Dielectric Properties and Characterisation of Titanium Dioxide Obtained by Different Chemistry Methods

We made comparison of titanium dioxide powders obtained from three syntheses including sol-gel and precipitation methods as well as using layered (tetramethyl)ammonium titanate as a source of TiO2. The obtained precursors were subjected to step annealing at elevated temperatures to transform into rutile form. The transformation was determined by Raman measurements in each case. The resulting products were characterised using Raman spectroscopy and dynamic light scattering. The main goal of the studies performed was to compare the temperature of the transformation in three titania precursors obtained by different methods of soft chemistry routes and to evaluate dielectric properties of rutile products by means of broadband dielectric spectroscopy. Different factors affecting the electrical properties of calcinated products were discussed. It was found that sol-gel synthesis provided rutile form after annealing at 850°C with the smallest particles size about 20 nm, the highest value of dielectric permittivity equal to 63.7, and loss tangent equal to 0.051 at MHz frequencies. The other powders transformed to rutile at higher temperature, that is, 900°C, exhibit lower value of dielectric permittivity and had a higher value of particles size. The correlation between the anatase-rutile transformation temperature and the size of annealed particles was proposed.This work was financially supported by the National Science Center (Poland) grant awarded by Decision no. DEC-2011/03/D/ST5/06074. The authors are grateful to Professor Adam Tracz from the Polish Academy of Science in Lodz for his help in performing SEM investigations

Molecular Mechanism of Mutant p53 Stabilization: The Role of HSP70 and MDM2

<div><p>Numerous p53 missense mutations possess gain-of-function activities. Studies in mouse models have demonstrated that the stabilization of p53 R172H (R175H in human) mutant protein, by currently unknown factors, is a prerequisite for its oncogenic gain-of-function phenotype such as tumour progression and metastasis. Here we show that MDM2-dependent ubiquitination and degradation of p53 R175H mutant protein in mouse embryonic fibroblasts is partially inhibited by increasing concentration of heat shock protein 70 (HSP70/HSPA1-A). These phenomena correlate well with the appearance of HSP70-dependent folding intermediates in the form of dynamic cytoplasmic spots containing aggregate-prone p53 R175H and several molecular chaperones. We propose that a transient but recurrent interaction with HSP70 may lead to an increase in mutant p53 protein half-life. In the presence of MDM2 these pseudoaggregates can form stable amyloid-like structures, which occasionally merge into an aggresome. Interestingly, formation of folding intermediates is not observed in the presence of HSC70/HSPA8, the dominant-negative K71S variant of HSP70 or HSP70 inhibitor. In cancer cells, where endogenous HSP70 levels are already elevated, mutant p53 protein forms nuclear aggregates without the addition of exogenous HSP70. Aggregates containing p53 are also visible under conditions where p53 is partially unfolded: 37°C for temperature-sensitive variant p53 V143A and 42°C for wild-type p53. Refolding kinetics of p53 indicate that HSP70 causes transient exposure of p53 aggregate-prone domain(s). We propose that formation of HSP70- and MDM2-dependent protein coaggregates in tumours with high levels of these two proteins could be one of the mechanisms by which mutant p53 is stabilized. Moreover, sequestration of p73 tumour suppressor protein by these nuclear aggregates may lead to gain-of-function phenotypes.</p> </div

Endogenous HSP70 protein levels in H1299 cells are sufficient for nuclear aggregation of p53R175H.

<p>(<b>A</b>) SK-BR-3, H1299 and MEF cells were seeded 24 hours prior to harvesting. Immunoblotting with HSP70-specific antibody revealed high levels of HSP70 in the cancer-derived SKBR3 and H1299 cell lines when compared to double knockout MEF (<i>Trp53^−/−, Mdm2^−/−</i>) cells. (<b>B</b>) H1299 cells were treated (or not) with a combination of two compounds –17-AAG (1 µM, HSP90 inhibitor) and VER155008 (10 µM, HSP70 inhibitor) – for 16 hours. Immunoblotting of cell lysates shows the accumulation of HSP70 protein upon treatment with 17-AAG and the decrease in p53 R175H protein level as a result of HSP70 inhibition by VER155008. (<b>C</b>) H1299 cells were transfected with plasmids encoding p53 R175H and optionally low or high amount of HSP70 and then treated with MG132 (2 µM) for 16 hours prior to fixation. Immunostaining with p53-specific antibody revealed that the endogenous HSP70 protein level is sufficient for induction of nuclear p53 R175H aggregate formation (left panel) and that an increase in HSP70 level results in appearance of cytoplasmic p53-containing speckles (middle and right panel). (<b>D</b>) Immunofluorescence of H1299 cells treated with 17-AAG further supports the notion that endogenous HSP70 shifts the equilibrium between various oligomeric states of mutated p53. The lower level of HSP70 (6 hours after 17-AAG treatment, middle panel) triggers p53 R175H nuclear aggregation, whereas the higher level of HSP70 (16 hours after 17-AAG treatment, right panel) causes the dissociation of the nuclear aggregates into smaller speckles and their relocalization to the cytoplasm. Scale bar 10 µm.</p

MDM2 and HSP70 colocalize with p53R175H in an aggressome.

