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The present study aimed to compare the absorption and transport patterns of three main betalains, betanin, vulgaxanthin I and indicaxanthin, into intestinal epithelial cells and to assess their distinct molecular effects on inflammatory and redox-related cell signalling in association with their radial scavenging potencies. All three betalains showed anti-inflammatory effects (5–80 μM), reflected by attenuated transcription of pro-inflammatory mediators such as cyclooxygenase-2 and inducible NO-synthase. Concomitant increases in antioxidant enzymes such as heme oxygenase-1 were only observed for betanin. Moreover, betanin uniquely demonstrated a potent dose-dependent radical scavenging activity in EPR and cell-based assays. Results also indicated overall low permeability for the three betalains with Papp of 4.2–8.9 × 10−7 cm s−1. Higher absorption intensities of vulgaxanthin and indicaxanthin may be attributed to smaller molecular sizes and greater lipophilicity. In conclusion, betanin, vulgaxanthin I and indicaxanthin have differentially contributed to lowering inflammatory markers and mitigating oxidative stress, implying the potential to ameliorate inflammatory intestinal disease. Compared with two betaxanthins, the greater efficacy of betanin in scavenging radical and promoting antioxidant response might, to some extent, compensate for its poorer absorption efficiency, as demonstrated by the Caco-2 cell model.

Recruitment of naturally occurring suppressive CD4+, CD25+, and FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the C-C chemokines CCL17 and CCL22. We have recently developed a series of potent, orally bioavailable small molecule antagonists of CCR4 that can block recruitment of Treg into the TME.

The effect of temperature (6, 15 and 30 °C) during ageing on the colour, phytochemical composition and bioactivity of roselle wine was investigated over 12 months. At the end of ageing, wines stored at 6 °C had the highest colour density and lowest polymeric anthocyanins. The initial concentration of most of the individual phenolic compounds decreased during ageing, with reduction of monomeric anthocyanins contributing to the formation of anthocyanin-derivatives (pyranoanthocyanins), eight of which were identified tentatively and reported here for the first time in roselle wine. The decrease in individual phenolic compounds did not affect inhibition of α-glucosidase (maltase) activity, which remained relatively low but stable throughout ageing. Diethyl succinate was the only volatile clearly influenced by ageing temperature, with the most pronounced effect at 30 °C (∼256 fold increase). In summary, the final concentrations of anthocyanins and diethyl succinate were the major compounds influenced by ageing temperature.

TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines which induces apoptotic cell death in a variety of tumor cell lines. It mediates its apoptotic effects through one of two receptors, DR4 and DR5, which are members of of the TNF receptor family, and whose cytoplasmic regions contain death domains. In addition, TRAIL also binds to 3 “decoy” receptors, DcR2, a receptor with a truncated death domain, DcR1, a glycosylphosphatidylinositol-anchored receptor, and OPG a secreted protein which is also known to bind to another member of the TNF family, RANKL. However, although apoptosis depends on the expression of one or both of the death domain containing receptors DR4 and/or DR5, resistance to TRAIL-induced apoptosis does not correlate with the expression of the “decoy” receptors. Previously, TRAIL has been described to bind to all its receptors with equivalent high affinities. In the present work, we show, by isothermal titration calorimetry and competitive enzyme-linked immunosorbent assay, that the rank order of affinities of TRAIL for the recombinant soluble forms of its receptors is strongly temperature dependent. Although DR4, DR5, DcR1, and OPG show similar affinities for TRAIL at 4 °C, their rank-ordered affinities are substantially different at 37 °C, with DR5 having the highest affinity (K D ≤ 2 nm) and OPG having the weakest (K D = 400 nm). Preferentially enhanced binding of TRAIL to DR5 was also observed at the cell surface. These results reveal that the rank ordering of affinities for protein-protein interactions in general can be a strong function of temperature, and indicate that sizeable, but hitherto unobserved, TRAIL affinity differences exist at physiological temperature, and should be taken into account in order to understand the complex physiological and/or pathological roles of TRAIL.

The Kennard–Stepanov relation connects the optical emission and absorption spectra of materials. It has proved useful in assessing the extent of equilibration of excited states of fluorescent systems. We propose a generalization of the theory that may help characterize the approach to equilibrium and we illustrate the method in model systems.

