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Epidemiological studies have suggested that long-term use of nonsteroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX) activity can moderate the onset or progression of Alzheimer's disease (AD). Thus it has been suggested that prostaglandin E2 (PGE2), a major end-product of COX, may play a pathogenic role in AD, but the involvement of PGE synthase (PGES), a terminal enzyme downstream from COX, has not been fully elucidated. Here we found that, among three PGES enzymes, only microsomal PGES-1 (mPGES-1) is induced, and its expression is associated with β-amyloid (Aβ) plaques in the cerebral cortex in human AD patients and in Tg2576 mice, a transgenic AD mouse model. Furthermore, to investigate whether mPGES-1 contributes to AD-like pathology, we bred mPGES-1-deficient mice with Tg2576 mice. We found that mPGES-1 deletion reduced the accumulation of microglia around senile plaques and attenuated learning impairments in Tg2576 mice. These results indicated that mPGES-1 is induced in the AD brain and thus plays a role in AD pathology. Blockage of mPGES-1 could form the basis for a novel therapeutic strategy for patients with AD. Inc. © 2013 Wiley Periodicals, Inc.

Aims : Secretory phospholipase A2 is associated with ischaemic injury in the human heart, but the distribution of type V secretory phospholipase A2 (sPLA2-V) remains unknown. The significance of sPLA2-V in myocardial infarction was investigated histopathologically. Methods : Sequential changes in the localization of sPLA2-V and its mRNA in myocardial tissues obtained from 30 autopsied hearts were examined by immunohistochemistry and in situ hybridization and compared with those of fibronectin, vascular endothelial growth factor (VEGF), interleukin (IL)-1β, tumour necrosis factor (TNF)-α, and cyclooxygenase-2 (COX-2). Results : No expression of sPLA2-V was observed in normal heart, but it was promptly expressed in wavy myofibres positive for fibronectin just after the onset of infarction. sPLA2-V was subsequently expressed in ischaemic cardiomyocytes around the lesion. The expression decreased at the granulation tissue and disappeared at the chronic stage with scar formation. The distribution of the signal for sPLA2-V mRNA paralleled that of the protein. Ischaemic myocytes around the lesion expressed VEGF, IL-1β, TNF-α and COX-2 at all stages. Conclusions : sPLA2-V production in myocardium is limited to the acute phase of infarction. sPLA2-V may play a dual role, acting both to remove degraded cell-membrane through cooperative activity with COX-2 in necrotic areas and to attack ischaemic myocytes around the lesion via degradation of membrane phospholipids.

Although group IIA secretory phospholipase A2 (sPLA2-IIA) is known to be abundantly present in the joints of patients with rheumatoid arthritis (RA), expression of other sPLA2s in this disease has remained unknown. In this study, we examined the expression and localization of six sPLA2s (groups IIA, IID, IIE, IIF, V and X) in human RA. Immunohistochemistry of RA sections revealed that sPLA2-IIA was generally located in synovial lining and sublining cells and cartilage chondrocytes, sPLA2-IID in lymph follicles and capillary endothelium, sPLA2-IIE in vascular smooth muscle cells, and sPLA2-V in interstitial fibroblasts. Expression levels of these group II subfamily sPLA2s appeared to be higher in severe RA than in inactive RA. sPLA2-X was detected in synovial lining cells and interstitial fibers in both active and inactive RA sections. Expression of sPLA2-IIF was partially positive, yet its correlation with disease states was unclear. Expression of sPLA2 transcripts was also evident in cultured normal human synoviocytes, in which sPLA2-IIA and -V were induced by interleukin-1 and sPLA2-X was expressed constitutively. Adenovirus-mediated expression of sPLA2s in cultured synoviocytes resulted in increased prostaglandin E2 production at low ng x mL(-1) concentrations. Thus, multiple sPLA2s are expressed in human RA, in which they may play a role in the augmentation of arachidonate metabolism or exhibit other cell type-specific functions.

