Precursor Of Platelet-activating Factor Research Articles

Platelet-activating factor induces acrosome reaction via the activation of extracellular signal-regulated kinase in human spermatozoa.

Platelet-activating factor (PAF) affects capacitation, acrosome reaction and fertilisation potential of spermatozoa. This study investigated the underlying mechanism(s) through which PAF regulated sperm function. Our data demonstrated that PAF dose-dependently induced, whilst lyso-PAF (PAF precursor) showed no effect on acrosome reaction of capacitated human spermatozoa. Treatment with PAF for 90min enhanced tyrosine phosphorylation and expression of extracellular signal-regulated protein kinases (ERK) 1 and 2 in human spermatozoa. Moreover, pre-treatment with the ERK inhibitor U0126 significantly and dose-dependently suppressed PAF-induced acrosome reaction. Therefore, PAF may be actively involved in the modulation of sperm acrosome reaction by interacting with ERK. The role of PAF in fertilisation warrants further investigation.

FAR2 is associated with kidney disease in mice and humans.

Mesangial matrix expansion is an important process in the initiation of chronic kidney disease, yet the genetic factors driving its development are unknown. Our previous studies have implicated Far2 as a candidate gene associated with differences in mesangial matrix expansion between mouse inbred strains. Consistent with the hypothesis that increased expression of Far2 leads to mesangial matrix expansion through increased production of platelet-activating factor precursors, we show that FAR2 is capable of mediating de novo platelet-activating factor synthesis in vitro and driven by the transcription factor NKX3.2. We demonstrate that knockdown of Far2 in mice delays the progression of mesangial matrix expansion with at least six months (equivalent to ~15 yr in human). Furthermore, we show that increased FAR2 expression in human patients is associated with diabetic nephropathy, lupus nephritis, and IgA nephropathy. Taken together, these results highlight FAR2's role in the development of mesangial matrix expansion and chronic kidney disease.

Influence of platelet-activating factor, lyso-platelet-activating factor and edelfosine on Langmuir monolayers imitating plasma membranes of cell lines differing in susceptibility to anti-cancer treatment: the effect of plasmalogen level

Three structurally related but differing in biological activities single-chained ether phospholipids (PAF (platelet-activating factor) and lyso-PAF) and an anti-cancer drug (edelfosine (ED)) were investigated in Langmuir monolayers imitating natural membranes. The aim of the undertaken experiments was to study the influence of these lipids on monolayers mimicking plasma membranes of cell lines differing in susceptibility to the anti-cancer activity of ED, i.e. promyelocytic leukaemia cells (HL-60) and promyeloblastic leukaemia cells (K-562). As these cells differ essentially in the cholesterol/phospholipid ratio and plasmalogen concentration in the membrane, we have carried out systematic investigations in artificial systems of various compositions. The results for model leukaemia cell membrane were compared with data acquired for systems imitating normal leucocytes. Our results show that the level of plasmalogens significantly modulates the influence of the single-chained phospholipids on the investigated systems. The experiments confirmed also that the interactions of ether lipids with a model membrane of HL-60 cells (in biological tests sensitive to ED) have opposite character when compared with K-562, being resistant to ED. Moreover, the values of the parameters characterizing monolayers serving as membrane models (strength of interactions, monolayers fluidity and morphology) proved both sensitivity of these cells to ED and lack of their susceptibility towards PAF. Interestingly, it has been found that lyso-PAF, which is usually described as an inactive precursor of PAF, displays a stronger effect on HL-60 model membranes than ED.

