Diacylglycerol kinase ζ generates dipalmitoyl-phosphatidic acid species during neuroblastoma cell differentiation

Phosphatidic acid (PA) is one of the phospholipids composing the plasma membrane and acts as a second messenger to regulate a wide variety of important cellular events, including mitogenesis, migration and differentiation. PA consists of various molecular species with different acyl chains at the sn-1 and sn-2 positions. However, it has been poorly understood what PA molecular species are produced during such cellular events. Here we identified the PA molecular species generated during retinoic acid (RA)-induced neuroblastoma cell differentiation using a newly established liquid chromatography/mass spectrometry (LC/MS) method. Intriguingly, the amount of 32:0-PA species was dramatically and transiently increased in Neuro-2a neuroblastoma cells 24–48 h after RA-treatment. In addition, 30:0- and 34:0-PA species were also moderately increased. Moreover, similar results were obtained when Neuro-2a cells were differentiated for 24 h by serum starvation. MS/MS analysis revealed that 32:0-PA species contains two palmitic acids (16:0 s). RT-PCR analysis showed that diacylglycerol kinase (DGK) δ and DGKζ were highly expressed in Neuro-2a cells. The silencing of DGKζ expression significantly decreased the production of 32:0-PA species, whereas DGKδ-siRNA did not. Moreover, neurite outgrowth was also markedly attenuated by the deficiency of DGKζ. Taken together, these results indicate that DGKζ exclusively generates very restricted PA species, 16:0/16:0-PA, and up-regulates neurite outgrowth during the initial/early stage of neuroblastoma cell differentiation.     

Lipid composition of Silybum marianum cell cultures treated with methyl jasmonate

Elicitation of cell cultures of Silybum marianum with methyl jasmonate (MeJA) increases the production and release of the secondary metabolite silymarin into the culture medium and this process seems to be dependent on phospholipase D activity and its product phosphatidic acid (PA). However, MeJA did not alter total membrane lipid content or overall fatty acid composition. A progressive increase in some galactolipids was observed with elicitation time. Phospholipids were mainly represented by phosphatidylcholine (PC) followed by phosphatidylethanolamine (PE) and phosphatidylinositol (PI). MeJA caused losses of PC species that contain two unsaturated acyl species, 36:5 and 36:6 and an increase in 36:2 species. A drop in the ratio of compounds with 18:3 in PI and PE was also observed. The presence of the lysophospholipids (LP) LPC (16:0, 18:3, 18:2, 18:1) and LPE (16:0, 18:3, 18:2, 18:1) and the high contents of PA, represented by the molecular species 34:3, 34:2 and 36:5 and 36:4, indicates high basal level of phospholipase activity in cultures and a high phospholipid turnover. MeJA treatment did not quantitatively alter these lipid classes.     

Diacylglycerol Kinase δ Phosphorylates Phosphatidylcholine-specific Phospholipase C-dependent,Palmitic Acid-containing Diacylglycerol Species in Response to High Glucose Levels

Decreased expression of diacylglycerol (DG) kinase (DGK) δ in skeletal muscles is closely related to the pathogenesis of type 2 diabetes. To identify DG species that are phosphorylated by DGKδ in response to high glucose stimulation, we investigated high glucose-dependent changes in phosphatidic acid (PA) molecular species in mouse C2C12 myoblasts using a newly established liquid chromatography/MS method. We found that the suppression of DGKδ2 expression by DGKδ-specific siRNAs significantly inhibited glucose-dependent increases in 30:0-, 32:0-, and 34:0-PA and moderately attenuated 30:1-, 32:1-, and 34:1-PA. Moreover, overexpression of DGKδ2 also enhanced the production of these PA species. MS/MS analysis revealed that these PA species commonly contain palmitic acid (16:0). D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), significantly inhibited the glucose-stimulated production of the palmitic acid-containing PA species. Moreover, PC-PLC was co-immunoprecipitated with DGKδ2. These results strongly suggest that DGKδ preferably metabolizes palmitic acid-containing DG species supplied from the PC-PLC pathway, but not arachidonic acid (20:4)-containing DG species derived from the phosphatidylinositol turnover, in response to high glucose levels.     

