Small molecule analysis and imaging of fatty acids in the zebra finch song system using time-of-flight-secondary ion mass spectrometry

Fatty acids are central to brain metabolism and signaling, but their distributions within complex brain circuits have been difficult to study. Here we applied an emerging technique, time-of-flight secondary ion mass spectrometry (ToF-SIMS), to image specific fatty acids in a favorable model system for chemical analyses of brain circuits, the zebra finch (Taeniopygia guttata). The zebra finch, a songbird, produces complex learned vocalizations under the control of an interconnected set of discrete, dedicated brain nuclei 'song nuclei'. Using ToF-SIMS, the major song nuclei were visualized by virtue of differences in their content of essential and non-essential fatty acids. Essential fatty acids (arachidonic acid and docosahexaenoic acid) showed distinctive distributions across the song nuclei, and the 18-carbon fatty acids stearate and oleate discriminated the different core and shell subregions of the lateral magnocellular nucleus of the anterior nidopallium. Principal component analysis of the spectral data set provided further evidence of chemical distinctions between the song nuclei. By analyzing the robust nucleus of the arcopallium at three different ages during juvenile song learning, we obtain the first direct evidence of changes in lipid content that correlate with progression of song learning. The results demonstrate the value of ToF-SIMS to study lipids in a favorable model system for probing the function of lipids in brain organization, development and function.     

Occurrence of dialkyl ether phospholipids in Stigmatella aurantiaca DW4.

We investigated the lipid composition of vegetative cells of Stigmatella aurantiaca. Four phospholipids were isolated and identified: phosphatidylethanolamine as the main component, phosphatidylglycerol, lysophosphatidylethanolamine in an exceptionally large amount (17%), and phosphatidylinositol (18 to 25%), rare in procaryotic cells. This composition did not change significantly during growth. The fatty acids of total lipids were found to be rather similar to those of other strains of myxobacteria; the main fatty acids found were unsaturated and branched. We noted a different fatty acid pattern for each phospholipid. The presence of unusual alkyl ether linkages, established by chemical hydrolysis and infrared spectroscopy, was unexpected in these bacteria. Diacyl ester, dialkyl ether, and monoacyl-monoalkyl structures were shown in phosphatidylethanolamine and phosphatidylglycerol. Lysophosphatidylethanolamine was essentially a monoacyl form, whereas phosphatidylinositol was a unique dialkyl ether phospholipid.     

Lipid and fatty acid composition of the fat body during the female reproductive cycle of Labidura riparia (Insecta Dermaptera)

During the reproductive cycle of the female Labidura riparia, cytological observations show cyclical modifications of lipid droplets in the periovarian adipocyte. Fat body lipids and their constitutive fatty acids are analyzed. The lipids are predominantly triacylglycerols, which increase after adult ecdysis during vitellogenic and non-vitellogenic periods. Small amounts of diacylglycerols and phospholipids are found. Diacylglycerols increase during vitellogenesis and decrease during the non-vitellogenic period. Cytological modifications of lipid droplets are probably related to diacylglycerol fluctuations. Gas-liquid chromatography of fatty acid methyl esters shows oleic acid to be the predominant fatty acid in total lipids and triacylglycerols; unsaturated acids are approximately twice as abundant as saturated acids all along the reproductive cycle. Fatty acid composition of diacylglycerols and phospholipids differs from triacylglycerols and total lipids composition. Palmitic, stearic, oleic and linoleic acids represent the major fatty acids; their relative amounts vary during the different periods of the reproductive cycle. The correlations between fat body lipid changes and ovarian development were discussed and compared with observations made on other insect species. Accepted: 23 April 1997     

Distribution of radioactivity in myelin lipids following subcutaneous injection of [14C]stearate