Group IID secretory phospholipase A2 (sPLA2-IID), a heparin-binding sPLA2 that is closely related to sPLA2-IIA, augments stimulus-induced cellular arachidonate release in a manner similar to sPLA2-IIA. Here we identified the residues of sPLA2-IID that are responsible for heparanoid binding, are and therefore essential for cellular function. Mutating four cationic residues in the C-terminal portion of sPLA2-IID resulted in abolition of its ability to associate with cell surface heparan sulfate and to enhance stimulus-induced delayed arachidonate release, cyclooxygenase-2 induction, and prostaglandin generation in 293 cell transfectants. As compared with several other group II subfamily sPLA2s, which were equally active on A23187- and IL-1-primed cellular membranes, sPLA2-IID showed apparent preference for A23187-primed membranes. Several human colon carcinoma cell lines expressed sPLA2-IID and sPLA2-X constitutively, the former of which was negatively regulated by IL-1. sPLA2-IID, but not other sPLA2 isozymes, was expressed in human cord blood-derived mast cells. The expression of sPLA2-IID was significantly altered in several tissues of mice with experimental inflammation. These results indicate that sPLA2-IID may be involved in inflammation in cell- and tissue-specific manners under particular conditions.

Although it is well known that the hippocampal CA1 subfield is highly vulnerable to ischemic injury, cellular mechanisms leading to this neuronal degeneration are not fully understood. Using organotypic cultures of rat hippocampal slices, we determined whether phospholipase A2 (PLA2) is activated in response to ischemic conditions (OGD; oxygen and glucose deprivation). The PLA2 activity in the pyramidal cell layer increased immediately following a 35-min exposure to OGD, which was likely to be mediated by selective activation of cytosolic Ca 2+ -dependent PLA2 subtype (cPLA2). This enhancement lasted for at least 24 h. Interestingly, no apparent increase was detected in the dentate gyrus. Twenty-four hours after the OGD exposure, neuronal death was detected mainly in the CA1 region of hippocampal slices. To examine whether the PLA2 activation is causally or protectively involved in the ischemic injury, we investigated the effect of pharmacological blockade of PLA2 on the OGD-induced neuronal death. The PLA2 inhibitor bromophenacyl bromide efficiently prevented the cell death in a concentration-dependent manner. Similar results were obtained for the selective cPLA2 inhibitor AACOCF3. However, the Ca 2+ -independent PLA2 inhibitor bromoenol lactone and the secretory PLA2 inhibitor LY311727 were virtually ineffective. These results suggest that cPLA2 plays a causative role in the neuronal death following OGD exposure. Thus, the present study may provide novel therapeutic targets for the development of neuroprotective agents.

By analyzing human embryonic kidney 293 cell transfectants stably overexpressing various types of phospholipase A2 (PLA2), we have shown that polyunsaturated fatty acids (PUFAs) preferentially activate type IIA secretory PLA2 (sPLA2-IIA)-mediated arachidonic acid (AA) release from interleukin-1 (IL-1)-stimulated cells. When 293 cells prelabeled with [3H]AA were incubated with exogenous PUFAs in the presence of IL-1 and serum, there was a significant increase in [3H]AA release (in the order AA>linoleic acid>oleic acid), which was augmented markedly by sPLA2-IIA and modestly by type IV cytosolic PLA2 (cPLA2), but only minimally by type VI Ca2+-independent PLA2, overexpression. Transfection of cPLA2 into sPLA2-IIA-expressing cells produced a synergistic increase in IL-1-dependent [3H]AA release and subsequent prostaglandin production. Our results support the proposal that prior production of AA by cPLA2 in cytokine-stimulated cells destabilizes the cellular membranes, thereby rendering them more susceptible to subsequent hydrolysis by sPLA2-IIA.

