The fatty acid profile changes in marine invertebrate larval cells during cryopreservation

The development of cryopreservation methods for embryonic cells and larvae of sea animals offers a great potential for marine biotechnology. Larval cells of bivalves and sea urchins were frozen to −196 °C using traditional cryoprotectants (Me₂SO and trehalose) and the cryoprotective mixture developed by us. In addition to Me₂SO and trehalose, this mixture contained an exogenous lipid extract from mussel tissues and antioxidants. A positive effect of antioxidants (α-tocopherol acetate, ascorbic acid or echinochrome, the quinoid pigment of sea urchins) on cell viability became significant only in the presence of exogenous lipids. Antioxidants added to cryoprotective mixtures did not reveal visible cryoprotective activity when used separately. To better understand the mechanism of the protective effect of exogenous lipids on cell membranes of sea animals, a comparative analysis of the fatty acid (FA) composition of total lipids in larval cells before and after freezing was carried out using a gas–liquid chromatography. The results indicate that freezing–thawing has direct effects on the FA composition of major lipid classes in marine invertebrate cells, and these effects can vary depending on the provenance of the cells. We have found that (I) both cell viability and the FA profile of cell lipids after cryopreservation depend on the cryoprotectants used; (II) an amount of saturated, monoenic and polyenic FAs changes significantly after cryopreservation. We assume that the addition of the exogenous lipid extract in form of liposomes could promote a renewal of disturbance areas and prevent from membrane damages during freezing–thawing.     

Effectors of fatty acid synthesis in hepatoma tissue culture cells

An investigation was undertaken to better understand the process of fatty acid synthesis in hepatoma tissue culture (HTC) cells. By comparing the findings to the normal liver some of the differences between normal and cancer tissue were defined. Incubation of the HTC cells in a buffered salt-defatted albumin medium showed that fatty acid synthesis was dependent upon the addition of substrate. The order of stimulation was glucose + pyruvate ～- glucose + alanine ～- glucose + lactate ～- pyruvate > glucose > alanine ? no additions. Fatty acid synthesis in HTC cells was decreased by oleate. In these respects HTC cells are similar to the liver; however, in contrast to the normal liver, N⁶, O²-dibutyryl cyclic adenosine 3′,5′-monophosphate (dibutyryl-cAMP) did not inhibit glycolysis or fatty acid synthesis. The cytoplasmic redox potential, as reflected by the lactate to pyruvate ratio, was found to be elevated compared to normal liver but unchanged by the addition of dibutyryl cAMP. Since higher rates of fatty acid synthesis are associated with lower lactate-to-pyruvate ratios in normal liver, it was expected that by decreasing the lactate-to-pyruvate ratio in HTC cells the rate of fatty acid synthesis would increase. One way to lower the lactate to pyruvate ratio is to increase the activity of the malate-aspartate shuttle. Stimulators of the hepatic malate-aspartate shuttle in normal liver (ammonium ion, glutamine, and lysine) had mixed effects on the redox state and fatty acid synthesis in HTC cells. Both ammonium ion and glutamine decreased the redox potential and increased the rate of fatty acid synthesis. Lysine was without effect on either process. Since NH₄Cl and glutamine stimulate the movement of reducing equivalents into the mitochondria and decrease the redox potential, then the stimulation of fatty acid synthesis by NH₄Cl and glutamine may be due to an increase in the movement of reducing equivalents into the mitochondria. However, if the shuttle were rate determining for fatty acid synthesis the rate from added lactate would be the same as from glucose alone but would be lower than from pyruvate which does not require the movement of reducing equivalents. This was not the case. Lactate and pyruvate gave comparable rates which were higher than glucose alone. Other possible sites of stimulation were investigated. The possibility that NH₄⁺ and glutamine stimulated fatty acid synthesis by activating pyruvate dehydrogenase was excluded by finding that dichloroacetate, an activator of pyruvate dehydrogenase, did not stimulate fatty acid synthesis when glucose was added. Stimulation by NH₄⁺ and glutamine at steps beyond pyruvate dehydrogenase was ruled out by the observation that NH₄⁺ caused no stimulation from added pyruvate. NH₄⁺ and glutamine did not alter the pentose phosphate pathway as determined by ¹⁴CO₂ production from [1-¹⁴C]- or [6-¹⁴C]glucose. Ammonium ion and glutamine increased glucose consumption and increased lactate and pyruvate accumulation. The increased glycolysis in HTC cells appears to be the explanation for the stimulation of fatty acid synthesis by NH₄⁺ and glutamine, even though glycolysis is much more rapid than fatty acid synthesis in these cells. The following observations support this conclusion. First, the percentage increase in glycolysis caused by NH₄⁺ or glutamine is closely matched by the percentage increase in fatty acid synthesis. Second, the malate-aspartate shuttle, the pentose phosphate pathway, and the steps past pyruvate are not limiting in the absence of NH₄⁺ or glutamine.     