Genetic Analysis of Mesangial Matrix Expansion in Aging Mice and Identification of Far2 as a Candidate Gene

Aging of the kidney is associated with renal damage, in particular mesangial matrix expansion (MME). Identifying the genes involved in this process will help to unravel the mechanisms of aging and aid in the design of novel therapeutic modalities aimed at prevention and regression. In this study, structural changes in glomeruli of 24 inbred mouse strains were characterized in male mice at 6, 12, and 20 months of age. Haplotype association mapping was used to determine genetic loci associated with the presence of MME at 20 months. This analysis identified a significant association with a 200-kb haplotype block on chromosome 6 containing Far2. Sequencing revealed that mouse strains with MME contain a 9-bp sequence in the 5' untranslated region of Far2 that is absent in most of the strains without MME. Real-time PCR showed a two-fold increase in the expression of Far2 in the kidneys of strains with the insert, and subsequent experiments performed in vitro with luciferase reporter vectors showed that this sequence difference causes differential expression of Far2. Overexpression of Far2 in a mouse mesangial cell line induced upregulation of platelet activating factor and the fibrotic marker TGF-β. This upregulation of MME-promoting factors may result, in part, from the FAR2-catalyzed reduction of fatty acyl-coenzyme A to fatty alcohols, which are possible precursors of platelet activating factor. Overall, these data suggest the identification of a novel pathway involved in renal aging that may yield therapeutic targets for reducing MME.

Using neurolipidomics to identify phospholipid mediators of synaptic (dys)function in Alzheimer's Disease

Not all of the mysteries of life lie in our genetic code. Some can be found buried in our membranes. These shells of fat, sculpted in the central nervous system into the cellular (and subcellular) boundaries of neurons and glia, are themselves complex systems of information. The diversity of neural phospholipids, coupled with their chameleon-like capacity to transmute into bioactive molecules, provides a vast repertoire of immediate response second messengers. The effects of compositional changes on synaptic function have only begun to be appreciated. Here, we mined 29 neurolipidomic datasets for changes in neuronal membrane phospholipid metabolism in Alzheimer's Disease (AD). Three overarching metabolic disturbances were detected. We found that an increase in the hydrolysis of platelet activating factor precursors and ethanolamine-containing plasmalogens, coupled with a failure to regenerate relatively rare alkyl-acyl and alkenyl-acyl structural phospholipids, correlated with disease severity. Accumulation of specific bioactive metabolites [i.e., PC(O-16:0/2:0) and PE(P-16:0/0:0)] was associated with aggravating tau pathology, enhancing vesicular release, and signaling neuronal loss. Finally, depletion of PI(16:0/20:4), PI(16:0/22:6), and PI(18:0/22:6) was implicated in accelerating Aβ42 biogenesis. Our analysis further suggested that converging disruptions in platelet activating factor, plasmalogen, phosphoinositol, phosphoethanolamine (PE), and docosahexaenoic acid metabolism may contribute mechanistically to catastrophic vesicular depletion, impaired receptor trafficking, and morphological dendritic deformation. Together, this analysis supports an emerging hypothesis that aberrant phospholipid metabolism may be one of multiple critical determinants required for Alzheimer disease conversion.

Opposing Effects of Platelet-Activating Factor and Lyso-Platelet-Activating Factor on Neutrophil and Platelet Activation

Platelet-activating factor (PAF) is a potent, bioactive phospholipid that acts on multiple cells and tissues through its G protein-coupled receptor (GPCR). PAF is not stored but is rapidly generated via enzymatic acetylation of the precursor 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphocholine (lysoPAF). The bioactivity of PAF is effectively and tightly regulated by PAF acetylhydrolases, which convert PAF back to lysoPAF. Previous studies report that lysoPAF is an inactive precursor and metabolite of PAF. However, lysoPAF has not been carefully studied in its own context. Here we report that lysoPAF has an opposing effect of PAF in the activation of neutrophils and platelets. Whereas PAF potentiates neutrophil NADPH oxidase activation, lysoPAF dose-dependently inhibits this function. Inhibition by lysoPAF is not affected by the use of a PAF receptor antagonist or genetic deletion of the PAF receptor gene. The mechanism of lysoPAF-mediated inhibition of neutrophils involves an elevation in the intracellular cAMP level, and pharmacological blockade of adenylyl cyclase completely reverses the inhibitory effect of lysoPAF. In addition, lysoPAF increases intracellular cAMP levels in platelets and inhibits thrombin-induced platelet aggregation, which can be reversed by inhibition of protein kinase A. These findings identify lysoPAF as a bioactive lipid with opposing functions of PAF and suggest a novel and intrinsic regulatory mechanism for balance of the potent activity of PAF.

