Search

Showing total 6 results
Start Over Topic biophysics Publication Year Range Last 50 years
6 results

1. 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

2. Deciphering the transport of elastic filaments by antagonistic motor proteins

Author

J. C. Dallon, Cécile Leduc, Stéphanie Portet, Sandrine Etienne-Manneville, University of Manitoba [Winnipeg], Polarité cellulaire, Migration et Cancer - Cell Polarity, Migration and Cancer, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Brigham Young University (BYU), S.P. was partly supported by an National Sciences and Engineering Research Concil of Canada (NSERC) Discovery Grant. S.E.-M. and C.L. were supported by the Centre National de Recherche Scientifique, La Ligue Contre le Cancer, and the Institut Pasteur., The authors would like to thank the Isaac Newton Institute for Mathematical Sciences for support and hospitality during the program 'Coupling Geometric PDE's with Physics for Cell Morphology, Motility and Pattern Formation' when the work on this paper was undertaken. S.P. would like to thank Institut Pasteur for support of her stay. S.P. would like to thank P. van den Driessche for discussions on similar matrices., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ANR-16-CE13-0019,SiFi2Net,Filaments intermédiaires: du filament unique au réseau(2016)

Subjects
MESH: Biological Transport, Dynein, Intermediate Filaments, MESH: Molecular Motor Proteins, macromolecular substances, Models, Biological, 01 natural sciences, 010305 fluids & plasmas, Slow motion, Protein filament, Motor protein, 0103 physical sciences, Molecular motor, Elasticity (economics), 010306 general physics, Intermediate filament, Physics, MESH: Kinetics, Molecular Motor Proteins, MESH: Models, Biological, Biological Transport, Elasticity, Kinetics, MESH: Intermediate Filaments, Biophysics, MESH: Elasticity, Kinesin, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie
Abstract

International audience; Intermediate filaments are long elastic fibers that are transported by the microtubule-associated motor proteins kinesin and dynein inside the cell. How elastic filaments are efficiently transported by antagonistic motors is not well understood and is difficult to measure with current experimental techniques. Adapting the tug-of-war paradigm for vesiclelike cargos, we develop a mathematical model to describe the motion of an elastic filament punctually bound to antagonistic motors. As observed in cells, up to three modes of transport are obtained; dynein-driven retrograde, kinesin-driven anterograde fast motions, and a slow motion. Motor properties and initial conditions that depend on intracellular context regulate the transport of filaments. Filament elasticity is found to affect both the mode and the efficiency of transport. We further show that the coordination of motors along the filament emerges from the interplay between intracellular context and elastic properties of filaments.

Published
2019

3. Stochastic modeling reveals how motor protein and filament properties affect intermediate filament transport

Author

Sandrine Etienne-Manneville, Stéphanie Portet, Cécile Leduc, J. C. Dallon, Brigham Young University (BYU), Polarité cellulaire, Migration et Cancer - Cell Polarity, Migration and Cancer, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), University of Manitoba [Winnipeg], This work was supported by: EPSRC grant numbers EP/K032208/1 and EP/R014604/1 and by La Ligue contre le cancer and the Institut Pasteur., The authors would like to thank the Isaac Newton Institute for Mathematical Sciences for support and hospitality during the programme 'Coupling Geometric PDE’s with Physics for Cell Morphology, Motility and Pattern Formation' when the work on this paper was undertaken., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ANR-16-CE13-0019,SiFi2Net,Filaments intermédiaires: du filament unique au réseau(2016)

Subjects
0301 basic medicine, Statistics and Probability, Materials science, [SDV]Life Sciences [q-bio], Dynein, Biological Transport, Active, Kinesins, Catch bond, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Microtubules, Models, Biological, General Biochemistry, Genetics and Molecular Biology, Quantitative Biology::Cell Behavior, Protein filament, Motor protein, Quantitative Biology::Subcellular Processes, 03 medical and health sciences, 0302 clinical medicine, Molecular motor, Vimentin, Intermediate filaments, Intermediate filament, Cytoskeleton, Stochastic Processes, General Immunology and Microbiology, Applied Mathematics, Dyneins, Molecular motors, General Medicine, Kinesin, Stochastic, 030104 developmental biology, Modeling and Simulation, Biophysics, General Agricultural and Biological Sciences, 030217 neurology & neurosurgery
Abstract

International audience; Intermediate filaments are a key component of the cytoskeleton. Their transport along microtubules plays an essential role in the control of the shape and structural organization of cells. To identify the key parameters responsible for the control of intermediate filament transport, we generated a model of elastic filament transport by microtubule-associated dynein and kinesin. The model is also applicable to the transport of any elastically-coupled cargoes. We investigate the effect of filament properties such as number of motor binding sites, length, and elasticity on motion of filaments. Additionally, we consider the effect of motor properties, i.e. off rates, on filament transport. When one motor has a catch bond off rate it dictates the motion, whereas when motors have the same type of off rate filaments can alternate between retrograde and anterograde motions. The elasticity of filaments optimizes the filament transport and the coordination of motors along the length of the filament.

