Facile synthesis of acid-labile polymers with pendent ortho esters

This work presents a facile approach for preparation of acid-labile and biocompatible polymers with pendent cyclic ortho esters, which is based on the efficient and mild reactions between cyclic ketene acetal (CKA) and hydroxyl groups. Three CKAs, 2-ethylidene-1,3-dioxane (EDO), 2-ethylidene-1,3-dioxolane (EDL), and 2-ethylidene-4- methyl-1,3-dioxolane (EMD) were prepared from the corresponding cyclic vinyl acetals by catalytic isomerization of the double bond. The reaction of CKAs with different alcohols and diols was examined using trace of p-toluenesulfonic acid as a catalyst. For the monohydroxyl alcohols, cyclic ortho esters were formed by simple addition of the hydroxyl group toward CKAs with ethanol showing a much greater reactivity than iso-propanol. When 1,2- or 1,3-diols were used to react with the CKAs, we observed the isomerized cyclic ortho esters besides the simple addition products. Biocompatible polyols, that is, poly(2-hydroxyethyl acrylate) (PHEA) and poly(vinyl alcohol) (PVA) were then modified with CKAs, and the degree of substitution of the pendent ortho esters can be easily tuned by changing feed ratio. Both the small molecule ortho esters and the CKA-modified polymers demonstrate the pH-dependent hydrolysis profiles, which depend also on the chemical structure of the ortho esters as well as the polymer hydrophobicity.     

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3′,5′-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I₂, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B₄ synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B₄ itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B₄ is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B₄ synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B₄ can limit their own activity in neutrophils.     

The identification by sequence homology of stage-specific sea urchin embryo histones H1

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I2, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B4 synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B4 itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B4 is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B4 synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B4 can limit their own activity in neutrophils.     

Ether phospholipids as inhibitors of the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase in macrophages

1-Alkylglycerophosphatide analogs which are known to activate macrophages to enhanced tumor cytotoxicity are structurally closely related to 1-acyl-sn-glycero-3-phosphocholine. In this study we have examined the influence of some of these compounds and of platelet-activating factor (PAF-acether, 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine) on the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) in homogenate of bone-marrow-derived murine macrophages. This enzyme is suggested to be involved in the control of the availability of the icosanoid precursor, arachidonic acid. Kinetic experiments revealed apparent Km and V values for 1-palmitoyl-sn-glycero-3-phosphocholine of 6.0 microM and 16.10 nmol/mg protein per min, respectively. When the 1-palmitoyl-sn-glycero-3-phosphocholine concentration was equal to Km, the enzyme was dose-dependently inhibited by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine with a 50% inhibition at 30 microM. The kinetic parameters in the presence of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (K'm = 10.0 microM, V' = 11.40 nmol X mg-1 X min-1) suggest that this alkyl phospholipid is a mixed-type inhibitor. All other alkyl analogs tested (1-O-methyl-2-O-octadecyl-rac-glycerol-3-phosphocholine, racemic PAF-acether, L-PAF-acether, D-1-O-hexadecyl-sn-glycero-3-phosphocholine, 1-O-octadecyl-rac-glycero-3-phosphocholine) inhibited the enzyme to various degrees. Arachidonic acid transfer to the 1-alkylglycerophosphatide analogs themselves could be ruled out under the assay conditions used. Therefore, we conclude that the arachidonoyl-CoA: 1-acyl-sn-glycero-3-phosphocholine acyltransferase can be inhibited by synthetic and naturally occurring ether phospholipids in homogenate of bone-marrow-derived murine macrophages.     