Blood fatty acids are an important parameter for the synthesis of brain myelin as exogenous stearic acid is needed: after subcutaneous injection to 18-day-old mice this labelled stearic acid is transported into brain myelin and incorporated into its lipids. However the acid is partly metabolized in the brain by elongation (thus providing very long chain fatty acids, mainly lignoceric acid) or by degradation to acetate units (utilized for synthesis of medium chain fatty acids as palmitic acid, and cholesterol). These metabolites are further incorporated into myelin lipids. The myelin lipid radioactivity increases up to 3 days; most of the activity is found in phospholipids; their fatty acids are labelled in saturated as well as in polyunsaturated homologues but sphingolipids, especially cerebrosides, contain also large amounts of radioactivity (which is mainly found in very long chain fatty acids, almost all in lignoceric acid). The occurrence of unesterified fatty acids must be pointed out, these molecules unlike other lipids, are found in constant amount (expressed in radioactivity per mg myelin lipid).     

Lipid composition of the electron transport membrane of Haemophilus parainfluenzae

The principal lipids associated with the electron transport membrane of Haemophilus parainfluenzae are phosphatidylethanolamine (78%), phosphatidylmonomethylethanolamine (0.4%), phosphatidylglycerol (18%), phosphatidylcholine (0.4%), phosphatidylserine (0.4%), phosphatidic acid (0.2%), and cardiolipin (3.0%). Phospholipids account for 98.4% of the extractible fatty acids. There are no glycolipids, plasmalogens, alkyl ethers, or lipo amino acid esters in the membrane lipids. Glycerol phosphate esters derived from the phospholipids by mild alkaline methanolysis were identified by their staining reactions, mobility on paper and ion-exchange column chromatography, and by the molar glycerol to phosphate ratios. Eleven diacyl phospholipids can be separated by two-dimensional thin-layer chromatography. Each lipid served as a substrate for phospholipase D, and had a fatty acid to phosphate ratio of 2:1. Each separated diacyl phospholipid was deacylated and the glycerol phosphate ester was identified by paper chromatography in four solvent systems. Of the 11 separated phospholipids, 3 were phosphatidylethanolamines, 2 were phosphatidylserines, and 2 were phosphatidylglycerols. Phosphatidylcholine, cardiolipin, and phosphatidic acid were found at a single location. Phosphatidylmonomethylethanolamine was found with the major phosphatidylethanolamine. Three distinct classes of phospholipids are separable according to their relative fatty acid compositions. (i) The trace lipids consist of two phosphatidylethanolamines, two phosphatidylserines, phosphatidylcholine, phosphatidic acid, and a phosphatidylglycerol. Each lipid represents less than 0.3% of the total lipid phosphate. These lipids are characterized by high proportions of the short (C(10) to C(14)) and long (C(19) to C(22)) fatty acids with practically no palmitoleic acid. (ii) The major phospholipids (93% of the lipid phosphate) are phosphatidylethanolamine, phosphatidylmonomethylethanolamine, and phosphatidylglycerol. These lipids contain a low proportion of the short (C(19)) fatty acids. Palmitic and palmitoleic acids represent over 80% of the total fatty acids. (iii) The fatty acid composition of the cardiolipin is intermediate between the other two classes. Both palmitoleic and the longer fatty acids represent a significant proportion of the total fatty acid.     

LIPID COMPOSITION OF CHLORARACHNIOPHYTES (CHLORARACHNIOPHYCEAE) FROM THE GENERA BIGELOWIELLA,GYMNOCHLORA, AND LOTHARELLA1

