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.     

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

Membrane phospholipid composition of hemocytes in the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum

The detailed sterol (free sterol proportions and compositions) and phospholipid (PL) compositions (relative proportions of PL classes and subclasses and their respective fatty acid (FA) compositions) of hemocyte membranes were investigated in two bivalve mollusks: the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum. Hemocyte membrane lipids of both species revealed similar general composition: i) their free sterol/PL ratio was above 0.4 and ii) their PL were predominated by the diacyl+alkyl forms of glycerophosphatidylcholine (PC), the plasmalogen form of glycerophosphatidylethanolamine (PE) and ceramide aminoethylphosphonate (CAEP). Free sterols were predominated by cholesterol in both species. Plasmalogen forms of PE and glycerophosphatidylserine (PS) represented 82-83% and 46-55% of total PE and PS, respectively. When compared to their respective diacyl+alkyl forms, plasmalogen forms of PE and PS were specifically enriched in non-methylene-interrupted (NMI) FA and 20:1n-11, suggesting a functional significance of these PL molecular species in bivalve hemocytes. Lysoglycerophosphatidylcholine (LysoPC) levels were found to be fairly high in hemocytes, accounting for about 8% of the PL. Some species-specific features were also found. LysoPC and glycerophosphatidylinositol (PI) FA compositions differed between Ruditapes philippinarum and Crassostrea gigas. CAEP proportion was higher in R. philippinarum than in C. gigas (14.5% and 27.9% of the PL, respectively). Hemolymph cell monolayer observations and flow-cytometric analyses revealed species-specific hemocyte morphology and sub-populations which could account for some of the observed species-specific membrane lipid compositions.     

Influence of dietary lipids upon lipids in larvae and adults of the dried-fruit moth

The dried-fruit moth was reared on three diets differing in quantity and types of fatty acids. Triacylglycerols (TAG) were the major lipid present in both larvae and adults and their fatty acid compositions reflected dietary fatty acids to some extent. However, larval and adult contained relatively high levels of 16:0, 16:1, and 18:1 even when these acids were not predominant in the dietary lipid. Diacylglycerols were present in larvae and adults at lower levels than TAG and their fatty acid compositions were similar to those of the corresponding TAG.Adults contained more phospholipid (PL) and cholesterol than larvae regardless of diet employed. Phosphatidyl-ethanolamine and phosphatidyl-choline were the major PL present with appreciable levels of diphosphatidyl-glycerol and lesser amounts of lysophosphatidyl-ethanolamine and choline, sphingomyelin, phosphatidyl-serine, and inositol being present. The higher level of PL noted in the adults were due to a general increase in all of the individual phospholipids and not to any specific PL. Dietary fatty acids did influence fatty acid compositions of larval and adult PL. However, there appeared to be compensations in the proportions of other fatty acids in the PL in that high 18:1 was accompanied by lowered levels of 18:2 and high 18:2 was accompanied by lowered levels of saturated fatty acids.In every case adult PL contained more polyenoic fatty acids than did larval PL. There were no apparent compensations in other fatty acids accompanying these changes.     

The highly unnatural fatty acid profile of cells in culture

The fatty acid profile of cells in culture are unlike those of natural cells with twice the monounsaturated (MUFA) and half the polyunsaturated fatty acids (PUFA) level (Mol%). This is not due to cell lines primarily being derived from cancers but is due to limited access to lipid and an inability to make PUFA de novo as vertebrate cells. Classic culture methods use media with 10% serum (the only exogenous source of lipid). Fetal bovine serum (FBS), the serum of choice has a low level of lipid and cholesterol compared to other sera and at 10% of media provides 2–3% of the fatty acid and cholesterol, 1% of the PUFA and 0.3% of the essential fatty acid linoleic acid (18:2n-6) available to cells in the body. Since vertebrate cell lines cannot make PUFA they synthesise MUFA, offsetting their PUFA deficit and reducing their fatty acid diversity. Stem and primary cells in culture appear to be similarly affected, with a rapid loss of their natural fatty acid compositions. The unnatural lipid composition of cells in culture has substantial implications for examining natural stems cell in culture, and for investigations of cellular mechanisms using cell lines based on the pervasive influence of fats.     

