Differential effect of platelet activating factor on 1-methyl-4-phenylpyridinium-induced cell death through regulation of apoptosis-related protein activation

Platelet activating factor (PAF) has been suggested to play a critical role in the pathogenesis of neurological disorders. We assessed the effect of PAF against the toxicity of 1-methyl-4-phenylpyridinium (MPP⁺), a parkinsonian toxin, in relation to apoptotic process. PAF exhibited differential effect against the MPP⁺ toxicity in differentiated PC12 cells depending on concentration. Treatment with 0.75 μM PAF significantly attenuated the MPP⁺-induced increase in Bax levels, decrease in Bid and Bcl-2 levels, and mitochondrial membrane potential loss that lead to the release of cytochrome c and subsequent caspase-3 activation. The inhibitory effect of PAF was not associated with nuclear factor-κB activation. In contrast, PAF at the concentrations greater than 2.5 μM exhibited a toxicity and additive effect on the MPP⁺ toxicity. The results show that PAF at low concentrations, which does not induce a significant toxicity, may prevent the MPP⁺ toxicity by suppressing the apoptosis-related protein activation and mitochondrial membrane permeability change that lead to the cytochrome c release and caspase-3 activation. The preventive effect seems to be associated with the inhibitory effect on the formation of reactive oxygen species and depletion of GSH. In contrast, PAF at higher concentrations may exhibit an additive toxic effect against the MPP⁺ toxicity by increasing apoptosis-related protein activation.     

Proliferating cell unclear antigen-associated factor (PAF15): A novel oncogene

Proliferating cell nuclear antigen (PCNA)-Associated Factor (PAF15) is a small protein containing a PCNA interacting motif and sequences for association with ubiquitin enzymes. In interaction with PCNA, PAF15 plays a key role in recruiting DNA replicative polymerase by double monoubiquitination at Lys¹⁵ and Lys²⁴. Under DNA damage conditions, PAF15 regulates the switch from DNA replicative polymerase to translesion synthesis polymerase in order to bypass the replication-blocking lesions. Overexpression of PAF15 promotes the repair of ultraviolet-induced DNA damage and prevents cell death, whereas attenuation of PAF15 decreases DNA replication and cell survival. Ectopic expression of PAF15 in mouse fibroblasts increases colony formation and tumourigenicity. PAF15 is aberrantly increased in various human malignancies with poor prognosis. Collectively, PAF15 may contribute to carcinogenesis and represents one of the potential therapeutic targets in the treatment of cancer.     

Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family

Four naturally occurring platelet-activating factor (PAF) analogs, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine, 1-hexade-canoyl-2-acetyl-sn-glycero-3-phosphocholine, 1-octadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, stimulated human neutrophils (PMN) to mobilize Ca²⁺, degranulate, and produce Superoxide anion. They were, respectively, 5-, 300-, 500-, and 4000-fold weaker than PAF in each assay; inhibited PMN-binding of [³H]PAF at concentrations paralleling their biological potencies; and showed sensitivity to the inhibitory effects of PAF antagonists. PAF and the analogs, moreover, desensitized PMN responses to each other but not to leukotriene B₄ and actually increased (or primed) PMN responses to N-formyl-MET-LEU-PHE. Finally, 5-hydroxyicosatetraenoate-enhanced PMN responses to PAF and the analogs without enhancing the actions of other stimuli. It stereospecifically raised each analog's potency by as much as 100-fold and converted a fifth natural analog, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine from inactive to a weak stimulator of PMN. PAF and its analogs thus represent a structurally diverse family of cell-derived phospholipids which can activate, prime, and desensitize neutrophils by using a common, apparently PAF receptor-dependent mechanism.     

Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (Δsse ^MGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. K_M and k _cat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of Δsse ^MGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses.     

