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AIMS: To determine the spread of different oomycete pathogens in hydroponic, soilless tomato growing systems and their impact on established microbial communities, as baseline studies prior to future introduction of microbial inoculants for disease suppression. METHODS AND RESULTS: The oomycete pathogens, Pythium group F, Pythium aphanidermatum and Phytophthora cryptogea, were introduced into small-scale recirculating tomato growing systems containing rockwool 6 weeks after set-up when roots were well-established. Two weeks later, half of the systems were switched over to run-to-waste. Pythium aphanidermatum spreads the fastest, Pythium group F the slowest and Ph. cryptogea was intermediate in its spread. The switch to run-to-waste had no effect on pathogen recovery. Microbial communities, monitored by dilution plating, were well-established at the first sampling, 6 weeks after set-up and although differences in community levels were found between experiments, changes during any one experiment were small, generally less than 1 log10 CFU g(-1) for bacteria. Pathogen introduction increased microbial community levels in roots but the switch to run-to-waste had no effect. Analysis of bacterial communities through amplification of a fragment of the 16S rRNA gene and DGGE profiling showed that different communities were established within each pathogen experiment and that different communities were established on roots, rockwool and in nutrient solutions. However, no significant changes in microbial profiles were found over time in any experiment. CONCLUSIONS: In these systems, the microbial communities were well-established 6 weeks after set-up and were resistant to biological and physical perturbation. SIGNIFICANCE AND IMPACT OF THE STUDY: The implication for microbial inoculation of such systems for disease suppression is that the micro-organisms would either have to be introduced very early during the set-up of the system or be able to replace an established but variable community.

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Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or -2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography-MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

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In Saccharomyces cerevisiae, phospholipase D (PLD), encoded by the SPO14 gene, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phosphatidic acid. SPO14 is essential for cellular differentiation during meiosis and is required for Golgi function when the normal secretory apparatus is perturbed (Sec14-independent secretion). We isolated specific alleles of SPO14 that support Sec14-independent secretion but not sporulation. Identification of these separation-of-function alleles indicates that the role of PLD in these two physiological processes is distinct. Analyses of the mutants reveal that the corresponding proteins are stable, phosphorylated, catalytically active in vitro, and can localize properly within the cell during meiosis. Surprisingly, the separation-of-function mutations map to the conserved catalytic region of the PLD protein. Choline and phosphatidic acid molecular species profiles during Sec14-independent secretion and meiosis reveal that while strains harboring one of these alleles, spo14S-11, hydrolyze phosphatidylcholine in Sec14-independent secretion, they fail to do so during sporulation or normal vegetative growth. These results demonstrate that Spo14 PLD catalytic activity and cellular function can be differentially regulated at the level of phosphatidylcholine hydrolysis.

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Porcine aortic endothelial cells have previously been shown to contain particularly high basal levels of polyunsaturated diacylglycerol (DAG) together with a very high degree of membrane-associated protein kinase C (PKC), which is largely insensitive to further activation (Pettitt, T. R., Martin, A., Horton, T., Liossis, C., Lord, J. M., and Wakelam, M. J. O. (1997) J. Biol. Chem. 272, 17354-17359). To investigate the possibility that the high polyunsaturated DAG levels were constitutively activating PKC, we transfected porcine aortic endothelial cells with two different forms of human diacylglycerol kinase, epsilon and zeta. In vitro, the former is specific for polyunsaturated structures, whereas the latter shows no apparent selectivity. Overexpression of DAGKepsilon specifically reduced the level of polyunsaturated DAG in the transfected cells while having little effect on the more saturated structures. It also caused the redistribution of PKCalpha and epsilon from the membrane to the cytosol. Overexpression of DAGKzeta caused a general reduction in DAG levels but had little effect on PKC distribution. These results for the first time show that DAGKepsilon specifically phosphorylates polyunsaturated DAG in vivo and that in so doing it regulates PKC localization and activity. This provides support for the proposal that it is the polyunsaturated DAGs that function as messengers and convincing evidence for DAGKepsilon being a physiological terminator of DAG second messenger signaling.