Fatty acid composition and antioxidant activity of Antarctic marine sponges of the genus <Emphasis Type="Italic">Latrunculia</Emphasis>

The fatty acid composition of marine cold-water sponges of genus Latrunculia, namely Latrunculia bocagei Ridley and Dendy, 1886, and Latrunculia biformis Kirkpatrick, 1907, for which no fatty acid data had been previously reported, has been examined. High levels of long-chain fatty acids (C_24–30) with high unsaturation levels (mainly polyunsaturation), and high incidence of branched- and odd-chain fatty acids in sponges are suggested to be partially connected with their specific cell membrane requirements to surmount the negative effects of low temperatures and to enable growth in extreme environments. Furthermore, variations in the fatty acid profile at the species level may reflect variations in compositions or quantities of symbiotic microorganisms that commonly represent up to 60 % of the sponge wet weight. The fatty acid compositions of lipid-rich sponge extracts from five Latrunculia specimens dredged from the Bransfield Strait near the Antarctic Peninsula, were obtained using supercritical carbon dioxide and analyzed using gas chromatography–mass spectrometry–flame ionization detection. Furthermore, the antioxidant activities of these extracts were determined using the photochemiluminescent method. Along with common fatty acids, the chemical analyses revealed very long-chain fatty acids (C22, C24). Differences were seen for the fatty acid levels between both species and different specimens of the same species. The observed differences do not seem connected to the habitat depth of the specimen, but rather indicate the variations within the associated microbiome. Furthermore, these sponge extracts showed antioxidant activities, confirming that they contain lipidic compounds that strongly scavenge free radicals.     

The fatty acids of some marine teleosts

The lipids of five mesopelagic species of myctophid, two mesopelagic species of stomia-toid, and one epipelagic species of Macrorhamphosidae from the eastern-North Atlantic have been examined by thin-layer chromatography and their fatty acid compositions have been determined by gas-liquid chromatography.     

Structural and functional conservation and divergence among acyl-CoA desaturases of two noctuid species, the corn earworm, Helicoverpa zea, and the cabbage looper, Trichoplusia ni

In this report, we describe the structural and functional analyses of four acyl-CoA desaturase-encoding cDNAs that we isolated from RNA expressed in the pheromone gland of the corn earworm, Helicoverpa zea. We deduced the homology relationships of the encoded proteins, designated HzPGDs1, HzPGDs2, HzPGDs3 and HzFBDs, to each other and to previously described desaturases of the cabbage looper moth, Trichoplusia ni, the fly, Drosophila melanogaster, and other more distantly related organisms. We also isolated genomic DNA fragments of the four H. zea desaturase-encoding genes, determined the locations of introns present in them, and compared them to conserved intron positions in reported desaturase genes of other species. We measured the levels of the four desaturase mRNAs in H. zea pheromone glands and larval fat bodies by RT-PCR. We established the functional identities of the deduced proteins HzPGDs1 and HzPGDs2, encoded by the two desaturase mRNAs that are differentially and abundantly expressed in pheromone glands of sexually mature adult H. zea females, by functional expression of their encoding cDNAs in a desaturase-deficient mutant, ole1, of the yeast Saccharomyces cerevisiae. We compared the unique unsaturated fatty acid profiles of HzPGDs1- and HzPGDs2-expressing transformants to those of strains expressing previously described Delta11 and Delta9 desaturases of T. ni.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Highly conserved piggyBac elements in noctuid species of Lepidoptera

