Changes In Molecular Species Research Articles

Computational Models on Pathological Redox Signalling Driven by Pregnancy: A Review.

Oxidative stress is associated with a myriad of diseases including pregnancy pathologies with long-term cardiovascular repercussions for both the mother and baby. Aberrant redox signalling coupled with deficient antioxidant defence leads to chronic molecular impairment. Abnormal placentation has been considered the primary source for reactive species; however, placental dysfunction has been deemed secondary to maternal cardiovascular maladaptation in pregnancy. While various therapeutic interventions, aimed at combating deregulated oxidative stress during pregnancy have shown promise in experimental models, they often result as inconclusive or detrimental in clinical trials, warranting the need for further research to identify candidates. The strengths and limitations of current experimental methods in redox research are discussed. Assessment of redox status and oxidative stress in experimental models and in clinical practice remains challenging; the state-of-the-art of computational models in this field is presented in this review, comparing static and dynamic models which provide functional information such as protein-protein interactions, as well as the impact of changes in molecular species on the redox-status of the system, respectively. Enhanced knowledge of redox biology in during pregnancy through computational modelling such as generation of Systems Biology Markup Language model which integrates existing models to a larger network in the context of placenta physiology.

Lipidomic Profiling of Host‐Pathogen Interactions Using Untargeted and Targeted Lipidomics Approaches in a Sepsis Cell Culture Model

Sepsis is a life‐threatening organ dysfunction caused by a dysregulated host response to an infection. When pathogens disseminate into the systemic blood circulation from a localized infection, the body initiates a complex series of immune and biochemical reactions. Neutrophils are the main leukocytes that are recruited to an infection site to eliminate bacteria. In sepsis, systemic neutrophil activation is also evident. One of the main pathologies of sepsis is the damage of the endothelial cell integrity by immune stimuli leading to leaky blood vessels. Although numerous research has been conducted on sepsis, the characteristic heterogeneity of sepsis has challenged the identification of specific biomarkers and treatments. We have designed an in vitro co‐culture model of endothelial cells, human neutrophils and, E.coli to identify lipid biomarkers of sepsis. The three cell types in the model allow us to dissect lipidome of individual cell systems under a cell culture sepsis‐like state. EA.hy926 cells were initially incubated with urosepsis E.Coli strain CFT073 and further incubated after adding neutrophils. We compared the lipidome of co‐culture samples with non‐co‐culture samples to identify altered lipid profiles. In addition, we have performed similar experiments with non‐pathogenic JM109 E.Coli strain. We first used an untargeted lipidomics platform with high‐resolution mass spectrometry in both positive and negative ion modes. Potential lipid biomarkers identified in untargeted lipidomics were furthered confirmed by a targeted lipidomics approach using shotgun lipidomics. In CFT073 co‐cultures, lipids were changed mainly in 4 lipid classes; phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine and triglycerides. Minor changes were observed in sphingomyelin, phosphatidylserine and phosphatidylglycerol classes. Several of the changes in molecular species in each lipid class included significant increases in PE 32:1, PE 34:1, PE 32:2, PC 36:0, PC 40:8 and PC 32:0. Lipid profiles were also altered in non‐pathogenic JM109 co‐cultures. Interestingly, there are marked species variations depending on E.coli strain used in co‐cultures such as PC 32:1, PC 36:1 are only elevated in CFT073 co‐cultures. Collectively, our studies demonstrate that lipid profiles are altered during inflammation initiated by bacterial exposure and subsequent neutrophil activation. We have also shown that pathogenicity of bacteria may lead to distinct alterations in lipidomes. These studies represent initial insights into the complex lipidomic alterations during host‐pathogen interactions and may provide new targets to examine as biomarkers and mediators during in vivo sepsis.Support or Funding InformationR01 GM115553, R01 GM129508, U01 ES026458, U01 ES027697, U01 ES028182

Endurance exercise training and high-fat diet differentially affect composition of diacylglycerol molecular species in rat skeletal muscle.

