Characterisation of Pseudomonas rhamnolipids

The Gram negative organism, Pseudomonas aeruginosa, is often found in the lungs of patients with cystic fibrosis and other forms of severe bronchiectasis, where it secretes a number of extracellular toxins including the mono- and dirhamnolipids. The principal monorhamnolipid from P. aeruginosa has previously been identified as rhamnosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rh-C10.C10). A number of related mono- and dirhamnolipids have been purified from cultures of a clinical isolate of P. aeruginosa and identified by fast atom bombardment and electron impact mass spectrometry: these contain the 3-hydroxyoctanoyl-3-hydroxydecanoate (C8.C10) and 3-hydroxydecanoyl-3-hydroxydodecanoate (C10.C12) homologues. Structural isomers were also present where the order of the lipid linkage was transposed (Rh-C10.C8 and Rh-C12.C10). Unsaturated mono- and dirhamnolipids containing the 3-hydroxydecanoyl-3-hydroxydodec-5-enoate (C10.C12:1) lipid were also present.     

Stimulation of CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis by incubation of rat hepatocytes with phospholipase A2

The effect of phospholipase A2 treatment of rat hepatocytes on CTP: phosphocholine cytidylyltransferase and phosphatidylcholine synthesis was investigated. Cytidylyltransferase is recovered from the cytosol and in a membrane-bound form with the microsomes. Digitonin treatment of cells causes rapid release into the medium of the cytosolic, but not the microsomal form of the cytidylyltransferase. Incubation of hepatocytes for 10 min with phospholipase A2 (0.9 units/dish) in the medium, resulted in a 33% decrease in the cytidylyltransferase activity released by digitonin treatment (2.5 +/- 0.15 nmol/min per mg compared to 3.9 +/- 0.10 nmol/min per mg in the control). In agreement with the digitonin experiments, incubation with 0.9 units/dish of phospholipase A2 resulted in a decrease in the cytidylyltransferase activity in the cytosol (from 4.3 +/- 0.10 nmol/min per mg to 2.6 +/- 0.14 nmol/min per mg) and a corresponding increase in the microsomal fraction (from 0.9 +/- 0.16 nmol/min per mg to 1.8 +/- 0.20 nmol/min per mg). The effect of phospholipase A2 on cytidylyltransferase translocation was concentration- and time-dependent. Incubation of hepatocytes in the presence of phospholipase A2 (0.9 units/dish) for 10 min prior to pulse-chase experiments resulted in an increase in radiolabel incorporation into phosphatidylcholine (from 2.4 +/- 0.02.10(-5) dpm/dish to 3.1 +/- 0.1.10(-5) dpm/dish) and a corresponding decrease in radiolabel associated with the choline (from 2.5 +/- 0.05.10(-5) to 1.4 +/- 0.03.10(-5) dpm) and phosphocholine fractions (from 8.5 +/- 0.07.10(-5) to 6.9 +/- 0.05.10(-5) dpm). We conclude that phospholipase A2 can cause a stimulation of CTP: phosphocholine cytidylyltransferase activity and phosphatidylcholine synthesis in cultured rat hepatocytes.     

Is methyl-branching in alpha-tocopherol's "tail" important for its in vivo activity? Rat curative myopathy bioassay measurements of the vitamin E activity of three 2RS-n-alkyl-2,5,7,8-tetramethyl-6-hydroxychromans

The vitamin E activity of the acetates of three 2RS-n-alkyl-2,5,7,8-tetramethyl-6-hydroxychroman analogs of alpha-tocopherol have been measured and compared directly with all-rac-alpha-tocopheryl acetate, or indirectly via 2R,4'R,8'R-alpha-tocopheryl acetate, using the rat curative myopathy, plasma pyruvate kinase assay. The analogs with alkyl chain lengths of 11 and 13 carbons have activities which not only do not differ significantly (p greater than 0.05) from each other but also do not differ from that of all-rac-alpha-tocopheryl acetate. This finding indicates that methyl branching in the phytyl tail at the 4', 8', and 12' positions has little if any influence upon vitamin E activity. Thus physical interactions involving the methyl branches of the phytyl tail and the polyunsaturated moieties of membrane phospholipids are unimportant in vivo, insofar as this bioassay is concerned. However, the length of the hydrocarbon tail is important. This is indicated by the result obtained with the acetate of the analog with an alkyl chain length of 15 carbon atoms which had only 15% of the activity of 2R,4'R,8'R-alpha-tocopheryl acetate, i.e., 22% of the activity of all-rac-alpha-tocopheryl acetate since this form is 1.47 times less active than 2R,4'R,8'R-alpha-tocopheryl acetate in the curative myopathy bioassay (Weiser, Vecchi, & Schlachter, Internat. J. Vit. Nutr. Res. 55:149-158, 1985).     

Effects of spaceflight on the proliferation of jejunal mucosal cells

The mitotic indices, villus heights, and crypt depths were determined in each of three jejunal regions (proximal, middle, and distal) for five animals each in the flight, vivarium, and synchronous groups. Because of the rapid turnover of intestinal mucosal cells and the delay in recovering the flight animals, it is not known whether the proliferation of jejunal mucosal cells is affected by microgravity conditions associated with spaceflight. However, since there were no consistent differences between animals in the flight group and those in the synchronous and vivarium control groups, it appears that any effects of microgravity on the turnover of jejunal mucosal cells are short-lived. Thus, this study represents an initial step in determining the effects of microgravity on the proliferation and turnover of intestinal mucosal cells.     

Cross-linkage sites in type I collagen fibrils studied by neutron diffraction

Cross-links in tendon collagen are essential for the biomechanical strength of healthy tissue. The nature and position of these cross-links has long been a subject for conjecture. We have approached this problem in a non-destructive manner, by studying neutron diffraction from collagen fibrils that have been specifically deuterated by reduction at keto-amine and Schiff base groups with sodium borodeuteride (NaB2H4). The intensities of the first 23 meridional reflections were recorded for both native and reduced tendons. These data were used to calculate the neutron-scattering density profile of the 67 nm (D) repeat of type I collagen fibrils in rat tail tendon. This approach not only succeeds in determining the location of the cross-linkage sites with respect to the fibril structure, as projected onto the fibre axis, but also presents a novel form of the isomorphous derivative solution to the phase problem.     

Fusaric acid and other metabolite production in Fusarium oxysporum f. sp. vasinfectum

A thin layer chromatography system is described for the analysis of fusaric acid and other secondary metabolites, to assist in the identification of pathogenic races of Fusarium oxysporum f. sp. vasinfectum . There were differences in the metabolite profiles of the races grown on different media.     

Angiotensin II-producing proteases from granulomatous tissue reaction in mice infected with Schistosoma mansoni

1. Angiotensin I hydrolases, Mr 140,000 and Mr 70,000 were separated by gel filtration from Tris-HCl buffer extract of hepatic granulomas developed in mice with schistosomiasis. Two enzymes had different substrate specificity. 2. Mr 140,000 hydrolase activity was inhibited by captopril as reported for angiotensin converting enzyme (ACE), while that of Mr 70,000 hydrolase activity was inhibited by potato carboxypeptidase inhibitor. 3. An intermediary, des-Leu10-angiotensin I and then angiotensin II were formed from angiotensin I by Mr 70,000 hydrolase. 4. The findings suggest that Mr 70,000 enzyme is tissue carboxypeptidase A, and it generates angiotensin II in granulomatous inflammation as does ACE.