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.     

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.     

Monitoring juvenile stocks of flatfish in the Wadden Sea and the coastal areas of the southeastern North Sea

Results are presented of an on-going monitoring program, started in 1970, of the demersal fish population in the Wadden Sea, Scheldt estuary, and along the continental coast between the Belgian-French border and Esbjerg. Particular attention is given to long-term trends in overall abundance and annual variations in spatial distribution of 0- and 1-group plaice and sole in relation to year-class strength and to variations in growth rate. Recruitment level in both plaice and sole appeared to be higher in the 1980s than in the 1970s. The most important nursery areas for plaice are the German Bight and the Wadden Sea, and the relative contributions of these areas to the total plaice stock in different years are relatively stable. In contrast, the most important nurseries for sole are along the continental coast, and the contribution of different parts of these nurseries vary considerably from year to year. Abundant year-classes of sole can originate from northern as well as from southern parts of the nurseries. Relations between survey results and year-class estimates from Virtual Population Analysis (VPA) for plaice are better compared to sole. No trends have been observed in mean length at age 0 and 1 during the years the surveys have been carried out. It is concluded that the quality of the continental coastal area and the Wadden Sea as nursery area for plaice and sole has not declined during the period of investigation. Presented at the VI International Wadden Sea Symposium (Biologische Anstalt Helgoland, Wattenmeerstation Sylt, D-2282 List, FRG, 1–4 November 1988)     

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.     

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.     

Ontogenetic trajectories and early shape differentiation of treehopper pronota (Hemiptera: Membracidae)

Membracids (family: Membracidae), commonly known as treehoppers, are recognizable by their enlarged and often elaborated pronota. Much of the research investigating the development and evolution of this structure has focused on the fifth instar to adult transition, in which the pronotum undergoes the largest transformation as it takes on adult identity. However, little is known about the earlier nymphal stages, the degree to which the pronotum develops at these timepoints, and how development has changed relative to the ancestral state. Here, we studied the nymphal stages and adults of five morphologically distinct membracid species and of Aetalion reticulatum (family: Aetalionidae), the outgroup which was used as an ancestral state proxy. We found that shape differentiation in the pronotum of membracids can start as early as the second instar stage. Most shape differentiation occurs within the nymphal stages and not in the embryo since the shape of the first-instar pronotum did not differ from the outgroup species in all but one species we investigated. We found the anterior–posterior axis of the pronotum elongated at a faster relative rate in membracid species than in A. reticulatum, which contributed to the development of exaggerated pronotal size. Finally, we found differences in the morphogenesis of shape across species. We suggest this is due to the developmental and evolutionary divergence of differential growth patterning of the dorsal surface of the pronotum, not only across species, but also between stages within the same species. This lability may contribute to the evolvability and diversification of the membracid pronotum.     

Gelation of liposome interior. A novel method for drug encapsulation.

Liposomes can be loaded with weak acids and bases, which exist in solutions in equilibrium with membrane permeable uncharged form, using various gradients across their membranes. Because in some cases the estimated drug concentration in the loaded liposomes exceeds their aqueous solubility we investigated the physical state of the liposome encapsulated anticancer drug Doxorubicin. X-Ray diffraction, electron microscopy, and test tube solubility experiments have shown that upon encapsulation the drug molecules form a gel-like phase.