The effects of N-3 polyunsaturated fatty acids on the generation of platelet-activating factor-acether by human monocytes

Human leukocytes generate platelet-activating factor (PAF-acether), a lipid mediator of inflammation, from membrane alkyl phospholipids through the release of arachidonic acid or other fatty acids at the 2-position and subsequent acetylation. Because it was previously demonstrated that fish oil fatty acids suppress human leukocyte arachidonic acid release and metabolism, separate experiments were conducted to investigate the effects of dietary fish oil supplementation and in vitro incubation with fish oil fatty acids on calcium ionophore-stimulated PAF-acether generation in human monocytes. In subjects on their regular diets, a 4-hr incubation of monocyte monolayers with an optimally effective concentration of arachidonic acid of 1 micrograms/ml resulted in a 64% increase of calcium ionophore-induced net PAF-acether generation from 7.75 +/- 0.78 ng/10(6) cells for untreated monolayers to 12.70 +/- 1.21 ng/10(6) cells (mean +/- SEM). Treatment of monolayers with eicosapentaenoic acid (EPA) at the optimal concentration of 1 micrograms/ml decreased net PAF-acether generation by 28%. However, treatment of monocyte monolayers with docosahexaenoic acid did not appreciably affect net PAF-acether generation. The changes in PAF-acether release with each fatty acid added in vitro paralleled those in total PAF-acether generation; the percentage PAF-acether release remained unaffected. Three weeks of dietary supplementation with 18 g MaxEPA daily, providing 3.2 g EPA did not affect the PAF-acether generation of calcium ionophore-stimulated human monocyte monolayers. However, 6 weeks of dietary supplementation resulted in a 47% decrease of net total PAF-acether generation and a concomitant 59% decline in net PAF-acether release; the percentage release of PAF-acether was not affected. Thus, whether added to the diet or introduced in vitro, fish oil-derived fatty acids suppress PAF-acether generation by human monocyte monolayers.     

Determination of the absolute stereospecificity of the enzymatic reduction of NAD+ analogs by means of the primary acid cyclization reaction

NADH, (3-acetylpyridine)ADH, (3-thionicotinamide)ADH and (pyridine-3-aldehyde)ADH were labeled with deuterium by the B specific enzyme lipoyl dehydrogenase. The primary acid rearrangement products, α-O^2′-6B cyclotetra-hydropyridine adenine dinucleotides, of the respective reduced coenzyme analogs were synthesized by incubation at pH 2–2.5. Analysis of the spin-spin coupling constants of the 220 MHz proton magnetic resonance spectra establishes that the structure of the ribose-tetrahydropyridine linkage of these analogs is the same as that of the primary acid product of NADH. All four specifically labeled dinucleotides possess the identical absolute configuration at the C4 position with respect to the deuterium label, establishing that the three coenzyme analogs are reduced by lipoyl dehydrogenase with the same absolute stereospecificity as NAD⁺. The primary acid reaction is described as a general method for the determination of the absolute configuration at the C4 position of specifically labeled β-1,3-substituted, 1,4 dihydropyridine nucleotides.     

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.     

Enzymatic resolution of 2-aryloxy-1-propanols via lipase-catalyzed enantioselective acylation using acid anhydrides as acyl donors

Pseudomonas sp. lipase-catalyzed enantioselective acylation procedure using acid anhydrides as acyl donors was exploited for the resolution of 2-aryloxy-1-propanols carrying different substituents on the benzene ring. These primary alcohols, which belong to primary alcohols with an oxygen atom at the stereocenter, were resolved generally with moderate to good enantioselectivity (E of up to 55) through the acylation with hexanoic anhydride in diisopropyl ether at 25 °C in a short reaction time. With the alcohol substrate, which gave a low enantioselectivity in the acylation at ordinary temperature, the selectivity proved to be enhanced by conducting the reaction at low temperature (−10 °C). By this acylation procedure employing the acid anhydride, enantiomerically pure (R)-2-phenoxy-1-propanol was prepared in a gram-scale reaction.     

Synthesis of Acyclic Nucleoside Analogs Related to Barbituric Acid

Abstract

The syntheses of the pyrimidine analogs 5,5-dihydroxymethyl-2,4,6-pyrimidineytrione I, 2-amino-5,5-dihydroxymethyl-4,6-pyrimidinedione II, 5,5-di(2-hydroxyethyl)-2,4,6-pyrimidinetrione III, and 2-amino-5,5-di(2-hydroxyethyl)-4,6-pyrimidinedione IV are described.     