We describe a method to generate monovalent quantum dots (QDs) using agarose gel electrophoresis. We passivated QDs with a carboxy-terminated polyethylene-glycol ligand, yielding particles with half the diameter of commercial QDs, which we conjugated to a single copy of a high-affinity targeting moiety (monovalent streptavidin or antibody to carcinoembryonic antigen) to label cell-surface proteins. The small size improved access of QD-labeled glutamate receptors to neuronal synapses, and monovalency prevented EphA3 tyrosine kinase activation.

A series of substrate analog inhibitors of 14 kDa PLA2 possessing replacements for the sn-3 phosphate moeity was prepared and evaluated. Tetrazole 26 possessed similar in vitro inhibition potency to phosphate-containing substrate analog inhibitors, but demonstrated superior cell permeability as monitored by LTC4 release in monocytes.

Interleukin 1beta (IL-1beta) up-regulates human rheumatoid synovial fibroblast (RSF) 85-kDa phospholipase A2 (PLA2) and mitogen-inducible cyclooxygenase (COX) II. Promoter regions for these genes contain a motif that closely resembles the "classic" NFkappaB consensus site. Immunoblot analysis identified NFkappaB1 (p50), RelA (p65), and c-Rel in RSF. Upon IL-1beta-stimulation, p65 and c-Rel but not p50 protein levels were reduced suggesting nuclear translocation. IL-1beta-induced RSF nuclear extracts contained a p65-containing complex, which bound to the classical NFkappaB consensus motif. An NFkappaB classical oligonucleotide decoy produced a concentration-dependent decrease in IL-1-stimulated PGE2 production (IC50 = approximately 2 microM), indicating a role of NFkappaB. Utilization of antisense technology showed that p65 but not p50 or c-Rel mediated IL-1beta-stimulated PGE2 formation. Treated RSF could not transcribe COX II or 85-kDa PLA2 mRNA, which reduced their respective proteins. Interestingly, stimulated IL-8 production was not inhibited by the classical NFkappaB decoy but was reduced by treatment with antisense to both p65 and c-Rel supporting preferential binding of c-Rel-p65 to the "alternative" IL-8 kappaB motif. Taken together, these data provide the first direct evidence for a role of p65 in COX II and 85-kDa PLA2 gene induction and support the IL-1 activation and participation of distinct NFkappaB protein dimers in RSF prostanoid and IL-8 formation.

The phospholipase A2 (PLA2) activities that are localized in the keratinocyte cytosolic and microsomal fractions were biochemically and pharmacologically characterized. The cytosol and to a lesser extent the microsome were sensitive to heat treatment and stable in the presence of sulfhydryl reducing agents. Both fractions were almost totally inactivated by reduction of pH to 2. The cytosolic activity demonstrated a sevenfold preference for arachidonic acid over oleic acid in the sn-2 position of substrate phospholipid and the microsome exhibited a fourfold preference. Neither the cytosol nor the microsome was inactivated by a neutralizing mouse monoclonal antibody 3F10 generated against recombinant human (rh) type II 14-kDa PLA2. Western immunoblot analysis of both fractions identified a high – molecular-mass protein in keratinocyte cytosol but not the microsome that migrated with rh 85-kDa PLA2. Neither the sytosol nor the microsome possessed immunoreactive bands that migrated with rh type II 14-kDa PLA2 when probed with monoclonal antibody 3F10. Further analysis of the cytosolic activity showed that it was activated by submicromolar concentrations of Ca2+ reduced by arachidonyl trifloromethylketone, a selective 85-kDa PLA2 inhibitor, but was unaffected by C-7 phosphonate phospholipid, a selective 14-kDa PLA2 transition state inhibitor. Taken together, the data supports the existence of a PLA2 activity in the cytosol that displays characteristics that are indistinguishable from those exhibited by the 85-kDa PLA2. Alternatively, both the cytosol and microsome were devoid of type II 14-kDa – like PLA2 activity. The failure of 12-epi scalaradial, a 14-kDa PLA 2 inhibitor, to modify A23187-stimulated keratinocyte prostaglandin E2 release, was consistent with the biochemistry and suggests that the 85-kDa PLA2 may play an important role in keratinocyte prostaglandin E2 formation.