Polyunsaturated fatty acid-containing phosphatidic acids selectively interact with L-lactate dehydrogenase A and induce its secondary structural change and inactivation

Phosphatidic acid (PA) consists of various molecular species that have different fatty acyl chains at the sn-1 and sn-2 positions; and consequently, mammalian cells contain at least 50 structurally distinct PA molecular species. However, the different roles of each PA species are poorly understood. In the present study, we attempted to identify dipalmitoyl (16:0/16:0)-PA-binding proteins from mouse skeletal muscle using liposome precipitation and tandem mass spectrometry analysis. We identified L-lactate dehydrogenase (LDH) A, which catalyzes conversion of pyruvate to lactate and is a key checkpoint of anaerobic glycolysis critical for tumor growth, as a 16:0/16:0-PA-binding protein. LDHA did not substantially associate with other phospholipids, such as phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphoinositides and cardiolipin at physiological pH (7.4), indicating that LDHA specifically bound to PA. Interestingly, 18:0/18:0-, 18:0/20:4- and 18:0/22:6-PA also interacted with LDHA, and their binding activities were stronger than 16:0/16:0-PA at pH 7.4. Moreover, circular dichroism spectrometry showed that 18:0/20:4- and 18:0/22:6-PA, but not 16:0/16:0- or 18:0/18:0-PA, significantly reduced the α-helical structure of LDHA. Furthermore, 18:0/20:4- and 18:0/22:6-PA attenuated LDH activity. Taken together, we demonstrated for the first time that LDHA is a PA-binding protein and is a unique PA-binding protein that is structurally and functionally controlled by associating with 18:0/20:4- and 18:0/22:6-PA.     

Selective enrichment of n-3 fatty acids in human plasma lipid motifs following intake of marine fish

Plasma levels of n-3 long chain polyunsaturated fatty acids (LCPUFA) are associated with a reduction in risk of cardiovascular disease and other chronic, age-related diseases like Alzheimer's disease. In this work, we tested the hypothesis that n-3 LCPUFA fatty acids in human plasma are incorporated into selective lipid species following intake of n-3 LCPUFA rich marine fish. To test this hypothesis, we performed lipidomic analysis on plasma samples from a clinical trial in which participants consumed increasing amounts of farmed Atlantic salmon (Salmo salar). Under basal conditions, n-3 and n-6 LCPUFA were selectively incorporated into plasma phosphatidylcholine (PC) species containing saturated fatty acids (SFA) versus unsaturated fatty acids as the complementary fatty acids. LCPUFA were incorporated into selective triacylglycerol (TAG) species with complementary diacylglyceryl environments of 34:1 or 34:2 (for 20:5 and 22:5) and 36:2>36:3>36:4 and 36:1 (for 20:4 and 22:6). High n-3 LCPUFA marine fish intake resulted in selective increases of PC SFA_n-3 LCPUFA species and LCPUFA-containing TAG species. Changes in cholesteryl esters and phosphatidylethanolamines also occurred following fish intake. Our results highlight the importance of discriminating phospholipid and TAG species and dietary background when evaluating lipidomic outcomes and disease associations.     

Characterization of polar lipids of oral isolates of Candida, Pichia and Saccharomyces by Fast Atom Bombardment Mass Spectrometry (FAB MS)

AIMS: To characterize fatty acid and phospholipid analogue profiles of oral yeasts. METHODS AND RESULTS: Twenty-seven strains of oral yeasts were cultured on SDA and lipids of freeze-dried cells were extracted and analysed by FAB MS. The most abundant carboxylate anion was m/z 281 (C18 : 1). The most intense phospholipid analogue ions were of PE, PG, PA and PI. Pichia etchellsii contained molecular species of PG and PE, whereas Saccharomyces cerevisiae had PA, PG and PE analogues. Mass spectra revealed that S. cerevisiae and Candida glabrata were distinct from one another and from the other species tested. CONCLUSION: Oral yeasts largely differ with respect to their polar lipids. It is concluded that oral yeast species have distinctive fatty acid and phospholipid analogue anion profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: FAB MS provided novel chemotaxonomic information.     