The Chlorarachniophyceae are unicellular eukaryotic algae characterized by an amoeboid morphology that may be the result of secondary endosymbiosis of a green alga by a nonphotosynthetic amoeba or amoeboflagellate. Whereas much is known about the phylogeny of chlorarachniophytes, little is known about their physiology, particularly that of their lipids. In an initial effort to characterize the lipids of this algal class, four organisms from three genera were examined for their fatty acid and sterol composition. Fatty acids from lipid fractions containing chloroplast‐associated glycolipids, storage triglycerides, and cytoplasmic membrane‐associated polar lipids were characterized. Glycolipid‐associated fatty acids were of limited composition, principally eicosapentaenoic acid [20:5(n‐3)] and hexadecanoic acid (16:0). Triglyceride‐associated fatty acids, although minor, were found to be similar in composition. The polar lipid fraction was dominated by lipids that did not contain phosphorus and had a more variable fatty acid composition with 16:0 and docosapentaenoic acid [22:5(n‐3)] dominant along with a number of minor C₁₈ and C₂₀ fatty acids. Crinosterol and one of the epimeric pair poriferasterol/stigmasterol were the sole sterols. Several genes required for synthesis of these sterols were computationally identified in Bigelowiella natans Moestrup. One sterol biosynthesis gene showed the greatest similarity to SMT1 of the green alga, Chlamydomonas reinhardtii. However, homologues to other species, mostly green plant species, were also found. Further, the method used for identification suggested that the sequences were transferred to a genetic compartment other than the likely original location, the nucleomorph nucleus.     

Identification, isolation and characterization of tetracosapolyenoic acids in lipids of marine coelenterates.

Several tetracosapolyenoic acids (TPA) were detected in lipids of different marine coelenterates. Two of these acids were isolated and their structures were confirmed by chemical and spectral methods as all-cis-6,9,12,15,18-tetracosapentaenoic and all-cis-6,9,12,15,18,21-tetracosahexaenoic acid. Their distribution among lipids of a number of species of different classes of coelenterates from the northern and tropical seas, among neutral and polar lipids of these organisms was investigated. Significant quantities of TPA were found in all of the Octacorallia species studied. In some cases the sum of TPA reaches the level of 20% of total lipid fatty acids. The fatty acid composition of different coelenterates is also discussed.     

Cancer cells incorporate and remodel exogenous palmitate into structural and oncogenic signaling lipids

De novo lipogenesis is considered the primary source of fatty acids for lipid synthesis in cancer cells, even in the presence of exogenous fatty acids. Here, we have used an isotopic fatty acid labeling strategy coupled with metabolomic profiling platforms to comprehensively map palmitic acid incorporation into complex lipids in cancer cells. We show that cancer cells and tumors robustly incorporate and remodel exogenous palmitate into structural and oncogenic glycerophospholipids, sphingolipids, and ether lipids. We also find that fatty acid incorporation into oxidative pathways is reduced in aggressive human cancer cells, and instead shunted into pathways for generating structural and signaling lipids. Our results demonstrate that cancer cells do not solely rely on de novo lipogenesis, but also utilize exogenous fatty acids for generating lipids required for proliferation and protumorigenic lipid signaling. This article is part of a special issue entitled Lipid Metabolism in Cancer.     

The preferential accumulation of cadmium in the head portion of the freshwater planarian, Dugesia japonica (Platyhelminthes: Turbellaria)

Free-living freshwater planarians are considered to have the potential for development as an experimental model for toxicological studies on xenobiotics, including metals. However, little was known about the distribution patterns of metals in the body of treated planarians. This study was conducted to determine the tissue distribution patterns of cadmium (Cd) in different body portions of the treated planarian, Dugesia japonica. Results showed that Cd accumulated in the head of planarians at a significantly higher concentration than in the tail. After examining the level of metallothionein (MT), we suggested that the tissue distribution pattern of Cd might be related to MT induction patterns. In contrast, in planarians treated with copper (Cu), neither the tissue accumulation of Cu nor the multiples of induction of MTs significantly differed between different portions. Furthermore, a higher Cd accumulation rate in the head of planarians caused more-severe oxidative stress to appear in this portion and also a higher susceptibility to a lethal concentration of Cd. Finally, both in vitro and in vivo acetylcholinesterase activities in both body portions of planarians were inhibited by Cd. The present study provides the first report that different metals are distributed in various body portions with different patterns in the planarian.     

