We have shown that both DNA topoisomerase (topo) IIalpha and beta are in vivo targets for etoposide using a new assay which directly measures topo IIalpha and beta cleavable complexes in individual cells after treatment with topo II targeting drugs. CCRF-CEM human leukemic cells were exposed to etoposide for 2 hr, then embedded in agarose on microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from drug-stabilized cleavable complexes remained associated with the DNA. The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the alpha or beta isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled slow scan charged coupled device camera was used to capture images. A dose-dependent increase in green immunofluorescence was observed when using antisera to either the alpha or beta isoforms of topo II, indicating that both isoforms are targets for etoposide. We have called this the TARDIS method, for trapped in agarose DNA immunostaining. Two key advantages of the TARDIS method are that it is isoform-specific and that it requires small numbers of cells, making it suitable for analysis of samples from patients being treated with topo II-targeting drugs. The isoform specificity will enable us to extend our understanding of the mechanism of interaction between topo II-targeting agents and their target, the two human isoforms.
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http://dx.doi.org/10.1124/mol.54.1.78 | DOI Listing |
Commun Biol
January 2025
Institute of biology, Plant Physiology Laboratory, Université de Neuchâtel, 2000, Neuchâtel, Switzerland.
Photosynthetic activity is established during chloroplast biogenesis. In this study we used 680 nm red light to overexcite Photosystem II and disrupt photosynthesis in two conditional mutants (var2 and abc1k1) which reversibly arrested chloroplast biogenesis. During biogenesis, chloroplasts import most proteins associated with photosynthesis.
View Article and Find Full Text PDFAdv Healthc Mater
January 2025
Max Bergmann Center of Biomaterials Dresden, Leibniz-Institut für Polymerforschung Dresden e. V., Hohe Str. 6, 01069, Dresden, Germany.
Cell-instructive polymer hydrogels are instrumental in tissue engineering for regenerative therapies. Implementing defined and selective responsiveness to external stimuli is a persisting challenge that critically restricts their functionality. Addressing this challenge, this study introduces a versatile, modular hydrogel system composed of four-arm poly(ethylene glycol)(starPEG)-peptide and glycosaminoglycan(GAG)-maleimide conjugates.
View Article and Find Full Text PDFAngew Chem Int Ed Engl
December 2024
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian, 350002, China.
Macrocycles represent one important class of functional molecules, and dynamic macrocycles with the potential of cleavability, adaptability, and topological conversion are challenging. Herein we report photoswitchable allosteric and topological control of dynamic covalent macrocycles and further the use in guest binding and mechanically interlocked molecules. The manipulation of competing ring-chain equilibria and bond formation/scission within reaction systems enabled light-induced structural regulation over dithioacetal and thioacetal dynamic bonds, accordingly realizing bidirectional switching between crown ether-like covalent macrocycles and their linear counterparts.
View Article and Find Full Text PDFCommun Chem
December 2024
Bioinformatics Research Group, University of Applied Sciences Upper Austria, Softwarepark 11, Hagenberg, 4232, Austria.
The field of crosslinking mass spectrometry has seen substantial advancements over the past decades, enabling the structural analysis of proteins and protein complexes and serving as a powerful tool in protein-protein interaction studies. However, data analysis of large non-cleavable crosslink studies is still a mostly unsolved problem due to its n-squared complexity. We here introduce an algorithm for the identification of non-cleavable crosslinks implemented in our crosslinking search engine MS Annika that is based on sparse matrix multiplication and allows for proteome-wide searches on commodity hardware.
View Article and Find Full Text PDFJ Colloid Interface Sci
December 2024
State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, College of Chemical Engineering, China University of Petroleum (East), 66 Changjiang West Road, Qingdao 266580, China.
Efficient intracellular delivery of native proteins remains a big challenge, which greatly hinders the development of protein therapy. Here, we report a generalizable peptide vector that can encapsulate and deliver various proteins to achieve efficient intracellular biocatalysis. The peptide was rationally designed to be cationic amphiphilic peptide that consist of four functional fragments, that is, a hydrophobic domain to promote molecular assembly, an enzyme-cleavable fragment to introduce stimuli-responsibility, several cationic arginine (Arg) residues to enhance cell interaction and transmembrane efficiency, and the cystine (Cys) residues with redox sensitivity to adjust the stability of the peptide/protein complexes as needed.
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