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71.
Matthias Rüdt Philipp Vormittag Nils Hillebrandt Jürgen Hubbuch 《Biotechnology and bioengineering》2019,116(6):1366-1379
Virus-like particles (VLPs) have shown great potential as biopharmaceuticals in the market and in clinics. Nonenveloped, in vivo assembled VLPs are typically disassembled and reassembled in vitro to improve particle stability, homogeneity, and immunogenicity. At the industrial scale, cross-flow filtration (CFF) is the method of choice for performing reassembly by diafiltration. Here, we developed an experimental CFF setup with an on-line measurement loop for the implementation of process analytical technology (PAT). The measurement loop included an ultraviolet and visible (UV/Vis) spectrometer as well as a light scattering photometer. These sensors allowed for monitoring protein concentration, protein tertiary structure, and protein quaternary structure. The experimental setup was tested with three Hepatitis B core Antigen (HBcAg) variants. With each variant, three reassembly processes were performed at different transmembrane pressures (TMPs). While light scattering provided information on the assembly progress, UV/Vis allowed for monitoring the protein concentration and the rate of VLP assembly based on the microenvironment of Tyrosine-132. VLP formation was verified by off-line dynamic light scattering (DLS) and transmission electron microscopy (TEM). Furthermore, the experimental results provided evidence of aggregate-related assembly inhibition and showed that off-line size-exclusion chromatography does not provide a complete picture of the particle content. Finally, a Partial-Least Squares (PLS) model was calibrated to predict VLP concentrations in the process solution. values of 0.947–0.984 were reached for the three HBcAg variants. In summary, the proposed experimental setup provides a powerful platform for developing and monitoring VLP reassembly steps by CFF. 相似文献
72.
Hernandez-Pigeon H Jean C Charruyer A Haure MJ Titeux M Tonasso L Quillet-Mary A Baudouin C Charveron M Laurent G 《The Journal of biological chemistry》2006,281(19):13525-13532
Ultraviolet (UV) radiation from the sun is widely considered as a major cause of human skin photoaging and skin cancer. Granzyme B (GrB) and perforin (PFN) are two proteins contained in granules and implicated in one of the mechanisms by which cytotoxic lymphocytes and natural killer cells exert their cytotoxicity against virus-infected, alloreactive, or transformed cells. The distribution of GrB and PFN in the skin has received little attention. However, Berthou and co-workers (Berthou, C., Michel, L., Soulie, A., Jean-Louis, F., Flageul, B., Dubertret, L., Sigaux, F., Zhang, Y., and Sasportes, M. (1997) J. Immunol. 159, 5293-5300) described that, whereas freshly isolated epidermal cells did not express GrB or PFN, keratinocyte growth to confluence was associated with GrB and PFN mRNA and protein synthesis. In this work, we have investigated the possible role of UV-B on GrB and PFN expression in keratinocytes. We found that UV-B induces GrB and PFN expression in these cells through redox-, epidermal growth factor receptor-, and mitogen-activated protein kinase-dependent signaling. Furthermore, under UV irradiation, keratinocytes acquire a significant cytotoxicity, which is GrB and PFN dependent, toward a variety of cellular targets including transformed T-lymphocytes, melanocytes, and keratinocytes. This phenomenon may have important functional consequences in the regulation of skin inflammatory response and in the emergence of cancer skin. 相似文献
73.
Schieb H Kratzin H Jahn O Möbius W Rabe S Staufenbiel M Wiltfang J Klafki HW 《The Journal of biological chemistry》2011,286(39):33747-33758
In this study, we report a detailed analysis of the different variants of amyloid-β (Aβ) peptides in the brains and the cerebrospinal fluid from APP23 transgenic mice, expressing amyloid precursor protein with the Swedish familial Alzheimer disease mutation, at different ages. Using one- and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry, we identified the Aβ peptides Aβ(1-40), -(1-42), -(1-39), -(1-38), -(1-37), -(2-40), and -(3-40) as well as minor amounts of pyroglutamate-modified Aβ (Aβ(N3pE)) and endogenous murine Aβ in brains from 24-month-old mice. Chemical modifications of the N-terminal amino group of Aβ were identified that had clearly been introduced during standard experimental procedures. To address this issue, we additionally applied amyloid extraction in ultrapure water. Clear differences between APP23 mice and Alzheimer disease (AD) brain samples were observed in terms of the relative abundance of specific variants of Aβ peptides, such as Aβ(N3pE), Aβ(1-42), and N-terminally truncated Aβ(2/3-42). These differences to human AD amyloid were also noticed in a related mouse line transgenic for human wild type amyloid precursor protein. Taken together, our findings suggest different underlying molecular mechanisms driving the amyloid deposition in transgenic mice and AD patients. 相似文献
74.
