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Stefan N Martin-Killias P Wyss-Stoeckle S Honegger A Zangemeister-Wittke U Plückthun A 《Journal of molecular biology》2011,413(4):826-843
Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (Kd = 68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting. 相似文献
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Wagner S Kufleitner J Zensi A Dadparvar M Wien S Bungert J Vogel T Worek F Kreuter J von Briesen H 《PloS one》2010,5(12):e14213
Background
Due to the use of organophosphates (OP) as pesticides and the availability of OP-type nerve agents, an effective medical treatment for OP poisonings is still a challenging problem. The acute toxicity of an OP poisoning is mainly due to the inhibition of acetylcholinesterase (AChE) in the peripheral and central nervous systems (CNS). This results in an increase in the synaptic concentration of the neurotransmitter acetylcholine, overstimulation of cholinergic receptors and disorder of numerous body functions up to death. The standard treatment of OP poisoning includes a combination of a muscarinic antagonist and an AChE reactivator (oxime). However, these oximes can not cross the blood-brain barrier (BBB) sufficiently. Therefore, new strategies are needed to transport oximes over the BBB.Methodology/Principal Findings
In this study, we combined different oximes (obidoxime dichloride and two different HI 6 salts, HI 6 dichloride monohydrate and HI 6 dimethanesulfonate) with human serum albumin nanoparticles and could show an oxime transport over an in vitro BBB model. In general, the nanoparticulate transported oximes achieved a better reactivation of OP-inhibited AChE than free oximes.Conclusions/Significance
With these nanoparticles, for the first time, a tool exists that could enable a transport of oximes over the BBB. This is very important for survival after severe OP intoxication. Therefore, these nanoparticulate formulations are promising formulations for the treatment of the peripheral and the CNS after OP poisoning. 相似文献5.
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
6.
Matthias Weiling Christian Lechtenfeld Felix Pfeiffer Lars Frankenstein Diddo Diddens Jian-Fen Wang Sascha Nowak Masoud Baghernejad 《Liver Transplantation》2024,14(5):2303568
The implementation of silicon (Si)-containing negative electrodes is widely discussed as an approach to increase the specific capacity of lithium-ion batteries. However, challenges caused by severe volume changes and continuous (re-)formation of the solid-electrolyte interphase (SEI) on Si need to be overcome. The volume changes lead to electrolyte consumption and active lithium loss, decaying the cell performance and cycle life. Herein, the additive 2-sulfobenzoic acid anhydride (2-SBA) is utilized as an SEI-forming electrolyte additive for SiOx-containing anodes. The addition of 2-SBA to a state-of-the-art carbonate-based electrolyte in high-voltage LiNi0.8Mn0.1Co0.1O2, NMC811||artificial graphite +20% SiOx pouch cells leads to improved electrochemical performance, resulting in a doubled cell cycle life. The origin of the enhanced cell performance is mechanistically investigated by developing an advanced experimental technique based on operando attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy. The operando ATR-FTIR spectroscopy results elucidate the degradation mechanism via anhydride ring-opening reactions after electrochemical reduction on the anode surface. Additionally, ion chromatography conductivity detection mass spectrometry, scanning electron microscopy, energy dispersive X-ray analysis, and quantum chemistry calculations are employed to further elucidate the working mechanisms of the additive and its degradation products. 相似文献
7.
Several studies demonstrated that visual filtering mechanisms might underlie both conflict resolution of the Flanker conflict and the control of the Garner effect. However, it remains unclear whether the mechanisms involved in the processing of both effects depend on similar filter mechanisms, such that especially the Garner effect is able to modulate filtering needs in the Flanker conflict. In the present experiment twenty-four subjects participated in a combined Garner and Flanker task during two runs of functional magnetic resonance imaging (fMRI) recordings. Behavioral data showed a significant Flanker but no Garner effect. A run-wise analysis, however, revealed a Flanker effect in the Garner filtering condition in the first experimental run, while we found a Flanker effect in the Garner baseline condition in the second experimental run. The fMRI data revealed a fronto-parietal network involved in the processing of both types of effects. Flanker interference was associated with activity in the inferior frontal gyrus, the anterior cingulate cortex, the precuneus as well as the inferior (IPL) and superior parietal lobule (SPL). Garner interference was associated with activation in middle frontal and middle temporal gyrus, the lingual gyrus as well as the IPL and SPL. Interaction analyses between the Garner and the Flanker effect additionally revealed differences between the two experimental runs. In the first experimental run, activity specifically related to the interaction of effects was found in frontal and parietal regions, while in the second run we found activity in the hippocampus, the parahippocampal cortex and the basal ganglia. This shift in activity for the interaction effects might be associated with a task-related learning process to control filtering demands. Especially perceptual learning mechanisms might play a crucial role in the present Flanker and Garner task design and, therefore, increased performance in the second experimental run could be the reason for the lack of behavioral Garner interference on the level of the whole experiment. 相似文献
8.