Thematic review series: skin lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis

The permeability barrier is required for terrestrial life and is localized to the stratum corneum, where extracellular lipid membranes inhibit water movement. The lipids that constitute the extracellular matrix have a unique composition and are 50% ceramides, 25% cholesterol, and 15% free fatty acids. Essential fatty acid deficiency results in abnormalities in stratum corneum structure function. The lipids are delivered to the extracellular space by the secretion of lamellar bodies, which contain phospholipids, glucosylceramides, sphingomyelin, cholesterol, and enzymes. In the extracellular space, the lamellar body lipids are metabolized by enzymes to the lipids that form the lamellar membranes. The lipids contained in the lamellar bodies are derived from both epidermal lipid synthesis and extracutaneous sources. Inhibition of cholesterol, fatty acid, ceramide, or glucosylceramide synthesis adversely affects lamellar body formation, thereby impairing barrier homeostasis. Studies have further shown that the elongation and desaturation of fatty acids is also required for barrier homeostasis. The mechanisms that mediate the uptake of extracutaneous lipids by the epidermis are unknown, but keratinocytes express LDL and scavenger receptor class B type 1, fatty acid transport proteins, and CD36. Topical application of physiologic lipids can improve permeability barrier homeostasis and has been useful in the treatment of cutaneous disorders.     

The Fatty Acid Composition of Paramecium aurelia Cells and Cilia: Changes with Culture Age

SYNOPSIS.
The fatty acids of whole cells and cilia from Paramecium tetraurelia strains 51s and d,95 and from Paramecium octaurelia strain 299s were identified. Ciliates were grown axenically in 3 types of culture media. More than 30 fatty acid species were identified and their structures determined by gas chromatography, mass spectrometry, argentation chromatography, hydro-genation, and fragmentation technics. The major fatty acids were hexadecanoic, octadecanoic, 9-octadecenoic, 9,12-octadecadi-enoic, 6,9,12-octadecatrienoic, and 5,8,11,14-eicosatetraenoic acids. Minor variations in fatty acid compositions were observed in cells grown in the different culture media as well as among the 3 strains. Major changes in fatty acid compositions occurred with culture age and cell density. The cells accumulated exogenous lipids in cytoplasmic vesicles. These lipids were utilized as culture age progressed. Both cellular volume and lipid content were greater in young than in older cultures. Fatty acid compositions of both whole cells and cilia changed with age and had a relative decrease in saturated, short-chained and odd-numbered carbon acids. Cilia lipids were enriched in long-chained, polyunsaturated acids as compared to lipids in whole cell extracts. Eicosatetra-enoic acid (arachidonic acid) increased to the greatest extent with age in both cellular and ciliary lipids, accounting for 20–60% of the total fatty acids in cilia. The age-related change in fatty acid composition in Paramecium is among the largest observed in eukaryotic organisms. It was concluded that some minor fatty acids found in Paramecium lipids were incorporated directly from certain culture media and that Paramecium had w 3, 6, and 9 pathways for polyunsaturated fatty acid biosynthesis.     

Phylogenomic analysis of lipid biosynthetic genes of Archaea shed light on the ‘lipid divide’

The lipid membrane is one of the most characteristic traits distinguishing the three domains of life. Membrane lipids of Bacteria and Eukarya are composed of fatty acids linked to glycerol‐3‐phosphate (G3P) via ester bonds, while those of Archaea possess isoprene‐based alkyl chains linked by ether linkages to glycerol‐1‐phosphate (G1P), resulting in the opposite stereochemistry of the glycerol phosphate backbone. This ‘lipid divide’ has raised questions on the evolution of microbial life since eukaryotes are thought to have evolved from the Archaea, requiring a radical change in membrane composition. Here, we searched for homologs of enzymes involved in membrane lipid and fatty acid synthesis in a wide variety of archaeal genomes and performed phylogenomic analyses. We found that two uncultured archaeal groups, i.e. marine euryarchaeota group II/III and ‘Lokiarchaeota’, recently discovered descendants of the archaeal ancestor leading to eukaryotes, lack the gene to synthesize G1P and, consequently, the capacity to synthesize archaeal membrane lipids. However, our analyses reveal their genetic capacity to synthesize G3P‐based ‘chimeric lipids’ with either two ether‐bound isoprenoidal chains or with an ester‐bound fatty acid instead of an ether‐bound isoprenoid. These archaea may reflect the ‘archaea‐to‐eukaryote’ membrane transition stage which have led to the current ‘lipid divide’.     