Marco Amante Matthias Schöller Pompeo Suma Agatino Russo 《Entomologia Experimentalis et Applicata》2017,163(3):251-264
Bethylidae is a family belonging to the insect order Hymenoptera and contains about 2 200 described species. Bethylids typically parasitize larvae of Lepidoptera and Coleoptera, including species that are serious pests of stored products. Here, we review the main characteristics of each of the bethylids reported as biological control agent of these pests. The biological characteristics and peculiarities are reported for each species, and the potential for their practical application is discussed. 相似文献
75.
76.
Holzapfel E Eisner G Alami M Barrett CM Buchanan G Lüke I Betton JM Robinson C Palmer T Moser M Müller M 《Biochemistry》2007,46(10):2892-2898
Translocation of twin-arginine precursor proteins across the cytoplasmic membrane of Escherichia coli requires the three membrane proteins TatA, TatB, and TatC. TatC and TatB were shown to be involved in precursor binding. We have analyzed in vitro a number of single alanine substitutions in tatC that were previously shown to compromise in vivo the function of the Tat translocase. All tatC mutants that were defective in precursor translocation into cytoplasmic membrane vesicles concomitantly interfered with precursor binding not only to TatC but also to TatB. Hence structural changes of TatC that affect precursor targeting simultaneously abolish engagement of the twin-arginine signal sequence with TatB and block the formation of a functional Tat translocase. Since these phenotypes were observed for tatC mutations spread over the first half of TatC, this entire part of the molecule must globally be involved in precursor binding. 相似文献
77.
E. Kathryn Morris Francois Buscot Christine Herbst Torsten Meiners Elisabeth Obermaier Nicole W. Wäschke Tesfaye Wubet Matthias C. Rillig 《Biodiversity and Conservation》2013,22(10):2193-2205
Arbuscular mycorrhizal fungi (AMF) provide a number of ecosystem services as important members of the soil microbial community. Increasing evidence suggests AMF diversity is at least partially controlled by the identities of plants in the host plant neighborhood. However, much of this evidence comes from greenhouse studies or work in invaded systems dominated by single plant species, and has not been tested in species-rich grasslands. We worked in 67 grasslands spread across the three German Biodiversity Exploratories that are managed primarily as pastures and meadows, and collected data on AMF colonization, AMF richness, AMF community composition, plant diversity, and land use around focal Plantago lanceolata plants. We analyzed the data collected within each Exploratory (ALB Schwäbische Alb, HAI Hainich-Dün, SCH Schorfheide-Chorin) separately, and used variance partitioning to quantify the contribution of land use, host plant neighborhood, and spatial arrangement to the effect on AMF community composition. We performed canonical correspondence analysis to quantify the effect of each factor independently by removing the variation explained by the other factors. AMF colonization declined with increasing land use intensity (LUI) along with concurrent increases in non-AMF, suggesting that the ability of AMF to provide protection from pathogens declined under high LUI. In ALB and HAI mowing frequency and percent cover of additional P. lanceolata in the host plant neighborhood were important for AMF community composition. The similar proportional contribution of land use and host neighborhood to AMF community composition in a focal plant rhizosphere suggests that the diversity of this important group of soil microbes is similarly sensitive to changes at large and small scales. 相似文献
78.
Maurer C Panahandeh S Jungkamp AC Moser M Müller M 《Molecular biology of the cell》2010,21(23):4151-4161
Twin-arginine-containing signal sequences mediate the transmembrane transport of folded proteins. The cognate twin-arginine translocation (Tat) machinery of Escherichia coli consists of the membrane proteins TatA, TatB, and TatC. Whereas Tat signal peptides are recognized by TatB and TatC, little is known about molecular contacts of the mature, folded part of Tat precursor proteins. We have placed a photo-cross-linker into Tat substrates at sites predicted to be either surface-exposed or hidden in the core of the folded proteins. On targeting of these variants to the Tat machinery of membrane vesicles, all surface-exposed sites were found in close proximity to TatB. Correspondingly, incorporation of the cross-linker into TatB revealed multiple precursor-binding sites in the predicted transmembrane and amphipathic helices of TatB. Large adducts indicative of TatB oligomers contacting one precursor molecule were also obtained. Cross-linking of Tat substrates to TatB required an intact twin-arginine signal peptide and disappeared upon transmembrane translocation. Our collective data are consistent with TatB forming an oligomeric binding site that transiently accommodates folded Tat precursors. 相似文献
79.
80.
Vielhauer O Zakhartsev M Horn T Takors R Reuss M 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(32):3859-3870
In the field of metabolomics, GC-MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or extracellular metabolites is nowadays mostly accomplished by application of diverse LC-MS techniques. Only few groups have so far adopted GC-MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive. Here we present an improved and simplified gas chromatography-isotope dilution mass spectrometry (GC-IDMS) protocol for the absolute determination of intra- and extracellular metabolite levels. Commercially available (13)C-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed. 相似文献