Myxozoan parasitism in waterfowl 总被引:2,自引:0,他引:2
Bartholomew JL Atkinson SD Hallett SL Lowenstine LJ Garner MM Gardiner CH Rideout BA Keel MK Brown JD 《International journal for parasitology》2008,38(10):1199-1207
Myxozoans are spore-forming, metazoan parasites common in cold-blooded aquatic vertebrates, especially fishes, with alternate life cycle stages developing in invertebrates. We report nine cases of infection in free-flying native and captive exotic ducks (Anseriformes: Anatidae) from locations across the United States and describe the first myxozoan in birds, Myxidium anatidum n. sp. We found developmental stages and mature spores in the bile ducts of a Pekin duck (domesticated Anas platyrhynchos). Spores are lens-shaped in sutural view, slightly sigmoidal in valvular view, with two polar capsules, and each valve cell has 14-16 longitudinal surface ridges. Spore dimensions are 23.1 microm x 10.8 microm x 11.2 microm. Phylogenetic analysis of the ssrRNA gene revealed closest affinity with Myxidium species described from chelonids (tortoises). Our novel finding broadens the definition of the Myxozoa to include birds as hosts and has implications for understanding myxozoan evolution, and mechanisms of geographical and host range extension. The number of infection records indicates this is not an incidental occurrence, and the detection of such widely dispersed cases suggests more myxozoans in birds will be encountered with increased surveillance of these hosts for pathogens. 相似文献
9.
Susanne Siebentritt Enrico Avancini Marcus Br Jakob Bombsch Emilie Bourgeois Stephan Buecheler Romain Carron Celia Castro Sebastien Duguay Roberto Flix Evelyn Handick Dimitrios Hariskos Ville Havu Philip Jackson Hannu‐Pekka Komsa Thomas Kunze Maria Malitckaya Roberto Menozzi Milos Nesladek Nicoleta Nicoara Martti Puska Mohit Raghuwanshi Philippe Pareige Sascha Sadewasser Giovanna Sozzi Ayodhya Nath Tiwari Shigenori Ueda Arantxa Vilalta‐Clemente Thomas Paul Weiss Florian Werner Regan G. Wilks Wolfram Witte Max Hilaire Wolter 《Liver Transplantation》2020,10(8)
10.
Kimia Ekramzadeh Chantal Brämer Thore Frister Jörg Fohrer Andreas Kirschning Thomas Scheper Sascha Beutel 《Biotechnology progress》2020,36(2):e2935
The patchoulol synthase (PTS) from Pogostemon cablin is a versatile sesquiterpene synthase and produces more than 20 valuable sesquiterpenes by conversion of the natural substrate farnesyl pyrophosphate (FPP). PTS has the potential to be used as a biocatalyst for the production of valuable sesquiterpenes such as (−)-patchoulol. The objective of the present study is to develop an efficient biotransformation and to characterize the biocatalytic mechanism of the PTS in detail. For this purpose, soluble PTS was prepared using an optimized cultivation protocol and continuous downstream process with a purity of 98%. The PTS biotransformation was then optimized regarding buffer composition, pH-value, and temperature for biotransformation as well as functional and kinetic properties to improve productivity. For the bioconversion of FPP, the highest enzyme activity was reached with the 2-(N-morphlino)ethanesulfonic acid (MES) buffer containing 10% (v/v) glycerol and 10 mM MgCl2 at pH 6.4 and 34°C. The PTS showed an unusual substrate inhibition for sesquiterpene synthases indicating an intermediate sesquiterpene formed in the active center. Deuteration experiments were used to gain further insights into the biocatalytic mechanism described in literature. Thus it could be shown that a second substrate binding site must be responsible for substrate inhibition and that further protonation and deprotonation steps are involved in the reaction mechanism. 相似文献