The cell-associated phospholipase A2 (PLA2) activities of the human platelet, neutrophil, and monocyte were simultaneously characterized, utilizing the biochemical differences observed between the 14 kDa (kilodalton), type II PLA2 isolated from inflammatory human synovial joint fluid (HSF) and the arachidonic acid (AA) specific, 85-kDa high molecular mass (HMM) PLA2 isolated from the cytosol of a U937 monocytic cell line. The HSF PLA2 can be distinguished from the HMM PLA2 by its resistance to acid treatment, sensitivity to a sulfhydryl reducing agent, lack of preference for the fatty acid on the sn-2 position of phospholipid substrate, and inhibition by the C-7 phosphonate–phospholipid transition-state PLA2 inhibitor. Evaluation of all three cell types revealed that HMM-like PLA2 activity was found predominantly in the cytosolic fractions, although detection in neutrophil cytosol required more concentrated preparations and the use of high specific activity [3H]AA-labeled Escherichia coli. HSF-PLA2-like activity was measured in microsomal and cytosolic fractions of all three cell types, but was found in neutrophil cytosol only after treatment with acid. Further HMM-PLA2-specific interfering agents in neutrophil cytosol were observed and exemplifies one problem in assigning the existence of this enzyme in crude broken cell preparations using activity measurements alone. The role that these two enzymes play in eicosanoid production of the respective cell types remains to be studied.Key words: phospholipase A2, arachidonic acid, neutrophil, platelet, monocyte, cytosol, microsome.

We present a new class of polymeric ligands for quantum dot (QD) water solubilization to yield biocompatible and derivatizable QDs with compact size (approximately 10-12 nm diameter), high quantum yields (50%), excellent stability across a large pH range (pH 5-10.5), and low nonspecific binding. To address the fundamental problem of thiol instability in traditional ligand exchange systems, the polymers here employ a stable multidentate imidazole binding motif to the QD surface. The polymers are synthesized via reversible addition-fragmentation chain transfer-mediated polymerization to produce molecular weight controlled monodisperse random copolymers from three types of monomers that feature imidazole groups for QD binding, polyethylene glycol (PEG) groups for water solubilization, and either primary amines or biotin groups for derivatization. The polymer architecture can be tuned by the monomer ratios to yield aqueous QDs with targeted surface functionalities. By incorporating amino-PEG monomers, we demonstrate covalent conjugation of a dye to form a highly efficient QD-dye energy transfer pair as well as covalent conjugation to streptavidin for high-affinity single molecule imaging of biotinylated receptors on live cells with minimal nonspecific binding. The small size and low serum binding of these polymer-coated QDs also allow us to demonstrate their utility for in vivo imaging of the tumor microenvironment in live mice.

The in vitro Ca 2+ dependencies of arachidonyl (AA)-selective high molecular mass phospholipase A 2 (HMM, 85 kDa-PLA 2 ) and human low molecular mass (LMM-Type II, 14 kDa)-PLA 2 were compared. When the LMM-PLA 2 and HMM-PLA 2 enzymes were examined for hydrolysis against [ 3 H]AA Escherichia coli in an ethyleneglycol-bis( β -aminoethyl ether) N , N , N , N ′-tetraacetic acid (EGTA)-free buffer system, neither enzyme demonstrated activity below 10 μM free Ca 2+ Beyond 11 μM Ca 2+ both enzyme activities increased steadily exhibiting 50% of maximal activity at 0.1 and 1.0 mM, respectively. Using EGTA-regulated free Ca 2+ buffers, both enzymes responded in a biphasic manner, achieving 50% of the maximum response by 0.5 μM Ca 2+ , stabilizing up to 0.1 mM, then further increasing with exposure to millimolar Ca 2+ concentrations. Replacement of [ 3 H]AA-labeled phosphatidylethanolamine vesicles for [ 3 H]AA E. coli or using Tris-HCl buffer instead of HEPES buffer did not alter these findings significantly. The presence of EGTA had a pronounced concentration-dependent effect on the activity of both the HMM- and LMM-PLA 2 enzymes but only in the range of 0 to 100 μM free Ca 2+ . EGTA (EC 50 ~ 200 μM) reduced the concentration of Ca 2+ required by PLA 2 to achieve 50% of maximal acylhydrolysis. In contrast, the Type I bovine pancreatic PLA 2 required millimolar Ca 2+ concentrations to elicit 50% of the maximal response in both EGTA-free or EGTA-containing systems, which is concordant with its extracellular role as a digestive enzyme. These data suggest that the LMM-Type II PLA 2 and HMM-PLA 2 ; are both activated at submicromolar, intracellularly relevant, Ca 2+ concentrations and therefore have the ability to contribute to cellular lipid metabolism.