Published
2019

4. Crystal structures of a pentameric ion channel gated by alkaline pH show a widely open pore and identify a cavity for modulation

Author

Marc Delarue, Catherine Van Renterghem, Zaineb Fourati, Pierre-Jean Corringer, Ludovic Sauguet, Haidai Hu, Ákos Nemecz, Dynamique structurale des Macromolécules / Structural Dynamics of Macromolecules, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU), Récepteurs Canaux - Channel Receptors, Á.N. and Z.F. were supported by Agence Nationale de Recherches Grant 13-BSV8-0020 'Pentagate.' H.H. was sponsored by the China Scholarship Council and Institut Pasteur., We thank Anaïs Menny for sharing expertise on plasmid constructions, the staff of the Crystallography Platform (PF6) in the Institut Pasteur for initial crystal screens, Reinis Reinholds Ruza for help in data collection, Jérôme Loc’h for help during structural refinement and picture preparation, Stacy Gellenoncourt for help in purification and crystallization of the sTeLIC R86A mutant, the staff of synchrotron beamlines in Soleil (Orsay) and European Synchrotron Radiation Facility (Grenoble) for data collection, Pierre Legrand (Soleil synchrotron) for suggestions on refinement in Buster, and Marc Gielen for suggestions during the paper preparation, ANR-13-BSV8-0020,Pentagate,Mécanismes d'activation et de désensibilisation d'un récepteur-canal pentamérique(2013), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Gating, Crystallography, X-Ray, Divalent, 03 medical and health sciences, chemistry.chemical_compound, Allosteric Regulation, Bacterial Proteins, [CHIM.CRIS]Chemical Sciences/Cristallography, structural biology, Binding site, crystallography, Ion channel, Tetramethylammonium, chemistry.chemical_classification, Multidisciplinary, ligand-gated ion channel, druggable cavity, Hydrogen-Ion Concentration, Ligand-Gated Ion Channels, electrophysiology, Transmembrane protein, Quaternary Ammonium Compounds, 030104 developmental biology, chemistry, Structural biology, PNAS Plus, Biophysics, Ligand-gated ion channel, Gammaproteobacteria
Abstract

International audience; Pentameric ligand-gated ion channels (pLGICs) constitute a widespread class of ion channels, present in archaea, bacteria, and eukaryotes. Upon binding of their agonists in the extracellular domain, the transmembrane pore opens, allowing ions to go through, via a gating mechanism that can be modulated by a number of drugs. Even though high-resolution structural information on pLGICs has increased in a spectacular way in recent years, both in bacterial and in eukaryotic systems, the structure of the open channel conformation of some intensively studied receptors whose structures are known in a nonactive (closed) form, such as Erwinia chrysanthemi pLGIC (ELIC), is still lacking. Here we describe a gammaproteobacterial pLGIC from an endo-symbiont of Tevnia jerichonana (sTeLIC), whose sequence is closely related to the pLGIC from ELIC with 28% identity. We provide an X-ray crystallographic structure at 2.3 Å in an active conformation, where the pore is found to be more open than any current conformation found for pLGICs. In addition, two charged restriction rings are present in the vestibule. Functional characterization shows sTeLIC to be a cationic channel activated at alkaline pH. It is inhibited by divalent cations, but not by quaternary ammonium ions, such as tetramethylammonium. Additionally, we found that sTeLIC is allosterically potentiated by aromatic amino acids Phe and Trp, as well as their derivatives, such as 4-bromo-cinnamate, whose cocrystal structure reveals a vestibular binding site equivalent to, but more deeply buried than, the one already described for benzodiazepines in ELIC.

Published
2018

5. Synchrotron FTIR microspectroscopy of Escherichia coli at single-cell scale under silver-induced stress conditions

Author

Claire Saulou, Muriel Mercier-Bonin, Isabelle Fourquaux, Paul Dumas, Claude Maranges, Frédéric Jamme, Muriel Cocaign-Bousquet, Laurence Girbal, Génie et Microbiologie des Procédés Alimentaires (GMPA), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de la Recherche Agronomique (INRA), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT), Centre de Microscopie Électronique Appliquée à la Biologie (CMEAB), Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), The authors gratefully acknowledge the 'BioPleasure' project of the Research National Agency (ANR-07-BLAN-0196-01) for funding., The research described in this paper was performed at the French national synchrotron facility SOLEIL (Gif-sur- Yvette, France), using the SMIS beamline (proposal no 20090556)., ANR-07-BLAN-0196,BIOPLEASURE,Plasma - surface engineering for biofilm prevention(2007), AgroParisTech-Institut National de la Recherche Agronomique (INRA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées, Institut National des Sciences Appliquées (INSA), Hôpital de Rangueil, CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]-Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Hôpital de Rangueil, and CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]

