A major drawback of angular SLI measurements is that pixels at the outer border of the image are illuminated under different polar angles θ than pixels in the center of the image. This asymmetric illumination at the image borders leads to asymmetries in the resulting SLI profiles so that peaks might not be detected and wrong/perpendicular fiber orientations are computed. Figure 6 shows angular SLI measurements of a coronal vervet monkey brain section (section 512, as shown in Menzel et al., 2021, Figure 8C) for different fields of view. The asymmetric illumination at the image borders becomes clearly visible in the maximum scattered light intensities (arrows in Figure 6A). The vector distribution maps in C show mirror-inverted regions at the borders of the corpus callosum: in regions that are not located at the image border (1,4), the reconstructed fiber orientations follow the course of the bundle as expected. At the image borders (2,3), some reconstructed fiber orientations run perpendicularly to the course of the bundle (highlighted by arrows). While angular SLI yields only line profiles, SLI scatterometry provides the full structural information of the scattering patterns, allowing to reliably determine the center of illumination for each image pixel (cf. Figure 1B) and to avoid artifacts caused by asymmetric illumination.
PilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 A resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. A panel of pilT mutations highlights the importance of the arginines, the PAS-like domain, the polar subunit interface, and the C-terminal helices for retraction. We present a model for ATP binding leading to dramatic PilT domain motions, engagement of the arginine wire, and subunit communication in this hexameric motor. Our conclusions apply to the entire type II/IV secretion ATPase family.
The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber.
Although not solely concerned with the role of women as artists in relation to the activities of the Pre-Raphaelite brotherhood (1850-1900), the show highlights the careers and achievements of several female artists. Iconic works are presented side by side with little-known pieces, with each protagonist being devoted a distinct section.
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