We report narrow-band absorption enhancement of semiconductor nanocrystals via Forster resonance energy transfer from cyanine J-aggregates. These J-aggregated dyes associate electrostatically with short quantum-dot (QD) surface ligands in solution. Energy transfer efficiencies approach unity for this light sensitization and result in a 5-fold enhancement in the QD excitation near the J-aggregate absorption maximum. Because a thin layer of J-aggregates attenuates the same amount of light (at peak absorbance) as a far thicker film of monomer dye, these absorption-enhanced materials may have applications in light-sensitizing applications such as photodetection and optical down-conversion.

Acetyl-CoA carboxylase (ACC) can be regulated in vitro via phosphorylation by a 5'-AMP-activated protein kinase. A potential intracellular role for this kinase has been studied in the Fao hepatoma cell by manipulating the intracellular adenine nucleotide pool with ATP-depleting agents. Three different ATP depletors, antimycin A, dinitrophenol, and sodium azide, all promote the rapid loss of ACC activity characterized by a marked reduction in enzyme Vmax, abolition of citrate-independent activity, an increase in the Ka for citrate and a reduction in the mass of a complex between the two major ACC isozymes. These effects persist through enzyme purification on monomeric avidin-Sepharose and are accompanied by an increase in 32P-content, both consistent with depletor-induced covalent enzyme modification. The effects of ATP depletors in intact cells are mimicked in vitro on phosphorylation of ACC by the 5'-AMP-activated protein kinase and are reversible on dephosphorylation. These data indicate that ACC activity is sensitive to the intracellular adenylate charge, but that changes in the state of enzyme phosphorylation, rather than direct allosteric regulation by adenine nucleotides, underly this mode of enzyme control. This kinase-mediated modulation provides a mechanism for altering the rate of fatty acid synthesis and, secondarily, fatty acid oxidation, depending on the rate of ATP generation from carbohydrate-derived precursors in several tissues in vivo.

The marine natural product, manoalide (MLD), was investigated to determine if this drug inhibited purified human synovial fluid phospholipase A2 (HSF-PLA2). Utilizing classical Michaelis-Menten kinetics, apparent Km and Vmax values for HSF-PLA2 of 1.34 mM and 0.47 mumol [3H]palmitic acid released/min/mg protein were obtained using dipalmitoylphosphatidylcholine (DPPC) as the substrate, and 38.0 microM and 18.8 mumol [3H]arachidonic acid released/min/mg protein with Escherichia coli as a natural substrate. These kinetic parameters were utilized subsequently to evaluate the inhibitory effects of manoalide on HSF-PLA2. Inhibition of HSF-PLA2 by MLD was concentration and time dependent with IC50 values of 0.2 and 0.02 microM for DPPC and E. coli respectively. Dialysis studies and examination of DPPC or E. coli hydrolysis versus enzyme concentration indicate that MLD is an irreversible inhibitor of HSF-PLA2. Substrate specificity was also examined in the absence and presence of MLD using dipalmitoylphosphatidylethanolamine (DPPE) as a substrate. MLD inhibited the hydrolysis of DPPE (greater than 90% inhibition at 2 microM), and preliminary results indicate that DPPC was more readily hydrolyzed than DPPE under the substrate conditions of the assay. While the cellular source of secreted HSF-PLA2 is unknown, these studies indicate that MLD can inactivate secreted phospholipase A2 isolated from patients with inflammatory joint disease.