Insulin resistance of peripheral muscle is implicated in the etiology of metabolic syndrome in obesity. Although accumulation of glycerolipids, such as triacylglycerol and diacylglycerol (DAG), in muscle contributes to insulin resistance in obese individuals, endurance-trained athletes also have higher glycerolipid levels but normal insulin sensitivity. We hypothesized that the difference in insulin sensitivity of skeletal muscle between athletes and obese individuals stems from changes in fatty acid composition of accumulated lipids. Here, we evaluated the effects of intense endurance exercise and high-fat diet (HFD) on the accumulation and composition of lipid molecular species in rat skeletal muscle using a lipidomic approach. Sprague-Dawley female rats were randomly assigned to three groups and received either normal diet (ND) in sedentary conditions, ND plus endurance exercise training, or HFD in sedentary conditions. Rats were fed ND or HFD between 4 and 12 wk of age. Rats in the exercise group ran on a treadmill for 120 min/day, 5 days/wk, for 8 wk. Soleus muscle lipidomic profiles were obtained using liquid chromatography/tandem mass spectrometry. Total DAG levels, particularly those of palmitoleate-containing species, were increased in muscle by exercise training. However, whereas the total DAG level in the muscle was also increased by HFD, the levels of DAG molecular species containing palmitoleate were decreased by HFD. The concentration of phosphatidylethanolamine molecular species containing palmitoleate was increased by exercise but decreased by HFD. Our results indicate that although DAG accumulation was similar levels in trained and sedentary obese rats, specific changes in molecular species containing palmitoleate were opposite.

Lipidomic analysis of psychrophilic yeasts cultivated at different temperatures

Analysis of polar lipids from eight psychrophilic yeasts (Cryptococcus victoriae, Cystofilobasidium capitatum, Holtermaniella wattica, Mrakiella aquatica, M. cryoconiti, Rhodotorula lignophila, Kondoa malvinella and Trichosporon aggtelekiense) grown at 4–28°C by hydrophilic interaction liquid chromatography/high resolution electrospray ionization tandem mass spectrometry determined 17 classes of lipids and identified dozens of molecular species of phospholipids including their regioisomers. Most of the yeasts were able to grow over the whole temperature range, reaching the highest biomass at 4 or 10°C. On temperature drop to 4°C, all eight strains showed a significant decrease of MUFA and a simultaneous increase of PUFA such as α-linolenic acid, the content of which in the biomass reached up to 20%. We also found alterations in the proportions of individual phospholipids (PI, PE and PC), the PC/PE-ratio decreasing with decreasing temperature. With increasing temperature the content of PoO-PC rose while that of LL-PC decreased, the drop in the content of LL-PC being nearly 100-fold while the content of PoO-PC increased more than twice.A change in temperature brought about changes in molecular species of PC (molecular species PO-PC versus OP-PC) as well as PE, i.e. PO-PE and OP-PE. The phase transition temperature of PO-PC differs from OP-PC by 7°C and the difference between PO-PE and OP-PE is some 10°C; we thus assume that the cell compensates for the adverse temperature effect by changing the fatty acids in the sn-1 and sn-2 positions.

Submergence induced changes of molecular species in membrane lipids in Arabidopsis thaliana

The composition of membrane lipids is sensitive to environmental stresses. Submergence is a type of stress often encountered by plants. However, how the molecular species of membrane lipids respond to submergence has not yet been characterised. In this study, we used a lipidomic approach to profile the molecular species of membrane lipids in whole plants of Arabidopsis thaliana that were completely submerged for three days. The plants survived one day of submergence, after which, we found that the total membrane lipids were only subtly decreased, showing significant decreases of monogalactosyldiacylglycerol (MGDG) and phosphatidylcholine (PC) and an increase of phosphatidic acid (PA); however, the basic lipid composition was retained. In contrast, three days of submergence caused plants to die, and the membranes deteriorated via the rapid loss of 96% of lipid content together with a 229% increase in PA. The turnover of molecular species from PG and MGDG to PA indicated that submergence-induced lipid changes occurred through PA-mediated degradation. In addition, molecular species of extraplastidic PG degraded sooner than plastidic ones, lyso-phospholipids exhibited various patterns of change, and the double-bond index (DBI) remained unchanged until membrane deterioration. Our results revealed the unique changes of membrane lipids upon submergence and suggested that the major cause of the massive lipid degradation could be anoxia.