Characterization of Membrane Lipidome Changes in Pseudomonas aeruginosa during Biofilm Growth on Glass Wool

Bacteria cells within biofilms are physiologically distinct from their planktonic counterparts. In particular they are more resistant to detrimental environmental conditions. In this study, we monitored the evolution of the phospholipid composition of the inner and outer membranes of P. aeruginosa during the biofilm formation (i.e., from 1-, 2-, to 6-day-old biofilm). Lipidome analyses were performed by electrospray ionization mass spectrometry. In addition to the lipidomic analysis, the fatty acid composition was analysed by gas chromatography/mass spectrometry. We found that the lipidome alterations of the inner and the outer membranes varied with the biofilm age. These alterations in phospholipid compositions reflect a higher diversity in sessile organisms than in planktonic counterparts. The diversity is characterized by the presence of PE 30∶1, PE 31∶0 and PG 31∶0 for the lower masses as well as PE 38∶1, 38∶2, 39∶1, 39∶2 and PG 38∶0, 38∶1, 38∶2, 39∶1, 39∶2 for the higher masses. However, this lipidomic feature tends to disappear with the biofilm age, in particular the high mass phospholipids tend to disappear. The amount of branched chains phospholipids mainly located in the outer membrane decreased with the biofilm age, whereas the proportion of cyclopropylated phospholipids increased in both membranes. In bacteria present in oldest biofilms, i.e., 6-day-old, the phospholipid distribution moved closer to that of planktonic bacteria.     

Common disbalance in the brain parenchyma of dementias: Phospholipid profile analysis between CADASIL and sporadic Alzheimer's disease

Sporadic Alzheimer's disease (SAD) is the most common form of dementia, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most frequent hereditary ischemic small vessel disease of the brain. Relevant biomarkers or specific metabolic signatures could provide powerful tools to manage these diseases. Therefore, the main goal of this study was to compare the postmortem frontal cortex gray matter, white matter and cerebrospinal fluid (CSF) between a cognitively healthy group and CADASIL and SAD groups. We evaluated 352 individual lipids, belonging to 13 lipid classes/subclasses, using mass spectrometry, and the lipid profiles were subjected to multivariate analysis to discriminate between the dementia groups (CADASIL and SAD) and healthy controls. The main lipid molecular species showing greater discrimination by partial least squares-discriminant analysis (PLS-DA) and a higher significance multivariate correlation (sMC) index were as follows: phosphatidylserine (PS) PS(44:7) and lysophosphatidylethanolamine (LPE) LPE(18:2) in gray matter (GM); phosphatidylethanolamine (PE) PE(32:2) and phosphatidylcholine PC PC(44:6) in white matter (WM), and ether PE (ePE) ePE(38:2) and ether PC (ePC) ePC(34:3) in CSF. Common phospholipid molecular species were obtained in both dementias, such as PS(44:7) and lyso PC (LPC) LPC(22:5) in GM, PE(32:2) in WM and phosphatidic acid (PA) PA(38:5) and PC(42:7) in CFS. Our exploratory study suggests that phospholipids (PLs) involved in neurotransmission alteration, connectivity impairment and inflammation response in GM, WM and CSF are a transversal phenomenon affecting dementias such as CADASIL and SAD independent of the etiopathogenesis, thus providing a possible common prodromal phospholipidic biomarker of dementia.     

Cauliflower (Brassica oleracea L.) Seed Vigour: Imbibition Effects

By means of a machine vision facility, the process of water-imbibitionin a small seeded brassica species was recorded as the visibleincrease in seed volume. Dry cauliflower seed (Brassica oleracea)showed an immediate rapid phase of imbibition upon the additionof water. This initial phase was associated with the reductionin seedling root growth resulting from the imbibition of coldwater, and the rate of uptake of water at 20?C was negativelycorrelated with subsequent seedling growth. Damage to the testaof dry seed resulted in an increased rate of imbibition anda corresponding decrease in seedling root growth measured onslant boards. This showed that the intact testa of cauliflowerseed is capable of acting as a barrier to water influx and thata high rate of water uptake is damaging to the embryo. Testadamage reduced percentage soil emergence of seeds, as did raisingthe soil moisture content during the imbibition period by wateringimmediately after sowing. Both these treatments were believedto increase imbibition rate. Conditions which encouraged a lowrate of water uptake also improved the rate and uniformity ofemergence. Correlation of mean imbibition rates (measured ina laboratory test) with soil-emergence indicated that the sensitivityto imbibition damage varied between seed lots and interactedwith the absolute rate of water influx to determine the finalpercentage emergence. This factor prevented reliable predictionof seedling performance from the ranked-order of measured imbibitionrates. The significance of these findings to the seed productionand modular transplant raising industries is discussed. Key words: Machine vision, imbibition rate, testa, soil moisture, vigour     

