Sulfatide Recognition by Colonization Factor Antigen CS6 from Enterotoxigenic Escherichia coli

The first step in the pathogenesis of enterotoxigenic Escherichia coli (ETEC) infections is adhesion of the bacterium to the small intestinal epithelium. Adhesion of ETEC is mediated by a number of antigenically distinct colonization factors, and among these, one of the most commonly detected is the non-fimbrial adhesin coli surface antigen 6 (CS6). The potential carbohydrate recognition by CS6 was investigated by binding of recombinant CS6-expressing E. coli and purified CS6 protein to a large number of variant glycosphingolipids separated on thin-layer chromatograms. Thereby, a highly specific binding of the CS6-expressing E. coli, and the purified CS6 protein, to sulfatide (SO₃-3Galβ1Cer) was obtained. The binding of the CS6 protein and CS6-expressing bacteria to sulfatide was inhibited by dextran sulfate, but not by dextran, heparin, galactose 4-sulfate or galactose 6-sulfate. When using recombinantly expressed and purified CssA and CssB subunits of the CS6 complex, sulfatide binding was obtained with the CssB subunit, demonstrating that the glycosphingolipid binding capacity of CS6 resides within this subunit. CS6-binding sulfatide was present in the small intestine of species susceptible to CS6-mediated infection, e.g. humans and rabbits, but lacking in species not affected by CS6 ETEC, e.g. mice. The ability of CS6-expressing ETEC to adhere to sulfatide in target small intestinal epithelium may thus contribute to virulence.     

AN ELECTROPHORETIC AND SEROLOGICAL INVESTIGATION OF SEED PROTEINS IN GALEOPSIS TETRAHIT L. (LABIATAE) AND ITS PUTATIVE PARENTAL SPECIES

Seed proteins from the tetraploid Galeopsis tetrahit L. and its putative parental species G. pubescens Bess. and G. speciosa Mill., were examined using disc gel electrophoresis (PAGE), Ouchterlony double diffusion, and Immunoelectrophoresis. Forty-three inbred cultivated lines of G. speciosa, G. pubescens, and G. tetrahit (both the naturally occurring species and an artificially produced hybrid) and two wild populations of G. tetrahit were examined. PAGE gels were stained for total protein or malate dehydrogenase. Although variability in the total protein pattern was detected within and between lines for all three species, there was much similarity among the taxa. No bands appeared in the hybrid that were not present in the parents. The band pattern for synthetic G. tetrahit was very similar to that of natural G. tetrahit suggesting that G. tetrahit originated from a cross in nature between G. pubescens and G. speciosa. The total protein PAGE results were analyzed using numerical taxonomic techniques. Although these analyses did not clearly separate the three species, the general tendency was toward grouping G. pubescens lines together, natural G. tetrahit lines together, and synthetic G. tetrahit lines together. Natural and synthetic G. tetrahit lines were also interspersed among each other along with some G. speciosa lines. The malate dehydrogenase pattern showed little variation among the three species. Qualitative serological methods did not distingush among the three species, but confirmed the similarity observed among the taxa in the results of electrophoresis.     

Towards an understanding of multimodal traits of female reproduction in chimpanzees

Primates - Although primates have long been regarded as microsmatic, recent studies indicate that olfaction is an important sensory mode of primate communication, e.g., in the context of...     

Down-Regulation of Small Rubber Particle Protein Expression Affects Integrity of Rubber Particles and Rubber Content in Taraxacum brevicorniculatum

The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP) has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1-5) were identified in the rubber-producing dandelion species Taraxacum brevicorniculatum. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic T. brevicorniculatum plants showed a 40-50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed in vivo, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in T. brevicorniculatum, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage.     

Proteomic analysis of latex from the rubber-producing plant Taraxacum brevicorniculatum

Many plants produce latex, a specialized, metabolically active cytoplasm. This is generally regarded as a defensive trait but latex may also possess additional functions. We investigated the role of latex in the dandelion species Taraxacum brevicorniculatum that contains considerable amounts of high-quality natural rubber by carrying out a comprehensive analysis of the latex proteome. We developed reliable protocols for the preparation of protein samples for one-dimensional gel electrophoresis, two-dimensional gel electrophoresis, and subsequent mass spectrometry analysis, which led to 278 unique identifications. A gene ontology classification system based on comparisons with known Arabidopsis thaliana root proteins showed that dandelion proteins involved in lipid metabolism and transport were enriched in the latex proteome, whereas those involved in stress responses were not. We also found that proteins involved in rubber biosynthesis were distributed among different fractions of the latex proteome.     

Interaction of extracellular S100A4 with RAGE prompts prometastatic activation of A375 melanoma cells

S100A4, a member of the S100 protein family of EF‐hand calcium‐binding proteins, is overexpressed in various tumour entities, including melanoma, and plays an important role in tumour progression. Several studies in epithelial and mesenchymal tumours revealed a correlation between extracellular S100A4 and metastasis. However, exact mechanisms how S100A4 stimulates metastasis in melanoma are still unknown. From a pilot experiment on baseline synthesis and secretion of S100A4 in human melanoma cell lines, which are in broad laboratory use, A375 wild‐type cells and, additionally, newly generated A375 cell lines stably transfected with human S100A4 (A375‐hS100A4) or human receptor for advanced glycation endproducts (A375‐hRAGE), were selected to investigate the influence of extracellular S100A4 on cell motility, adhesion, migration and invasion in more detail. We demonstrated that A375 cells actively secrete S100A4 in the extracellular space via an endoplasmic reticulum‐Golgi‐dependent pathway. S100A4 overexpression and secretion resulted in prometastatic activation of A375 cells. Moreover, we determined the influence of S100A4‐RAGE interaction and its blockade on A375, A375‐hS100A4, A375‐hRAGE cells, and showed that interaction of RAGE with extracellular S100A4 contributes to the observed activation of A375 cells. This investigation reveals additional molecular targets for therapeutic approaches aiming at blockade of ligand binding to RAGE or RAGE signalling to inhibit melanoma metastasis.     

Ejectisins: tough and tiny polypeptides are a major component of cryptophycean ejectisomes

Fragments of discharged ejectisomes were isolated from two Cryptomonas and a Chroomonas species by detergent treatment followed by Percoll density gradient centrifugation. The fragments withstand high concentrated detergent solutions, reducing agents and freeze-thawing. Disintegration was achieved in 6 M guanidine hydrochloride. Reassembly into long, filamentous, ejectisome-like structures occurred after dialysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the polypeptide patterns of isolated ejectisome fragments and of reconstituted ejectisome-like structures were dominated by polypeptides with relative molecular weights of approximately 6 kDa. The polypeptides were not glycosylated and did not cross-react with antisera directed against recombinant Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis. A polyclonal antiserum directed against reconstituted, ejectisome-like filaments cross-reacted with the 6-kDa polypeptides and immunolabeled extruded ejectisome filaments. Twenty amino acid residues, obtained by N-terminal amino acid sequence analysis, matched to polypeptide sequences deduced from cDNA sequences of the cryptophyte Guillardia theta. The term “ejectisins” is introduced for the 6-kDa polypeptides which represent a major component of cryptophycean ejectisomes.