All fatty acids are not equal: discrimination in plant membrane lipids

Plant membrane lipids are primarily composed of 16-carbon and 18-carbon fatty acids containing up to three double bonds. By contrast, the seed oils of many plant species contain fatty acids with significantly different structures. These unusual fatty acids sometimes accumulate to >90% of the total fatty acid content in the seed triacylglycerols, but are generally excluded from the membrane lipids of the plant, including those of the seed. The reasons for their exclusion and the mechanisms by which this is achieved are not completely understood. Here we discuss recent research that has given new insights into how plants prevent the accumulation of unusual fatty acids in membrane lipids, and how strict this censorship of membrane composition is. We also describe a transgenic experiment that resulted in an excessive buildup of unusual fatty acids in cellular membranes, and clearly illustrated that the control of membrane lipid composition is essential for normal plant growth and development.     

Isolation of planarian X-ray-sensitive stem cells by fluorescence-activated cell sorting

The remarkable capability of planarian regeneration is mediated by a group of adult stem cells referred to as neoblasts. Although these cells possess many unique cytological characteristics (e.g. they are X-ray sensitive and contain chromatoid bodies), it has been difficult to isolate them after cell dissociation. This is one of the major reasons why planarian regenerative mechanisms have remained elusive for a long time. Here, we describe a new method to isolate the planarian adult stem cells as X-ray-sensitive cell populations by fluorescence-activated cell sorting (FACS). Dissociated cells from whole planarians were labeled with fluorescent dyes prior to fractionation by FACS. We compared the FACS profiles from X-ray-irradiated and non-irradiated planarians, and thereby found two cell fractions which contained X-ray-sensitive cells. These fractions, designated X1 and X2, were subjected to electron microscopic morphological analysis. We concluded that X-ray-sensitive cells in both fractions possessed typical stem cell morphology: an ovoid shape with a large nucleus and scant cytoplasm, and chromatoid bodies in the cytoplasm. This method of isolating X-ray-sensitive cells using FACS may provide a key tool for advancing our understanding of the stem cell system in planarians.     

Identification of immunological reagents for use in the study of freshwater planarians by means of whole-mount immunofluorescence and confocal microscopy

In recent years, interest in planarians as a model system for the study of metazoan regeneration, adult stem cell biology, and the evolution of metazoan body plans has been growing steadily. The availability of RNA interference (RNAi), BrdU-labeling of planarian stem cells, and thousands of planarian cDNA sequences soon to be released into public databases has opened planarians to molecular dissection. However, the successful application of large-scale RNAi-based screens, for example, will depend in part on the availability of markers to characterize the resulting phenotypes. Given the paucity of antibodies available for the study of planarian biology, we have screened various public and commercial antibody resources to identify immunoreagents capable of cross-reacting with planarian tissues. Here we report the identification and characterization of 33 such antibodies recognizing a wide variety of tissues in freshwater planarians.     

A tale of two lipids: Lipid unsaturation commands ferroptosis sensitivity

Membrane lipids play important roles in the regulation of cell fate, including the execution of ferroptosis. Ferroptosis is a non-apoptotic cell death mechanism defined by iron-dependent membrane lipid peroxidation. Phospholipids containing polyunsaturated fatty acids (PUFAs) are highly vulnerable to peroxidation and are essential for ferroptosis execution. By contrast, the incorporation of less oxidizable monounsaturated fatty acids (MUFAs) in membrane phospholipids protects cells from ferroptosis. The enzymes and pathways that govern PUFA and MUFA metabolism therefore play a critical role in determining cellular sensitivity to ferroptosis. Here, we review three lipid metabolic processes—fatty acid biosynthesis, ether lipid biosynthesis, and phospholipid remodeling—that can govern ferroptosis sensitivity by regulating the balance of PUFAs and MUFAs in membrane phospholipids.     