Phospholipid and phospholipase changes by jasmonic acid during stolon to tuber transition of potato

Potato tuber formation starts with the stolon swelling and is regulated by jasmonates. The cascade of events leading to tuber formation is not completely understood. The aim of this study was to evaluate phospholipid composition and phospholipase activities during four stages of stolon-to-tuber transition of Solanum tuberosum L., cv. Spunta, and involvement of phosphatidic acid (PA) in stolon cell expansion during early stages. Effects of jasmonic acid (JA) treatment on phospholipid content and activation of phospholipase D (PLD) (EC 3.1.4.4) and phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP₂-PLC) (EC 3.1.4.3) were studied in the early stages (first stage, hooked apex stolon; second stage, initial swelling stolon) of tuberization. All the phospholipid species identified, phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), PA, and cardiolipin (CL), decreased as tuber formation progressed. PLD and PLC were activated in control tissues at an early stage. JA treatment caused a decrease of PC and PS in first stage stolons, accumulation of PA in second stage stolons, and modification of PLD and PLC activities. PA increased stolon cell area in the first and second stages. These findings indicate that phospholipid catabolism is activated from the early stages of tuber formation, and that JA treatment modifies the pattern of phospholipid (PC, PS, and PA) composition and phospholipase (PLD and PLC) activity. These phospholipids therefore may play a role in activation of an intracellular mechanism that switches the developmental fate of stolon meristem cells, causing differentiation into a tuber.     

Thermotolerance is developmentally dependent in germinating wheat seed

During the initial 9 to 12 hours of imbibition, the imbibing wheat (Triticum aestivum L.) seed was found to exhibit substantial tolerance to high temperature relative to later times of imbibition. Tolerance was assessed by seed viability and seedling growth. This initial high temperature tolerance gradually declines with increasing time of seed imbibition. A range of 2 hour heat pretreatments (38-42°C) prior to imposition of a 2 hour heat shock (51-53°C) during this same 9 to 12 hour interval was unable to increase survival or seedling growth over that of seed that did not receive a pretreatment. However, after 9 to 12 hours of imbibition the pretreatment provided both increased survival and increased seedling growth, measured 120 hours later, i.e., classical thermotolerance could be acquired. This response is called a `thermotolerance transition.' Isolated embryos responded in a similar manner using a 2,3,5-triphenyltetrazolium chloride assay for viability determination following heat treatments. The high temperature tolerance during early imbibition indicates that the thermotolerance transition involves the loss of an existing thermotolerance coincident with acquiring the ability to become thermotolerant following heat pretreatment. Despite the inability to acquire thermotolerance, heat shock protein synthesis was induced by heat shock immediately upon imbibition of wheat seed or isolated embryos. Developmentally regulated heat shock proteins of 58 to 60, 46, 40, and 14 kilodaltons were detected at 1.5 hours of imbibition following heat shock, but were absent or greatly reduced by 12 hours. Constitutive synthesis of 70 and 90 kilodalton hsp groups appeared to be greater at 1.5 hours of imbibition than at 12 hours of imbibition.     

Molecular species of phospholipid in rats in primary and transplanted fibrosarcomas induced by soybean oil containing tocopherol acetate.

When soybean oil containing tocopherol acetate was given to rats once a week subcutaneously for 10-12 months, it caused the development of fibrosarcomas at the injection site in 11 of 15 rats. A tumor produced in this manner proved eminently transplantable into other rats. The molecular species of phospholipid subclasses were determined in primary and transplanted tumors. The molecular species composition of the phospholipid subclasses in both types of tumors were similar. The percentages of diacyl and alkylacyl glycerophosphocholine (GPC) were 90-93 and 6-8% of total phosphatidylcholine, respectively. The percentages of diacyl and alkenylacyl glycerophosphoethanolamine (GPE) were 51 and 45%, respectively, of total phosphatidylethanolamine (PE). Diacyl and alkylacyl GPC species containing arachidonic acid (20:4) composed about 15-16 and 37-40% of each subclass, respectively. Diacyl and alkenylacyl GPE species containing 20:4 composed about 38-40 and 56-60% of each subclass, respectively. Disaturated species of diacyl and alkylacyl GPC composed about 22-24 and 13% of each subclass, respectively, whereas these species of PE composed less than 2%. The fatty acid composition of the other tumor phospholipids was analyzed.     

