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End date: 2024 × Start date: 2020 × DDC: 500 × Type: article × Subject: metabolism × Subject: procedures ×

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    Monoclonal Antibodies 13A4 and AC133 Do Not Recognize the Canine Ortholog of Mouse and Human Stem Cell Antigen Prominin-1 (CD133)
    (San Francisco, California, US : PLOS, 2016) Thamm, Kristina; Graupner, Sylvi; Werner, Carsten; Huttner, Wieland B.; Corbeil, Denis; Nabi, Ivan R
    The pentaspan membrane glycoprotein prominin-1 (CD133) is widely used in medicine as a cell surface marker of stem and cancer stem cells. It has opened new avenues in stem cell-based regenerative therapy and oncology. This molecule is largely used with human samples or the mouse model, and consequently most biological tools including antibodies are directed against human and murine prominin-1. Although the general structure of prominin-1 including its membrane topology is conserved throughout the animal kingdom, its primary sequence is poorly conserved. Thus, it is unclear if anti-human and -mouse prominin-1 antibodies cross-react with their orthologs in other species, especially dog. Answering this issue is imperative in light of the growing number of studies using canine prominin-1 as an antigenic marker. Here, we address this issue by cloning the canine prominin-1 and use its overexpression as a green fluorescent protein fusion protein in Madin-Darby canine kidney cells to determine its immunoreactivity with antibodies against human or mouse prominin-1. We used immunocytochemistry, flow cytometry and immunoblotting techniques and surprisingly found no cross-species immunoreactivity. These results raise some caution in data interpretation when anti-prominin-1 antibodies are used in interspecies studies.
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    Spatially resolved spectroscopic differentiation of hydrophilic and hydrophobic domains on individual insulin amyloid fibrils
    ([London] : Macmillan Publishers Limited, part of Springer Nature, 2016) Deckert-Gaudig, Tanja; Kurouski, Dmitry; Hedegaard, Martin A. B.; Singh, Pushkar; Lednev, Igor K.; Deckert, Volker
    The formation of insoluble β-sheet-rich protein structures known as amyloid fibrils is associated with numerous neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. A detailed understanding of the molecular structure of the fibril surface is of interest as the first contact with the physiological environment in vivo and plays a decisive role in biological activity and associated toxicity. Recent studies reveal that the inherent sensitivity and specificity of tip-enhanced Raman scattering (TERS) renders this technique a compelling method for fibril surface analysis at the single-particle level. Here, the reproducibility of TERS is demonstrated, indicating its relevance for detecting molecular variations. Consequently, individual fibrils are systematically investigated at nanometer spatial resolution. Spectral parameters were obtained by band-fitting, particularly focusing on the identification of the secondary structure via the amide III band and the differentiation of hydrophobic and hydrophilic domains on the surface. In addition multivariate data analysis, specifically the N-FINDR procedure, was employed to generate structure-specific maps. The ability of TERS to localize specific structural domains on fibril surfaces shows promise to the development of new fibril dissection strategies and can be generally applied to any (bio)chemical surface when structural variations at the nanometer level are of interest.
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    Nonlinear Structured Illumination Using a Fluorescent Protein Activating at the Readout Wavelength
    (San Francisco, California, US : PLOS, 2016) Lu-Walther, Hui-Wen; Hou, Wenya; Kielhorn, Martin; Arai, Yoshiyuki; Nagai, Takeharu; Kessels, Michael M.; Qualmann, Britta; Heintzmann, Rainer
    Structured illumination microscopy (SIM) is a wide-field technique in fluorescence microscopy that provides fast data acquisition and two-fold resolution improvement beyond the Abbe limit. We observed a further resolution improvement using the nonlinear emission response of a fluorescent protein. We demonstrated a two-beam nonlinear structured illumination microscope by introducing only a minor change into the system used for linear SIM (LSIM). To achieve the required nonlinear dependence in nonlinear SIM (NL-SIM) we exploited the photoswitching of the recently introduced fluorophore Kohinoor. It is particularly suitable due to its positive contrast photoswitching characteristics. Contrary to other reversibly photoswitchable fluorescent proteins which only have high photostability in living cells, Kohinoor additionally showed little degradation in fixed cells over many switching cycles.