Subjects
In situ, Analytical chemistry, medicine.disease_cause, Biochemistry, Analytical Chemistry, 03 medical and health sciences, Stress, Physiological, Spectroscopy, Fourier Transform Infrared, medicine, Escherichia coli, Fourier transform infrared spectroscopy, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein secondary structure, 030304 developmental biology, 0303 health sciences, biology, Ionic silver, 030306 microbiology, Chemistry, Synchrotron FTIR-ATR microspectroscopy, Single-cell scale, [CHIM.MATE]Chemical Sciences/Material chemistry, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Antibacterial effect, Transmission electron microscopy, Attenuated total reflection, Ultrastructure, Biophysics, Silver Nitrate, Single-Cell Analysis, [SPI.SIGNAL]Engineering Sciences [physics]/Signal and Image processing, Synchrotrons, Bacteria
Abstract

International audience; The present work was focused on elucidating biochemical changes in the model bacterium Escherichia coli exposed to ionic silver mediated stress, at a single-cell scale. In order to achieve this, in situ synchrotron Fourier-transform infrared (sFTIR) microspectroscopy was performed, for the first time, on individual cells by attenuated total reflectance (ATR) combined with the use of zinc-selenide hemisphere for high spatial resolution. In a first part, the potential of the method was evaluated on bacteria subjected to a lethal 100 μM AgNO3 concentration for 2 h compared to untreated 100 % viable cells. Differences in cell composition were assessed for the C–H stretching and protein spectral regions, indicating that the inhibitory action was targeted against both fatty acids and proteins. Transmission electron microscopy (TEM) confirmed morphological damages of the cell ultrastructure. The relevance of ATR-sFTIR microspectroscopy for highlighting the heterogeneity in Ag+-mediated effects within a given bacterial population was also pointed out. In a second part, cells were exposed to sub-lethal Ag+ concentrations (

Published
2013

6. Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168

Author

Samir Bejar, Kameleddine Bouchaala, Wacim Bejar, Mounira Ben Farhat, Ameny Farhat-Khemakhem, Radhouane Kammoun, Ines Boukhris, Emmanuelle Maguin, Hichem Chouayekh, Université de Sfax, MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Tunisian Government (Contrat Programme CBS-LMB, project of Valorization of Research Results 'Overproduction, formulation and assessment of the efficiency of a novel thermostable phytase as feed additive in poultry diets'), CMCU project 'Chouayekh/Maguin' (2007-2009) [07G0922], AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), This research was endorsed by the Tunisian Government (Contrat Programme CBS-LMB and the project of Valorization of Research Results 'Overproduction, formulation and assessment of the efficiency of a novel thermostable phytase as feed additive in poultry diets') as well as the CMCU project no 07G0922 'Chouayekh/Maguin' (2007-2009). The authors wish to express their sincere gratitude to Dr Thorsten Eggert, Mrs Rozenn Dervyn and Mr Amin Mrabet for their valuable collaboration. The authors would also like to thank Mr Maher Siala ELT supervisor and teacher of English at the Faculty of Sciences of Sfax for carefully proofreading the present paper., and Chouayekh, Hichem

Subjects
0106 biological sciences, Original, [SDV]Life Sciences [q-bio], Biophysics, Cloning vector, Bacillus subtilis, 01 natural sciences, Applied Microbiology and Biotechnology, Microbiology, law.invention, 03 medical and health sciences, Plasmid, law, 010608 biotechnology, Bacillus subtilis 168, Phytase, overexpression, multimeric DNA forms, experimental designs, thermostability, Yeast extract, 030304 developmental biology, Thermostability, 0303 health sciences, biology, biology.organism_classification, Biochemistry, Recombinant DNA, Heterologous expression
Abstract

To attempt cost-effective production of US417 phytase in Bacillus subtilis, we developed an efficient system for its large-scale production in the generally recognized as safe microorganism B. subtilis 168. Hence, the phy US417 corresponding gene was cloned in the pMSP3535 vector, and for the first time for a plasmid carrying the pAMβ1 replication origin, multimeric forms of the resulting plasmid were used to transform naturally competent B. subtilis 168 cells. Subsequently, a sequential optimization strategy based on Plackett-Burman and Box-Behnken experimental designs was applied to enhance phytase production by the recombinant Bacillus. The maximum phytase activity of 47 U ml-1 was reached in the presence of 12.5 g l-1 of yeast extract and 15 g l-1 of ammonium sulphate with shaking at 300 rpm. This is 73 fold higher than the activity produced by the native US417 strain before optimization. Characterization of the produced recombinant phytase has revealed that the enzyme exhibited improved thermostability compared to the wild type PHY US417 phytase strengthening its potential for application as feed supplement. Together, our findings strongly suggest that the strategy herein developed combining heterologous expression using a cloning vector carrying the pAMβ1 replication origin and experimental designs optimization can be generalized for recombinant proteins production in Bacillus.

Published
2012