Increased production of the ether-lipid platelet-activating factor in intestinal epithelial cells infected by Salmonella enteritidis

When exposed to enteric pathogens intestinal epithelial cells produce several cytokines and other proinflammatory mediators. To date there is no evidence that the ether-lipid platelet-activating factor (PAF) is one of these mediators. Our results revealed a significant increase in PAF production by human colonic tissue 4 h after infection by enterohemorrhagic Escherichia coli (EHEC) or Salmonella enteritidis. PAF is produced in the gut by cells of the immune system in response to bacterial infection. To determine whether the epithelial cells of colonic mucosa might also modulate PAF levels, we carried out PAF quantification and analysis of the enzymes involved in PAF synthesis in 5-day-old (undifferentiated) or 28-day-old (differentiated) Caco-2 cell cultures. Infection of undifferentiated Caco-2 cells with either bacterium had no effect on PAF levels, whereas in differentiated cells, infection by S. enteritidis increased PAF levels. Following infection by S. enteritidis, there were no changes in the activity of dithiothreitol-insensitive choline phosphotransferase. However, the enzymes of the remodeling pathway cytosolic phospholipase A 2, which catalyzes the formation of the PAF precursor lysoPAF, and lysoPAF acetyltransferase, are activated in the infected epithelial cells. This response is Ca 2+-dependent.

A Single Enzyme Catalyzes Both Platelet-activating Factor Production and Membrane Biogenesis of Inflammatory Cells: CLONING AND CHARACTERIZATION OF ACETYL-CoA:LYSO-PAF ACETYLTRANSFERASE

Platelet-activating factor (PAF) is a potent proinflammatory lipid mediator eliciting a variety of cellular functions. Lipid mediators, including PAF are produced from membrane phospholipids by enzymatic cascades. Although a G protein-coupled PAF receptor and degradation enzymes have been cloned and characterized, the PAF biosynthetic enzyme, aceyl-CoA:lyso-PAF acetyltransferase, has not been identified. Here, we cloned lyso-PAF acetyltransferase, which is critical in stimulus-dependent formation of PAF. The enzyme is a 60-kDa microsomal protein with three putative membrane-spanning domains. The enzyme was induced by bacterial endotoxin (lipopolysaccharide), which was suppressed by dexamethasone treatment. Surprisingly, the enzyme catalyzed not only biosynthesis of PAF from lyso-PAF but also incorporation of arachidonoyl-CoA to produce PAF precursor membrane glycerophospholipids (lysophosphatidylcholine acyltransferase activity). Under resting conditions, the enzyme prefers arachidonoyl-CoA and contributes to membrane biogenesis. Upon acute inflammatory stimulation with lipopolysaccharide, the activated enzyme utilizes acetyl-CoA more efficiently and produces PAF. Thus, our findings provide a novel concept that a single enzyme catalyzes membrane biogenesis of inflammatory cells while producing a prophlogistic mediator in response to external stimuli.

1‐O‐alkyl‐2‐(ω‐oxo)acyl‐sn‐glycerols from shark oil and human milk fat are potential precursors of PAF mimics and GHB