<p>(<b>A</b>) Immunostaining of MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) transfected with plasmids encoding p53 R175H, HSP70 and optionally MDM2 (lower panel) shows that only in the presence of MDM2, A11 antibodies (labelling amyloid-oligomers) recognize aggregates composed of mutant p53 and HSP70. (<b>B</b>) Immunostaining of MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) transfected with plasmids encoding p53R175H, MDM2 and HSP70 revealed recruitment of both <b>(1)</b> the E3 ubiquitin ligase and <b>(2)</b> the chaperone to the large inclusion body. (<b>3)</b> The inclusion is formed at the centrosome marked by the presence of gamma-tubulin, suggesting that the large inclusion body containing p53 R175H is an aggresome. (<b>4</b>) Disruption of microtubules with nocodazole treatment impairs protein transport and results in the disappearance of the aggresome; smaller, scattered cytoplasmic p53-containing aggregates form instead. Scale bar 10 µm.</p

MDM2 and HSP70/HSPA family members change the localization and aggregation state of EYFP-p53 R175H in MEFs.

<p>(<b>A</b>) MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) were transfected with various combinations of plasmids encoding EYFP-p53 R175H, MDM2, HSC70, WT HSP70 and HSP70 K71S, as labelled above. Cells shown in 9–16 were treated with MG132 (2,5 µM) for 16 hours before imaging. Detailed description in text. Scale bar 10 µm. (<b>B</b>) MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>) were transfected with plasmid encoding EYFP-p53 R175H together with HSP70 and optionally MDM2 (lower panel). 24 hours post-transfection cells were treated with MG132 and scanned using Olympus ScanR Station widefield fluorescent microscope (time-lapse imaging) for additional 16 hours. 0 h represents 8 hours post-transfection, subsequent frames were acquired every 1 hour. The obtained images were analysed by ScanR Analysis software.</p

Aggregation propensity of p53 is conformation-dependent and promoted by HSP70.

<p>(<b>A</b>) Unfolding of p53 induces its nuclear aggregation<b>.</b> H1299 cells were transfected with plasmids encoding WT p53 (1, 3, 5) or p53 V143A (2, 4) and the next day incubated for 24 hours at the indicated temperature. In case of 42°C treatment, cells were incubated for 23 hours at 37°C and for 1 hour at 42°C. Small nuclear p53-containing aggregates can be seen in 4 and 5. Scale bar 10 µm. (<b>B</b>) HSP70 delays the disappearance of severely unfolded p53 temperature sensitive mutant V143A. Lysates from H1299 cells, transfected with plasmids encoding for indicated proteins, were subjected to p53 conformation-specific immunoprecipitation and analysed by western blotting. For WT conformation two exposures of the same blot are shown.</p

HSP70 regulates CHIP- and MDM2-dependent degradation of p53 R175H in opposite ways.

<p>Analysis of mutant p53 degradation was performed in double knockout MEF cells (<i>Trp53^−/−, Mdm2^−/−</i>). (<b>A</b>) CHIP-mediated degradation is accelerated by HSP70. (<b>B</b>) HSP70 WT partially inhibits MDM2-dependent p53 R175H degradation. ANOVA statistical test was carried out, where P values were counted for 3 independent experiments. (<b>C)</b> The steady-state level of p53 R175H is increased or decreased upon co-expression of WT or K71S HSP70, respectively. MEF cells were transfected with plasmids encoding p53 R175H and MDM2 together with increasing amounts of WT HSP70. Additionally, for the highest concentration of WT HSP70 titration of HSP70 K71S was applied. 24 hours later the cells were harvested and analysed by immunoblotting with p53 specific antibody (DO-1). The graph depicts the densitometric analysis performed using Image Quant software. Mean and standard deviation (s.d.) of two independent experiments are shown. (<b>D</b>) Overexpression of HSP70 downregulates the level of MDM2-dependent ubiquitination of p53 R175H<b>.</b> Cell based ubiquitination was carried out in MEF cells treated with MG132 (10 µM) for 4 hours. Mutant p53 protein was immunoprecipitated from the lysate using DO-1 antibody and analysed by western blotting using HA tag-specific antibody.</p