Utilization of Amygdalin during Seedling Development of Prunus serotina

Cotyledons of mature black cherry (Prunus serotina Ehrh.) seeds contain the cyanogenic diglucoside (R)-amygdalin. The levels of amygdalin, its corresponding monoglucoside (R)-prunasin, and the enzymes that metabolize these cyanoglycosides were measured during the course of seedling development. During the first 3 weeks following imbibition, cotyledonary amygdalin levels declined by more than 80%, but free hydrogen cyanide was not released to the atmosphere. Concomitantly, prunasin, which was not present in mature, ungerminated seeds, accumulated in the seedling epicotyls, hypocotyls, and cotyledons to levels approaching 4 [mu]mol per seedling. Whether this prunasin resulted from amygdalin hydrolysis remains unclear, however, because these organs also possess UDPG:mandelonitrile glucosyltransferase, which catalyzes de novo prunasin biosynthesis. The reduction in amygdalin levels was paralleled by declines in the levels of amygdalin hydrolase (AH), prunasin hydrolase (PH), mandelonitrile lyase (MDL), and [beta]-cyanoalanine synthase. At all stages of seedling development, AH and PH were localized by immunocytochemistry within the vascular tissues. In contrast, MDL occurred mostly in the cotyledonary parenchyma cells but was also present in the vascular tissues. Soon after imbibition, AH, PH, and MDL were found within protein bodies but were later detected in vacuoles derived from these organelles.     

Dynamic lipidomic insights into the adaptive responses of Saccharomyces cerevisiae to the repeated vacuum fermentation

Vacuum fermentation is utilized in a wide range of life science industries and biomedical R&D. Little is known, however, on the effects of the vacuum on the yeast, and in particular, on the yeast lipidome that plays a central role in maintaining cell membrane and other vital (yeast) cell functions. The present study evaluated the adaptive responses of Saccharomyces cerevisiae to repeated vacuum fermentation by lipidomic analysis. We employed gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI/MS(n)) to quantify a total of 13 intermediate sterols and 139 phospholipid species of yeast cells. Principal components analysis found that the PI (phosphatidylinositol) 26:0, PI 28:0, PE (phosphatidylethanolamine) 32:1, and PE 34:1 were potential biomarkers to distinguish the vacuum fermentation process. Quantitative analysis showed that vacuum fermentation increased the synthesis of PI and the PC (phosphatidylcholine) species with short saturated acyl chains. The synthesis of PC via CDP-choline and turnover of PC were enhanced, instead of formation via methylation of PE. Additionally, increased PI at the expense of PE and PG (phosphatidylglycerol) was associated with enhancement of ethanol productivity. Vacuum fermentation caused eburicol accumulation, suggesting that vacuum can activate the branch of the ergosterol biosynthesis pathway. Eburicol decrease and PI increase contributed to recovery of cellular activities with oxygenating treatment. Ethanol productivity was increased by sixfold in vacuum-treated cells. These observations may allow the development of future mechanistic approaches to optimization of yeast fermentation under vacuum for bioindustry and life science applications. In particular, our findings on changes in lipid molecular species and the ergosterol biosynthesis pathway elucidate the defense responses of yeast cell membranes during the repeated vacuum fermentation, which by extension, provided an important lead insight on how best to protect the cell membranes from the extreme long-term stress conditions.     

Phospholipid molecular species of Bacteroides

The fatty acid composition, and types of polar lipid (PL) present, have been well-studied in Bacteroides. Nothing is known, however, of the detailed structures of individual phospholipid and molecular species. The aim of this study was to determine molecular weights and putative identities of individual phospholipid molecular species present in Bacteroides. Thirteen culture collection strains were harvested, washed and freeze-dried. Polar lipids were extracted and separated by conventional fast atom bombardment mass spectrometry (FAB MS). For each strain, hundreds of polar lipid peaks were seen. Nineteen major anions in the range m/z 209–299 were separated. The most intense of these was consistent with the expected presence of the C_15:0 anion. Other anions were attributable to saturated, mono-unsaturated, di-unsaturated, tri-unsaturated and hydroxy-carboxylate ions. Twenty-two major anion peaks were recorded in the range m/z 505–722. These were consistent with the presence of analogues of phosphatidic acid (PA), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). The most intense peaks included those consistent with the presence of PE(30:0), PG(29:1), PG(30:1), PG(28:1), PE(31:0), PE(OH-30:0), PG(31:1) and PE(OH-31:0). This combination of PL molecular species is unique to Bacteroides and has not been reported in other organisms so far examined.     

