Intracellular DNA Dotaz Zobrazit nápovědu
-- Plasmid Copy Number 39 -- Expression of Heterologous Proteins 40 -- Expression Modes 47 -- Intracellular DNA Probes Informative for Diagnosis and Identification 232 -- Introduction 232 -- Single Copy DNA Probes and Their Use 236 -- Methods for Labeling DNA Probes 248 -- Conclusion 251 -- References 252 -- Chapter Plasmid Stability of Recombinant DNA Microorganisms 316 -- Introduction 316 -- General Properties of Product Recovery and Purification: An Overview 357 -- Introduction 357 -- Intracellular versus Secreted
619 s.
- Klíčová slova
- DNA,
- MeSH
- rekombinantní DNA MeSH
DNA nanodevices have been developed as platforms for the manipulation of gene expression, delivery of molecular payloads, and detection of various molecular targets within cells and in other complex biological settings. Despite efforts to translate DNA nanodevices from the test tube (in vitro) to living cells, their intracellular trafficking and functionality remain poorly understood. Herein, quantitative and super-resolution microscopy approaches were employed to track and visualise, with nanometric resolution, the molecular interactions between a synthetic DNA nanosensor and transcription factors in intracellular compartments. Specifically, fluorescence resonance energy transfer microscopy, fluorescence correlation spectroscopy, fluorescence lifetime imaging microscopy and multicolour single-molecule localisation microscopy were employed to probe the specific binding of the DNA nanosensor to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). We monitored the mobility, subcellular localisation and degradation of the DNA nanosensor inside living prostate cancer PC3 cells. Super-resolution imaging enabled the direct visualisation of the molecular interactions between the synthetic DNA nanosensors and the NF-κB molecules in cells. This study represents a significant advance in the effective detection as well as understanding of the intracellular dynamics of DNA nanosensors in a complex biological milieu.
- MeSH
- DNA vazebné proteiny MeSH
- DNA MeSH
- lidé MeSH
- NF-kappa B * genetika metabolismus MeSH
- rezonanční přenos fluorescenční energie MeSH
- signální transdukce * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The DNA damage response factor 53BP1 functions at the intersection of two major double strand break (DSB) repair pathways--promoting nonhomologous end-joining (NHEJ) and inhibiting homology-directed repair (HDR)--and integrates cellular inputs to ensure their timely execution in the proper cellular contexts. Recent work has revealed that 53BP1 controls 5' end resection at DNA ends, mediates synapsis of DNA ends, promotes the mobility of damaged chromatin, improves DSB repair in heterochromatic regions, and contributes to lethal mis-repair of DSBs in BRCA1-deficient cells. Here we review these aspects of 53BP1 and discuss new data revealing how 53BP1 is loaded onto chromatin and uses its interacting factors Rif1 and PTIP to promote NHEJ and inhibit HDR.
Several studies have demonstrated that zinc is required for the optimal functioning of the skin. Changes in intracellular zinc concentrations have been associated with both improved protection of skin cells against various noxious factors as well as with increased susceptibility to external stress. Still, little is known about the role of intracellular zinc in hexavalent chromium (Cr(VI))-induced skin injury. To address this question, the effects of zinc deficiency or supplementation on Cr(VI)-induced cytotoxicity, oxidative stress, DNA injury and cell death were investigated in human diploid dermal fibroblasts during 48 h. Zinc levels in fibroblasts were manipulated by pretreatment of cells with 100 microM ZnSO4 and 4 or 25 microM zinc chelator TPEN. Cr(VI) (50, 10 and 1 microM) was found to produce time- and dose-dependent cytotoxicity resulting in oxidative stress, suppression of antioxidant systems and activation of p53-dependent apoptosis which is reported for the first time in this model in relation to environmental Cr(VI). Increased intracellular zinc partially attenuated Cr(VI)-induced cytotoxicity, oxidative stress and apoptosis by enhancing cellular antioxidant systems while inhibiting Cr(VI)-dependent apoptosis by preventing the activation of caspase-3. Decreased intracellular zinc enhanced cytotoxic effects of all the tested Cr(VI) concentrations, leading to rapid loss of cell membrane integrity and nuclear dispersion--hallmarks of necrosis. These new findings suggest that Cr(VI) as a model environmental toxin may damage in deeper regions residing skin fibroblasts whose susceptibility to such toxin depends among others on their intracellular Zn levels. Further investigation of the impact of Zn status on skin cells as well as any other cell populations exposed to Cr(VI) or other heavy metals is warranted.