Polyunsaturated fatty acid metabolism in neuroblastoma cells in culture.

Neuroblastoma cell cultures took up linoleic and linolenic acids at approximately equal rates, and incorporated them into a variety of lipid fractions, principally cellular phospholipids. Linoleic acid was preferentially incorporated into choline phosphoglycerides, while most of the radioactivity derived from linolenic acid entered ethanolamine phosphoglycerides. There was no evidence for direct transfer of fatty acids between these two phosphoglyceride fractions. When, after the addition of cytosine arabinoside, cell division was arrested, the entry of labelled fatty acids into ethanolamine and serine phosphoglycerides was reduced, suggesting that these lipids are involved in the formation of new cell membranes. In the ethanolamine phosphoglyceride fraction, phosphatidal ethanolamine (plasmalogen) was the principal acceptor for the higher polyunsaturated fatty acids of the φ 3 series. The ratio of labelled fatty acids entering ethanolamine plasmalogens to that entering ethanolamine phosphoglycerides increased following the addition of cytosine arabinoside, suggesting plasmalogens to be involved in formation of cell processes. The first step in the metabolism of both linoleic and linolenic acid was the addition of a two-carbon unit. Conversion of linoleic acid to higher polyunsaturated fatty acids was slower than the conversion of linolenic acid to its higher analogues. This contrasted with the behaviour of dissociated cultures of normal brain cells which were able to form higher analogues of linoleic and linolenic acids at nearly equal rates.     

The phospholipids of lamellar body material from fetal rabbit lung tissue in culture. Unusual phosphatidylcholine fatty acid profile

Tissue pieces of rabbit fetal lung, 23 days gestation, were cultured for 7 days in serum-free medium to obtain lamellar body material for phospholipid analysis. Cultures were maintained in culture medium without serum and (1) with no added hormones (control cultures), (2) with thyroxine (1 x 10(-7) M), (3) with cortisol (1 x 10(-7) M) and (4) with thyroxine plus cortisol (1 x 10(-7) M each). The hormonal response was evaluated by measuring the quantity of lamellar body material isolated from the tissue pieces after the 7-day culture period. Compared to control cultures, more lamellar body material was recovered from cultures treated with cortisol (180% of control) and with thyroxine plus cortisol (250% of control). Cultures treated with thyroxine alone yielded the same amount of lamellar body material as the controls. Hormone treatment produced only minor changes in the glycerophospholipid profile of the lamellar body material. A small but significant increase in the percentage of phosphatidylglycerol and a small but significant decrease in phosphatidylinositol were found in lamellar body material from cultures treated with thyroxine and thyroxine plus cortisol. The disaturated phosphatidylcholine content of the lamellar body material from culture was 28% of the total lamellar body phospholipid and was not affected by hormone treatment. This disaturated phosphatidylcholine content was low compared to the disaturated phosphatidylcholine of lamellar body material from adult lung (46%). The low proportion of disaturated phosphatidylcholine was due to the unusual presence of palmitoleic acid (16:1(cis-9)), which was more than one-fourth of the total fatty acid of the lamellar body phosphatidylcholine. It is possible that an abnormal delta 9 fatty acid desaturation activity was expressed in the lung tissue in vitro, which resulted in the high incorporation of the 16:1 fatty acid into lamellar body phosphatidylcholine.     

Variation of fatty acid profile with solar cycle in outdoor chemostat culture ofIsochrysis galbana ALII-4

Outdoor chemostat cultures of the marine microalgaIsochrysis galbana at constant dilution rate (0.034 h^–1 ) have been carried out under different weather conditions. Steady-state biomass concentrations were 1.61±0.03 kg m^–3 in May and 0.95±0.04 kg m^–3 in July, resulting in biomass output rates of 0.54 kg m^–3 d^–1 and 0.32 kg m^–3 d^–1 in May and July, respectively. Two patterns of daily variation with the solar cycle were observed in the fatty acid content. Saturated and mono-unsaturated fatty acids (16:0 and 16:1n7) show significant variation with the solar cycle, associated with short-term changes in environmental factors. Palmitic and palmitoleic acids are generated during daylight and consumed during the dark period. However, polyunsaturated fatty acids do not show a significant response to the solar cycle and their changes are associated with long-term variation in environmental factors. The maximum EPA productivity was obtained in May, 14.1 g m^–3 d^–1, which is close to that found in the literature for indoor continuous cultures. Nonetheless, the outdoor EPA content (up to 2.61 % d.wt) was lower than the indoor EPA content from a previous study (5% d.wt).     

