Soluble Leukocyte-Associated Ig-Like Receptor-1 in Amniotic Fluid Is of Fetal Origin and Positively Associates with Lung Compliance

The soluble form of the inhibitory immune receptor leukocyte-Associated Ig-like Receptor-1 (sLAIR-1) is present in plasma, urine and synovial fluid and correlates to inflammation. We and others previously showed inflammatory protein expression in normal amniotic fluid at term. We hypothesized that sLAIR-1 is present in amniotic fluid during term parturition and is related to fetal lung function development. sLAIR-1 was detectable in all amniotic fluid samples (n=355) collected during term spontaneous deliveries. First, potential intra-uterine origins of amniotic fluid sLAIR-1 were explored. Although LAIR-1 was expressed on the surface of amniotic fluid neutrophils, LAIR-1 was not secreted upon ex vivo neutrophil stimulation with LPS, or PMA/ionomycin. Cord blood concentrations of sLAIR-1 were fourfold lower than and not related to amniotic fluid concentrations and placentas showed no or only sporadic LAIR-1 positive cells. Similarly, in post-mortem lung tissue of term neonates that died of non-pulmonary disorders LAIR-1 positive cells were absent or only sporadically present. In fetal urine samples, however, sLAIR-1 levels were even higher than in amniotic fluid and correlated with amniotic fluid sLAIR-1 concentrations. Second, the potential relevance of amniotic fluid sLAIR-1 was studied. sLAIR-1 concentrations had low correlation to amniotic fluid cytokines. We measured neonatal lung function in a convenient subset of 152 infants, using the single occlusion technique, at a median age of 34 days (IQR 30-39). The amniotic fluid concentration of sLAIR-1 was independently correlated to airway compliance (ρ=0.29, P=.001). Taken together, we show the consistent presence of sLAIR-1 in amniotic fluid, which originates from fetal urine. Concentrations of sLAIR-1 in amniotic fluid during term deliveries are independent from levels of other soluble immune mediators. The positive association between concentrations of amniotic fluid sLAIR-1 and neonatal lung compliance suggests that amniotic fluid sLAIR-1 may be useful as a novel independent marker of neonatal lung maturation.     

Detection of phospholipid peroxides in biological samples

Peroxidation of membrane lipids has been hypothesized to play a key role in various types of tissue degeneration and pathology. Lipid peroxides are formed when oxygen reacts with an unsaturated fatty acid chain. Virtually all of the unsaturated fatty acids in biological systems are bound by ester linkages in phospholipids or triglycerides. Phospholipid and triglyceride peroxides are primary products of lipid peroxidation and have rarely been measured. Most of the commonly used methods for detection of lipid peroxidation are based on detection of malondialdehyde or other chemical species that are derived from oxidized fatty acids. This review presents an overview of recently developed methods aimed at identifying and measuring oxidized phospholipids and triglycerides which are direct evidence of the occurrence of lipid peroxidation in vivo.     

Inhibition of platelet-activating factor biosynthesis via the acetyltransferase by arachidonic and oleic acids in ionophore A23187-stimulated bovine neutrophils

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation and allergy that is synthesized by several inflammatory cells including neutrophils. Addition of exogenous arachidonic acid to ionophore A23187-stimulated bovine neutrophils led to the inhibition of PAF biosynthesis assayed by incorporation of [3H]acetate into PAF and by bioassay; under the same conditions, leukotriene B4 (LTB4) formation was not decreased. The activities of the PAF metabolism enzymes indicated that the PAF synthesis inhibition by arachidonic acid is mediated via the acetyltransferase inhibition which is the last enzyme of the PAF formation. Another unsaturated fatty acid, oleic acid, exhibited the same inhibitory effect on [3H]acetate-PAF formation; however, the saturated stearic acid did not lead to any inhibition. These findings suggest that liberation of unsaturated fatty acids from membrane phospholipids, as a consequence of phospholipase A2 activation, would modulate PAF formation via inhibition of the acetyltransferase. In addition, the utilization of arachidonic acid oleic acids in activated neutrophils furnishes an easy means of blocking PAF synthesis in order to understand the role of this mediator in cellular processes.     

Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min^–1.mg protein^–1 in the presenceof 1 µM fru-2,6-P₂. The pH optima is similar for boththe forward and reverse reactions (pH 7.5–7.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P₂. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P₂ stimulates activity by increasing V_max and increasingthe affinity for fru-6-P, fru-1,6-P₂ and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986)     

A comparison between dopamine-stimulated adenylate cyclase and 3H-SCH 23390 binding in rat striatum

Methods for measuring 3H-SCH 23390 binding and dopamine (DA) stimulated adenylate cyclase (AC) were established in identical tissue preparations and under similar experimental conditions. Pharmacological characterization revealed that both assays involved interaction with the D1 receptor or closely associated sites. In order to investigate whether the binding sites for 3H-SCH 23390 and DA in fact are identical, the antagonistic effects of a variety of pharmacologically active compounds were examined. Surprisingly, the Ki-values obtained from Schild-plot analysis of the antagonism of DA-stimulated AC, were 80-240 times higher than the Ki-values obtained from competition curves of 3H-SCH 23390 binding. Since both assays were performed under identical conditions, the differences in Ki-values indicate the possibility of different binding sites for DA and 3H-SCH 23390 or, that DA and 3H-SCH 23390 label different states of the same receptor.     

Formation of multilamellar vesicles by addition of tannic acid to phosphatidylcholine-containing small unilamellar vesicles

Tannic acid induces aggregation and formation of multilamellar vesicles when added to preparations of small unilamellar vesicles, specifically those containing phosphatidylcholine. Aggregation and clustering of vesicles was demonstrated by cryo-electron microscopy of thin films and by freeze-fracture technique. Turbidity measurements revealed an approximately one-to-one molar ratio between tannic acid and phosphatidylcholine necessary for a fast and massive aggregation of the small unilamellar vesicles. When tannic acid-induced aggregates were dehydrated and embedded for conventional thin-section electron microscopy, multilamellar vesicles were retrieved in thin sections. It is concluded from morphological studies, as well as previous tracer studies, that tannic acid, at least to a great extent, prevents the extraction of phosphatidylcholine. Multilamellar vesicles were also observed in tannic acid-treated vesicles prepared from total lipid extracts from either rabbit or rat hearts. Substantially more multilamellar vesicles were retrieved in the rabbit vesicle preparation. This difference can probably be explained by the difference in the proportion of the plasmalogen phosphatidylcholine, and possibly the content of sphingomyelin, in lipid extracts of rabbit and rat hearts. It is concluded that the dual effect (reduced extraction and aggregation) of tannic acid on phosphatidylcholines should be taken into consideration when tannic acid is used in tissue preparation.     

Molecular-cytogenetic characterization of a higher plant centromere/kinetochore complex

The centromeric region of a telocentric field bean chromosome that resulted from centric fission of the metacentric satellite chromosome was microdissected. The DNA of this region was amplified and biotinylated by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR)/linker-adapter PCR. After fluorescence in situ hybridization (FISH) the entire chromosome complement of Vicia faba was labelled by these probes except for the nucleolus organizing region (NOR) and the interstitial heterochromatin, the chromosomes of V. sativa and V. narbonensis were only slightly labelled by the same probes. Dense uniform labelling was also observed when a probe amplified from a clearly delimited microdissected centromeric region of a mutant of Tradescantia paludosa was hybridized to T. paludosa chromosomes. Even after six cycles of subtractive hybridization between DNA fragments amplified from centromeric and acentric regions no sequences specifically located at the field bean centromeres were found among the remaining DNA. A mouse antiserum was produced which detected nuclear proteins of 33 kDa and 68 kDa; these were predominantly located at V. faba kinetochores during mitotic metaphase. DNA amplified from the chromatin fraction adsorbed by this serum out of the sonicated total mitotic chromatin also did not cause specific labelling of primary constrictions. From these results we conclude: (1) either centromere-specific DNA sequences are not very conserved among higher plants and are — at least in species with large genomes — intermingled with complex dispersed repetitive sequences that prevent the purification of the former, or (2) (some of) the dispersed repeats themselves specify the primary constrictions by stereophysical parameters rather than by their base sequence.     

Lipase-Catalyzed Regioselective Acylation and Deacylation of Glucose Derivatives

In the development of an efficient synthesis of 1-O-decanoyl-2,3,4,6-tetra-O-acetyl-β-D-glucose (β-2) several lipase-based approaches have been explored. Among five immobilized Upases tested, the lipase from Candida antarctica proved particularly efficient for catalyzing selective hydrolysis in the 1-position of 1,2,3,4,6-penta-O-acetyl-β-D-glucose (β-1). Using triethylamine as catalyst, the hydrolysis product 2,3,4,6-tetra-O-acetyl-D-glucose (3) can be esterified with decanoyl chloride to form β-2 selectively, thereby providing an efficient chemo-enzymatic synthesis starting from readily available raw materials. Attempts to produce β-2 from β-1 by lipase-catalyzed interesterification or to esterify 3 with decanoic acid using a lipase as catalyst were unsuccessful. The latter finding was explained by the hemiacetal OH group of glucose being unable to act as nucleophile in the lysis of the lipase acyl-enzyme intermediate. Furthermore, β-2 was found to bee a too bulky substrate to fit into the active site of any of the lipases tested.