Purification and characterization of membrane-bound quinoprotein cyclic alcohol dehydrogenase from Gluconobacter frateurii CHM 9

A quinoprotein catalyzing oxidation of cyclic alcohols was found in the membrane fraction for the first time, after extensive screening among aerobic bacteria. Gluconobacter frateurii CHM 9 was finally selected in this study. The enzyme tentatively named membrane-bound cyclic alcohol dehydrogenase (MCAD) was found to occur specifically in the membrane fraction, and pyrroloquinoline quinone (PQQ) was functional as the primary coenzyme in the enzyme activity. MCAD catalyzed only oxidation reaction of cyclic alcohols irreversibly to corresponding ketones. Unlike already known cytosolic NAD(P)H-dependent alcohol-aldehyde or alcohol-ketone oxidoreductases, MCAD was unable to catalyze the reverse reaction of cyclic ketones or aldehydes to cyclic alcohols. MCAD was solubilized and purified from the membrane fraction of the organism to homogeneity. Differential solubilization to eliminate the predominant quinoprotein alcohol dehydrogenase (ADH), and the subsequent two steps of column chromatographies, brought MCAD to homogeneity. Purified MCAD had a molecular mass of 83 kDa by SDS-PAGE. Substrate specificity showed that MCAD was an enzyme oxidizing a wide variety of cyclic alcohols. Some minor enzyme activity was found with aliphatic secondary alcohols and sugar alcohols, but not primary alcohols, differentiating MCAD from quinoprotein ADH. NAD-dependent cytosolic cyclic alcohol dehydrogenase (CCAD) in the same organism was crystallized and its catalytic and physicochemical properties were characterized. Judging from the catalytic properties of CCAD, it was apparent that CCAD was distinct from MCAD in many respects and seemed to make no contributions to cyclic alcohol oxidation.     

Modification at the 5-Position of 4-Deoxypyridoxol and α4-Norpyridoxol

In an attempt to relate structure to anticoccidial activity, a number of 5-modified analogs of 4-deoxypyridoxol (4-DOP) and α⁴-norpyridoxol have been synthesized and their biological activities examined. The compounds prepared include the 5-(3-hydroxypropyl), 5-(2-hydroxyethyl), 5-(1-hydroxyethyl), formyl and acetyl analogs of 4-DOP, and 5-(3-hydroxypropyl), formyl, ethoxycarbonyl, carbamoyl and hydroxyl analogs of α⁴-norpyridoxol. Among these compounds, 4-deoxyisopyridoxal and α⁴-norisopyridoxal were found to exhibit anticoccidal activity.     

Synthesis,characterization, and reactions of 2′-, 3′-, and 5′-(2-bromoethyl)-AMP: Potential affinity labels for nucleotide binding sites in enzymes

Two new adenosine analogs, 2′-(2-bromoethyl) adenosine monophosphate and 3′-(2-bromoethyl) adenosine monophosphate, were synthesized, purified by semipreparative high-pressure liquid chromatography, and completely characterized. A new synthesis of 5′-(2-bromoethyl) adenosine monophosphate is presented which facilitates the preparation of radioactive reagent with label either in the ethyl group or the purine ring of the nucleotide derivative. The reactive moiety of these derivatives, a bromoalkyl group, has the ability to react with the nucleophilic side chains of several amino acids. The second-order, pH-independent rate constants for reaction with the side chains of the amino acids cysteine, lysine, histidine, and tyrosine were determined as 3×10^?4, 6×10^?6, 3×10^?7, and <1×10^?7 M^?1 sec^?1, respectively. These data could be use in estimating the rate enhancement observed in modification of a protein by these affinity-labeling reagents. 5′-(S-(2-hydroxyethyl)cysteine) adenosine monophosphate, the derivative expected from exhaustive digestion of protein in which a cysteinyl residue is modified by 5′-(2-bromoethyl) adenosine monophosphate, and S-2-hydroxyethyl)cysteine, the derivative anticipated upon acid hydrolysis of such a modified protein, were synthesized, characterized, and their elution positions from an amino acid analyzer determined. These bromoethyl AMP derivatives are potential affinity labels for enzymes that bind 2′-, 3′-, or 5′-nucleotides such as TPN, coenzyme A, or ADP, respectively.     