Microsomal Phospholipid Molecular Species Alterations during Low Temperature Acclimation in Dunaliella

A detailed analysis of the low temperature-induced alterations of Dunaliella salina (UTEX 1644) microsomal membrane lipids was carried out. Microsomal membranes were isolated from cells grown at 30 degrees C, from cells shifted to 12 degrees C for 12 hours, and from cells acclimated to 12 degrees C. Fatty acid analyses of the major lipid classes demonstrated significant changes in the fatty acid composition of phosphatidylcholinemine (PE) and phosphatidylglycerol (PG) but not phosphatidylcholine (PC) during the initial 12 hours at low temperature. These changes did not entail enhanced desaturation of linoleic acid. Subsequent to 12 hours, the proportions of linolenic acid increased in all phospholipids.Molecular species analyses of the phospholipids demonstrated that the most immediate changes following a shift to low temperature were limited to several molecular species of PE and PG. The changes observed in PE included a decrease in C(30) species and concomitant increases in C(34) and C(36) species. Compositional changes associated with PG entailed the emergence of a new molecular species (18:1/18:1) not found at 30 degrees C. The retailoring of molecular species resulted in an increase in the number of species having two unsaturated acyl chains and did not reflect a simple enhancement of desaturase activity as suggested by the fatty acid analysis. We conclude that the initial alterations in response to low temperature stress involve discrete changes in certain molecular species. These and further alterations of molecular species following acclimation to low temperature would appear to augment increases in acyl chain desaturation as a means of modifying membrane properties in response to low temperature stress.     

Cerebral Phospholipid Content and Na+, K+-ATPase Activity During Ischemia and Postischemic Reperfusion in the Mongolian Gerbil

Using bilateral carotid artery occlusion in adult gerbils we examined the effects of ischemia and ischemia/reperfusion on cerebral phospholipid content and Na+,K+-ATPase (EC 3.6.1.3) activity. In contrast to the large changes in phospholipid content and membrane-bound enzyme activity that have been observed in liver and heart tissues, we observed relatively small changes in the cerebral content of total phospholipid, phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE) following ischemic intervals of up to 240 min. Following 15 min of ischemia the cerebral content of sphingomyelin (SM) was decreased to less than 50% of control values but returned to near-normal levels with longer ischemic periods. Significant decreases in the cerebral content of phosphatidylinositol (PI) and phosphatidic acid (PA) were observed following shorter intervals of ischemia (15-45 min). Na+,K+-ATPase activity of cerebral homogenates prepared from the brains of gerbils subjected to 30-240 min of ischemia was decreased but significantly different from control activity only after 30 min of ischemia (-29%, p less than or equal to 0.05). With the exception of PS, reperfusion for 60 min following 60 min of ischemia resulted in marked increases in cerebral phospholipid content with PC, SM, PI, and PA levels exceeding and PE levels equal to preischemic values. Longer periods of reperfusion (180 min) resulted in decreases in cerebral phospholipid content toward (PC, SM, PI, and PA) or below (PE) preischemic levels. In contrast, the cerebral content of PS significantly decreased during reperfusion (-51% at 60 min, p less than or equal to 0.05) and remained below preischemic values even after 180 min of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of the Activity of Purified Tonoplast H+-ATPase from Mung Bean (Vigna radiata L.) Hypocotyls by Various Lipids