The piggyBac transposable element was originally discovered in a Trichoplusia ni cell line and nearly identical elements were subsequently discovered in the tephritid fly, Bactrocera dorsalis. This suggested the existence of piggyBac in additional insects and this study shows highly conserved, though not identical, piggyBac sequences in the noctuid species Heliocoverpa armigera, H. zea, and Spodoptera frugiperda, as well as new piggyBac sequences from the T. ni organismal genome. Genomic piggyBac elements could not be unambiguously identified in several other moth species indicating a discontinuous presence of piggyBac in the Lepidoptera. Most sequences have greater than 95% nucleotide identity to the original IFP2 piggyBac, except for a more diverged sequence in S. frugiperda, having approximately 78% identity. Variants of 1.3 and 0.8kb sequences found in both H. armigera and H. zea most likely became established by interbreeding, supporting the notion that the species are conspecific. None of the independent piggyBac sequences isolated from T. ni larval genomes are identical to IFP2, though all have an uninterrupted reading frame with the potential for encoding a functional transposase. The piggyBac sequences from T. ni and the Helicoverpa species, as well as those previously reported from B. dorsalis, all share three common nucleotide substitutions resulting in a single amino acid substitution in the transposase. This suggests that the original IFP2 piggyBac is a related variant of a predecessor element that became widespread. The existence of conserved piggyBac elements, some of which may have been transmitted horizontally between lepidopteran species, raises important considerations for the stability and practical use of piggyBac transformation vectors.     

Incorporation and modification of dietary fatty acids in gill polar lipids by two bivalve species Crassostrea gigas and Ruditapes philippinarum

Two bivalve species Crassostrea gigas and Ruditapes philippinarum were fed eight weeks with three mono-specific algae diets: T-Isochrysis galbana, Tetraselmis suecica, Chaetoceros calcitrans, selected on the basis of their polyunsaturated fatty acid (PUFA) composition. The incorporation and the modification of dietary fatty acids in C. gigas and R. philippinarum gill lipids were analysed and compared. Essential PUFA (20:4n-6, 20:5n-3 and 22:6n-3) and non-methylene interrupted PUFAs (known to be synthesised from monounsaturated precursors) contents of gill polar lipid of both species were greatly influenced by the dietary conditioning. Interestingly, oysters and clams responded differentially to the mono-specific diets. Oysters maintained higher 20:5n-3 level and higher 22:2j/22:i and n-7/n-9 ratio in gill polar lipids than clams. To better discriminate dietary and species influences on the fatty acid composition, a Principal Component Analysis followed by a MANOVA on the two most explicative components was performed. These statistical analyses showed that difference in fatty acid compositions attributable to species were just as significant as the diet inputs. The differences of gill fatty acid compositions between oysters and clams are speculated to result of an intrinsic species characteristic and perhaps of a group characteristic: Fillibranch vs. Eulamellibranch.     

Differences in tissue-specific lipid composition between embryos of wild and captive breeding alligators (Alligator mississippiensis)

Fertile eggs obtained from alligators reared in captivity typically exhibit high rates of embryonic mortality. Also, the fatty acid composition of the yolk lipid of the captive eggs is markedly different from that observed in eggs from wild alligators, possibly as a result of differences in maternal diet in the two situations. The fatty acid compositions of tissue lipids during the embryonic development of wild and captive alligators were compared. The lipids of liver, adipose tissue and heart of the two types of embryo displayed fatty acid profiles which generally reflected the acyl compositions of the respective yolks. Thus the lipids from these tissues of the captive embryos contained markedly higher proportionate levels of linoleic and linolenic acids, lower levels of palmitoleic acid, and, in general, lower levels of docosahexaenoic acid and other C20 and C22 polyunsaturates, in comparison to the values for the wild embryos. In contrast, the fatty acid composition of the brain phosphoglycerides was very similar in the two types of embryo. Thus, at least in those embryos which had survived during the developmental period studied, the brain was able to maintain a relatively constant fatty acid composition, in spite of major differences between the wild and captive eggs in the proportions of the various fatty acids supplied from the yolk. It is suggested that a major cause of embryonic mortality in the captive embryos could be a failure to maintain an adequate level of docosahexaenoic acid in the developing brain.     