Modification of the fatty acid composition of individual phospholipids and neutral lipids after infection of the simian erythrocyte by Plasmodium knowlesi

Using capillary gas-liquid chromatography, we have analyzed the alteration in the total fatty acid, phospholipid and neutral lipid compositions of the monkey erythrocyte, after infection by the malarial parasite Plasmodium knowlesi. Data based on fatty acid quantitation show that the phospholipid composition is altered, with particularly large increases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the most abundant phospholipids in normal and P. knowlesi-schizont-infected cells. Unesterified fatty acids were found to be less abundant in infected cells. The total fatty acid content of the cell is increased 6-fold during infection, and total fatty acid composition is also changed: the infected cells are richer in palmitate (+23%), oleate (+29%) and linoleate (+89%), but contained less stearate (-27%) and arachidonate (-40%). The determination of the fatty acid composition of individual phospholipids, neutral lipids and unesterified fatty acids showed that choline-containing phospholipids (PC and sphingomyelin) were not as altered in their fatty acid pattern as anionic phospholipids (PE, phosphatidylserine (PS) and phosphatidylinositol (PI) and lysophosphatidylcholine (lysoPC). Specific alterations in the fatty acid compositions of individual phospholipids were detected, whereas the rise in linoleic acid was the only change during infection that was recovered in each phospholipid (except PC), neutral lipid and unesterified fatty acids. The fatty acid composition of the neutral lipids and unesterified fatty acids was particularly modified: the only rise in arachidonic acid level was observed in these lipid classes after infection. The total plasmalogen level of the erythrocyte is decreased in infected cells (-60%), but their level is increased in PI.     

Biophysical properties of tear film lipid layer II. Polymorphism of FAHFA

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered class of endogenous lipids that consist of two acyl chains connected through a single ester bond. Being a unique species of FAHFAs, (O-acyl)-ω-hydroxy fatty acids (OAHFAs) differ from other FAHFAs in that their hydroxy fatty acid backbones are ultralong and their hydroxy esterification is believed to be solely at the terminal (ω-) position. Only in recent years with technological advances in lipidomics have OAHFAs been identified as an important component of the tear film lipid layer (TFLL). It was found that OAHFAs account for approximately 4 mol% of the total lipids and 20 mol% of the polar lipids in the TFLL. However, their biophysical function and contribution to the TFLL is still poorly understood. Here we studied the molecular biophysical mechanisms of OAHFAs using palmitic-acid-9-hydroxy-stearic-acid (PAHSA) as a model. PAHSA and OAHFAs share key structural similarities that could result in comparable biophysical properties and molecular mechanisms. With combined biophysical experiments, atomic force microscopy observations, and all-atom molecular dynamics simulations, we found that the biophysical properties of a dynamic PAHSA monolayer under physiologically relevant conditions depend on a balance between kinetics and thermal relaxation. PAHSA molecules at the air-water surface demonstrate unique polymorphic behaviors, which can be explained by configurational transitions of the molecules under various lateral pressures. These findings could have novel implications in understanding biophysical functions that FAHFAs, in general, or OAHFAs, specifically, play in the TFLL.     

Nature of DNA-bound fatty acids in Pseudomonas aurantiaca

The existence of Pseudomonas aurantiaca DNA-bound fatty acids and lipids is presented in this work. The isolation of DNA was carried out by two different procedures, namely, phenol and detergent-based phenol isolation in order to prove the presence of DNA-bound lipids. The lipid content of DNA is expressed in terms of fatty acid profile. A high level of 16:0, 18:0 and 18:1 is characteristic for tightly bound DNA lipids. On the other hand, the fatty acids such as 14:1, iso14:0 and iso16:0 are found in trace amounts only in DNA lipid fraction, but these fatty acids are not found in the whole-cell lipids. Absolutely no 3-hydroxy fatty acids were found in DNA lipids. However, both C16 and C18 species represent the main fatty acids of whole-cell and DNA-bound lipids. The presence of DNA-bound lipids even under tough treatment of DNA allows to conclude that these lipids represent a special pool among cellular lipids.