3D Visualization of Human Blood Vascular Networks Using Single-Domain Antibodies Directed against Endothelial Cell-Selective Adhesion Molecule (ESAM)

High-quality three-dimensional (3D) microscopy allows detailed, unrestricted and non-destructive imaging of entire volumetric tissue specimens and can therefore increase the diagnostic accuracy of histopathological tissue analysis. However, commonly used IgG antibodies are oftentimes not applicable to 3D imaging, due to their relatively large size and consequently inadequate tissue penetration and penetration speed. The lack of suitable reagents for 3D histopathology can be overcome by an emerging class of single-domain antibodies, referred to as nanobodies (Nbs), which can facilitate rapid and superior 2D and 3D histological stainings. Here, we report the generation and experimental validation of Nbs directed against the human endothelial cell-selective adhesion molecule (hESAM), which enables spatial visualization of blood vascular networks in whole-mount 3D imaging. After analysis of Nb binding properties and quality, selected Nb clones were validated in 2D and 3D imaging approaches, demonstrating comparable staining qualities to commercially available hESAM antibodies in 2D, as well as rapid and complete staining of entire specimens in 3D. We propose that the presented hESAM-Nbs can serve as novel blood vessel markers in academic research and can potentially improve 3D histopathological diagnostics of entire human tissue specimens, leading to improved treatment and superior patient outcomes

Structural aspects of the cytochromeb 6 f complex; structure of the lumen-side domain of cytochromef

The following findings concerning the structure of the cytochrome b(6)f complex and its component polypeptides, cyt b(6), subunit IV and cytochrome f 1. Comparison of the amino acid sequences of 13 and 16 cytochrome b(6) and subunit IV polypeptides, respectively, led to (a) reconsideration of the helix lengths and probable interface regions, (b) identification of two likely surface-seeking helices in cyt b(6) and one in SU IV, and (c) documentation of a high degree of sequence invariance compared to the mitochondrial cytochrome. The extent of identity is particularly high (88% for conserved and pseudo- conserved residues) in the segments of cyt b(6) predicted to be extrinsic on the n-side of the membrane. 2. The intramembrane attractive forces between trans-membrane helices that normally stabilize the packing of integral membrane proteins are relatively weak. 3. The complex isolated in dimeric form has been visualized, along with isolated monomer, by electron microscopy. The isolated dimer is much more active than the monomer, is the major form of the complex isolated and purified from chloroplasts, and is inferred to be a functional form in the membrane. 4. The isolated cyt b(6)f complex contains one molecule of chlorophyll a. 5. The structure of the 252 residue lumen-side domain of cytochrome f isolated from turnip chloroplasts has been solved by X-ray diffraction analysis to a resolution of 2.3 Å