Evidence for nuclear internalisation of biocompatible [60]fullerene1)

Huang, Feiran; Mackeyev, Yuri; Watson, Erin; Cheney, Matthew; Wilson, Lon J.; Suh, Junghae

doi:10.1515/nano.0012.00039

Cited by 2 publications

(2 citation statements)

References 12 publications

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“…While the ability of light chains to localise to perinuclear compartments have been noted [ 22 ], to the best of our knowledge a demonstration of intranuclear localisation of light chain variable domain has not previously been documented by such methods presented here. Similar staining patterns have however, been documented previously for other fluorescently tagged proteins and small molecules [ 42 , 43 ].…”

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confidence: 85%

Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli

2018

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Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is amyloidogenic in vivo, but LEN adopts a stable dimer, displaying amyloidogenic properties only under destabilising conditions in vitro. We present here a refined and reproducible periplasmic expression and purification protocol for SMA and LEN that improves on existing methods and provides high yields of pure protein (10-50mg/L), particularly suitable for structural studies that demand highly concentrated and purified proteins. We confirm that recombinant SMA and LEN proteins have structure and dimerization capabilities consistent with the native proteins and employ fluorescence to probe internalization and cellular localization within cardiomyocytes. We propose periplasmic expression and simplified chromatographic steps outlined here as an optimized method for production of these and other variable light chain domains to investigate the underlying mechanisms of light chain amyloidosis. We show that SMA and LEN can be internalised within cardiomyocytes and were observed to localise to the perinuclear area, assessed by confocal microscopy as a possible mechanism for underlying cytotoxicity and pathogenesis associated with amyloidosis.

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Section: Resultssupporting

confidence: 85%

Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli

2018

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“…For this study, we created a water soluble, traceable malonodi serinolamide-derivatized C 60 by covalently attaching C 60 -ser to the fluorophore PromoFluor-633 (PF), with excitation/emission above 600 nm, to produce C 60 -serPF particles, which are only few nm in size (MW circa 2.8 kDa) [20]. To create larger NPs with similar surface chemistry, C 60 -ser and C 60 -serPF were conjugated to the surface of 200 nm SiO 2 NPs (Fig.…”

Section: Resultsmentioning

confidence: 99%

The influence of cell and nanoparticle properties on heating and cell death in a radiofrequency field

et al. 2017

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The use of non-invasive radiofrequency (RF) energy to induce mild thermal and non-thermal effects in cancer tissue is under study as an adjuvant to chemo, radio or immuno therapy. This study examines cell specific sensitivities to RF exposure and the potential of nanoparticles to elevate heating rates or enhance biological effects. Increases in the heating rate of water in an RF field operating at 13.56 MHz (0.004–0.028 °C/s) were positively correlated with concentration of hybrid nanoparticles (1–10 mg/ml) consisting of water soluble malonodiserinolamide [60]fullerene (C60-ser) conjugated to the surface of mesoporous silica nanoparticles (SiO2-C60). The heating rate of highly conductive cell culture media (0.024 °C/s) was similar to that of the highest concentration of nanoparticles in water, with no significant increase due to addition of nanoparticles at relevant doses (<100 μg/ml). With respect to cell viability, anionic (SiO2 and SiO2-C60) or neutral (C60) nanoparticles did not influence RF-induced cell death, however, cationic nanoparticles (4–100 μg/ml) caused dose-dependent increases in RF-induced cell death (24–42% compared to RF only). The effect of cell type, size and immortalization on sensitivity of cells to RF fields was examined in endothelial (HUVEC and HMVEC), fibroblast (primary dermal and L939) and cancer cells (HeLa and 4T1). While the state of cellular immortalization itself did not consistently influence the rate of RF-induced cell death compared to normal cell counter parts, cell size (ranging from 7 to 30 μm) negatively correlated with cell sensitivity to RF (21–97% cell death following 6 min irradiation). In summary, while nanoparticles do not alter the heating rate of biologically-relevant solutions, they can increase RF-induced cell death based on intrinsic cytotoxicity; and cells with smaller radii, and thereby greater surface membrane, are more susceptible to cell damage in an RF field than larger cells.

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Evidence for nuclear internalisation of biocompatible [60]fullerene1)

Cited by 2 publications

References 12 publications

Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli

Isolation and purification of recombinant immunoglobulin light chain variable domains from the periplasmic space of Escherichia coli

The influence of cell and nanoparticle properties on heating and cell death in a radiofrequency field

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