Glycosphingolipid expression in spontaneously aborted fetuses and placenta from blood group p women. Evidence for placenta being the primary target for anti-Tja-antibodies

A 12-week-old fetus and one 17-week-old fetus + placenta were obtained after spontaneous abortions from two women of blood group p. The 17-week-old fetus was dissected into intestine, liver, brain and residual tissue. Nonacid glycosphingolipid fractions were prepared from the tissues. Glycolipid characterization was carried out using thin layer chromatography immunostained with monoclonal antibodies and bacteria and by¹H NMR spectroscopy and mass spectrometry. In the placental fraction substantial amounts of globotetraosylceramide (P-antigen) and globotriaosylceramide (P^k-antigen) were identified. In contrast, the fetuses contained only trace amounts of these structures, as revealed by immunostaining. These results indicate that the primary target for the antibodies of the anti-Tj^a serum is the placenta tissue, resulting in termination of the pregnancy.     

Sensitive detection of isoglobo and globo series tetraglycosylceramides in human thymus by ion trap mass spectrometry

Glycosphingolipids serve as ligands for receptors involved in signal transduction and immune recognition, as exemplified by isoglobotrihexosylceramide, an antigenic ligand for T cell receptors. Mechanistic studies on the regulation of isoglobotrihexosylceramide require biochemical measurement of its lysosomal precursor, isoglobotetraglycosylceramide. It remains a challenge to distinguish between complex tetraglycosylceramide glycosphingolipid isomers with the same sugar components but diverse internal linkages. Here we established a simple and sensitive method to separate globo- and isoglobotetraglycosylceramide by MS5 ion trap mass spectrometry, and report the identification of isoglobotetraglycosylceramide in a CHO cell line transfected by iGb3 synthase, as well as in human thymus.     

Biosynthesis of photodynamically active rubellins and structure elucidation of new anthraquinone derivatives produced by Ramularia collo-cygni

Here we present the first isolation of the anthrachinone derivative rubellin A out of mycelium and culture filtrate of Ramularia collo-cygni. Furthermore, two compounds, rubellin E and 14-dehydro rubellin D were isolated and their structures elucidated. In comparison to the other rubellins, rubellin A shows increased photodynamic oxygen activation. By incorporating both [1-(13)C]-acetate and [2-(13)C]-acetate into the rubellins, we showed that such anthraquinone derivatives were biosynthesised via the polyketide pathway. The labelling pattern after being fed [U-(13)C(6)]-glucose proved the existence of fungal folding mode of the poly-beta-keto chain. The ability to produce rubellins is not limited to R. collo-cygni in the anamorph genus Ramularia.     

Saccharomyces cerevisiae porin pore forms complexes with mitochondrial outer membrane proteins Om14p and Om45p

Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites.     

Preparation and Properties of Recombinant Rat and Human Procholecystokinin57 –95

Recombinant human progastrin_6–80 binds two ferric ions with an apparent dissociation constant of 2.2 ± 0.1 μM [Baldwin (2004) Protein J 23:65–70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not they bound ferric ions. Recombinant rat and human procholecystokinin_57–95 were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant procholecystokinin_57–95 did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated that binding to procholecystokinin_57–95 was at least 40–fold weaker than the binding of ferric ions to progastrin_6–80.