Transformation of thiodiglycol by resting cells of Alcaligenes xylosoxydans PGH10

A new strain of Alcaligenes xylosoxydans able to aerobically cometabolize thiodiglycol, the primary hydrolysis product of sulfur mustard, was isolated and tested in a laboratory scale stirred tank reactor. The strain, named PGH10, cannot use TDG as sole carbon and energy source for growth, but resting cells previously grown on either rich broth or defined mineral media efficiently metabolize this compound through [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid as intermediates. Degradation of TDG by PGH10 is shown to take place at late exponential and stationary phase but is not triggered by carbon exhaustion. Cultures pregrown to saturation for 48 h in the absence of TDG can be stored and used for degradation of TDG, reducing significantly the time required to achieve the reduction of the compound concentration to undetectable levels. Degradation can take place in buffered media with no carbon source added, although best results were obtained in mineral media supplemented with citrate or fructose. Oxidation to [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid was proposed to be catalyzed by a butanol-dehydrogenase activity. Inhibition of TDG transformation in the presence of several alcohols is also shown.     

Acute effects of intratracheal administration of platelet-activating factor in baboons

The bronchomotor effect of intratracheal administration of PAF-acether (60 micrograms X kg -1) was investigated in 37 curarized baboons mechanically ventilated with constant volume and frequency. PAF-acether caused an immediate bronchoconstriction as assessed by a marked increase in peak inspiratory pressure with no change in static pulmonary compliance and chest X-rays. There was a concomitant fall in arterial PO2 and a significant increase in ventilated unperfused lung zones. A decrease of circulating platelets and leucocytes was also observed. Local anesthesia with lidocaine and atropine did not prevent PAF-acether-induced bronchoconstriction although both markedly reduced the bronchial response to histamine. Albuterol significantly reduced the bronchial response to PAF-acether. Pretreatment with aspirin (80 mg X kg -1 iv) did not prevent the bronchoconstriction caused by PAF-acether, and intravenous or intratracheal arachidonic acid caused no bronchial response. Thus the role of cyclooxygenase metabolites of arachidonic acid in PAF-acether-induced bronchoconstriction is unlikely. In conclusion, an acute bronchoconstriction probably not triggered by stimulation of irritant receptors of the airways and associated with aggregation of platelet takes place subsequent to intratracheal administration of PAF-acether. These data suggest that PAF-acether might play a role in the pathogenesis of human asthma.     

Regulation of PAF-acether (platelet-activating factor) biosynthesis in cultured human vascular endothelial cells stimulated with thrombin

The regulating mechanisms of PAF-acether (platelet-activating factor) biosynthesis in cultured human vascular endothelial cells stimulated with thrombin were investigated. The formation of PAF-acether was maximal at 5 min after stimulation and gradually decreased for up to 30 min. Thrombin induced a rapid 3-4-fold increase in the activity which was maximal by 1 min after stimulation and returned progressively to basal level within 10 min. The thrombin-induced enhancement in acetyltransferase activity was due to an increase of the Vmax of the acetylation reaction without a significant effect on the apparent Km of the enzyme for acetyl-CoA. Human endothelial cells also exhibited a basal PAF-acether acetylhydrolase activity which was not altered upon thrombin stimulation. The pretreatment with 2 mM phenylmethylsulfonyl fluoride (PMSF), a serine proteinase inhibitor reported to block the acetylhydrolase, induced about 2-times more PAF-acether production in response to 2.5 U/ml thrombin stimulation. However, this enhancement of PAF-acether formation seems to be not only due to the inhibition of the acetylhydrolase, but also to the influences on the activities of the acetyltransferase and other enzymes such as phospholipase A2. These results suggest a key role for acetyltransferase and acetylhydrolase in the regulation of PAF-acether formation and catabolism in thrombin-stimulated human endothelial cells.     