Methotrexate (MTX) resistance in mitoxantrone-selected MCF7/MX cells and in MTX-selected CEM/MTX cells is associated with reduced drug accumulation, albeit caused by different mechanisms. In addition, in both resistant cell lines the proportion of active long-chain MTX-polyglutamate (MTX-PG) metabolites is reduced relative to that in the respective parental cell line. Previous studies by others have implied that increased lysosomal uptake could affect the rate of MTX-PG hydrolysis, and hence the length distribution of the polyglutamate chains. However, in the two cell line pairs studied, the number of lysosomes per cell was not different between the corresponding parental and resistant cells. Instead, we observed a two- to three-fold increased facilitative uptake of MTX-Glu 4 by the lysosomes from these two independently derived MTX-resistant cell lines, compared to uptake by lysosomes from their corresponding parental cells. Enhanced lysosomal uptake of MTX-Glu 4 was reflected in an increased maximal uptake velocity, without a change in the apparent substrate affinity. In addition, the rate of MTX efflux from lysosomes from CEM/MTX cells was two-fold faster than from lysosomes from CEM cells. Consistent with this observation, the relative amount of short-chain MTX-Glu 1+2 species, as a fraction of the total amount of all MTX-Glu 1−4 species combined, was only half as large in lysosomes from CEM/MTX cells as in lysosomes from CEM cells. Together, these results suggest the possibility that increased lysosomal uptake, and hence enhanced sequestration of MTX-PGs in resistant cells, contributes to the development of high-level MTX resistance by decreasing the cytosolic levels of MTX-PGs.

Marine natural products provide a rich source of chemical diversity that can be used to design and develop new, potentially useful therapeutic agents. We report here that scytonemin, a pigment isolated from cyanobacteria, is the first described small molecule inhibitor of human polo-like kinase, a serine/threonine kinase that plays an integral role in regulating the G(2)/M transition in the cell cycle. Scytonemin inhibited polo-like kinase 1 activity in a concentration-dependent manner with an IC(50) of 2 microM against the recombinant enzyme. Biochemical analysis showed that scytonemin reduced GST-polo-like kinase 1 activity in a time-independent fashion, suggesting reversibility, and with a mixed-competition mechanism with respect to ATP. Although scytonemin was less potent against protein kinase A and Tie2, a tyrosine kinase, it did inhibit other cell cycle-regulatory kinases like Myt1, checkpoint kinase 1, cyclin-dependent kinase 1/cyclin B, and protein kinase Cbeta2 with IC(50) values similar to that seen for polo-like kinase 1. Consistent with these effects, scytonemin effectively attenuated, without chemical toxicity, the growth factor- or mitogen-induced proliferation of three cell types commonly implicated in inflammatory hyperproliferation. Similarly, scytonemin (up to 10 microM) was not cytotoxic to nonproliferating endotoxin-stimulated human monocytes. In addition, Jurkat T cells treated with scytonemin were induced to undergo apoptosis in a non-cell cycle-dependent manner consistent with its activities on multiple kinases. Here we propose that scytonemin's dimeric structure, unique among natural products, may be a valuable template for the development of more potent and selective kinase inhibitors used for the treatment of hyperproliferative disorders.

The enzyme coenzyme A-independent transacylase (CoA-IT) has been demonstrated to be the key mediator of arachidonate remodeling, a process that moves arachidonate into 1-ether-containing phospholipids. Blockade of CoA-IT by reversible inhibitors has been shown to block the release of arachidonate in stimulated neutrophils and inhibit the production of eicosanoids and platelet-activating factor. We describe novel inhibitors of CoA-IT activity that contain a beta-lactam nucleus. beta-Lactams were investigated as potential mechanism-based inhibitors of CoA-IT on the basis of the expected formation of an acyl-enzyme intermediate complex. Two beta-lactams, SB 212047 and SB 216754, were shown to be specific, time-dependent inhibitors of CoA-IT activity (IC50 = 6 and 20 microM, respectively, with a 10-min pretreatment time). Extensive washing and dilution could not remove the inhibition, suggesting it was irreversible. In stimulated human monocytes, SB 216754 decreased the production of eicosanoids in a time-dependent manner. In an in vivo model of phorbol ester-induced ear inflammation, SB 216754 was able to inhibit indices of both edema and cell infiltration. Taken together, the results support two hypotheses: 1) CoA-IT activity is important for the production of inflammatory lipid mediators in stimulated cells and in vivo and 2) the mechanism by which CoA-IT acts to transfer arachidonate is through an acyl-enzyme intermediate.

