RESUMEN
Bioreduction of spin labels and polarizing agents (generally stable radicals) has been an obstacle limiting the in-cell applications of pulsed electron paramagnetic resonance (EPR) spectroscopy and dynamic nuclear polarization (DNP). In this work, we have demonstrated that two semiquinone methide radicals (OXQMâ and CTQMâ ) can be easily produced from the trityl-based quinone methides (OXQM and CTQM) via reduction by various reducing agents including biothiols and ascorbate under anaerobic conditions. Both radicals have relatively low pKa's and exhibit EPR single line signals at physiological pH. Moreover, the bioreduction of OXQM in three cell lysates enables quantitative generation of OXQMâ which was most likely mediated by flavoenzymes. Importantly, the resulting OXQMâ exhibited extremely high stability in the E.coli lysate under anaerobic conditions with 76- and 14.3-fold slower decay kinetics as compared to the trityl OX063 and a gem-diethyl pyrrolidine nitroxide, respectively. Intracellular delivery of OXQM into HeLa cells was also achieved by covalent conjugation with a cell-permeable peptide as evidenced by the stable intracellular EPR signal from the OXQMâ moiety. Owing to extremely high resistance of OXQMâ towards bioreduction, OXQM and its derivatives show great application potential in in-cell EPR and in-cell DNP studies for various cells which can endure short-term anoxic treatments.
Asunto(s)
Indolquinonas , Oxidación-Reducción , Humanos , Células HeLa , Espectroscopía de Resonancia por Spin del Electrón , Indolquinonas/química , Anaerobiosis , Benzoquinonas/química , Benzoquinonas/metabolismo , Compuestos de Tritilo/química , Escherichia coli/metabolismo , Marcadores de SpinRESUMEN
Electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) is a powerful tool in protein structural research. Nitroxides are highly suitable spin labeling reagents, but suffer from limited stability, particularly in the cellular environment. Herein we present the synthesis of a maleimide- and an azide-modified tetraethyl-shielded isoindoline-based nitroxide (M- and Az-TEIO) for labeling of cysteines or the noncanonical amino acid para-ethynyl-l-phenylalanine (pENF). We demonstrate the high stability of TEIO site-specifically attached to the protein thioredoxin (TRX) against reduction in prokaryotic and eukaryotic environments, and conduct double electron-electron resonance (DEER) measurements. We further generate a rotamer library for the new residue pENF-Az-TEIO that affords a distance distribution that is in agreement with the measured distribution.
Asunto(s)
Alquinos/química , Aminoácidos/química , Cisteína/química , Óxidos de Nitrógeno/química , Azidas/química , Espectroscopía de Resonancia por Spin del Electrón , Isoindoles/química , Marcadores de Spin , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMEN
The emergence and the dissemination of multidrug-resistant bacteria constitute a major public health issue. Among incriminated Gram-negative bacteria, Pseudomonas aeruginosa has been designated by the WHO as a critical priority threat. During the infection process, this pathogen secretes various virulence factors in order to adhere and colonize host tissues. Furthermore, P. aeruginosa has the capacity to establish biofilms that reinforce its virulence and intrinsic drug resistance. The regulation of biofilm and virulence factor production of this micro-organism is controlled by a specific bacterial communication system named Quorum Sensing (QS). The development of anti-virulence agents targeting QS that could attenuate P. aeruginosa pathogenicity without affecting its growth seems to be a promising new therapeutic strategy. This could prevent the selective pressure put on bacteria by the conventional antibiotics that cause their death and promote resistant strain survival. This review describes the QS-controlled pathogenicity of P. aeruginosa and its different specific QS molecular pathways, as well as the recent advances in the development of innovative QS-quenching anti-virulence agents to fight anti-bioresistance.
RESUMEN
El estudio de la biología y cerebro humano desde miradas complejas ha permitido una comprensión más amplia de las maneras en que funciona la máquina humana, a la vez se ha sistematizado el conocimiento alrededor de las formas en que puede ser afectado/manipulado por acciones del medio externo. De esta forma conocer el funcionamiento cerebral interno, la biología y sus efectos comportamentales es un poder que ha venido surgiendo ya bien para producir técnicas de dominación como el miedo o renovadas acciones de bioresistencias, todo mediado por nuevas formas de asumir el conocimiento desde las ciencias de la complejidad.
The study of biology and human brain from complex perspectives has allowed a broader understanding of the ways in which the human machine works, atthe same time knowledge has been systematized around the ways in which it can be affected / manipulated by environmental actions external. In this way knowing the internal brain functioning, biology and its behavioral effects is a powerthat has been emerging well to produce techniques of domination such as fear or renewed actions of bioresistencias, all mediated by new ways of taking knowledge from the sciences of complexity.
O estudo da biologia e do cérebro humano de perspectivas complexas permitiu uma compreensão mais ampla das maneiras como a máquina humana funciona, ao mesmo tempo em que o conhecimento foi sistematizado em torno das formas em que pode ser afetado / manipulado por ações ambientais externo Desta forma, conhecer o funcionamento do cérebro interno, a biologia e seus efeitos comportamentais é um poder que vem emergindo bem para produzir técnicas de dominação, como medo ou ações renovadas de bioresistencias, todas mediadas por novas formas de tirar conhecimento das ciências de complexidade.