Esterification reactions catalyzed by lipases in microemulsions: the role of enzyme localization in relation to its selectivity

The activity of lipases from Rhizopus delemar, Rhizopus arrhizus, and Penicillium simplicissimum entrapped in microemulsions formulated by bis-(2-ethylhexyl)sulfo-succinate sodium salt (AOT) in isooctane has been studied in esterification reactions of various aliphatic alcohols with fatty acids. The effect of the nature of the fatty acids (chain length) and of the alcohols (primary, secondary, or tertiary; chain length; cyclic structures) on the lipase activities was investigated in relation to the reverse micellar structure. The lipases tested showed a selectivity regarding the structure of the substrates used when hosted in the AOT/isooctane microemulsion systems. Penicillium simplicissimum lipase showed higher reaction rates in the esterification of long chain alcohols as well as secondary alcohols. Primary alcohols had a low reaction rate and tertiary a very slow rate of esterification. Long chain fatty acids were better catalyzed as compared to the shorter ones. Rhizopus delemar and R. arrhizus lipases showed a preference for the esterification of short chain primary alcohols, while the secondary alcohols had a low rate of esterification and the tertiary ones could not be converted. The reaction of medium chain length fatty acids was also better catalyzed than in the case of the long ones. The observed lipase selectivity appeared to be related to the localization of the enzyme molecule within the micellar microstructure due to the hydrophobic/hydrophilic character of the protein. The reverse micellar structural characteristics, as well as the localization of the enzyme, were examined by fluorescence quenching measurements and spectroscopical studies. (c) 1993 John Wiley & Sons, Inc.     

Fungal Reduction of C6 α,β-Unsaturated Carboxylic Acids

The metabolism of sorbic acid (trans-2,trans-4-hexadienoic acid) and its related compounds by Mucor sp. A-73 was investigated. Sorbic acid was reduced by this fungus to trans-4-hexenol (more than 90% yield). In a series of hexamonoenoic acids, carboxyl groups and α,β-double bond were reduced, but β,γand γ,δ double bonds were hardly reduced. The reduction of cis-2-hexenoic acid was slower than that of the corresponding trans isomer. Sorbic alcohol, one of α,β-unsaturated alcohols, was converted well to trans-4-hexenol by the fungus. These results showed that this fungus could carry out two independent reductions: (i) carboxyl group→alcohol, (ii) α,β-unsaturated alcohol→αβ-saturated one. Furthermore, α,β-unsaturated alcohols were temporarily detected in the course of fungal reductions of some α,β-unsaturated acids. The fact suggested that the reduction of α,β-unsaturated acids to α,β-saturated alcohols was initiated by the reaction (i) and followed by (ii). The biological hydrogenation of α,β-unsaturated alcohols is a new reaction.     

Antigen-initiated release of platelet-activating factor (PAF-acether) from mouse bone marrow-derived mast cells sensitized with monoclonal IgE

Mouse bone marrow mast cells sensitized with monoclonal IgE and activated with specific antigen released 2.8 +/- 0.5 ng of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (PAF-acether)/ 10(6) cells. The PAF-acether was identified by its ability to aggregate fully aspirin-treated washed rabbit platelets in the presence of an adenosine diphosphate (ADP)-scavenger complex, by its co-chromatography with [3H]-labeled semi-synthetic PAF-acether and synthetic 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, and by its inactivation by phospholipases A2, C, and D and not by lipase A1. The antigen-initiated release of PAF-acether, leukotriene C4 (LTC4), and leukotriene B4 (LTB4), and the secretion of the granule marker beta-hexosaminidase were not diminished by washing the cells before challenge, indicating that they were due to the interaction of antigen with the IgE fixed on the cell membrane and not to phagocytosis of immune complexes formed in the fluid phase. The parallel antigen-induced dose-response relationship, along with the superimposable time-course of the extracellular appearance, of beta-hexosaminidase, PAF-acether, and both leukotrienes indicated that the origin of these diverse mediators was from a common cell type with IgE-Fc receptors. Ethanol extraction of antigen-stimulated bone marrow-derived mast cells revealed the early transient appearance of a cell-associated platelet-aggregating activity, the action of which on platelets, like PAF-acether, was independent of ADP and arachidonic acid metabolism. The cell-associated activity contained a novel product that eluted at 13 min during high performance liquid chromatography (HPLC) (solvent hexane:n-propanol:water, 46:46:8), permitting resolution from PAF-acether and lyso-PAF-acether (1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which eluted at 29 min and 30 min, respectively. The cell-associated material, which differs from lyso-PAF-acether, the putative precursor of PAF-acether, in being active in the bioassay on platelets may represent a newly recognized intermediate in the generation of PAF-acether. As the transiently present cell-associated intermediate has not been previously recognized, its detection may depend upon the relatively unique properties of the bone marrow-derived mast cell system in which IgE-dependent activation leading to product generation is complete within 5 min.(ABSTRACT TRUNCATED AT 400 WORDS)     

