Simultaneous Display of Multiple Foreign Peptides in the FliD Capping and FliC Filament Proteins of the Escherichia coli Flagellum

The bacterial flagellum is composed of more than 20 different proteins. The filament, which constitutes the major extracellular part of the flagellum, is built up of approximately 20,000 FliC molecules that assemble at the growing distal end of the filament. A capping structure composed of five FliD molecules located at the tip of the filament promotes polymerization of FliC. Lack of FliD leads to release of the subunits into the growth medium. We show here that FliD can be successfully used in bacterial surface display. We tested various insertion sites in the capping protein, and the optimal region for display was at the variable region in FliD. Deletion and/or insertion at other sites resulted in decreased formation of flagella. We further developed the technique into a multihybrid display system in which three foreign peptides are simultaneously expressed within the same flagellum, i.e., D repeats of FnBPA from Staphylococcus aureus at the tip and fragments of YadA from Yersinia enterocolitica as well as SlpA from Lactobacillus crispatus along the filament. This technology can have biotechnological applications, e.g., in simultaneous delivery of several effector molecules.     

Access to Awareness for Faces during Continuous Flash Suppression Is Not Modulated by Affective Knowledge

It is a controversially debated topic whether stimuli can be analyzed up to the semantic level when they are suppressed from visual awareness during continuous flash suppression (CFS). Here, we investigated whether affective knowledge, i.e., affective biographical information about faces, influences the time it takes for initially invisible faces with neutral expressions to overcome suppression and break into consciousness. To test this, we used negative, positive, and neutral famous faces as well as initially unfamiliar faces, which were associated with negative, positive or neutral biographical information. Affective knowledge influenced ratings of facial expressions, corroborating recent evidence and indicating the success of our affective learning paradigm. Furthermore, we replicated shorter suppression durations for upright than for inverted faces, demonstrating the suitability of our CFS paradigm. However, affective biographical information did not modulate suppression durations for newly learned faces, and even though suppression durations for famous faces were influenced by affective knowledge, these effects did not differ between upright and inverted faces, indicating that they might have been due to low-level visual differences. Thus, we did not obtain unequivocal evidence for genuine influences of affective biographical information on access to visual awareness for faces during CFS.     

Globule Leukocyte And Mast Cell in Bile Ducts of Cattle Naturally Infected with Liver Flukes

In n previous report dealing with the pathology of bovine fascio-liasis the author described an unknown cell type in the epithelium of bile ducts. The histological and histochemical investigations published in this paper suggest that the cell may be considered a globule leukocyte. Globule leukocytes are rare in uninfected livers but are occurring in abundance in main bile ducts of cattle with spontaneous fascioliasis and also in small perilobular ducts in dicrocoeliasis. Liver fluke infection causes an increase in the population of subepithelial mast cells. Mast cell and globule leukocyte present similarities In their cytochemical properties. However, at low pH toluldinc blue shows a stronger but Alcian blue a weaker affinity for mast cells than for globule leukocytes.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)