Scalaradial, a marine natural product with anti-inflammatory activity, has been shown to be a selective inhibitor of 14 kDa type II phospholipase A2(PLA2). We have examined the inhibition by scalaradial (0.1 nM to 10 microM) of neutrophil function (degranulation) in response to receptor-mediated activation [N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), 30 nM; leuokotriene B4 (LTB4), 100 nM; platelet-activating factor (PAF), 100 nM] and non-receptor-mediated stimuli [A23187 (1 microM) and thapsigargin (100 nM)]. Furthermore, we evaluated the ability of scalaradial to inhibit the increase in intracellular Ca2+ in response to fMLP, LTB4, A23187, and thapsigargin as well as its ability to prevent either fMLP- or LTB4-mediated elevation in inositol phosphate production (InsP). Scalaradial was a potent inhibitor of both receptor- (IC50 = 50-200 nM) and non-receptor- (IC50 = 40-900 nM) mediated degranulation. Although scalaradial inhibited the mobilization of Ca2+ induced by fMLP, LTB4, and PAF, it did not affect the maximal Ca2+ levels attained with A23187 or thapsigargin. Neutrophil-binding studies with [3H]fMLP and [3H]LTB4 would suggest that the effect of scalaradial on agonist-induced degranulation and increase in intracellular Ca2+ was not at the receptor level because 50-fold higher concentrations were required to have a significant effect on the binding of these agonists. To determine if scalaradial affected phosphatidylinositol selective phospholipase C (PI-PLC) activity, assays were conducted to monitor fMLP- and LTB4-induced formation of InsPs using myo-[3H]inositol-labeled U-937 cells. In these cells, 2.5 to 9-fold higher concentrations of scalaradial were required to inhibit PI-PLC activity than to inhibit agonist-induced degranulation of neutrophils, suggesting that the effects of scalaradial on Ca2+ and degranulation are not the sole result of blocking receptor activation of PI-PLC. Results obtained with receptor-mediated stimuli suggest that scalaradial may have direct effects on Ca2+ channels and InsP turnover, but inhibition of intracellular Ca2+ levels was not required for scalaradial to block degranulation since scalaradial was capable of inhibiting degranulation produced by either A23187 or thapsigargin, without changing the maximal Ca2+ levels obtained with these two stimuli. These results demonstrate that scalaradial can inhibit degranulation in the presence of micromolar intracellular Ca2+ concentration, thus supporting the hypothesis that a 14 kDa PLA2 may be important in the regulation of neutrophil degranulation.

Studies with therapeutic inhibitors or gene-deficient mice have implicated IL-18 in the pathogenesis of several animal models of inflammatory or autoimmune disease. Conflicting results have been obtained, however, concerning the role of IL18 vs. IL-18R in experimental autoimmune encephalomyelitis (EAE), a commonlyused model of multiple sclerosis. In order to better understand these discrepancies, we compared the development of MOG35-55 -induced EAE in C57Bl/6 wild type mice or C57Bl/6 mice deficient in IL-18, IL-18Rα, IL-18Rβ, or IL18/IL-18Rα (double-knockout). Female IL-18-deficient mice were protected from severe disease compared to wild type controls, however male IL-18-deficient mice were generally not protected. Mice deficient in either of the IL-18 receptor chains, or deficient in both IL-18 and IL-18Rα, exhibited disease scores similar to or, in some instances, worse than wild type controls. In addition, prophylactic treatment of wild type mice with a mAb to either IL-18 or IL-18Rα did not alter disease scores compared with those of isotype control treated mice . In the course of evaluating these lines, we performed high density microsatellite analyses to confirm their congenic C57Bl/6 backgrounds and discovered that further back-crossing was required to eliminate non-C57Bl/6 loci from the IL-18Rα-deficient line . In spite of the back-crossing, closely-linked regions of chromosome 1 remained and these regions, derived from the 129 strain, contained numerous immune system genes that could influence the outcome in this model. In conclusion, these results suggest that IL-18 contributes to the development of EAE, but the degree and manner of this contribution appears to be influenced by both gender and genetic background differences. Abstract