This study examines the feasibility that peroxidation and lipolysis of 1-O-alkyl-2,3-diacyl-sn-glycerols (DAGE) found in shark liver oil and human milk fat constitutes a potential source of dietary precursors of platelet activating factor (PAF) mimics and of gamma-hydroxybutyrate (GHB). Purified DAGE were converted into 1-O-alkyl-2-acyl-sn-glycerols by pancreatic lipase, without isomerization, and transformed into 1-O-alkyl-2-oxoacyl-sn-glycerols by mild autooxidation. The various core aldehydes without derivatization, as well as the corresponding dinitrophenylhydrazones, were characterized by chromatographic retention time and diagnostic ions by online electrospray mass spectrometry. Core aldehydes of oxidized shark liver oil yielded 23 molecular species of 1-O-alkyl-sn-glycerols with short-chain sn-2 oxoacyl groups, ranging from 4 to 13 carbons, some unsaturated. Autooxidation of human milk fat yielded 1-O-octadecyl-2-(9-oxo)nonanoyl-sn-glycerol, as the major core aldehyde. Because diradylglycerols with short fatty chains are absorbed in the intestine and react with cytidine diphosphate-choline in the enterocytes, it is concluded that formation of such PAF mimics as 1-O-alkyl-2-(omega-oxo)acyl-sn-glycerophosphocholine from unsaturated dietary DAGE is a realistic possibility. Likewise, a C4 core alcohol produced by aldol-keto reduction of a C4 core aldehyde constitutes a dietary precursor of the neuromodulator and recreational drug GHB, which has not been previously pointed out.

Platelet-activating factor in cirrhotic liver and hepatocellular carcinoma

Platelet-activating factor (PAF) is a pro-inflammatory and angiogenic lipid mediator. Here we aimed to investigate levels of PAF, lyso-PAF (the PAF precursor), phospholipase A(2) (PLA(2), the enzymatic activity generating lyso-PAF), acetylhydrolase activity (AHA, the PAF degrading enzyme) and PAF receptor (PAF-R) transcripts in cirrhotic liver and hepatocellular carcinoma (HCC). Twenty-nine patients with HCC were enrolled in this study. Cirrhosis was present in fourteen patients and seven had no liver disease. Tissue PAF levels were investigated by a platelet-aggregation assay. Lyso-PAF was assessed after its chemical acetylation into PAF. AHA was determined by degradation of [(3)H]-PAF. PLA(2) levels were assessed by EIA. PAF-R transcripts were investigated using RT-PCR. Elevated amounts of PAF and PAF-R transcripts 1 (leukocyte-type) were found in cirrhotic tissues as compared with non-cirrhotic ones. Higher amounts of PAF and PAF-R transcripts 1 and 2 (tissue-type) were found in HCC tissues as compared with non-tumor tissues. PLA(2), lyso-PAF and AHA levels were not changed in cirrhotic tissues and HCC. While the role of PAF is currently unknown in liver physiology, this study suggests its potential involvement in the inflammatory network found in the cirrhotic liver and in the angiogenic response during HCC.

Platelet-activating factor and human thyroid cancer

Platelet-activating factor (PAF) is a pro-inflammatory and angiogenic lipid mediator involved in several types of cancer in humans. The levels of PAF, lyso-PAF (the PAF precursor), phospholipase A2 activity (PLA2, the enzymatic activity implicated in lyso-PAF formation) and acetylhydrolase activity (AHA, the PAF-degrading enzyme) were investigated in various diseased thyroid tissues. Control and diseased tissue of patients with a hyperplastic goitre (n = 14), a benign adenoma (n = 12) and a papillary thyroid carcinoma (n = 15) were investigated. PAF receptor transcripts were found in the human thyroid tissue. PAF, lyso-PAF, PLA2 and AHA were present in control thyroid tissues, their levels being significantly correlated with each other, suggesting tiny regulations of the PAF metabolic pathways inside the thyroid gland. PAF, lyso-PAF, PLA2 and AHA levels remained unchanged in diseased tissues of patients with a hyperplastic goitre, a benign adenoma and a papillary thyroid carcinoma. No difference was found between PAF, lyso-PAF, PLA2 and AHA levels with respect to the TNM tumour status and the histological sub-type of papillary thyroid carcinoma. No correlation was found between tissue PAF levels and those of vascular endothelial growth factor and basic fibroblast growth factor, two angiogenic growth factors involved in thyroid cancer and that mediate their effect through PAF release in breast and colorectal cancer. PAF, PAF receptor transcripts and the enzymatic activities implicated in PAF production and degradation are present in the thyroid gland. While the physiological role of PAF is presently unknown in thyroid physiology, this study highlights no evidence for a potentially important role of PAF during human thyroid cancer, a result that markedly differs from breast and colorectal ones.

