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Start Over Topic microscopy, fluorescence
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1. Determination of cellulase colocalization on cellulose fiber with quantitative FRET measured by acceptor photobleaching and spectrally unmixing fluorescence microscopy.

Author

Wang L, Wang Y, and Ragauskas AJ

Subjects
Animals, Cellulase chemistry, Cellulose chemistry, Fluorescence Resonance Energy Transfer instrumentation, Fluorescent Dyes chemistry, Fluorescent Dyes metabolism, Fungal Proteins chemistry, Fungal Proteins metabolism, Microscopy, Fluorescence instrumentation, Molecular Structure, Cellulase metabolism, Cellulose metabolism, Fluorescence Resonance Energy Transfer methods, Microscopy, Fluorescence methods, Photobleaching
Abstract

The determination of cellulase distribution on the surface of cellulose fiber is an important parameter to understand when determining the interaction between cellulase and cellulose and/or the cooperation of different types of cellulases during the enzymatic hydrolysis of cellulose. In this communication, a strategy is presented to quantitatively determine the cellulase colocalization using the fluorescence resonance energy transfer (FRET) methodology, which is based on acceptor photobleaching and spectrally unmixing fluorescence microscopy. FRET monitoring of cellulase colocalization was achieved by labeling cellulases with an appropriate pair of FRET dyes and by adopting an appropriate FRET model. We describe here that the adapted acceptor photobleaching FRET method can be successfully used to quantify cellulase colocalization regarding their binding to a cellulose fiber at a resolution <10 nm. This developed quantitative FRET method is promising for further studying the interactions between cellulase and cellulose and between different types of cellulases.

Published
2012
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2. Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and intracellular distribution

Author

Luigi Messori, Peter Schumacher, Pierluigi Orioli, Felix Kratz, Clemens Unger, Heinz H. Fiebig, Thomas Roth, D. H. Paper, Paula C.A. Rodrigues, Rolf Mülhaupt, and Ulrich Beyer

Subjects
Clinical Biochemistry, Pharmaceutical Science, Antineoplastic Agents, Polyethylene glycol, Biochemistry, Polyethylene Glycols, chemistry.chemical_compound, Drug Stability, Amide, Drug Discovery, PEG ratio, medicine, Tumor Cells, Cultured, Animals, Humans, Doxorubicin, Molecular Biology, Maleimide, Tumor Stem Cell Assay, Organic Chemistry, DNA, Hydrogen-Ion Concentration, chemistry, Doxorubicin Preparation, Microscopy, Fluorescence, Biophysics, Molecular Medicine, Cattle, Drug carrier, Ethylene glycol, medicine.drug
Abstract

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to alpha-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), alpha,omega-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or alpha-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02-1.5 microm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.

Published
2000