Rapid Production of Platelet-activating Factor Is Induced by Protein Kinase Cα-mediated Phosphorylation of Lysophosphatidylcholine Acyltransferase 2 Protein

Platelet-activating factor (PAF), a potent proinflammatory lipid mediator, is synthesized rapidly in response to extracellular stimuli by the activation of acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAFAT). We have reported previously that lyso-PAFAT activity is enhanced in three distinct ways in mouse macrophages: rapid activation (30 s) after PAF stimulation and minutes to hours after LPS stimulation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2) was later identified as a Ca²⁺-dependent lyso-PAFAT. However, the mechanism of rapid lyso-PAFAT activation within 30 s has not been elucidated. Here we show a new signaling pathway for rapid biosynthesis of PAF that is mediated by phosphorylation of LPCAT2 at Ser-34. Stimulation by either PAF or ATP resulted in PKCα-mediated phosphorylation of LPCAT2 to enhance lyso-PAFAT activity and rapid PAF production. Biochemical analyses showed that the phosphorylation of Ser-34 resulted in augmentation of V_max with minimal K_m change. Our results offer an answer for the previously unknown mechanism of rapid PAF production.     

A new method for evaluating defense of microalgae against protozoan grazing

Body-size spectrum has proved to be a highly informative indicator to summarize the functional structure of a community at taxon-free resolution. In this study, an approach based on body-size spectrum of protozoan communities was used to detect the defense of microalgae against protozoan grazing. The biofilm-dwelling protozoan communities were used as a test predator system, and two algal species, Chlorella sp. and Nannochloropsis oceanica, were employed as test microalgae. A nine-day bioassay test was carried out by exposing biofilm-dwelling protozoan communities to a gradient of concentrations 10⁰ (control), 10⁴, 10⁵, 10⁶, and 10⁷ cell ml⁻¹ of both microalgae, respectively. Results showed that both algal species represented strong defense effects on the test predator system at different levels of concentration. The body-size distinctness of the protozoan assemblages showed a sharp decrease at high concentration level more than 10⁶ cell ml⁻¹ in both algal treatments. Based on the paired body-size distinctness indices of the protozoa, ellipse tests demonstrated that the body-size spectrum showed an increasing trend of departure from the expected pattern with increasing concentrations of both test algae. Thus, it is suggested that the body-size spectrum of protozoa may be used as a useful indicator to identify the defense of microalgae against protozoan grazing.     

Characterization of3H-labelled platelet activating factor receptor complex solubilized from rabbit platelet membranes

Rabbit platelet membranes, preincubated with³H-labeled platelet activating factor ([³H]PAF), were solubilized with 2% digitonin. Sedimentation of the detergent extract in a sucrose density gradient revealed a major labeled component with a sedimentation coefficient (s_20,ω) of 10.5 S, which was substantially diminished when an excess of unlabeled PAF or L-652,731, (trans-2,5-bis(3,4,5-trimethoxyphenyl)tetrahydrofuran), (PAF antagonist) was present in the preincubation mixture, suggesting that the 10.5 S component is a specific receptor-bound [³H]PAF complex. Gel filtration of the [³H]PAF-receptor complex on Sephacryl S-300 revealed a single radiolabeled fraction with an apparent Stokes' radius of 4.9 nm. The apparent molecular weight and the frictional ratio of the agonist-receptor complex were computed to be 220 000 and 1.13, respectively. Dissociation of [³H]PAF from the radioligand-receptor complex was facilitated by Na⁺ and Li⁺, whereas K⁺ and Cs⁺ were ineffective. The guanine nucleotide, GTP, was also found to promote the dissociation in a manner that is additive with the effect of Na⁺, suggestive of the coupling of a guanine nucleotide binding protein to the solubilized PAF-receptor complex.     