Role of aminoalcoholphosphotransferases 1 and 2 in phospholipid homeostasis in Arabidopsis.

Aminoalcoholphosphotransferase (AAPT) catalyzes the synthesis of phosphatidylcholine (PC) and phosphotidylethanolamine (PE), which are the most prevalent membrane phospholipids in all eukaryotic cells. Here, we show that suppression of AAPTs results in extensive membrane phospholipid remodeling in Arabidopsis thaliana. Double knockout (KO) mutants that are hemizygous for either aapt1 or aapt2 display impaired pollen and seed development, leading to embryotic lethality of the double KO plants, whereas aapt1 or aapt2 single KO plants show no overt phenotypic alterations. The growth rate and seed yield of AAPT RNA interference (RNAi) plants are greatly reduced. Lipid profiling shows decreased total galactolipid and phospholipid content in aapt1-containing mutants, including aapt1, aapt1/aapt1 aapt2/AAPT2, aapt1/AAPT1 aapt2/aapt2, and AAPT RNAi plants. The level of PC in leaves was unchanged, whereas that of PE was reduced in all AAPT-deficient plants, except aapt2 KO. However, the acyl species of PC was altered, with increased levels of C34 species and decreased C36 species. Conversely, the levels of PE and phosphatidylinositol were decreased in C34 species. In seeds, all AAPT-deficient plants, including aapt2 KO, displayed a decrease in PE. The data show that AAPT1 and AAPT2 are essential to plant vegetative growth and reproduction and have overlapping functions but that AAPT1 contributes more than AAPT2 to PC production in vegetative tissues. The opposite changes in molecular species between PC and PE and unchanged PC level indicate the existence of additional pathways that maintain homeostatic levels of PC, which are crucial for the survival and proper development of plants.

Tumor necrosis factor-alpha is a potential target for the neuroprotector Dimebon

Since 1983, Dimebon (Dimebolin) is used clinically in Russia as an antihistamine drug. Recent interest in Dimebolin is associated with its therapeutic effect in patients with Alzheimer’s disease. Animal studies have shown that Dimebon activity is realized via multiple mechanisms. Our experiments performed on the fibroblast cell culture L929 and C57Bl mice have been shown that Dimebon may block cytotoxic signals induced by the proinflammatory cytokines, tumor necrosis factor α (TNFα). Dimebon (10 μg/mL) protected mouse fibroblast cells L929 against toxic action of TNFα. Pretreatment of mice with Dimebon prevented development of changes in molecular species of sphingomyelins and galactosylceramides induced by a single dose administration of TNAα. Dimebon itself did not induce changes in sphingolipids of the investigated brain structures.

Plant adaptation to frequent alterations between high and low temperatures: remodelling of membrane lipids and maintenance of unsaturation levels

One major strategy by which plants adapt to temperature change is to decrease the degree of unsaturation of membrane lipids under high temperature and increase it under low temperature. We hypothesize that this strategy cannot be adopted by plants in ecosystems and environments with frequent alterations between high and low temperatures, because changes in lipid unsaturation are complex and require large energy inputs. To test this hypothesis, we used a lipidomics approach to profile changes in molecular species of membrane glycerolipids in two plant species sampled from alpine screes and in another two plant species grown in a growth chamber, with the temperature cycling daily between heat and freezing. We found that six classes of phospholipid and two classes of galactolipid showed significant changes, but the degree of unsaturation of total lipids and of three lysophospholipid classes remained unchanged. This pattern of changes in membrane lipids was distinct from that occurring during slow alterations in temperature. We propose two types of model for the adaptation of plants to temperature change: (1) remodelling of membrane lipids but maintenance of the degree of unsaturation are used to adapt to frequent temperature alterations; and (2) both remodelling and changes in the degree of unsaturation to adapt to infrequent temperature alterations.