Rats were fed either a stock diet or purified diets containing either 10% by weight corn oil or linseed oil for 8 weeks. Splenocytes from rats on the stock ration were cultured for 48 hours in media containing either serum from the rats fed stock diet, corn oil or linseed oil, or fetal bovine serum (FBS). Populations were cultured without stimulation and with stimulation by leucogglutinin (PHA), and the production of prostaglandin F2 alpha by the cells was determined. The serum from the corn oil-fed rats differed markedly in composition from that of the linseed oil-fed rats notably in the higher level of arachidonic acid (20:4 omega 6) and lower level of timnodonic acid (20:5 omega 3). FBS also had a lower level of 20:4 omega 6. These changes were reflected in the fatty acid composition of the spleen choline glycerophosphatide. PGF2 alpha production was significantly depressed in the medium from linseed oil-fed rats compared to the corn oil group. This effect was due to the competition of 20:5 omega 3 for the cyclooxygenase. Viability of cells was better in rat serum than in FBS. The study shows that when studying functions in cultured cells obtained from rats fed different dietary fats the dietary effect will be abrogated or modified by use of FBS in the medium.

It has been reported that leukotriene B4 can translocate calcium across model membranes (Serhan et. al., (1982) J. Biol. Chem., 257: 4746). Such ionophoretic behavior could account for its biological effects. We have examined the effect of chromatographically pure leukotriene B4 on Ca2+ permeability when added exogeneously at 3 μM to phosphatidylcholine liposomes and when incorporated at 5 mole % in the lipid mixture used to prepare liposomes. No effect was observed with either procedure. An oxidized preparation of leukotriene B4 stimulated calcium permeability, however, suggesting that oxidation may account for the previously reported ionophoretic behavior of leukotriene B4.

The LTD4 antagonist, Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methanesulfonamide), was assessed for its ability to modulate arachidonic acid metabolism in several inflammatory cells. In A23187-stimulated rat neutrophils, Wy-48,252 effectively inhibited the conversion of exogenous [14C]arachidonic acid to radiolabeled 5-hydroxyeicosatetraenoic acid (5-HETE) and thromboxane B2 (TxB2) (IC50 = 2 and 9.1 microM, respectively). Synthesis of immunoreactive leukotriene B4 (LTB4) (IC50 = 4.6 microM) and TxB2 (IC50 = 3.3 microM) from endogenous substrate by these cells in the absence of [14C]arachidonic acid was similarly reduced. Wy-48,252 also reduced leukotriene C4 (LTC4) and PGE2 synthesis by zymosan-activated mouse peritoneal macrophages (IC50 = 4.4 and 4.3 microM, respectively). 5-Lipoxygenase (5-LO) catalyzed reactions in human neutrophils, lung mast cells and basophils activated by various stimuli were dose dependently inhibited by Wy-48,252 while PGD2 synthesis by lung mast cells was inhibited at 100 microM. By contrast, 12-LO, 15-LO, phosphodiesterase activity and histamine release from mast cells and basophils were unaffected by Wy-48,252. These data suggested that the LTD4 antagonist, Wy-48,252, also inhibited the synthesis of eicosanoids, a feature that may contribute to its pharmacological actions in vivo.

Semipurified diets containing ratios of alpha-linolenic acid (18:3 omega 3) to linoleic acid (18:2 omega 6) of 1/32, 1/7, 1/1, and 3.5/1 in the form of corn oil, soybean oil, soybean/linseed oil mix and linseed oil were fed to rats for 2 months. The first 3 diets were fed to another group of rats for 4 months and to a group through the second generation. Fatty acid analysis of liver and spleen ethanolamine glycerophosphatide revealed that, as the level of 18:3 omega 3 in the diet increased, the elongated, desaturated metabolites of the omega 6 series decreased and the omega 3 series increased. Noteworthy was the depression in the amount of the precursor of the 2-series prostaglandins (PG) as the omega 3 levels increased. Synthesis of PG by liver of rats fed 2 or 4 months markedly decreased, but at 2 months in thymus and spleen, it showed a trend toward decreasing only. Brain slices showed no decrease in PGF2 alpha synthesis after 4 months, but did decrease significantly after feeding the diets to the second generation. Synthesis of PGE2 by spleen homogenate from the second generation also significantly decreased. The replacement of omega 6 series fatty acids by omega 3 series is explained by the effective competition of 18:3 omega 3 over 18:2 omega 6 for the delta 6 desaturase. Depressions in PG synthesis by high dietary 18:3 omega 3 is explained by the competitive inhibition of the PG synthetase complex by 20:5 omega 3 as well as by the decreased levels of 20:4 omega 6.