Effect of 1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-acether) on leukocytes I. Analysis of the in vitro migration of human neutrophils

The effect of synthetic 1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-acether) and of 1-O-octadecyl-sn-glycero-3-phosphocholine (lyso-PAF-acether) on human neutrophil migration was studied in modified Boyden chambers, with the following results: (1) By checker-board analysis and deactivation experiments, the factors are chemokinetic at low (10^?8 M) and chemotactic at higher concentrations (10^?6 M), with lyso-PAF-acether being less potent at all concentrations. (2) Cross-deactivation occurs between the two PAF compounds, but not with two other chemotactic factors, suggesting a specific, common receptor for the PAFs on the neutrophil membrane. (3) Other chemotactic substances may act as potentiating or additive factors to the PAF compounds. (4) Inhibition of arachidonic acid turnover during chemotaxis by compound BW 755 C enhances leukocyte chemotaxis towards the PAF compounds and towards other chemotactic factors. The data suggest that PAF and its lyso-derivate may contribute in a unique and potent fashion to leukocyte accumulation at inflammatory sites.     

Effect of platelet-activating factor (PAF) on sodium calcium exchange in cardiac sarcolemmal vesicles

Summary The effects of platelet-activating factor (PAF) on Na⁺-dependent calcium uptake in myocardial sarcolemmal vesicles were examined in order to clarify its mechanism of inotropic action on the heart. PAF (40 and 20 µM) significantly inhibited Na⁺-Ca²⁺ exchange by 61% and 37%, respectively. Both initial rate of exchange and maximal exchange were inhibited. The Km for the reaction was not altered but Vmax was lowered 55% by PAF. Lyso-PAF inhibited Na⁺-Ca²⁺ exchange to a similar degree as PAF. CV-3988, a specific PAF receptor antagonist, failed to diminish the inhibitory effect of PAF on Na⁺-Ca²⁺ exchange, suggesting that the effect of PAF on Na⁺-Ca ²⁺ exchange is not via a receptor mechanism. The passive permeability of sarcolemmal vesicles to Ca²⁺ was markedly elevated after PAF treatment. However, this effect could not account for the decrease in Na⁺-Ca²⁺ exchange. Interestingly, passive Ca²⁺ binding to cardiac sarcolemma was increased by 40 µM PAF. This study indicates that a depression of Na⁺-Ca²⁺ exchange probably does not play a role in the negative inotropic effect of PAF on the myocardium under physiological conditions. Its mechanism of action on Na⁺-Ca²⁺ exchange is discussed.     

Effect of detritus supply on trophic relationships within experimental benthic food webs. II. Microbial responses,fate and composition of decomposing detritus

The dynamics of bacterial, protozoan and fungal populations were examined in a long-term (40–55 wk) microcosm experiment designed to assess the effects of detritus supply on meiofauna-polychaete (Capitella capitata (Type I) Fabricius) interactions. Bacterial and protozoan numbers and bacterial growth rates were inversely correlated with population fluctuations of the polychaete at low (50 mg N · m^?2 · day ^?1) detritus supply, but did not correlate with fluctuating polychaete densities at two higher (100 and 150 mg N · m^?2 · day^?1) ration levels of detritus. Bacterial and protozoan numbers and bacterial growth rates did not correlate with standing amounts of detritus or with fungi or meiofauna at any of the detritus rations. Fungi were associated primarily with aggregates of detritus particles and fecal pellets produced by C. capitata.Labile (fiber-free) organic matter did not correlate with microbes or meiofauna, but was inversely correlated with population fluctuations of the polychaete C. capitata at all three ration levels of detritus. Polychaete fecal pellets accounted for most of the refractory matter in the tanks with C. capitata and did not accumulate in the sediments, suggesting that fecal pellets were continually being produced, broken apart and decomposed.Our experiments suggest that contradictions in previous studies on the effects of macroconsumers on microbes, especially bacteria, can be explained as a failure to consider the effects of detritus supply on microbial growth rates.     