When rat polymorphonuclear neutrophils (PMN) were treated with N -formyl-Met-Leu-Phe (fMLP), the release of arachidonic acid in preference to other fatty acids was observed. Levels of arachidonic acid reached a plateau within 5 min, and were accompanied by an ∼ 4-fold increase in in vitro phospholipase (PL) A 2 and PLD activities in PMN lysates. Treatment of PMN with ethanol (an inhibitor of PLD-mediated phosphatidic acid formation), propranolol (a phosphatidic acid phosphatase inhibitor), or 4-bromophenacylbromide (a PLA 2 inhibitor), each suppressed fMLP-stimulated arachidonate release. Treatment with RHC-80267 (a diacylglycerol lipase inhibitor), however, had no such effect. The cytosolic PLA 2 (cPLA 2 ) inhibitor, arachidonoyl trifluoromethyl ketone, suppressed PLA 2 activity in PMN homogenates and arachidonate release by fMLP-treated PMN. These results suggest that fMLP-elicited arachidonate release is mediated by cPLA 2 but not diacylglycerol lipase, and that the activation of cPLA 2 is downstream of the PLD-dependent signaling pathway.

The alkaline phospholipase A2 (PLA2) was purified from nuclei of rat ascites hepatoma cells (AH7974) by column chromatography with a Sephacryl S-300 column and an immunoadsorbent using anti-group II PLA2 monoclonal antibody. From these two columns, the alkaline PLA2 was eluted in parallel with a 17-kDa protein which is reactive to another antigroup II PLA2 polyclonal antibody. Approximately 80% of nuclear PLA2 was inhibited by this antibody. The alkaline PLA2 was found in association with the chromatin fraction among subnuclear fractions. By an immunocytochemical staining, the nuclei of AH7974 were stained more strongly than other parts of cells with anti-group II PLA2 antiserum.

Evidence has accumulated to suggest that a wide variety of mammalian cells and tissues express a cytosolic phospholipase A2 with arachidonoyl preference (cPLA2). Purified rabbit platelet-derived cPLA2, as well as the human recombinant enzyme originally identified in the monocytic leukemic cell line U937, exhibit significant lysophospholipase activity. Several series of experiments indicated that a single protein mediated both activities. Treatment of the purified enzyme with p-bromophena-cylbromide or an anti-(rabbit platelet cPLA2) monoclonal antibody, RHY-5, suppressed the activity of phospholipase A2 without any appreciable effect on lysophospholipase activity, suggesting that the domain(s) required for phospholipase A2 activity may be located separately from that for lysophospholipase activity. Lysophospholipase activity was appreciably detected above the critical micellar concentration of the substrate. Lysophosphatidylcholine was also hydrolyzed efficiently when it was incorporated into liposomes made of dialkylphosphatidylcholine. The hydrolysis of lysophospholipid was dependent on the fatty acid bound at the sn1 position; the relative rates of hydrolysis of 1-oleoyllysophosphatidylcholine, 1-palmitoyllysophosphatidylcholine, and 1-stearoyllysophosphatidylcholine were 23, 8, and 1, respectively. A similar order of reactivity was observed with lysophospholipid incorporated into dialkylphosphatidylcholine liposomes. cPLA2 may function not only as an arachidonate liberation enzyme but also as an enzyme responsible for degradation of certain molecular species of lysophospholipids formed in membranes.

The extracellular form of 14-kDa group II phospholipase A2 has been found to accumulate at various types of inflammatory sites. In the present paper, we have studied the possible role of the extracellular 14-kDa group II phospholipase A2 in the process of prostaglandin production in activated rat mast cells. When mast cells obtained from the peritoneal cavity of rats were sensitized with IgE, challenged with antigen and then exposed to extracellular 14-kDa group II phospholipase A2, appreciable release of prostaglandin D2 was observed. Generation of prostaglandin D2 was dependent on the concentration of the phospholipase A2 as well as that of the antigen, while no appreciable prostaglandin D2 generation was observed with cells in the absence of the antigen. No histamine release was observed under the same conditions. Phosphatidylcholine in mast cell membranes was appreciably hydrolyzed to liberate free arachidonic acid when mast cells were incubated with 14-kDa group II phospholipase A2 added exogenously in the presence of the antigen. Both the generation of prostaglandin D2 and the release of arachidonic acid were retarded by inhibitors specific to 14-kDa group II phospholipase A2. Thus, 14-kDa group II phospholipase A2 may function in the process of inflammation by acting on IgE-antigen-primed mast cells, which are not fully activated, to generate eicosanoids.