Molecular characterization of immune inhibitor A, a secreted virulence protease from Bacillus thuringiensis

The gene for the secreted neutral metalloprotease, immune inhibitor A (InA), from Bacillus thuringiensis var. alesti has been cloned and sequenced. The deduced amino acid sequence has been confirmed by partial amino acid sequencing. The central part of the amino acid sequence showed similarity to the active site in thermolysin. Southern and Western blots show that InA-related sequences are common among other B. thuringiensis subspecies. In Western blots, 17 out of 25 tested species gave a positive signal. Culture filtrates from subspecies expressing InA were toxic when injected in Trichoplusia ni larvae, whereas filtrate from a strain negative in Western blot had no effect when injected. The LD50 dose of purified InA protein injected in T. ni larvae was 12.5 +/- 2.5 ng per mg of larval body weight.     

Changes in tissue and mitochondrial membrane composition during rapid growth, maturation and aging in rainbow trout, Oncorhynchus mykiss

Membrane compositions, particularly of mitochondria, could be critical factors in the mechanisms of growth and aging processes, especially during phases of high oxidative stress that result in molecular damage. In the present study, liver and mitochondrial membrane phospholipid (PL) compositions were analyzed in rainbow trout during its four first years of life, a period characterized by rapid growth and high oxidative stress. Specifically, farmed fish of three ages (1-, 2- and 4-years) were studied, and PL compositions of whole liver and liver mitochondria, and fatty acid compositions of individual PL classes were determined. Liver mitochondrial membranes showed a PL composition different to that of the whole tissue suggesting adaptation of cell and subcellular membranes to specific functions. Individual PL had characteristic fatty acid compositions that were similar in whole liver and mitochondrial membranes. Whole liver and mitochondria showed increased lipid peroxidation with age along with changes in membrane PL fatty acid compositions. Most PL classes showed similar changes in fatty acid composition among the age groups, with reduced proportions of docosahexaenoic acid (DHA) and, generally, concomitantly increased levels of monounsaturated fatty acids, which together resulted in reduced peroxidation index (PIn). However, total polyunsaturated fatty acid (PUFA) content did not change significantly with age due to increased eicosapentaenoic acid, docosapentaenoic acid and, in most PL, increased n−6 PUFA. These results suggest there may be oxidation of PL DHA with compensatory mechanisms to maintain membrane fluidity and function. However, modification of fatty acid composition of specific PLs, such as cardiolipin, could affect the electron transport chain efficiency and propagate the oxidative reaction throughout the cell. In addition, both the content and fatty acid composition of sphingomyelin, which has been suggested as a possible mediator of cell dysfunction and apoptosis, changed with age differently to the other PL classes. Moreover, these changes showed different trends between mitochondria and whole liver. These data suggest there is marked oxidative stress associated with rapid growth and maturation in rainbow trout. Changes observed in membrane lipids point to their possible participation in the processes involved in this species response to oxidative stress and damage accumulation rate.     

Fatty acid composition remains stable across trophic levels in a gall wasp community

Acquiring sufficient nutrients is particularly important for insects that are unable to synthesize certain nutrient types de novo, as is the case for numerous parasitoid species that do not synthesize lipids. The lipid reserves of parasitoids are acquired from a single host during larval development. This imposes constraints on the quantity and quality of available lipids. In the present study, the lipid dynamics throughout the trophic cascade are investigated by measuring lipogenic ability, modifications in fatty acid composition and host exploitation efficiency in species at different trophic positions within the community of parasitoids associated with the gall wasp Diplolepis rosae L. (Hymenoptera: Cynipidae). The results obtained show that lipid levels remain stable or decline after feeding in all species, indicating that none of the wasps synthesize lipids. Fatty acid composition is highly similar between the gall wasp, parasitoid and hyperparasitoid species, with the exception of the parasitoid Orthopelma mediator Thunberg (Hymenoptera: Ichneumonidae). The divergence of fatty acid composition in O. mediator suggests that this species is able to modify its fatty acid composition after the consumption of host lipids. The efficiency of exploitation of host resource, in terms of dry body mass acquired, varies among the species (41–70%), although it is high overall compared with the efficiencies reported in other animals. Hence, for parasitoid wasps that lack lipid synthesis capabilities, the efficiency of host exploitation is high and fatty acids are consumed directly from the host without modification, leading to stable fatty acid compositions throughout the trophic cascade.     