Analysis of cell membrane aminophospholipids as isotope-tagged derivatives

Glycerophosphoethanolamine (GPEtn) and glycerophosphoserine (GPSer) lipids were reacted with a multiplexed set of differentially isotopically enriched N-methylpiperazine acetic acid N-hydroxysuccinimide ester reagents, which place isobaric mass labels at a primary amino group. The resulting derivatized aminophospholipids were isobaric and chromatographically indistinguishable but yielded positive reporter ions (m/z 114 or 117) after collisional activation that could be used to identify and quantify individual members of the multiplex set. The chromatographic and mass spectrometric response of N-methylpiperazine amide-tagged aminophospholipids was probed using glycerophosphoethanolamine and glycerophosphoserine lipid standards. The [M+H]+ of each tagged aminophospholipid shifted 144 Da, and during collision-induced dissociation the major fragmentation ion was either m/z 114 or 117. This mode of detecting aminophospholipids was useful for an unbiased analysis of plasmalogen GPEtn lipids. Molecular species information on the esterified fatty acyl substituents was obtained by collisional activation of the [M-H]- ions. The isotope-tagged reagents were used to assess changes in the distribution of GPEtn lipids after exposure of liposomes made from phospholipids extracted from RAW 264.7 cells to Cu2+/H2O2 to illustrate the ability of these reagents to aid in the mass spectrometric identification of aminophospholipid changes that occur during biological stimuli.

Changes in molecular species of triacylglycerols in developing cotton seeds under salt stress

Cotton plants (Gossypium hirsutum L. cv. S4) were grown and irrigated with increasing salt concentrations: 0, 50, 100 and 150 mM NaCl. Lipids from developing seeds, at stages of 3, 4, 5 and 6 weeks after anthesis, were analysed. High salt dose (150 mM NaCl) affected triacylglycerol levels drastically at the end of ripening. HPLC analysis of triacylglycerols in control seeds showed ten molecular species. Palmitodilinolein (PLL) is the major fraction, representing about 25% of total triacylglycerols. The levels of trilinolein (LLL), palmitolinoleo-olein (PLO) and oleodilinolein (OLL) molecules varied between 13 and 16% of the total. The other molecular species: triolein (OOO), dioleopalmitin (POO) and dipalmito-olein (POP) are minor and do not exceed 5%. Moreover, the composition of triacylglycerol molecular species was almost constant during developing stages of control seeds. However, amounts of triacylglycerol species LLL, OLL and palmitodilinolein (PLL), as expressed on a dry-matter basis (mg/g), decreased severely under the highest NaCl concentration, while contents of triacylglycerol species OOO, POO, POP were unchanged. These findings confirm our previous results, which indicated that the amount of linoleic acid in cotton seeds was reduced by salt stress and that the amount of oleic acid remained unchanged.

Changes in molecular species of triacylglycerols in developing cotton seeds under salt stress

Changes in molecular species of triacylglycerols in developing cotton seeds under salt stress

Dietary cholesterol induces changes in molecular species of hepatic microsomal phosphatidylcholine.