Protective immunity against lethal anaphylactic reaction in Toxoplasma gondii-infected mice by DNA vaccination with T. gondii-derived heat shock protein 70 gene

Toxoplasma gondii-derived heat shock protein 70 (T.g.HSP70) was proven to induce lethal anaphylactic reaction in T. gondii-infected mice through platelet-activating factor (PAF)-mediated, but not classical IgE-dependent, pathway via TLR4/MyD88 signal pathway. The effector cells generating PAF and causing T.g.HSP70-induced anaphylactic reaction were CD11b⁺ and CD11c⁺ cells, although the reaction was enhanced by marked IFN-γ production by CD11b⁺, CD11c⁺, CD4⁺ and CD8⁺ splenocytes. In the present study, the effects of T.g.HSP70 gene vaccine targeting peripheral dendritic cells were evaluated against T.g.HSP70-induced anaphylactic reaction in T. gondii-infected mice. C57BL/6 mice receiving T.g.HSP70 gene vaccine showed prolonged survival. Platelets of peripheral blood, which completely disappeared during the T.g.HSP70-induced anaphylactic reaction, were partially restored with the T.g.HSP70 gene vaccination. The T.g.HSP70-induced marked production of PAF and IFN-γ from splenocytes of infected mice during the T.g.HSP70-induced anaphylactic reaction was shown to decrease after the T.g.HSP70 gene vaccination. Thus, T.g.HSP70 gene vaccine induced protective immunity against T.g.HSP70-induced PAF-mediated lethal anaphylactic reaction in T. gondii-infected mice.     

Autoradiographic localization of platelet-activating factor (PAF) binding sites in the rabbit endometrium during the peri-implantation period

Summary This communication describes the use of in-vivo and in-vitro autoradiography to map specific platelet-activating factor (PAF) receptors in the rabbit uterus. Specific [³H]PAF uptake was predominantly localized on epithelial, but not on stromal or myometrial cells. Very few silver grains were associated with the luminal epithelial cells in the uterus of the estrous rabbit, primarily because of the non-differentiated state of the epithelium. In the differentiated pregnant uterus, significantly more [³H]PAF was bound to the glandular epithelial cells, with the stromal cells binding consistently significantly less. The highest density of silver grains was observed at the implantation sites on day 7 of pregnancy. There was no apparent difference in [³H]PAF C18:0 uptake between the epithelial cells at the inter-implantation zone on day 7 and on day 6. Bound [³H]PAF was displaceable by lyso-PAF, U66985, CV3988, but not U66982, L652,731, SRI 63,441 or the inactive PAF isomer, oleoyl PAF. Bovine serum albumin (BSA) significantly inhibited tissue uptake of [³H]PAF C18:0. Intraluminally administered [³H]PAF C18:0 and intravenously injected [³H]methylcarbamyl-PAF, a non-metabolizable PAF analog, penetrated the implanted blastocyst and bound to the embryoblast. This event was reproducible in vitro with pre-implantation blastocysts from day-6 pregnant rabbits, which suggests that uterine-derived PAF may translocate into the blastocyst after attachment.     

Topical Application of a Platelet Activating Factor Receptor Agonist Suppresses Phorbol Ester-Induced Acute and Chronic Inflammation and Has Cancer Chemopreventive Activity in Mouse Skin

Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-Kit ^W-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-Kit ^W-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development.     

The Antifungal Activity of the Penicillium chrysogenum Protein PAF Disrupts Calcium Homeostasis in Neurospora crassa