Publisher Summary This chapter discusses the recent advances in the design and evaluation of inhibitors of phospholipase A 2 (PLA 2 ). PLA 2 refers to a large class of acylhydrolytic enzymes that specifically act at the sn-2 position of phospholipid substrate. Substrate sources are derived from two broad categories: 1) synthetic PL or 2) natural “membrane” lipid forms. The use of PLA 2 administration as the initiating inflammatory insult are the most documented and well characterized models in terms of evaluating the proinflammatory activity of PLA 2 . The activity of PLA 2 is greatly enhanced when the substrate is above its critical micelle concentration (CMC). The importance of comparing inhibitor activity against the same enzyme can be illustrated with manoalide. Its IC 50 value for inhibition of PLA 2 hydrolysis has been measured against a number of enzymes and varies over almost three log units. The inhibitory effects of these retinoids on the release and metabolism of AA from rat peritoneal macrophages challenged with A23187, zymosan, and 12-O-tetradecanoate phorbol-13-acetate (TPA) have also been studied. Several other natural products have also been reported to inhibit PLA 2 . Gossypol, a male non-steroidal antifertility agent, has been shown, at 100 μM, to completely inhibit intact human spermatozoa PLA 2 hydrolysis of monolayers of phosphatidylglycerol. A three-dimensional study of the bovine pancreatic PLA 2 X-ray structure, with the goal of identifying novel compounds that fit both sterically and electronically into the active site, has resulted in the identification of a series of potent in vitro inhibitors. Another study of the same X-ray structure has resulted in the synthesis of long chain alkylamine inhibitors. Although there has been considerable activity in this field over the last decade, much work remains before a clinical candidate can emerge.

Analysis of diet-induced fatty acid changes in the major phospholipids of various immune cell populations has not been previously documented, particularly modifications induced by dietary alpha-linolenic acid. Rats were fed purified diets containing either 10% corn oil (CO), 10% linseed oil (LO) or 10% soybean oil-linseed mixture (SL) for 8 weeks. The alpha-linolenic to linoleic acid ratios of the diets were 1:32, 1:1 and 3:1, respectively. Fatty acid analysis of cell populations isolated from he spleen, thymus, thoracic cavity and peripheral blood phospholipids showed increases in omega 3 fatty acids accompanied by decreases in the omega 6 fatty acids when diets high in alpha-linolenic to linoleic acid ratios were fed. The extent of change observed was dependent on the magnitude of the alpha-linolenic to linoleic acid ratio. Both magnitude of change and the specific fatty acids altered varied with the cell population examined.

Sprague-Dawley rats were fed one of three purified diets--10% corn oil, 10% hydrogenated coconut oil, or 10% linseed oil--through two generations. At 60-80 days of age the animals were sacrificed. The fatty acyl composition of phosphatidylcholine, phosphatidylethanolamine, plasmalogen phosphatidylethanolamine, and combined phosphatidylinositol/phosphatidylserine from cerebral cortex and isolated cerebral microvessels was determined. Brain slice prostaglandin F2 alpha or microvascular prostacyclin synthesis was also measured. Major changes were noted in the fatty acid profiles, most dramatically in the phosphatidylethanolamine and ethanolamine plasmalogen fractions, with an active rise in docosahexaenoic acid resulting from linseed oil feeding. A depression in prostaglandin F2 alpha synthesis was seen in brain slices of hydrogenated coconut oil- and linseed oil-fed rats. Such a depression was also observed in microvascular prostaglandin synthesis at basal and stimulated levels but not in control incubations. The potential importance of these findings to cerebral microcirculation and hemostasis is discussed.