carbon fatty acids to odd-carbon fatty acids was observed as the carbon number of the alkane substrate increased. Unsaturated fatty acids were found to comprise a significant percentage of all profiles. Analysis of unsaturated fatty acids showed all odd- and even-carbon acids analyzed were Delta(9) monounsaturated fatty acids.     

Saccharomyces cerevisiae和Yarrowia lipolytica对自由饱和脂肪酸的选择性吸收及胞内积累特性研究

利用产油微生物生产特殊功能、高附加值的脂肪酸,具有良好的开发利用前景。以酿酒酵母(S.cerevisiae)和解脂耶氏酵母(Y.lipolytica)为出发菌株,以链长C4-C18的单一自由饱和脂肪酸作为唯一碳源,探究了两类酵母吸收利用、积累脂肪酸情况及胞内脂肪酸组成情况。结果表明:当碳链长C≤10时不能被利用,而且抑制细胞的生长,特别是当碳链长C≤8时,细胞很快被杀死;当碳链长C=11时,对细胞的生长有一定的抑制作用,菌体长势缓慢;碳链长C≥12时,对细胞生长没有影响;脂肪酸利用速度,偶数C脂肪酸奇数C脂肪酸;Nile red全细胞脂类染色显示,S.cerevisiae胞内脂质主要集中于胞内周边膜部位,Y.lipolytica主要以脂质体形式存在胞内,及少部分在胞内周边膜部位;GC/Mass脂类成分分析表明,菌株S.cerevisiae S228C BY4741-pox1和S.cerevisiae S228C BY4741-pox1,3可以积累培养基添加的相应脂肪酸,而其他供试菌积累的脂肪酸链长C≥16,没有检测到培养基含有相应的脂肪酸。这些结果为利用酵母生产特殊功能脂肪酸,及开发特色高附加值油脂提供了有意义的参考。     

Accumulation of N-3 Polyunsaturated Fatty Acids by Cultured Human Y79 Retinoblastoma Cells

Abstract: The metabolism of the n-3 class of polyunsaturated fatty acids, which occur in relatively high quantities in neural tissues, was studied in human Y79 retinoblastoma cells. These cells contained low levels of n-3 polyunsaturates when grown in culture media supplemented with fetal bovine serum. The cells readily incorporated preformed docosahexaenoic acid (22:6 n-3) into phospholipids, but human skin fibroblasts did this to a similar extent. When 10 to 30 μmol/ml linolenic acid (18:3 n-3) was added, the cells also accumulated 22:6 in phospholipids. The capacity to convert appreciable amounts of 18:3 to 22:6 appears to be a unique property of the retinoblastoma cells as compared with other continuously cultured cell lines. More 18:3 than linoleic acid (18:2 n-6) was incorporated into phospholipids by the retinoblastoma cultures, and 18:3 was channeled to a larger extent into the ethanolamine glycerophospholipid fraction. These findings indicate that retinoblastoma cells handle n-3 polyunsaturated fatty acids in a manner very similar to neural tissue in vivo . Based on the results obtained with this model system, it appears that three processes may contribute to the accumulation of 22:6 in retina and neural tissue: increased ability to incorporate 18:3, the capacity to convert 18:3 to 22:6, and channeling of 18:3 and its metabolites into ethanolamine glycerophospholipids.     

BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS : I. Protein Amino Acids

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with ¹⁴CO₂ for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of ¹⁴CO₂ into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with ¹⁴CO₂ in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.     

Fluorescent n-3 and n-6 Very Long Chain Polyunsaturated Fatty Acids: THREE-PHOTON IMAGING IN LIVING CELLS EXPRESSING LIVER FATTY ACID-BINDING PROTEIN*

Despite the considerable beneficial effects of n-3 and n-6 very long chain polyunsaturated fatty acids (VLC-PUFAs), very little is known about the factors that regulate their uptake and intracellular distribution in living cells. This issue was addressed in cells expressing liver-type fatty acid-binding protein (L-FABP) by real time multiphoton laser scanning microscopy of novel fluorescent VLC-PUFAs containing a conjugated tetraene fluorophore near the carboxyl group and natural methylene-interrupted n-3 or n-6 grouping. The fluorescent VLC-PUFAs mimicked many properties of their native nonfluorescent counterparts, including uptake, distribution, and metabolism in living cells. The unesterified fluorescent VLC-PUFAs distributed either equally in nuclei versus cytoplasm (22-carbon n-3 VLC-PUFA) or preferentially to cytoplasm (20-carbon n-3 and n-6 VLC-PUFAs). L-FABP bound fluorescent VLC-PUFA with affinity and specificity similar to their nonfluorescent natural counterparts. Regarding n-3 and n-6 VLC-PUFA, L-FABP expression enhanced uptake into the cell and cytoplasm, selectively altered the pattern of fluorescent n-6 and n-3 VLC-PUFA distribution in cytoplasm versus nuclei, and preferentially distributed fluorescent VLC-PUFA into nucleoplasm versus nuclear envelope, especially for the 22-carbon n-3 VLC-PUFA, correlating with its high binding by L-FABP. Multiphoton laser scanning microscopy data showed for the first time VLC-PUFA in nuclei of living cells and suggested a model, whereby L-FABP facilitated VLC-PUFA targeting to nuclei by enhancing VLC-PUFA uptake and distribution into the cytoplasm and nucleoplasm.     

Lipid Metabolism of Rumen Ciliates and Bacteria: I. Uptake of Fatty Acids by Isotricha prostoma and Entodinium simplex

Washed suspensions of the ruminal ciliates, Isotricha prostoma and Entodinium simplex, concentrated C¹⁴-labeled oleic, palmitic, stearic, and linoleic acids within the cells during short incubation periods. Radioautographs demonstrated that oleic acid-1-C¹⁴ was hydrogenated to stearic acid by I. prostoma, and Warburg manometric data showed that the sodium salts of oleic, valeric, caproic, and acetic acids, and methyl myristate, methyl laurate, and the triglyceride tributyrin stimulated fermentation of I. prostoma. The total lipid and free fatty acid contents of I. prostoma were determined.