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A fluorescent tRNA derivative labeled at 3′-O position of the ultimate adenosine residue by reaction, under mild conditions, of tRNA with isatoic anhydride [3,1-benzoxazine- 2,4(1H)-dione] was obtained. The labeling selectivity was determined by several criteria: digestion with RNase, followed by HPLC of the digest, produces only one labeled nucleoside, identified as 3′-O-anthraniloyladenosine; the ratio of the absorbance at 260 nm to 332 nm also suggests a 1:1 molar ratio between the nucleic acid and the fluorophore; finally, the incapacity of the labeled tRNA to be charged by the specific aminoacyltransferase further demonstrates the engagement of the 3′-O position. Although the 3′-O-anthraniloyl-labeled tRNA does not seem to be functionally active, as far as the aminoacyl charging activity is concerned, surprisingly we found that it is able to form the ternary complex with elongation factor Tu (EF-Tu) and GTP with an affinity consistently higher than uncharged tRNA. From fluorescence anisotropy measurements the ternary complex dissociation constant was estimated as 73 nM for Escherichia coli and 140 nM for yeast anthraniloyl-tRNAPhe. These results may be interpreted in terms of the particular structure of the anthraniloyl group that makes the labeled tRNA similar to an aminoacyl-tRNA.

A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholipids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl- cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A2 and PAF-acetylhydrolase, on single phospholipids or their mixtures.

A sensitive method for determining platelet-activating factor acetylhydrolase (PAF-AH) activity in human serum, using high-performance liquid chromatography (HPLC) with a fluorimetric detection, is described. The method is based on the derivatization with 7-diethylaminocoumarin-3-carbonylazide of the 2-lysoPAF, by-product of PAF-AH activity, extracted from the reaction mixture by phase partition into organic solvents. After 3 h of derivatization, the fluorescent derivatives were analyzed by HPLC on a reversed-phase column. The mobile phase was made up with a gradient between head solvent, composed of methanol:water (80:20, v/v) containing 0.25 g/liter choline chloride, and chloroform. Fluorescence detection was at excitation wavelength of 400 nm and at emission wavelength of 480 nm. The described chromatographic procedure is able to resolve and simultaneously quantitate the fluorescent derivatives of the C:18 and C:16 2-lysoPAF. The comparison with the classical radiometric determination of PAF-AH activity demonstrates that the herein described procedure is suitable for study of enzyme kinetics and changes occurring in physiological conditions such as pregnancy.

Porins and lipopolysaccharide stimulate platelet activating factor synthesis by human mesangial cells. Porins, a family of hydrophobic proteins located in the outer membrane of the cell wall of gram-negative bacteria and lipopolysaccharide (LPS), were shown to stimulate the synthesis of platelet activating factor (PAF), a phospholipid mediator of inflammation and endotoxic shock, by cultured human glomerular mesangial cells (MC). The synthesis of PAF induced by porins was rapid (peak at 20 min) and independent either from contamination by LPS or from generation of an endotoxin-induced cytokine such as tumor necrosis factor (TNF) since it was not prevented by cycloheximide, an inhibitor of protein synthesis or anti-TNF blocking antibodies. LPS also stimulated PAF synthesis by MC. However, the kinetic of PAF synthesis induced by LPS was biphasic with an early and transient peak at 10 minutes and a second and sustained peak at three to six hours. This second peak required an intact protein synthesis and was prevented by anti-TNF antibodies, suggesting the dependency on LPS-induced synthesis of TNF. Experiments with labeled precursors demonstrated that in MC, either after stimulation with porins or LPS, PAF was synthetised via the remodeling pathway that involves acetylation of 1-0-alkyl-sn-glyceryl-3-phosphorylcholine (2-lyso-PAF) generated from 1-0-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins and LPS, indeed, induced PLA2-dependent mobilization of [ 14 C]-arachidonic acid that was inhibited by p-bromodiphenacylbromide (PBDB). PBDB, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase. The addition of 2-lyso-PAF restored PAF synthesis. The activity of acetyl CoA:2-lyso-PAF acetyltransferase was increased in porin- as well as in LPS-stimulated MC and, after cell preincubation with [ 3 H]-acetyl CoA, [ 3 H]-acetyl was incorporated in the newly synthetised PAF. The activation of PAF synthesis by porins was dependent on extracellular Ca 2+ . Porins by forming trans-membrane channels determined a sustained influx of 45 Ca 2+ into the cytosol. The inhibitory effect of trifluoperazine, an inhibitor of Ca 2+ -calmodulin complexes, on PAF synthesis by porinstimulated MC suggested that calmodulin mediated the Ca 2+ -dependent activation of enzymes involved in PAF synthesis.

Rat mesangial cells stimulated with calcium ionophore A23187 and phagocytosis were shown to produce platelet-activating factor (PAF), a mediator of inflammation and endotoxic shock. In the study presented here, the cultured human mesangial but not epithelial cells synthetized PAF not only in response to calcium ionophore A23187 and phagocytosis of immunoglobulin G-coated latex beads, but also after stimulation with cytokines such as tumor necrosis factor-alpha and interleukin-1 beta. PAF synthetized after stimulation with A23187 and to a lesser extent with phagocytosis was partially released. In contrast, PAF synthesized by stimulation with tumor necrosis factor-alpha and interleukin-1 beta remained cell associated. Experiments with labeled precursors demonstrated that PAF was synthetized via the remodeling pathway that involves the activation of phospholipase A2 and of an acetyl-coenzymeA:2-lyso-PAF acetyltransferase. Synthetic inhibitors of serine proteases as well as plasma alpha 1-proteinase inhibitor inhibited the activation of phospholipase A2 detected as release of (14C) arachidonic acid and the activation of acetyl-CoA:2-lyso-PAF acetyltransferase at concentrations 100-fold lower than those present in plasma. This raises the question about the ability of mesangial cells to synthetize PAF in vivo. However, the inhibitory effect of plasma alpha 1-proteinase inhibitor may be abrogated by oxidative inactivation due to a concomitant stimulation of mesangial cell respiratory burst or in zones of close contact among cells or matrix, which have been shown to exclude antiproteinases.

Porins, a family of hydrophobic proteins located in the outer membrane of cell-wall of Gram-negative bacteria, were shown to stimulate the synthesis and release of platelet-activating factor (PAF), a 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphorylcholine mediator of inflammation and endotoxic shock produced by polymorphonuclear neutrophils. PAF synthesis was independent either from contamination by LPS or generation of TNF. Experiments with labeled precursors demonstrated that PAF was synthesized via the remodeling pathway that involves acetylation of 1-O-alkyl-sn-glyceryl-3-phosphorylcholine generated from 1-O-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins, indeed, induced a sustained PLA2-dependent mobilization of [14C]arachidonic acid that was inhibited by p-bromodiphenacylbromide. p-Bromodiphenacylbromide, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase. The addition of 2-lyso-PAF restored PAF synthesis. The activity of acetyl CoA:2-lyso-PAF acetyltransferase was transiently increased in porin-stimulated PMN and the [3H]acetyl group was incorporated in the synthetized PAF after cell preincubation with [3H]acetyl CoA. The activation of PAF synthesis by porins as well as its release were dependent on extracellular Ca2+. Porins by forming trans-membrane channels determined a sustained influx of 45Ca2+ into the cytosol. As shown by inhibitors of Ca(2+)-calmodulin complexes, calmodulin mediated the Ca(2+)-dependent activation of enzymes involved in PAF synthesis.