The antifungal protein PAF from Penicillium chrysogenum exhibits growth-inhibitory activity against a broad range of filamentous fungi. Evidence from this study suggests that disruption of Ca²⁺ signaling/homeostasis plays an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca²⁺ concentrations counteracted PAF toxicity toward PAF-sensitive molds. By using a transgenic Neurospora crassa strain expressing codon-optimized aequorin, PAF was found to cause a significant increase in the resting level of cytosolic free Ca²⁺ ([Ca²⁺]_c). The Ca²⁺ signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA [bis-(aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid], a Ca²⁺ selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted PAF induced perturbation of Ca²⁺ homeostasis. These results indicate that extracellular Ca²⁺ was the major source of these PAF-induced effects. The L-type Ca²⁺ channel blocker diltiazem disrupted Ca²⁺ homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and accentuating the change in Ca²⁺ signatures in response to external stimuli. Notably, both PAF and diltiazem increased the [Ca²⁺]_c resting level. However, experiments with an aequorin-expressing Δcch-1 deletion strain of N. crassa indicated that the L-type Ca²⁺ channel CCH-1 was not responsible for the observed PAF-induced elevation of the [Ca²⁺]_c resting level. This study is the first demonstration of the perturbation of fungal Ca²⁺ homeostasis by an antifungal protein from a filamentous ascomycete and provides important new insights into the mode of action of PAF.The secreted antifungal protein PAF from Penicillium chrysogenum is a small-molecular-mass (6.2 kDa), cationic, and cysteine-rich peptide that inhibits the growth of numerous filamentous fungi (14–16, 21). It belongs to a family of antifungal peptides which show—despite considerable amino acid homology—significant differences in species specificity and modes of action (reviewed in reference 27). Importantly, the primary structures of these antifungals show no similarity to those from higher eukaryotes, e.g., plants, insects, or mammals (see reference 28 for a detailed review on parallels with and differences between PAF and antimicrobial proteins from higher eukaryotes and their mechanisms of action). Apart from the Aspergillus giganteus-derived antifungal protein AFP (19, 45; reviewed in reference 29), PAF is one of the best-studied peptides of this protein family. We have shown that PAF causes rapid hyperpolarization of the plasma membrane at hyphal tips, increased K⁺ efflux, induction of oxidative stress, and apoptotic cell death (21, 25) and that PAF is internalized by hyphae of PAF-sensitive fungi (33). Furthermore, we have shown that PAF interferes with at least two signaling cascades, the protein kinase C/mitogen-activated protein (MAP) kinase and the cyclic AMP (cAMP)/protein kinase A pathways, which play a role in mediating PAF toxicity (5). However, it still has to be elucidated in more detail how these PAF-dependent effects are linked.Recent evidence indicated that the ionic strength of the growth medium interferes with the antifungal activity of PAF (21). It is possible that Ca²⁺ ions may play a major role in influencing protein toxicity in an analogous way to the Ca²⁺-dependent mode of action of antifungal plant defensins. Supplementation of the test medium with low concentrations of CaCl₂ (1 to 5 mM) reversed the antifungal activity of plant defensins (34, 43–44). The defensins RsAFP2 from the seeds of Raphanus sativus and DmAMP1 from the seeds of Dahlia merckii induced K⁺ efflux and Ca²⁺ uptake in Neurospora crassa and caused alkalinization of the growth medium (46). Another seed defensin, MsDef1 from Medicago sativa, was reported to cause Ca²⁺ influx and the inhibition of mammalian L-type Ca²⁺ channels, similar to the Ustilago maydis killer toxin KP4 (13, 40).Ca²⁺ is a universal intracellular second messenger in eukaryotic cells (4, 36). In fungi, there is evidence for Ca²⁺ signaling regulating numerous processes, including spore germination, tip growth, hyphal branching, sporulation, infection structure differentiation, and circadian clocks, as well as responses to osmotic stress, heat shock, mechanical stimuli, oxidative stress, and electrical fields (7, 11, 17, 20, 22, 30–31, 38–39, 41, 49). Ca²⁺ signaling typically involves transient increases in intracellular Ca²⁺ concentrations originating from the extracellular medium and/or mobilization from internal stores (4, 23, 36–37). Little was known about Ca²⁺ dynamics in filamentous fungi until a new method based on the Ca²⁺-sensitive photoprotein aequorin was adapted, allowing routine and easy measurement of intracellular calcium dynamics in these organisms (3, 30).The aim of our study was to investigate the effect of PAF on the level of cytosolic free Ca²⁺ ([Ca²⁺]_c) in the PAF-sensitive target organism N. crassa to characterize its effects on stimulus-specific Ca²⁺ signatures and to define the Ca²⁺ sources responsible for the perturbation of Ca²⁺ homeostasis. By obtaining [Ca²⁺]_c measurements in living cells expressing the bioluminescent Ca²⁺ reporter aequorin, we provide novel insights into the mode of action of this biotechnologically promising antifungal protein.     