After 21 days on a diet containing 1 g% cholesterol and 0.5 g% cholic acid, rats had an increased content of cholesterol in liver microsomal lipids. In liver, both cholesterol content and delta9 desaturase activity increased, whereas delta6 and delta5 desaturase activities decreased. These changes correlated with increases in oleic, palmitoleic, and linoleic acids and decreases in arachidonic and docosahexenoic acids in total microsomal lipids. Similar fatty acid changes were found in phosphatidylcholine (PC), the principal lipid of the microsomal membrane. In PC the predominant molecular fatty acid species (67% of the total) in the control rats were 18:0/20:4, 16:0/20:4, and 16:0/18:2; and they mainly determined the contribution of PC to the biophysical and biochemical properties of the phospholipid bilayer. The cholesterol diet decreased specifically the 18:0/20:4 species, and to a lesser extent, 16:0/20:4 and 18:0/22:6. The 18:1-containing species, especially 18:1/18:2 and less so 16:0/18:1 and 18:1/20:4, were increased. A new 18:1/18:1 species appeared. The independent effects of the presence of cholesterol and change of the fatty acid composition of the phospholipid bilayer of liver microsomes on the packing were studied by fluorescence methods using 6-lauroyl-2,4-dimethylaminonaphthalene, 1,6-diphenyl-1,3,5-hexatriene and 1-(4-trimethylammonium phenyl)-6-phenyl-1,3,5-hexatriene, which test different parameters and depths of the bilayer. Data showed that the increase of cholesterol in the membrane, and not the change of the fatty acid composition of phospholipids, was the main determinant of the increased bulk packing of the bilayer. The increase of fluid oleic- and linoleic-containing species almost compensated for the drop in 20:4- and 22:6-containing molecules. But the most important effect was that the general drop in essential n-6 and n-3 polyunsaturated fatty acids meant that this endogenous source for the needs of the animal decreased.

Roasting influences on molecular species of triacylglycerols in sesame seeds (Sesamum indicum)

Sesame seeds were roasted at different temperatures (180–220 °C) using a domestic electric oven. Molecular species and fatty acid distributions of triacylglycerols (TAGs) isolated from total lipids in the sesame seeds were analysed by a combination of argentation thin-layer chromatography (TLC) and gas–liquid chromatography. A modified argentation TLC procedure, developed to optimise the separation of the complex mixture of total TAGs, provided 11 different groups of TAGs, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester analysis was performed to determine the composition of each zone. Eleven molecular species of TAGs were still detected in the sesame seeds following roasting treatment. Dilinoleolein (33.7–35.8%), palmitoleolinolein (20.3–22.8%), dioleolinolein (15.0–15.4%) and trilinolein (8.8–10.7%) were the main components during roasting. However, roasting for 10 min at 220 °C caused a significant decrease (P < 0.05) not only in molecular species containing more than four double bonds, but also in the amount of diene and triene species present in TAGs (with a few exceptions). These results suggest that no significant changes in molecular species or fatty acid distribution of TAGs would occur within 25 min of roasting at 180 °C, ensuring that a good-quality product would be attained. © 2000 Society of Chemical Industry

Molecular Defects in Sarcolemmal Glycerophospholipid Subclasses in Diabetic Cardiomyopathy

A. Vecchini, F. Del Rosso, L. Binaglia, N. S. Dhalla and V. Panagia. Molecular Defects in Sarcolemmal Glycerophospholipid Subclasses in Diabetic Cardiomyopathy. Journal of Molecular and Cellular Cardiology (2000)32, 1061–1074. Although still scarcely studied, the phospholipid component of the cell membrane is of absolute importance for cell function. Experimental evidence indicates that individual molecular species of a given phospholipid can influence specific membrane functions. We have examined the changes in molecular species of diacyl and alkenylacyl choline/ethanolamine glycerophospholipid subclasses and those of phosphatidylserine in purified cardiac sarcolemma of healthy and streptozotocin-induced insulin dependent diabetic rats without or with insulin treatment. The relative content of plasmalogens increased in all the phospholipid classes of diabetic sarcolemma under study. Phosphatidylcholine and phosphatidylethanolamine were mostly enriched with molecular species containing linoleic acid in sn -2 position and deprived of the molecular species containing arachidonic acid. The molecular species of phosphatidylserine containing either arachidonic or docosahexaenoic acid were less abundant in membranes from diabetic rats than in membranes from controls. Insulin treatment of diabetic rats restored the species profile of phosphatidylethanolamine and overcorrected the changes in molecular species of phosphatidylcholine. The results suggest that the high sarcolemmal level of plasmalogens and the abnormal molecular species of glycerophospholipids may be critical for the membrane dysfunction and defective contractility of the diabetic heart.