We investigated the effects of platelet-activating factor (PAF) on guinea pig peritoneal macrophages. Specific and high-affinity binding sites for PAF were detected on guinea pig peritoneal macrophages. Scatchard analysis of PAF binding revealed high affinity binding sites (7.9 x 10(4)/cell) with a dissociation constant of 2.3 x 10(-10) M. When treated with 10(-9)-10(-5) M PAF, guinea pig peritoneal macrophages released hydrogen peroxide into the medium in a time-dependent manner. The release reaction upon stimulation with 10(-5) M PAF reached a plateau within 30 min and the extent of release was twice as high as that when stimulated by N-formyl-L-methionyl-leucyl-L-phenylalanine (fMLP; 2 microM)-treated cells. Neither lysoPAF nor the PAF enantiomer was effective. PAF-induced H2O2 release was inhibited specifically by PAF antagonists, suggesting that PAF activated macrophages through binding to specific sites. Lysosomal enzyme (N-acetyl-beta-D-glucosaminidase) was released from guinea pig peritoneal macrophages upon treatment with 10(-5) M PAF for 60 min. Guinea pig peritoneal macrophages were treated with PAF for 8 hr and the conditioned medium was examined for cytokines. The medium exhibited cytocidal activity against mouse fibroblast L929 cells [tumor necrosis factor (TNF) activity], and this activity was comparable to that detected after treatment of cells with the bacterial lipopolysaccharide (LPS). Furthermore, the same conditioned medium also showed colony-stimulating factor (CSF) activity. Generation of these cytokines was stereospecific. Our findings suggest that PAF is a unique macrophage activator that potentiates both respiratory burst/lysosomal enzyme release (early-phase response) and monokine production/glucose consumption (late-phase response).

New antitumor alkylglycerophospholipids, in which primarily the phosphocholine moiety of the platelet activating factor (PAF) molecule was modified, were synthesized from 1-alkyl-2-substituted glycerols by introducing polar head phosphoryl groups having methylene bridges of various lengths (from 2 to 14 carbons). They were tested for PAF agonistic activity and antitumor properties. In a series of 1-octadecyl-2-acetoacetylglycerophospholipids (1a-f), an increase in the length of the methylene bridge separating the phosphate and trimethylammonio group in the polar head side chain at position 3 of the glycerol backbone resulted in a progressive decrease in PAF agonistic activity and a characteristic change in antitumor activity against human promyelocytic leukemia cells (HL-60). Maximal potency was obtained with the compound having a decamethylene bridge (1e, IC50 value=1.5 μg/ml). Thus, alkylphospholipids possessing a decanmethylene bridge and a variety of substituents at position 2 (1g-n) were synthesized. They showed potent inhibitory activity with IC50 values ranging from 0.4 to 1.9 μg/ml, depending on the nature of the 2-substituent in the phospholipid molecule. In in vivo tests of the present series of alkylglycerophospholipids (1a-n), using mice bearing sarcoma 180 and mice with mammary carcinoma MM46 (both cells and compounds were given i.p.), 1-octadecyl-2-acetoacetyl-3-glyceryl ω-trimethylammoniodecyl phosphate (1e) showed the most potent life-prolonging effect. The structure-activity relationships are discussed.

The endogeneous ligand for the cannabinoid receptor, arachidonylethanolamide (anandamide) and its analogues, oleinylethanolamide, palmitylethanolamide and eicosapentaenoylethanolamide, were derivatized with a fluorogenic reagent, 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminsulpho ny1-2,1,3- benzoxadiazole (DBD-COCl). They were separated on a reversed phase HPLC with a mobile phase of acetonitrile:water. The fluorometric detection of the derivatives was made at 560 nm with excitation at 450 nm and the detection limits for anandamide was 20 fmol on column. The structures of DBD-CO-ethanolamides were confirmed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS).