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Phosphatidic acid generation through activation of diacylglycerol kinase alpha has been implicated in interleukin-2-dependent T-lymphocyte proliferation. To investigate this lipid signaling in more detail, we characterized the molecular structures of the diradylglycerols and phosphatidic acids in the murine CTLL-2 T-cell line under both basal and stimulated conditions. In resting cells, 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol subtypes represented 44 and 55% of total diradylglycerol, respectively, and both showed a highly saturated profile containing primarily 16:0 and 18:1 fatty acids. 1-O-Alk-1'-enyl-2-acylglycerol represented 1-2% of total diradylglycerol. Interleukin-2 stimulation did not alter the molecular species profiles, however, it did selectively reduce total 1-O-alkyl-2-acylglycerol by over 50% at 15 min while only causing a 10% drop in 1,2-diacylglycerol. When radiolabeled CTLL-2 cells were challenged with interleukin-2, no change in the cellular content of phosphatidylcholine nor phosphatidylethanolamine was observed thereby ruling out phospholipase C activity as the source of diradylglycerol. In addition, interleukin-2 failed to stimulate de novo synthesis of diradylglycerol. Structural analysis revealed approximately equal amounts of 1,2-diacyl phosphatidic acid and 1-O-alkyl-2-acyl phosphatidic acid under resting conditions, both containing only saturated and monounsaturated fatty acids. After acute (2 and 15 min) interleukin-2 stimulation the total phosphatidic acid mass increased, almost entirely through the formation of 1-O-alkyl-2-acyl species. In vitro assays revealed that both 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol were substrates for 1,2-diacylglycerol kinase alpha, the major isoform in CTLL-2 cells, and that the lipid kinase activity was almost totally inhibited by R59949. In conclusion, this investigation shows that, in CTLL-2 cells, 1,2-diacylglycerol kinase alpha specifically phosphorylates a pre-existing pool of 1-O-alkyl-2-acylglycerol to form the intracellular messenger 1-O-alkyl-2-acyl phosphatidic acid.

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Cross-linking of the Ag receptors on B cells induces DNA synthesis and proliferation. Butanol trap experiments suggest that one or more phospholipase D activities play a key role in this process. Although phosphatidylcholine-phospholipase D has been shown to play a central role in the transduction of proliferative responses for a wide variety of calcium-mobilizing receptors, we show that the Ag receptors are not coupled to this phospholipase. In addition, phosphatidylcholine-phospholipase D is not stimulated under conditions that mimic T cell-dependent B cell activation. In contrast, ATP, which inhibits surface Ig (sIg)-mediated DNA synthesis in murine B cells via P2-purinoceptors, activates phosphatidylcholine-phospholipase D. Phosphatidylcholine-phospholipase D is therefore associated with antiproliferative signal transduction in mature B cells, but it does not transduce early signals associated with sIg-mediated growth arrest or apoptosis in immature B cells. Mitogenic stimulation of sIg is, however, coupled to a novel nonphosphatidylcholine-hydrolyzing phospholipase D activity. The resultant sIg-generated phosphatidic acid, unlike the phosphatidylcholine-derived phosphatidic acid generated via the purinoceptors, is converted to diacylglycerol. These data provide the first evidence that while the novel sIg-coupled phospholipase D and resultant diacylglycerol generation may play a role in B cell survival and proliferation, phosphatidylcholine-phospholipase D may transduce, via phosphatidic acid, negative immunomodulatory signals in mature B lymphocytes.

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Prostaglandin F2alpha, platelet-derived growth factor (PDGF) and calcium ionophore A23187 stimulated the rapid (within 25 s) generation of polyunsaturated 1,2-diacylglycerol (DAG) species, in particular 18:0/20:3n-9, 18:0/20:4n-6 and 18:0/20:5n-3, in Swiss 3T3 fibroblasts. This was followed by a second sustained phase characterised by saturated, monounsaturated and diunsaturated DAG species derived, at least partially, from a phospholipase D/phosphatidate phosphohydrolase-linked pathway. This could be directly activated by phorbol ester. Assay of rat brain protein kinase C (PKC) in lipid vesicles showed that first phase, polyunsaturated-enriched DAG isolated from Swiss 3T3 cells was a more potent activator of kinase activity compared to that achieved with DAG from control or 5 min stimulated cells. Thus activation of distinct members of the phospholipase C family leads to the rapid and almost identical generation of polyunsaturated DAG species which are capable of preferentially activating protein kinase C (PKC).