Influence of testosterone on polyunsaturated fatty acid biosynthesis in Sertoli cells in culture

Sertoli cells play a central role in spermatogenesis, its development and regulation. They are target cells for androgen action in the seminiferous tubules. The Sertoli cell is considered to be the most important cell type in the testis with regard to essential fatty acid metabolism. We studied the response to testosterone of cultured Sertoli cells from immature rats by determining the fatty acid composition of total cellular lipids as well as by the biosynthesis of polyunsaturated fatty acids. Fatty acid methyl esters were analysed by gas liquid chromatography and radiochromatography. Two doses of testosterone were tested (150 and 300 ng ml(-1)). Significant differences were found in fatty acids derived from total cellular lipids after 8 days in culture in the presence of testosterone (300 ng ml(-1), for 48 h). Compared to controls, the hormone produced a significant increase of 16:1 and 18:1 n-9, and of 18:2 n-6, and a decrease of 20:4 and 22:5 n-6 in total cellular lipids. The decrease in the n-6 fatty acid ratios 20:4/20:3, 20:4/18:2 and 24:5/24:4, and the increase in 18:1n-9/18:0 and 16:1n-9/16:0 ratios were taken as an indirect signal of testosterone effects on Delta5, Delta6 and Delta9 desaturase activities. The drop in Delta5 and Delta6 desaturase activities was corroborated by analysing the transformation of [1-14C]20:3 n-6 into its higher homologues. We concluded that testosterone modifies the fatty acid pattern of cultured Sertoli cells, and this hormone is involved in polyunsaturated fatty acid biosynthesis, modulating Delta5 and Delta6 desaturases activity.     

Carnitine uptake and fatty acid utilization by diploid cells aging in culture

Uptake of carnitine by cultured human fetal lung flbroblasts (WI-38 and IMR-90) and by smooth muscle cells from calf aorta and from human uterus was found to be temperature dependent and saturable. IMR-90 cells showed an apparent K_m of 6–8 μM and a V of 21–28 pmol/h/10⁶ cells for l-carnitine. Transport was abolished by N-ethylmaleimide and was inhibited variably by octanoyl-d-carnitine, d-carnitine, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Although WI-38 and IMR-90 cells accumulate lipids as they age in culture, they take up carnitine as rapidly as do smooth muscle cells of aorta and uterus that do not exhibit such accumulation. Comparison of the rates of carnitine uptake by IMR-90 fibroblasts during the logarithmic phase of growth shows no difference between “young” and “old” cultures. In contrast, when confluent or postconfluent monolayers were compared and uptake expressed as a function of cell number, cells grown from late passages took up carnitine more rapidly than did cells grown from early passages. However, when account was taken of cell size, and carnitine expressed as a function of cell volume, the differences in carnitine uptake between early and late passages were no longer apparent for the confluent or postconfluent monolayers examined. Moreover, late passage fibroblasts took up and oxidized radioactive palmitate at least as rapidly as did cells from early passages. Our results suggest that accumulation of lipid in aging fibroblasts is not due to decreased carnitine uptake or fatty acid oxidation.     

Lipases in organic solvents: The fatty acid chain length profile

Summary Competitive lipase-catalysed hydrolyses of triacylglycerols were used for the quantitative determination of the fatty acid profile of several lipases. In contrast to previously described methods a complete and reliable activity profile for triacylglycerols containing fatty acid residues with chain lengths from C₄ to C₁₈₁ can be obtained in one single experiment.     

Carcinogenesis alters fatty acid profile in breast tissue

Cancerogenesis is associated with cell membrane changes. The aim of this study was to investigate whether breast tissues with different degrees of cancer involvement have different fatty acid profiles. Fourteen breast cancer patients with a mean age of 61 years were recruited. Morphological features of the tumoral specimens were characterized. Approximately 60 % of patients had invasive ductal carcinoma, and 80 % were ER positive; 65 % were PR positive; and 65 % were HER2 negative. The segments with confirmed cancer had significantly less amounts of total lipids as compared with the corresponding grossly normal or interface tissues. The fatty acid profile in cancer tissue was significantly different from that in other tissues. Fatty acid composition of five classes of phospholipids revealed the variations between cancer tissue and the other two segments. A transition of changes in fatty acid composition in these fractions of phospholipids was observed. The interface tissue had intermediate amounts of several fatty acids including palmitic acid, stearic acid, and arachidonic acid. Interestingly, we observed significantly higher amounts of the n-3 fatty acid DHA in cancer tissue as compared to the other two tissues. Data from this study will provide evidence that biochemical changes particularly phospholipid composition may take place well in advance prior to morphological changes. Should this theory be confirmed by larger studies, deviation of phospholipid composition from normal values can be used as markers of susceptibility of tissue to cancer development.     