H⁺-ATPase was solubilized from the tonoplast of mung bean (Vignaradiata L.) hypocotyls and purified by fast protein liquid chromatographyon a Mono Q ion-exchange column. The purified ATPase hardlycontained any phospholipid, but it did contain 10 to 15 moleculesof sterol and 25 to 30 molecules of glycolipid per ATPase molecule,and it had little activity without exogenously added phospholipids.Each individual polar head group, acylglyceride and fatty acidthat constituted a phospholipid was incapable by itself of activatingthe ATPase. Sterols and cerebroside had little activating effect.Maximal activation of ATPase was noted with asolectin or variousmolecular species of phosphatidylcholine (PC) at 0.005% to 0.01%(w/v). The activation by the various molecular species of PCwas dependent on the length and degree of unsaturation of fattyacyl chains. PC with two saturated and long fatty acyl chainsof more than 18 carbon atoms failed entirely to activate theATPase. PC, PS and PG with 1-palmitoyl (16:0)-2-oleoyl(18:1)fatty acyl chains all activated ATPase to nearly the same extentas asolectin, but the activation by PE and PA with the samefatty acyl composition was 52% and 15% of that by asolectin,respectively. The molecular species of PC with phase-transitiontemperatures below 50C activated ATPase, as determined at 38C.The dependence on temperature of the activation by the molecularspecies of PC indicated that the activation of the ATPase beganclose to the temperature of the phase transition of the PC added.These data indicate that phospholipids in the liquid-crystallinephase are essential for the catalytic activity of the ATPase. (Received June 4, 1992; Accepted January 18, 1993)     

Substrate preference of stress-activated phospholipase D in Chlamydomonas and its contribution to PA formation

In response to various environmental stress conditions, plants rapidly form the intracellular lipid second messenger phosphatidic acid (PA). It can be generated by two independent signalling pathways via phospholipase D (PLD) and via phospholipase C (PLC) in combination with diacylglycerol kinase (DGK). In the green alga Chlamydomonas, the phospholipid substrates for these pathways are characterized by specific fatty acid compositions. This allowed us to establish: (i) PLD's in vivo substrate preference; and (ii) PLD's contribution to PA formation during stress signalling. Accordingly, G-protein activation (1 micro m mastoparan), hyperosmotic stress (150 mm NaCl) and membrane depolarization (50 mm KCl) were used to stimulate PLD, as monitored by the accumulation in 5 min of its unique transphosphatidylation product phosphatidylbutanol (PBut). In each case, PBut's fatty acid composition specifically matched that of phosphatidylethanolamine (PE), identifying this lipid as PLD's favoured substrate. This conclusion was substantiated by analysing the molecular species by electrospray ionization-mass spectrometry (ESI-MS/MS), which revealed that PE and NaCl-induced PBut share a unique (18 : 1)2-structure. The fatty acid composition of PA was much more complex, reflecting the different contributions from the PLC/DGK and PLD pathways. During KCl-induced stress, the PA rise was largely accounted for by PLD activity. In contrast, PLD's contribution to hyperosmotic stress-induced PA was less, being approximately 63% of the total increase. This was because the PLC/DGK pathway was activated as well, resulting in phosphoinositide-specific fatty acids and molecular species in PA.     

Spatially regulated genes expressed during seed germination and postgerminative development are activated during embryogeny

Summary We investigated the control of genes expressed primarily during seed germination and postgerminative development in Brassica napus L. We identified cloned mRNA sequences which became prevalent within 1 day after the start of imbibition and were at low or undetectable levels in immature embryos, dry seeds, and leaves. Most postgermination-abundant mRNAs accumulated primarily, though not exclusively, in different parts of the seedling. Of the 14 cloned mRNAs, 8 were prevalent in cotyledons, 2 were abundant in seedling axes, and 4 were approximately equally distributed in both parts. We showed that although these mRNAs reached maximal levels in seedlings, the spatially regulated mRNAs were also detected at distinct embryonic stages; mRNAs prevalent in seedling axes accumulated primarily during early embryogenesis while cotyledon-abundant mRNA concentration increased during late embryogeny. We conclude that the temporal and spatial regulation of gene expression in seedlings reflects similarities and differences in the physiological functions of cotyledons and axes. Furthermore, the regulated expression of cotyledon-abundant genes during late embryogeny suggests that the mRNAs and possibly proteins may accumulate in preparation for subsequent seedling growth. Similarities in the accumulation of cotyledon-abundant mRNAs may indicate coordinate regulation of this gene set.Abbreviations DAF days after flowering - DAI days after the start of imbibition - HAI hours after the start of imbibition - kb kilobase(pairs)