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In eukaryotes, many receptor agonists use phospholipase-generated lipids as intracellular messengers. Receptor occupation stimulates the production of polyunsaturated 1,2-diacylglycerols by phosphatidylinositol-4,5-bisphosphate specific phospholipases C and/or of mono-unsaturated and saturated phosphatidates by phospholipase-D-catalysed phosphatidylcholine breakdown. The primary phospholipase products are rapidly metabolized: polyunsaturated 1,2-diacylglycerols are converted to polyunsaturated phosphatidates by diacylglycerol kinase; mono-unsaturated and saturated phosphatidates are dephosphorylated to give mono-unsaturated and saturated 1,2-diacylglycerols by phosphatidate phosphohydrolase. The phospholipase-generated polyunsaturated 1,2-diacylglycerols and mono-unsaturated and saturated phosphatidates appear to be intracellular messengers, whereas their immediate metabolites probably do not have signalling functions.

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Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive.

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PLD is regulated by the small GTP binding proteins Rho and Arf, though predominantly by the latter. The PA product of PLD activation is an activator of Rho-regulated actin stress fibre formation and in invasive cells of MMP-9 synthesis and activation. Together this may explain the increased invasion of cells in response to PA.

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Bombesin induces the down-regulation of protein kinase C-delta (PKC-delta) and PKC-epsilon in Swiss 3T3 cells. Simultaneous addition of transforming growth factor beta 1 (TGF beta 1) selectively blocks PKC-delta down-regulation at mid-S-phase, whereas PKC-epsilon levels continue to decline. Northern blot analysis shows that PKC-epsilon levels could be controlled in part at the level of transcription; PKC-delta mRNA levels remained constant at these later times. Bombesin induces a sustained elevation of some species of diacylglycerol (DAG), consistent with the observed loss of PKC-delta and PKC-epsilon. Interestingly, the combination of bombesin and TGF-beta 1 produces an even greater DAG response. Flow cytometric analysis demonstrates that bombesin induces only 15% of the cells to enter the cell cycle, in contrast to the combination of TGF beta 1 plus bombesin which induces 75-80% of the cells to progress through the cycle. The protection of PKC-delta from down-regulation under conditions of sustained DAG elevation correlates with the mitogenic response and implies that the down-regulation process itself is regulated. Consistent with this, it is demonstrated that bombesin plus TGF beta 1 protects PKC-delta from phorbol ester-induced down-regulation.

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Stimulation of 3T3 fibroblasts with epidermal growth factor (EGF) results in an increase in 1,2-diacylglycerol (DAG) mass which is maximal at 25 s, declining at 1 min and returning to basal levels by 30 min. No changes in alkylacylglycerol or alkenylacylglycerol were detected. Three species account for most of this mass increase: 18:0/20:5,n-3, 18:0/20:4,n-6 and 18:0/20:3,n-9. These species are characteristic of the phosphoinositides; however, previous work failed to detect any EGF-stimulated rise in inositol phosphates in these cells [Cook and Wakelam (1992) Biochem. J. 285, 247-253]. This ruled out phosphoinositide hydrolysis by phospholipase C, but raised the possibility of phospholipase D/phosphatidate phosphohydrolase-catalysed hydrolysis of phosphatidylinositol. The inclusion of butanol in the incubation medium failed to block the diacylglycerol changes, indicating that the phospholipase D pathway is not involved and that DAG must be derived from another source, probably via phospholipase C-catalysed hydrolysis of a phosphatidylcholine pool that is particularly rich in these species. The tyrosine kinase inhibitor ST-271 almost abolished the elevation in 18:0/20:5,n-3, 18:0/20:4, n-6 and 18:0/20:3,n-9 at 25 s, but only reduced the rise in total DAG mass by about 50%. The protein kinase C (PKC) inhibitor Ro-31-8220 increased DAG levels at all time points but had no effect on the species profiles. This provides additional evidence for PKC-mediated regulation of cell-surface EGF receptors, since the inhibition of PKC would increase the availability and/or ligand binding affinity of receptors at the plasma membrane and hence increase and prolong the response to EGF.

