Hans-Henning Schott

Chemical composition of Litomosoides carinii microfilarial sheaths

Litomosoides carinii microfilariae were exsheathed by freezing and thawing, and the sheaths were separated by filtration. Samples of pure sheaths thus obtained were hydrolyzed, methanolyzed or oxidized with nitric acid under pressure at 300 degrees C, respectively, and were analyzed for amino acids, sugars, fatty acids or for metal ions and phosphorus. Almost 75% of the sheath dry weight could thus be accounted for. Amino acids (55 weight %) were the major constituents, and amongst these glutamine and proline (approximately 11% each). The detection of 2% cysteine/cystine indicated the possible presence of disulfide crosslinks. Besides amino acids, approximately 8% of sugars--roughly equimolar amounts of (N-acetyl)galactosamine and uronic acids--1.5% of monovalent cations (Na+ and K+) and 9.5% of phosphate were detected. No appreciable amounts of fatty acids, neutral sugars, neuraminic acid, or (N-acetyl)glucosamine (i.e. no chitin) were found.

The sheaths of Brugia microfilariae: isolation and composition.

Burgia malayi andB. pahangi microfilariae were isolated from the blood of infectedMastomys natalensis, and were exsheathed by freezing, thawing and agitation. Pure sheaths were obtained by a filtration procedure. The sheaths were found to contain about 95 mol% of amino acids, with proline, glutamic acid/glutamine, alanine, cysteine/cystine and glycine being the major components, and 5 mol% of carbohydrates, notably (N-acetyl)galactosamine, but no (N-acetyl)glucosamine.

Litomosoides carinii: extraction of the microfilarial sheath components and antigenicity of the sheath fractions

Microfilarial sheaths ofLitomosoides carinii were isolated and extracted with 2% sodium dodecyl sulfate (SDS) and 5% 2-mercaptoethanol (2ME). Extraction with SDS alone did not alter the ultrastructure of the sheaths and yielded five polypeptides (27–67 kDa) that were not recognized by antibodies of infected hosts but reacted with antibodies to host-serum proteins. 2ME treatment caused partial solubilization of the sheaths (45% as determined by amino acid analysis), which could be further improved by combining 2ME with SDS. The remainder showed filamentous/threadlike structures on electron microscopic examination. As compared with whole sheaths, the insoluble proportion was markedly enriched in alanine and cysteine but contained less galactosamine, serine, and threonine. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of 2ME/SDS-extractable components showed 12–16 bands of 14>120 kDa. A predominant component had an apparent molecular mass of 22 kDa. Two bands (42 and 120 kDa) could be stained with Coomassie blue but showed “negative” staining when gels were stained with silver. Several components (but not the 22-kDa polypeptide) bore phosphocholine epitopes. Apart from the negatively staining bands, most of the 2ME-soluble sheath components were recognized by antibodies ofL. carinii-infectedMastomys coucha. Except for several polypeptides that had been unspecifically recognized by IgM, the antibody response to sheath components started at the end of the prepatent period.

Litomosoides carinii microfilarial sheaths: Partial amino-acid sequences of several major polypeptide constituents

Isolated sheaths from Litomosoides carinii microfilariae were disintegrated by reduction with dithiothreitol and were 14C-carboxymethylated. Five major sheath proteins thus solubilized were purified by size exclusion chromatography and reversed-phase HPLC (rpHPLC). Proteolytic fragments of complete sheaths and of the single sheath proteins were isolated by rpHPLC and were N-terminally sequenced. A library of 27 partial sheath polypeptide sequences was thus established, 21 of which could be assigned to three L. carinii sheath structural genes (shp1,2, and 3/3a) isolated on the basis of this and of previous amino acid sequence information. The remaining peptides document the presence of at least one additional major sheath constituent.

Determination of anthranilate, 3-hydroxyanthranilate and 2,3-dihydroxybenzoate in the enzyme assay of anthranilate hydroxylase by thin-layer and high-performance liquid chromatography

Abstract A high-peformance liquid chromatography technique using UV detection is reported for the assay of anthranilate 2,3-dioxygenase (deaminating) activity. The substrate anthranilate, the main product 2,3-dihydroxybenzoate and the by-product 3-hydroxyanthranilate are determined quantitatively in the presence of biolgical material. After addition of benzoic acid as internal standard, the substances of interest are separated isocratically on a reversed-hase C 18 column with a detection sesitivity of 0.5 nmol and retention times of 3.5–10 min. A thin-layer chromatographic and fluorimetric method is also described.