4-Methylumbelliferyl-β-N-Acetylglucosaminide Hydrolysis by a High-Affinity Enzyme, a Putative Marker of Protozoan Bacterivory

Hydrolysis of an artificial fluorogenic substrate, 4-methylumbelliferyl-β-N-acetylglucosaminide, has been studied in a monoculture predator-prey system with either a flagellate (Bodo saltans) or a ciliate (Cyclidium sp.) fed upon pure bacterial culture (Aeromonas hydrophila or Alcaligenes xylosoxidans). Aeromonas hydrophila produced a low-affinity β-N-acetylglucosaminidase-like enzyme (K_m, 100 μmol liter^-1) but Alcaligenes xylosoxidans did not. Inoculation of both bacterial strains with bacterivorous protozoa induced the occurrence of another, high-affinity, β-N-acetylglucosaminidase-like enzyme (K_m, <0.5 μmol liter^-1). The latter enzyme showed significant, close correlations with total grazing rates of both B. saltans (r² = 0.96) and Cyclidium sp. (r² = 0.89) estimated by using uptake of fluorescently labelled bacteria. Further significant correlations between several protozoan parameters and kinetic parameters of this enzyme suggest its likely protozoan origin. If both types of enzyme occurred together, they could be satisfactorily distinguished by using kinetic data analysis. Hence, measurements of β-N-acetylglucosaminidase-like activities might be promising to use to improve estimations of protozoan bacterivory.     

Characterization of the enzymatic hydrolysis of acetate from alkylacetylglycerols in the de novo pathway of PAF biosynthesis

This report describes the partial characterization of the enzymatic activity responsible for the hydrolysis of acetate from 1-alkyl-2-acetyl-sn-glycerol, the immediate precursor in the de novo synthesis of PAF (platelet-activating factor or 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by Ehrlich ascites cells. The highest acetylhydrolase activity for this neutral lipid was associated with the membrane fractions from Ehrlich ascites cells (> 90% of total activity); only a minimal level of activity (< 10%) was observed in the cytosol which contrasts with the cytosolic site of PAF acetylhydrolase in normal cells. Hydrolysis of 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction at pH 7.5 and 37°C gave apparent values for K_m and V_max of 45 μM and 179 nmol/min per mg protein, respectively. Hydrolysis of acetate from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by the membrane fraction was not affected by 5 mM concentrations of Ca⁺², Mg⁺² or EDTA, but was significantly inhibited (80% reduction) by 10 mM NaF. Based on differences in both the subcellular distribution and response to inhibition by NaF, the neutral lipid acetylhydrolase does not appear to be the same enzyme that hydrolyzes acetate from platelet-activating factor. In contrast to inhibition of diacylglycerol lipase by p-chloromercuribenzoate and N-ethylmaleimide, we found no significant inhibition of acetate hydrolysis from 1-[³H]hexadecyl-2-acetyl-sn-glycerol by either of these compounds. Also, p-nitrophenyl acetate (a nonspecific esterase substrate) failed to inhibit acetate hydrolysis of 1-[³H])hexadecyl-2-acetyl-sn-glycerol. Our studies of this enzyme would indicate that it may play an important role in regulating the levels of platelet-activating factor synthesized by the de novo pathway via hydrolysis of the immediate precursor of PAF.     