Platelet-activating factor acts on cortisol secretion by perfused guinea-pig adrenals via calcium-/phospholipid-dependent mechanisms

Bilateral adrenals of the guinea pig were perfused in situ with an artificial medium equilibrated with 95% O2/5% CO2. Platelet-activating factor (PAF) induced biphasic cortisol responses, which reached a maximum at 10 nM PAF and declined at 100 nM. The effect of the PAF receptor antagonists CV-3988 and CV-6209 on PAF-stimulated cortisol secretion was examined. Prior exposure of adrenal glands to 10 microM CV-3988 or a simultaneous incubation with 10 microM CV-6209 abolished the cortisol response to 10 nM PAF. Lyso-PAF (a PAF precursor and breakdown product) did not affect cortisol secretion. Concentrations of 5-12.5 microM 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a protein kinase C (PKC) inhibitor, abolished subsequent cortisol secretion in response to 10 nM PAF. N-[2-(Methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H-8), a protein kinase A inhibitor, was less effective. A calcium ionophore (A23187) at 3.3 and 10 microM increased cortisol secretion, but the activator of PKC, l-alpha-1-oleoyl-2-acetyl-sn-3-glycerol (OAG), at 50 microM had no effect. When infused simultaneously, OAG (50 microM) and A23187 (3.3 microM) stimulated cortisol secretion synergistically. The secretory response of cortisol to repeated infusions of adrenocorticotrophin (100 pg/ml) or forskolin (10 microM) was essentially reproducible. By contrast, cortisol secretion in response to repeated infusions of PAF (10 nM) or OAG plus A23187 was not reproducible and the second response was diminished compared with the first. Our findings suggest that PAF plays a role in the regulation of steroidogenesis via a mechanism mediated by the PAF receptor and PKC.

Cloning, Genomic Organization, and Characterization of a Human Cholinephosphotransferase

A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated using in vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeast CPT1 gene in its interaction with SEC14 and affect cell growth.

Modulation of PAF production by incorporation of arachidonic acid and eicosapentaenoic acid in phospholipids of human leukemic monocyte-like cells THP-1

Stimulated leukocytes generate platelet-activating factor (PAF) from membrane 1- O-alkyl-2-acyl-sn-glycerophosphocholine through hydrolysis of fatty acid and subsequent acetylation at the sn2 position of glycerol. Since the enzymes involved in the hydrolysis step of PAF biosynthesis have relative selectivity for arachidonic acid (AA), the fatty acid composition of PAF precursors might modulate PAF production. We studied the effect of AA and eicosapentaenoic acid (EPA) incorporation on PAF biosynthesis, by measuring the incorporation of [ 3H]acetate, in Ca 2+ ionophore (A23187)-stimulated human leukemic monocyte-like cells, THP-1. Supplementation of THP-1 with AA (25 μM, 1 week) or EPA (25 μM, 1 week) led to their efficient incorporation, in comparable quantities and with similar distributions, into phosphatidylcholine and phosphatidylethanolamine, and to a lesser extent into phosphatidylinositol. THP-1 cells supplemented with AA or with EPA synthetized similar amounts of PAF and of acyl analog of PAF under resting condition. However, AA-supplemented cells responded to A23187 stimulation by important raises of PAF (+125.71%) and of acyl analog of PAF (+381.75%) productions, whereas the same stimulation had little effect or no effect at all in cells supplemented with EPA. These results show that both EPA and AA may influence PAF production through their incorporation into PAF precursors, indicating that PAF production might be modulated by the fatty acid composition of its precursors.

Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2.

Phospholipase A2 (PLA2) enzymes are critical regulators of prostaglandin and leukotriene synthesis and can directly modify the composition of cellular membranes. PLA2 enzymes release fatty acids and lysophospholipids, including the precursor of platelet-activating factor, PAF, from phospholipids. Free fatty acids, eicosanoids, lysophospholipids and PAF are potent regulators of inflammation, reproduction and neurotoxicity. The physiological roles of the various forms of PLA2 are not well defined. The cytosolic form, cPLA2, preferentially releases arachidonic acid from phospholipids and is regulated by changes in intracellular calcium concentration. We have now created 'knockout' (cPLA2-/-) mice that lack this enzyme, in order to evaluate its physiological importance. We find that cPLA2-/- mice develop normally, but that the females produce only small litters in which the pups are usually dead. Stimulated peritoneal macrophages from cPLA2-/- animals did not produce prostaglandin E2 or leukotriene B4 or C4. After transient middle cerebral artery occlusion, cPLA2-/- mice had smaller infarcts and developed less brain oedema and fewer neurological deficits. Thus cPLA2 is important for macrophage production of inflammatory mediators, fertility, and in the pathophysiology of neuronal death after transient focal cerebral ischaemia.

Evidence that 85 kDa phospholipase A 2 is not linked to CoA-independent transacylase-mediated production of platelet-activating factor in human monocytes

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from l-O-alkyl-2-arachidonoyl- sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC 50S of 10 and 12 µM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A 2 (PLA 2), and AACOCF 3, an inhibitor of 85 kDa PLA 2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF 3 had no effect on A23187-induced PAF formation at concentrations as high as 3 µM. Further, depletion of 85 kDa PLA 2 using antisense (SB 7111, 1 µM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA 2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA 2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC 50s of 2 and 0.5 µM, respectively), suggesting a key role of 14 kDa PLA 2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 µM) had a greater effect on the production of 1-O-alkyl (−80%) than of 1-acyl (−14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA 2, but not rh 85 kDa PLA 2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA 2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA 2-like activity, and not 85 kDa PLA 2, is coupled to monocyte CoA-IT-induced PAF production.

Presence and metabolism of lyso platelet-activating factor in human bone marrow

Lyso platelet-activating factor (PAF) is the precursor of PAF, an inflammatory phospholipid molecule present in human bone marrow. The present study shows that in healthy volunteers lyso PAF concentrations are significantly lower ( P=0.0001, Mann-Whitney U-test) in bone marrow plasma (594 ± 67 ng/ml, n = 47) than in blood plasma (1448 ± 99 ng/ml, n = 31). Marrow plasma lyso PAF concentrations are similar in patients with lymphoid and nonlymphoid malignancies as compared with controls. Freshly isolated mononuclear marrow cells and cultures of marrow stromal cells contain lyso PAF. Experiments with [ 3H]lyso PAF indicate that human mononuclear bone marrow cells and marrow stromal cells actively acylate lyso PAF into a 1-alkyl analogue of phosphatidylcholine. Results of this investigation indicate: (1) that lyso PAF is present in human marrow cells and plasma; and (2) that marrow cells and stromal cells metabolize it, thus suggesting their role in the regulation of lyso PAF amounts in human bone marrow.

Transgenic mice expressing rabbit C-reactive protein are resistant to endotoxemia.

C-reactive protein (CRP), the prototypic acute-phase reactant in humans, is synthesized in liver in response to a wide variety of inflammatory stimuli. We have generated a line of transgenic mice that express rabbit CRP from the rat phosphoenolpyruvate carboxykinase (PEPCK) promoter in response to gluconeogenic signals. Here we show that transgenic mice expressing high levels of CRP were partially protected from a lethal challenge of bacterial lipopolysaccharide compared with littermates in which CRP expression had been suppressed. Similar protection was observed with challenges from platelet-activating factor (PAF) and the combination of tumor necrosis factor alpha (TNF-alpha) plus interleukin 1beta, but not with TNF-alpha alone. We further demonstrate that although PAF was able to bind CRP, the mechanism by which CRP provides protection probably does not involve sequestration of PAF. The biologically inactive precursor of PAF, lyso-PAF, also bound CRP but did not render the transgenic mice sensitive to PAF when CRP-expressing animals were simultaneously challenged with PAF and an excess of lyso-PAF. These results suggest that CRP functions in vivo by modulating host defense systems.