New cardiolipin analogs synthesized by phospholipase D-catalyzed transphosphatidylation

Cardiolipin (CL) and related diphosphatidyl lipids are hardly accessible because of the complexity of their chemical synthesis. In the present paper, the transphosphatidylation reaction catalyzed by phospholipase D (PLD) from Streptomyces sp. has been proven as an alternative enzyme-assisted strategy for the synthesis of new CL analogs. The formation of this type of compounds from phosphatidylcholine was compared for a series of N- and C2-substituted ethanolamine derivatives as well as non-charged alcohols such as glycerol and ethylene glycol. The rapid exchange of the choline head group by ethanolamine derivatives having a low molecular volume (diethanolamine and serinol) gave rise to an efficient production of the corresponding CL analogs. In contrast, the yields were comparably low in the reaction with bulky nitrogenous acceptor alcohols (triethanolamine, tris(hydroxymethyl)aminomethane, tetrakis(hydroxyethyl)ammonium) or the non-charged alcohols. Therefore, a strong dependence of the conversion of the monophosphatidyl to the diphosphatidyl compound on steric parameters and the head group charge was concluded. The enzyme-assisted strategy was used for the preparation of purified diphosphatidyldiethanolamine and diphosphatidylserinol.     

Introduction of Aromatic Ring-Containing Substituents in Cyclic Nucleotides Is Associated with Inhibition of Toxin Uptake by the Hepatocyte Transporters OATP 1B1 and 1B3

Analogs of the cyclic nucleotides cAMP and cGMP have been extensively used to mimic or modulate cellular events mediated by protein kinase A (PKA), Exchange protein directly activated by cAMP (Epac), or protein kinase G (PKG). We report here that some of the most commonly used cyclic nucleotide analogs inhibit transmembrane transport mediated by the liver specific organic anion transporter peptides OATP1B1 and OATP1B3, unrelated to actions on Epac, PKA or PKG. Several cAMP analogs, particularly with 8-pCPT-substitution, inhibited nodularin (Nod) induced primary rat hepatocyte apoptosis. Inhibition was not mediated by PKA or Epac, since increased endogenous cAMP, and some strong PKA- or Epac-activating analogs failed to protect cells against Nod induced apoptosis. The cAMP analogs inhibiting Nod induced hepatocyte apoptosis also reduced accumulation of radiolabeled Nod or cholic acid in primary rat hepatocytes. They also inhibited Nod induced apoptosis in HEK293 cells with enforced expression of OATP1B1 or 1B3, responsible for Nod transport into cells. Similar results were found with adenosine analogs, disconnecting the inhibitory effect of certain cAMP analogs from PKA or Epac. The most potent inhibitors were 8-pCPT-6-Phe-cAMP and 8-pCPT-2′-O-Me-cAMP, whereas analogs like 6-MB-cAMP or 8-Br-cAMP did not inhibit Nod uptake. This suggests that the addition of aromatic ring-containing substituents like the chloro-phenyl-thio group to the purines of cyclic nucleotides increases their ability to inhibit the OATP-mediated transport. Taken together, our data show that aromatic ring substituents can add unwanted effects to cyclic nucleotides, and that such nucleotide analogs must be used with care, particularly when working with cells expressing OATP1B1/1B3, like hepatocytes, or intact animals where hepatic metabolism can be an issue, as well as certain cancer cells. On the other hand, cAMP analogs with substituents like bromo, monobutyryl were non-inhibitory, and could be considered an alternative when working with cells expressing OATP1 family members.