1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OMe) has been reported to possess definite antitumor activity in vivo. Twenty-two alkyl lysophospholipid analogs were chemically synthesized, and their antitumor activity against mouse experimental tumors (Sarcoma 180, MM46, P388) was examined. Among them, 1-O-octadecyl-2-O-acetoacetyl-rac-glycerol-3-phosphocholine was found to show antitumor activity similar to ET-18-OMe with less acute toxicity. Intravenous injection of the ET-18-OMe withsn-3 configuration retarded the subcutaneous growth of Sarcoma 180 cells effectively, while the growth inhibition by thesn-1 isomer was much less effective. This stereospecificity was similar to that observed in their activities as platelet-activating factor (PAF) agonists. The acetoacetyl compound, another PAF agonist, showed similar stereospecific antitumor action in vivo. These findings suggest that some alkyl lysophospholipids may activate host cells to a cytostatic stage against tumor cells in vivo through binding to a PAF receptor. Our preliminary results indicated that the responsible cells under these conditions might be primarily immature macrophages present in the bone marrow. No appreciable or even adverse stereospecificity was observed in the different sets of experiments where the activity of ET-18-OMe against MM46 tumor cells in vivo or the direct cytotoxicity against human promyelocytic leukemia HL-60 cells in vitro was examined. Under, some conditions, the antitumor activity of ET-18-OMe in vivo may be revealed through direct cytotoxicity and/or modulation of the host defense system by “nonspecific” mechanisms. Some alkylphospholipids without PAF activity may also show antitumor activity through similar, “nonspecific” mechanisms.

In vitro chemical labeling of DNA and RNA has received considerable attention [ 1 ] because the so-labeled polynucleotides may be used for hybridization studies, sequence analysis, and other investigations of biochemical interest. The principle so far employed is the hydrogen-tritium (H + 3H) exchange at the 8-positions of purines which occurs on heating a solution of nucleic acids in [3H]H20. We recently reported that bisulfite catalyzes the H -+ 3H (2H) exchange at position 5 of pyrimidine nucleotides CMP [2,3] and UMP [4j. The catalysis is believed to occur through reversible formation of a type of adduct between the pyrimidine nucleotides and bisulfite, 5,6-dihydropyrimidine nucleotide 6sulfonate. For cytosine nucleotide, bisulfite also brings about easy deamination of the 4-NH2 group at pH 4-6 [3,5-71, but at pH 7.5 the H-exchange reaction exclusively takes place [2]. For the labeling of UMP, not only bisulfite ion but also an amine is required, which apparently promotes the cleavage of the C(S) H bond by donating electrons to the hydrogen [4] . The method was successfully applied to the labeling of uridylyl (3’5’)cytidine with tritium, without concomitant degradation of the dinucleoside phosphate. In contrast to the easy back exchange (3H + H) of 8-[3H] purines in H20, the 5-[3H]pyrimidines do not lose the isotope under usual reaction conditions. We now report the results of the ammonium bisulfite-catalyzed tritium labeling of DNA and RNA.

Synthese enantioselective de S- et R-[trimethylammonio-2ethyl] phosphate de (O-acetyl-3 desoxy-1 O-palmityl-2) tetritol

The bisulflte-mediated deamination of 5-methylcytosine yielded a diastereomeric mixture of 5, 6-dihydrothymine-6-sulfonate. One of the diastereomers was identical with the thymine-bisulfite adduct formed by treatment of thymine with bisulfite, and therefore was assigned the structure in which the hydrogen at position 5 and the sulfonate at position 6 are trans. The rates of degradation of this adduct into thymine in aqueous solutions were determined, and it was found that the adduct was considerably stable at pH 6.9, the half life being 13 hr at 37°, whereas at pH 8.9 it degrades with a half life of 70 min. The other diastereomer, in which the 5-H and 6-SO3- are cis, was stable in mildly alkaline conditions, as might be expected from the difficulty in undergoing elimination. This isomer rapidly generated thymine at pH 13.8.