Impact of Water Column Acidification on Protozoan Bacterivory at the Lake Sediment-Water Interface

Although the impact of acidification on planktonic grazer food webs has been extensively studied, little is known about microbial food webs either in the water column or in the sediments. Protozoon-bacterium interactions were investigated in a chronically acidified (acid mine drainage) portion of a lake in Virginia. We determined the distribution, abundance, apparent specific grazing rate, and growth rate of protozoa over a pH range of 3.6 to 6.5. Protozoan abundance was lower at the most acidified site, while abundance, in general, was high compared with other systems. Specific grazing rates were uncorrelated with pH and ranged between 0.02 and 0.23 h^-1, values similar to those in unacidified systems. The protozoan community from an acidified station was not better adapted (P = 0.95) to low-pH conditions than a community from an unacidified site (multivariate analysis of variance on growth rates for each community incubated at pHs 4, 5, and 6). Both communities had significantly lower (P < 0.05) growth rates at pHs 4 and 5 than at pH 6. Reduced protozoan growth rates coupled with high grazing rates and relatively higher bacterial yields (ratio of bacterial-protozoan standing stock) at low pH indicate reduced net protozoan growth efficiency and a metabolic cost of acidification to the protozoan community. However, the presence of an abundant, neutrophilic protozoan community and high bacterial grazing rates indicates that acidification of Lake Anna has not inhibited the bacterium-protozoon link of the sediment microbial food web.     

Studies on ether-phospholipids of vascular smooth muscle cells. Identification of a rapid Ca2+-dependent hydrolysis of alkyl-phosphatidylethanolamine promoted by endothelin-1

We have investigated the metabolism of 1-O-[³H]octadecyl-sn-glycero-3-phosphocholine ([³H]lyso PAF) and [³H]myristic acid in secondary cultures of aortic smooth muscle cells (SMC) to characterize the origin of second messengers generated upon stimulation with endothelin-1 (ET-1). When cells were labelled with [³H]lyso PAF, we observed a transfer of the label from phosphatidylcholine (PC) to phosphatidylethanolamine (PE). In contrast, incubation with [³H]myristate labelled mainly PC. Both precursors were incorporated into all PC and PE subclasses. However, [³H]lyso PAF labelled mainly alkyl-subclasses while [³H]myristate was associated with diacyl-subclasses. Using these specific labelling procedures, we have shown that ET-1 induced a strong hydrolysis of PE. This hydrolysis was specific for alkyl-PE with a maximum after 5 s of stimulation. We have also observed an extracellular Ca²⁺-dependent increase in diglyceride (DG), phosphatidic acid (PA) and mainly triglyceride (TG) concomitant to alkyl-PE hydrolysis. Thus, alkyl-DG generated from alkyl-PE appears to be a major product in ET-1 stimulation of SMC. These results suggest a new level of complexity in the signal transduction cascade involving a specificity for phospholipid subclasses.     

The association of MDR1 C3435T and G2677T/A polymorphisms with plasma platelet-activating factor levels and coronary artery disease risk in Turkish population

Increased levels of peripheral proinflammatory mediators can contribute to the development of coronary artery disease (CAD). Platelet activating factor (PAF) is an important proinflammatory mediator and plasma levels of PAF correlate with transmembrane transporter multidrug resistant 1 P-glycoprotein (MDR1 Pgp) expression and activity. MDR1 polymorphisms can affect the expression and activity of Pgp and plasma PAF levels. Therefore, we investigated the possible relationship between MDR1 C3435T and G2677T/A polymorphisms and plasma PAF levels and the risk of CAD. The study population consisted of 198 patients angiographically documented CAD, including 113 cases with at least 1 coronary artery with ≥ 50% luminal diameter stenosis and 85 control subjects with strictly normal coronary angiograms. Genotypes of the MDR1 C3435T and G2677T/A polymorphisms were determined by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP). Plasma PAF levels were detected by enzyme-linked immunosorbent assay (ELISA). There were no significant differences among plasma PAF levels in regard to MDR1 C3435T and G2677T polymorphisms in CAD patients and controls. No statistically significant difference was found for the genotypic and allelic distributions of the polymorphisms in the MDR1 gene between the patients and the control subjects. Furthermore, analysis of MDR1 haplotypes did not show any associations with increased plasma PAF levels and risk of CAD. Our results suggest that plasma PAF levels are not associated with MDR1 gene polymorphisms. There is no association between MDR1 C3435T and G2677T/A polymorphisms and the risk of CAD in Turkish patients.