- MeSH
- antioxidancia metabolismus MeSH
- apoptóza účinky záření MeSH
- buněčná membrána účinky léků MeSH
- chrom farmakologie MeSH
- DNA biosyntéza MeSH
- fibroblasty cytologie účinky léků MeSH
- financování organizované MeSH
- glutathionperoxidasa metabolismus MeSH
- glutathionreduktasa metabolismus MeSH
- kaspasa 3 MeSH
- kaspasy metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- metalothionein metabolismus MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- oxidační stres účinky záření MeSH
- poškození DNA genetika účinky léků MeSH
- proliferace buněk účinky léků MeSH
- škára cytologie účinky léků MeSH
- viabilita buněk účinky léků MeSH
- zinek metabolismus MeSH
- Check Tag
- lidé MeSH
The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.
- MeSH
- DNA metabolismus MeSH
- endonukleasy chemie genetika metabolismus MeSH
- enzymy opravy DNA chemie genetika metabolismus MeSH
- intracelulární signální peptidy a proteiny fyziologie MeSH
- lysin metabolismus MeSH
- mutace MeSH
- oprava DNA * MeSH
- poškození DNA MeSH
- protein-serin-threoninkinasy fyziologie MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus fyziologie MeSH
- sumoylace * MeSH
- ubikvitinligasy fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Efficient delivery of stabilized nucleic acids (NAs) into cells and release of the NA payload are crucial points in the transfection process. Here we report on the fabrication of a nanoscopic cellular delivery carrier that is additionally combined with a label-free intracellular sensor device, based on biocompatible fluorescent nanodiamond particles. The sensing function is engineered into nanodiamonds by using nitrogen-vacancy color centers, providing stable non-blinking luminescence. The device is used for monitoring NA transfection and the payload release in cells. The unpacking of NAs from a poly(ethyleneimine)-terminated nanodiamond surface is monitored using the color shift of nitrogen-vacancy centers in the diamond, which serve as a nanoscopic electric charge sensor. The proposed device innovates the strategies for NA imaging and delivery, by providing detection of the intracellular release of non-labeled NAs without affecting cellular processing of the NAs. Our system highlights the potential of nanodiamonds to act not merely as labels but also as non-toxic and non-photobleachable fluorescent biosensors reporting complex molecular events.
- MeSH
- buňky HT-29 MeSH
- DNA * MeSH
- lidé MeSH
- luminiscence MeSH
- myši inbrední DBA MeSH
- nanodiamanty * MeSH
- transfekce * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
This work is focused on the function of the microtubule and actin networks in plasmid DNA transport during liposomal transfection. We observed strong binding of plasmid DNA-lipid complexes (lipoplexes) to both networks and directional long-range motion of these lipoplexes along the microtubules. Disruption of either of these networks led to the cessation of plasmid transport to the nucleus, a decreased mobility of plasmids, and accumulation of plasmid DNA in large aggregates at the cell periphery. Our findings show an indispensable but different role of both types of cytoskeleton, actin and microtubular, in the processes of gene delivery.
- MeSH
- aktiny fyziologie metabolismus MeSH
- bicyklické sloučeniny heterocyklické farmakologie MeSH
- biologický transport účinky léků MeSH
- cytochalasin D farmakologie MeSH
- DNA genetika metabolismus MeSH
- fibroblasty cytologie metabolismus účinky léků MeSH
- financování organizované MeSH
- imunoblotting MeSH
- kultivované buňky MeSH
- lidé MeSH
- mikrotubuly fyziologie metabolismus MeSH
- plazmidy genetika metabolismus MeSH
- rekombinantní fúzní proteiny genetika metabolismus MeSH
- thiazolidiny farmakologie MeSH
- transfekce MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