Fatty Acid Compositions of Paracolons: Arizona, Citrobacter, and Providencia

The fatty acid compositions of stationary-phase cultures of Arizona arizonae, Citrobacter freundii, Providencia alcalifaciens, Providencia stuartii, and Providencia sp. were studied. The major fatty acids of A. arizonae, C. freundii, and Providencia were 16:0, 16:1, 17:cyclopropane, and 19:cyclopropane. The fatty acid compositions of the two strains of A. arizonae examined were similar to each other, but the three strains of C. freundii differed from one another in their fatty acid compositions. In both A. arizonae and C. freundii, the relative quantities of saturated, unsaturated, and cyclopropane fatty acids were similar to those which have been found in stationary-phase cultures of other members of the Enterobacteriaceae. The three strains of Providencia also differed from one another in their fatty acid compositions. In all three strains, the total quantity of unsaturated fatty acids was larger and that of the cyclopropane fatty acids was smaller than those found in stationary-phase cultures of other enteric bacteria.     

Variations in the Lipid Compositions and in the Lipid Molecular Species of Lipomyces starkeyi Cultivated in the Glucose Sufficient and the Glucose Deficient Media

The lipid compositions, fatty acid compositions, positional distributions of fatty acids in glycerides, and molecular species of phospholipids of L. starkeyi, cultured in the glucose sufficient and the glucose deficient media were compared.

Under the glucose sufficient condition, the triglyceride content increased, accompanied by the remarkable increase of C_16:0–C_18:1–C_18:0. The phospholipid content also increased with the variations of the compositions of molecular species in phosphatidylethanolamine and phosphatidylcholine.

Under the glucose deficient condition, the triglyceride content remarkably decreased, especially in C_18:1–C_18:1–C_18:1. The compositions of phospholipid molecular species were considerably different from those of the glucose sufficient condition.     

Screening of Red Algae Filaments As a Potential Alternative Source of Eicosapentaenoic Acid

Lipids were extracted by a supercritical fluid extraction method from 10 species of filamentous red algae obtained from culture collections and their fatty acid compositions were determined. The fatty acid profiles of the 10 species were similar. The major fatty acids were 16:0, 20:4ω6 and 20:5ω3 (eicosapentaenoic acid, EPA), which amounted to over 70% of the total fatty acids. The highest EPA content (29.8 mg/L), as a percentage of total fatty acids, was produced by Liagora boergesenii filaments, which has good potential for EPA mass production in pilot plants.     

The relationship between the fatty acid profiles of the yolk and the embryonic tissue lipids: A comparison between the lesser black backed gull (Larus fuscus) and the pheasant (Phasianus colchicus)

Although substantial information is available regarding the fatty acid composition of lipids of the yolk and of the developing tissues of the chicken embryo, there is little knowledge on this topic for other avian species. The aim of the present study was to compare the yolk and embryonic tissue fatty acid profiles for a species selecting its food in the wild (the lesser black backed gull) with one fed on a standard commercial diet (the commercially reared pheasant). The fatty acid compositions of the yolk lipids were determined, and major differences were observed between the two species. In particular, the phospholipid of the gull yolk was enriched in 20:4n-6 and 22:6n-3 (18.8 and 7.1%, respectively, by weight of total fatty acids) in comparison with the pheasant (4.0 and 4.1%, respectively). The fatty acid compositions of the embryonic tissues were determined using eggs incubated in the laboratory. For the liver and heart, the fatty acid composition of the lipids in the two species reflected the initial yolk composition, with the gull tissue lipids generally containing higher proportions of 20:4n-6 and 22:6n-3 than those of the pheasant. In contrast, the fatty acid profiles of the brain phospholipid were essentially identical in the two species, with 20:4n-6 and 22:6n-3 comprising approximately 9 and 17%, respectively, of total fatty acids in both cases.