Changes in PAF (platelet-activating factor) production during cell cycle of yeast Saccharomyces cerevisiae.

Yeast Saccharomyces cerevisiae cells were cultured synchronously and the change of platelet-activating factor (PAF) production during the cell cycle was investigated at each phase of the cycle. The basal PAF contents of diploid AKU4103 cells in G1 and M phases were higher than those of cells in S phase. Both diploid and haploid strains showed the same level of PAF production in response to the calcium ionophore A23187. A23187-stimulated PAF productions of cells in G1 and M phases were about 20 times higher than that of cells in S phase. The contents of PAF precursor in G1 and M phases cells of AKU4103 were higher than those in S phase cells, and the ratio of A23187-stimulated PAF to the precursor was highest in G1 phase cells. We also examined the change in a PAF-synthesizing enzyme, acetyltransferase, activity during the cell cycle using a microsomal fraction. Specific activity was the highest at G1 phase, and total activity was higher at M phase. The enzyme activities of cells in S phase of strains AKU4103 and RAY-3Aa were one-third and one-tenth of those in G1 phase of corresponding cells, respectively. These results suggest that PAF production was higher at G1 and M phases and lower at S phase, and changes in PAF productivity during cell cycle were related to the precursor contents and the synthesizing enzyme activities in those cells. These data suggest that PAF may control the cell cycle phase in budding yeast.

Platelet-Activating Factor Biosynthesis Induced by Various Stimuli in Human HaCaT Keratinocytes

Platelet-activating factor (PAF) is a potent inflammatory mediator that is thought to play a role in cutaneous inflammation. These studies used mass spectrometry to examine the molecular species of PAF precursor glycerophosphocholine lipids (GPC) as well as the biosynthesis of PAF and other sn-2 acetyl-GPC in a human keratinocyte-derived cell line (HaCaT keratinocytes). Approximately 28% of HaCaT keratinocyte GPC consisted of 1-alkyl species, and the relative amounts of the sn-1 alkyl constituents of the PAF precursor 1-alkyl-2-acyl-GPC were as follows: hexadecyl > octadecenyl > octadecyl. Ionophore (A23187)-stimulated HaCaT keratinocytes synthesized both PAF (1-hexadecyl, 1-octadecenyl, and 1-octadecyl species) and less potent 1-acyl analogs (1-palmitoyl, 1-oleoyl, and 1-stearoyl species). PAF production was rapid and maximal by 10 min. The major species of sn-2acetyl-GPC at 2.5 min were 1-hexadecyl-2-acetyl-GPC (2.2 ng/10(6) cells) and 1-palmitoyl-2-acetyl-GPC (2.4 ng/10(6) cells). HaCaT keratinocytes also synthesized PAF and 1-acyl PAF analogs when stimulated with the peptide growth factor endothelin-1 and the nonhydrolyzable PAF receptor agonist carbamyl-PAF. Both 1-hexadecyl-2- acetyl-GPC and 1-palmitoyl-2-acetyl-GPC stimulated intracellular calcium mobilization in HaCaT cells, indicating that these sn-2 acetyl-GPC act in autocrine fashion. These studies revealed that the human keratinocyte-derived cell line HaCaT can synthesize significant amounts of PAF and 1-acyl analogs in vitro from both nonspecific (A23187) and specific (endothelin-1, carbamyl-PAF) stimulation, suggesting a role for this inflammatory lipid mediator in keratinocyte pathophysiology.