Roasting influences on molecular species of triacylglycerols in sesame seeds (Sesamum indicum)

Sesame seeds were roasted at different temperatures (180–220 °C) using a domestic electric oven. Molecular species and fatty acid distributions of triacylglycerols (TAGs) isolated from total lipids in the sesame seeds were analysed by a combination of argentation thin-layer chromatography (TLC) and gas–liquid chromatography. A modified argentation TLC procedure, developed to optimise the separation of the complex mixture of total TAGs, provided 11 different groups of TAGs, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester analysis was performed to determine the composition of each zone. Eleven molecular species of TAGs were still detected in the sesame seeds following roasting treatment. Dilinoleolein (33.7–35.8%), palmitoleolinolein (20.3–22.8%), dioleolinolein (15.0–15.4%) and trilinolein (8.8–10.7%) were the main components during roasting. However, roasting for 10 min at 220 °C caused a significant decrease (P < 0.05) not only in molecular species containing more than four double bonds, but also in the amount of diene and triene species present in TAGs (with a few exceptions). These results suggest that no significant changes in molecular species or fatty acid distribution of TAGs would occur within 25 min of roasting at 180 °C, ensuring that a good-quality product would be attained. © 2000 Society of Chemical Industry

Microwave roasting and molecular species of triacylglycerols in soybean embryonic axes

AbstractEmbryonic axes were separated from soybeans roasted in a microwave oven. Molecular species and fatty acid distributions of triacylglycerols (TAG) isolated from total lipids in the embryonic axis were analyzed by a combination of argentation thin‐layer chromatography (TLC) and gas‐liquid chromatography. A modified argentation‐TLC procedure, developed to optimize the separation of the complex mixture of total TAG, provided 15 different groups of TAG, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester analysis was performed to determine the composition of each band. Fifteen molecular species of TAG were still found in the embryonic axes following roasting treatment. Microwave roasting for 6 min did not change the molecular species of the embryonic axis TAG (with a few exceptions), nor cause a loss of unsaturated fatty acids. However, microwave roasting for 12 min caused a significant decrease (P&lt;0.05) not only in molecular species containing more than four double bonds but also in the amount of diene and triene species present in TAG. These results suggest that no significant changes in molecular species or fatty acid distribution of TAG would occur within 6 min of microwave roasting, ensuring that a good quality product would be attained.

Changes in molecular species of triacylglycerols during frying

The loss of specific molecular species of triacylglycerols from sunflower, high-oleate sunflower and palm oils has been investigated in commercial frying operations and simulated frying experiments. The non-oxidized triacylglycerols were isolated and molecular species separated by silver ion highperformance liquid chromatography. Linoleate-containing species were lost more rapidly than those containing oleate, as expected. However, all species were liable to oxidation and those containing oleate were lost more rapidly than might have been anticipated. It is suggested that oxidation of linoleate is the probable initiation step, but then the reaction can be propagated readily to all unsaturated species.

Molecular species of phospholipids in a murine stem-cell line responsive to erythropoietin

The generation of the lipid signalling molecules, diacylglycerol (DAG) and phosphatidic acid (PA), has been implicated in the transduction events essential for proliferation of murine B6SUt.EP stem cells responsive to erythropoietin (EPO). Some of the responses were rapid and transient while others were slower and sustained. In an attempt to better understand the biphasic nature of DAG and PA appearance in response to EPO, an analysis of the molecular species of DAG, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PA in control and EPO-treated B6SUt.EP cells was made by HPLC and TLC. Fifteen to eighteen species were identified, which were increased non-uniformly by 0.2 unit/ml EPO. Greater increases (x6) were observed in 16:0,20:4 and 18:0,20:4 DAGs than in other species. The molecular species profiles of the stimulated DAGs were compared with the profiles of molecular species contained in the phospholipids. Comparison of the increase in DAG species caused by EPO with the molecular species present in PC and PI showed both PI and PC as the source of the fast DAG accumulation and only PC as the source of the slow DAG accumulation. PE was involved in both phases. We found a consistent formation of ethanolamine over time, in larger amounts than choline, providing strong evidence that, in addition to PC, PE is a major substrate. In addition, changes in molecular species of PA in response to EPO suggest that PI cannot account for the mass of PA formed during the first 30 s incubation with EPO, nor for PA formed during 30 min with EPO. It is concluded that the majority of PA was formed by a direct action of phospholipase D on PC.

