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Manipulating the Intracellular Trafficking of Nucleic Acids
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
Kathleen E. B Meyer, Lisa S. Uyechi, Francis C. Szoka
Microinjection, cell permeabilization, and isolated nuclei have been used to study the properties of nucleocytoplasmic transport. Microinjection allows the introduction of material into either the cytoplasmic or nuclear compartments in a living cell. This has been a powerful technique to study diffusion of macromolecules through the cytoplasm, the role of compartmentalization on expression of plasmid DNA, and the factors that regulate macromolecular transport into the nucleus. Digitonin has been effectively employed to permabilize the cell plasma membrane and introduce the substrate and supplemental cytosolic factors to replace proteins which escape while the membranes are compromised (developed by Adam and Gerace; see Ref. 115). This is a common method used to study the requirements for import of proteins into the nucleus. Isolated or reconstituted nuclei have also been used for studying nuclear transport in vitro and permit stringent control of the transport environment. More recently, genetic manipulation via deletion and complementation have been employed to determine the necessity of specific nuclear pore proteins for viability, growth, and transport.
Carnitine-acylcarnitine translocase deficiency
Published in William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop, Atlas of Inherited Metabolic Diseases, 2020
William L. Nyhan, Georg F. Hoffmann, Aida I. Al-Aqeel, Bruce A. Barshop
The fundamental defect in CACT may be demonstrated in cultured fibroblasts [1]. In the first patient, activity was barely detectable at 0.8 percent of the normal mean. Prior incubation with digitonin, to increase permeability, indicated the fibroblast assay to be linear with time and protein and that, with this assay, the activity in the patient studied was zero [3]. In 12 patients, the activity was less than 1 percent of control in all but one [4]. In that patient, the one with the mild phenotype [16], activity ranged from 3 to 6.8 percent of normal. The enzyme can be assayed in fresh lymphocytes and in amniocytes [3, 4].
Cholesterol in the Walls of Odontogenic Cysts: A Histochemical and Ultrastructural Study
Published in Roger M. Browne, Investigative Pathology of the Odontogenic Cysts, 2019
J. R. Garrett, K. M. S. Winstone
Use of the perchloric acid naphthoquinone (PAN) method for demonstrating cholesterol15 showed positive staining in those sections that contained birefringent material under crossed polars. The amount of pigment formed corresponded to the amount of this birefringent material. Sections showing no birefringent lipid crystals gave no staining with the PAN method. Sections pretreated with digitonin16 which forms cholesterol digitonide with free cholesterol only, and then treated with acetone to wash out all other lipids still showed a positive PAN reaction, albeit usually reduced. It can be inferred from these results that cholesterol had been present in the free state and any decrease in staining after the digitonin treatment was attributable to some of the cholesterol being present in an esterified form.
Deletion of SDF-1 or CXCR4 regulates platelet activation linked to glucose metabolism and mitochondrial respiratory reserve
Published in Platelets, 2022
Yi Li, Ziqian Feng, Luochen Zhu, Ni Chen, Qin Wan, Jianbo Wu
For the experiment of permeabilized platelets, routine respiration was established. To change membrane permeability, digitonin was added. Then, state 2 respiratory rate was initiated by injection of glutamate (5 mM) and malate (5 mM). ADP (1 mM) was added to start OXPHOS through complex I (OXPHOS CI). Furthermore, succinate (10 mM), which is oxidized by complex II (CII), was added to achieve the simultaneous input of electrons through both complex I (CI) and CII (OXPHOS CI+CII). Next, oligomycin (2 μg/mL), ATP synthase inhibitor, was added to measure proton leakage over the mitochondrial membrane (LEAK CI+CII). Titration of FCCP (1uM each step) was applied to induce maximum uncoupled respiration of the ETS (ETS CI+CII). Inhibition of Complex I by rotenone (1 μM) revealed the ETS capacity supported by succinate through Complex II alone (ETS CII). Finally, addition of a complex III inhibitor antimycin-A (1 µg/mL) determined the ROX.
Unfolding the Role of Splicing Factors and RNA Debranching in AID Mediated Antibody Diversification
Published in International Reviews of Immunology, 2021
Ankit Jaiswal, Amit Kumar Singh, Anubhav Tamrakar, Prashant Kodgire
AID was also found physically in the regions of nucleus augmented with splicing factors. To illustrate the localization of AID in the subnuclear structures, high-resolution confocal microscopy was used [70]. Subsequently, it was found that AID was co-localized with fibrillarin in the exportin 1 inhibitor (blocks nuclear export) treated cells [70]. Fibrillarin is a nucleolar protein and is the marker for nucleoli. Similarly, AID and AID1-186 were also co-localized with fibrillarin in the digitonin treated cells. As digitonin is a membrane permeabilizer, if proteins are soluble then it can easily diffuse out of the nucleus and cytoplasm, whereas proteins that are structurally bounded, they cannot diffuse. Thus, AID and AID1-186 are indeed physically associated with nucleoli and subnuclear domain [70].
Diethyldithiocarbamate encapsulation reduces toxicity and promotes leishmanicidal effect through apoptosis-like mechanism in promastigote and ROS production by macrophage
Published in Journal of Drug Targeting, 2020
João Paulo Assolini, Fernanda Tomiotto-Pellissier, Bruna Taciane da Silva Bortoleti, Manoela Daiele Gonçalves, Claudia Stoeglehner Sahd, Amanda Cristina Machado Carloto, Paulo Emilio Feuser, Arthur Poester Cordeiro, Sergio Marques Borghi, Waldiceu Aparecido Verri, Claudia Sayer, Pedro Henrique Hermes de Araújo, Idessania Nazareth Costa, Ivete Conchon-Costa, Milena Menegazzo Miranda-Sapla, Wander Rogério Pavanelli
The determination of cell membrane integrity was performed according to Miranda-Sapla et al. [19]. Promastigotes forms of L. amazonensis (106 cells/mL) in 24-well plates were treated with IC50 (0.33 µM) of DETC for 24 h at 24 °C. The parasites were collected and washed with PBS and directly incubated with propidium iodide (PI) (Sigma, St. Louis, MO, USA) (0.50 µg/mL) for 5 min, according to the manufacturer’s instructions. Immediately thereafter, we analysed the parasites using a fluorescence microplate reader (Victor X3, PerkinElmer, Finland) at an excitation wavelength of 480 nm and emission of 580 nm. Digitonin (Sigma, St. Louis, MO, USA) (40.0 µM) was used as a positive control. We normalised the fluorescence values obtained to the total number of cells from the treatment.