Beta-adrenergic stimulation of fatty acid release from brown fat cells differentiated in monolayer culture

The ability of brown adipocytes differentiated in monolayer culture to respond to norepinephrine was investigated. It was found that fatty acid release in confluent brown adipocytes in monolayer culture was induced by norepinephrine, thus these cells were hormone-sensitive. After confluence, the rate of fatty acid release successively declined. The norepinephrine-stimulated fatty acid release was inhibited by propranolol, but not by phentolamine, indicating a mediation via beta-adrenergic receptors. It was concluded that there exist in the brown adipose tissue of nonfetal rats preadipocytes which possess the ability to express in culture a fully developed beta-adrenergic lipolytic response.     

Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.     

Bicuculline induces free fatty acid release from phospholipids in Neuro-2A cells in culture

This study characterizes free fatty acid release in a neuroblastoma cell line (Neuro-2A), a potential model system for the study of factors that control phospholipase A₂ in neurons. Two compounds, bicuculline (an antagonist at -aminobutyric acid receptors), and A23187 (a Ca²⁺ ionophore), were examined. The release of endogenous fatty acids and the turnover of radiolabeled arachidonic and docosahexaenoic acids were measured. The cells actively incorporated radiolabeled fatty acids into various glycerolipid pools. Both endogenous fatty acids and radiolabeled fatty acids were released from glycerolipids in a time-dependent manner. Phosphatidylcholine was a major source of released fatty acids. Release of free fatty acids was markedly stimulated by both bicuculline and A23187. We conclude that the Neuro-2A cell contain phospholipase activity that is sensitive to Ca²⁺ ionophore and bicuculline, and may provide a good system for further studies on the regulation of phospholipase A₂ in neurons.Abbteviations 160 palmitic acid - 180 stearic acid - 181 oleic acid - 182 linoleic acid - 183 linolenic acid - 204 arachidonic acid - 226 docosahexaenoic acid - DG diacylglycerol - FAME fatty acid methyl ester - FFA free fatty acid - GABA -aminobutyric acid - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - TG triacylglycerol     

Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells

In this study we demonstrate that polysialyltransferases are capable of accepting unnatural substrates in terminally differentiated human neurons. Polysialyltransferases catalyze the glycosylation of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA). The unnatural sialic acid analog, N-levulinoyl sialic acid (SiaLev), was incorporated into cell surface glycoconjugates including PSA by the incubation of cultured neurons with the metabolic precursor N-levulinoylmannosamine (ManLev). The ketone group within the levulinoyl side chain of SiaLev was then used as a chemical handle for detection using a biotin probe. The incorporation of SiaLev residues into PSA was demonstrated by protection from sialidases that can cleave natural sialic acids but not those bearing unnatural N-acyl groups. The presence of SiaLev groups on the neuronal cell surface did not impede neurite outgrowth or significantly affect the distribution of PSA on neuronal compartments. Since PSA is important in neural plasticity and development, this mechanism for modulating PSA structure might be useful for functional studies.     

The fatty acid profile of vegetative Azotobacter vinelandii ATCC 12837: growth phase-dependence

Fatty acids of Azotobacter vinelandii ATCC 12837 were determined at various times during aerobic vegetative growth at 30°C to provide baseline data for studying the effects of chemical agents on the organism’s survival and fatty acid biosynthesis. Palmitate (16:0) was the highest at 36.7±4.3 mol% (mean±SD) after the first 5 h in fresh culture, decreasing slightly to 33.4±2.6 mol% at 49 h. The other fatty acids were therefore each normalized as a ratio of 16:0. At 5 h, as a ratio of 16:0, myristate (14:0) was 0.14±0.06, palmitoleate (16:1cΔ^9–10) 0.13±0.06, oleate (18:1cΔ^9–10) 0.21±0.12, cis-vaccenate (18:1cΔ^11–12) 0.30±0.17 and stearate (18:0) 0.68±0.02. As the growth phase advanced to 49 h, 14:0 and 16:1cΔ^9–10 increased, 18:1cΔ^9–10 decreased and cis-vaccenate reciprocally increased, whereas 18:0 decreased. These suggest that the saturated fatty acid biosynthesis pathway yielded 16:0 and 18:0 in the 5-h lag period. By desaturation, 18:0 formed the unsaturated fatty acid (UFA) 18:1cΔ^9–10. As the culture aged, the anaerobic UFA biosynthesis pathway formed 16:1cΔ^9–10, which was elongated to 18:1cΔ^11–12. These fatty acid alterations represent a homeoviscous adaptation, modulating the microbe’s membrane lipid viscosity for optimal cellular function.