Mechanism for Stabilization of Fish Actomyosin by Sodium Lactate

The mechanism for stabilization of actomyosin (AM) by sodium lactate (SL) was studied by comparing physical properties (viscosity, density, surface tension, and water activity) of SL and sucrose solutions. Relatively high intrinsic viscosity [η] and Huggins factor (k‘) values showed SL is an effective water structure-maker. Thermal aggregation studies showed that AM stability increased up to 15% SL; higher concentrations of SL destabilized the AM. Surface tension and water activity measurements indicated that the transition from stabilization to destabilization and from increasing to decreasing surface tension coincided with a change in molecular species in the SL solutions. The decrease in surface tension (and AM stability) with increasing SL concentration above the optimum is presented to be due to formation of the lactate dimer lactoyl lactate. This molecule is amphiphilic and would be expected to preferentially absorb at an interface (with for example air or protein) and thus lower surface tension. Ke...

Isoprenoid-mediated changes in the glycerophospholipid molecular species of the sterol auxotrophic fungus Lagenidium giganteum.

The mosquito pathogenic fungus Lagenidium giganteum (Oomycetes: Lagenidiales) is a sterol auxotroph that can grow vegetatively in the absence of these compounds, but requires an exogenous source of sterols to enter its sexual and asexual reproductive cycles. Electrospray mass spectrometry (MS) and electrospray MS/MS were used to examine three major glycerophospholipid molecular species--glycerophosphocholine (GPC), glycerophosphoethanolamine (GPE) and glycerophosphoinositol (GPI)--from fungal mycelium and nuclei grown in defined medium with and without isoprenoids which induce (cholesterol and ergosterol) or do not induce (squalene, cholestane) reproduction. Testosterone supplementation of defined media inhibited growth of L. giganteum, so the effect of this steroid on phospholipid metabolism could not be assessed. Mycelium grown in defined media supplemented with these isoprenoids produced significantly different quantities of total phospholipid relative to unsupplemented media and to each other, ranging from a mean of 292 micrograms phosphate per g wet weight for cholesterol-supplemented media to 56 micrograms phosphate per g wet weight for mycelium grown in the presence of squalene. A very large percentage of the GPC (69-80 mol%) and GPI (74-79 mol%) molecular species from mycelia and nuclei contained ether linkages. GPE molecular species had 13-20 mol% ether-containing moieties. The elevated levels of ether lipids may be related to the sterol auxotrophic nature of the fungus. Isoprenoid supplementation of defined growth media resulted in many significant changes in molecular species for all three lipid classes. Significant differences (P < 0.05) in the percentage of total cell ether lipids in GPC and GPE were generated by isoprenoid supplements to culture media. Mycelium grown in the presence of the two sterols which induce asexual and sexual reproduction in L. giganteum, cholesterol and ergosterol, had a significantly greater percentage of ether-containing GPE moieties. The glycerolipid species from nuclei isolated from cultures grown with cholesterol and ergosterol were similar to the composition of nuclei isolated from fungus cultured in defined medium without any supplement or supplemented with squalene. The nuclear membrane from mycelia grown in cholestane-supplemented media, however, had a very different glycerophospholipid composition relative to either whole cells or nuclei from cells grown on other media. It appears that one of the reasons that cyclic isoprenoids such as cholestane do not induce fungal reproduction is that they drastically alter the nuclear membrane glycerophospholipid composition.