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We have developed procedures for the analysis of endogenous diradylglycerol (DRG) molecular species using derivatization with 3,5-dinitrobenzoyl chloride. The introduction of this strong chromatophore enabled us to separate less than 1 nmol of DRG into its three classes (diacylglycerol, alkylacylglycerol and alkenylacylglycerol) using a combination of h.p.l.c. and t.l.c. followed by reversed-phase h.p.l.c. to resolve these classes into their component molecular species. When applied to Swiss 3T3 mouse fibroblasts stimulated with bombesin for 25 s, 5 min or 30 min, subtle time-dependent changes in the DRG patterns were observed, with only certain polyunsaturated 1,2-diacyglycerol species [18:0/20:3(n-9), 18:0/20:4(n-6), 18:0/20:4(n-3), 18:0/20:5(n-3), 18:1(n-9)/20:3(n-9), 18:1(n-9)/20:4(n-6), 16:0/22:6(n-3), 18:0/20:3(n-6) and 16:0/20:5(n-3)] showing significant agonist-stimulated increases. The amounts of the first six species were all raised at 25 s, whereas all except the latter two were elevated at 5 min. By 30 min these last species were also increased but 18:0/20:3(n-9) had returned to basal levels. Overall DRG levels, as measured by total molecular-species peak area, remained effectively constant. No changes in the amount or species profile of 1-alkyl-2-acylglycerol were observed. Comparison of these species with the acyl-chain structure of phospholipids supports the idea that inositol lipids could be the source of DRG at early stimulation times, but phosphatidylcholine appears to be a phospholipase substrate at all times. These results indicate sequential activation of several phospholipases with different substrate specificities and/or access to different phospholipid pools. They also suggest that only polyunsaturated DRGs act as second messengers and that changes in the relative amounts of these species may trigger activation of different proteins and/or isoforms (e.g. the different isoforms of protein kinase C).

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The migration-inducing abilities of both synthetic lipoxin A4 (LXA4) and leukotriene B4 (LTB4) for rainbow trout neutrophils were examined with an in vitro assay. LXA4 caused enhanced migration of these cells with a three- to fourfold greater response than that observed toward LTB4 at most concentrations tested. Checkerboard assays showed that synthetic LXA4 was chemokinetic for trout leukocytes, while LTB4 was chemotactic. The chemokinetic ability of LX synthesized by rainbow trout macrophages maintained in short-term culture was also determined. These cells produced both 4-and 5-series LX, namely LXA4, LXA5, 11-trans-LXA4, 11-trans-LXA5, 7-cis-11-trans-LXA4 and 6(S)-LXA4. Although greater migration was found to LXA4 than to most of the other 4-series positional isomers, at least at some concentrations, this did not exhibit the degree of stereospecificity previously reported in some studies for mammalian leukocytes. Little difference was found between the chemokinetic responses of rainbow trout leukocytes to 4- and 5-series LX. These findings suggest that LXs are important migration-inducing molecules in trout and have a relatively long evolutionary history.

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Rainbow trout macrophages maintained in short term culture when incubated with either calcium ionophore, A23187, or opsonized zymosan synthesize a range of lipoxygenase products including lipoxins and leukotrienes. These cells are unusual in that they generate more lipoxin than leukotriene following such challenge. The main lipoxin synthesized was lipoxin (LX) A4. This compound was identified by cochromatography with authentic standard during reversephase high performance liquid chromatography, by ultra violet spectral analysis, radiolabeling following incorporation of [14C]arachidonic acid substrate into macrophage phospholipids, and gas chromatography electron impact mass spectrometry of the methyl ester, trimethylsilyl ether derivative. Other 4-series lipoxins synthesized by trout macrophages were identified as 11-trans-LXA4, 7-cis-11-trans-LXA4, and 6(S)-LXA4. These cells also produced 5-series lipoxins tentatively identified as LXA5, 11-trans-LXA5 and possibly 6(S)-LXA5. No LXB4 or LXB5 was, however, detected. The dynamics of leukotriene and lipoxin release were also determined. Lipoxin generation was slower than leukotriene generation the latter reaching a maximum after 30 min of exposure to ionophore (5 microM, 18 degrees C) compared with 45 min for the former.

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