The mutagenicity and DNA-damaging activity of cyclic aliphatic sulfuric acid esters.

The cyclic aliphatic sulfuric acid esters 1,2-ethylene sulfate (ESF), 1,3-propylene sulfate (PSF) and 1,3-butylene sulfate (BSF) have been tested for their mutagenic and DNA-damaging activity. Mutagenicity of the compounds was established with his-auxotrophic indicator strains of Salmonella typhimurium using the in vitro plate test and the host-mediated assay technique with mice as host animals. The DNA-damaging activity was tested in a repair test with Proteus mirabilis mutants defective in DNA repair.In the repair test with a set of P. mirabilis strains (PG713 hcr^?rec^?: PG273 hcr⁺rec⁺) PSF and BSF showed a preferential growth inhibition of the repair-defective strain suggesting DNA-damaging activity of these chemicals. No such activity was found for ESF using the same concentrations of 5 and 15 μmol/plate.All cyclic sulfates revert the tester strain TA1535 of S. typhimurium in vitro indicating their ability to induce base substitutions. Compared with the reference compounds dimethyl sulfate (DMS), diethyl sulfate (DES), 1,3-propane sulfone (PPS) and 1,4-butane sulfone (BTS) the mutagenic activity in the plate test can be described as follows: PPS > PSF > BSF > BTS > ESF > DES > DMS.Dose-response studies in the host-mediated assay with tester strain TA1950 of S. typhimurium as genetic indicator system revealed a linear dosedependency of mutagenic activity. For PPS and PSF the lowest effective dose (LED) has been established as 10 μmol/kg. The LED for BSF and BTS was 50 μmol/kg, DMS and DES were mutagenic in doses of 2500 μmol/kg, while ESF was only weakly mutagenic with a LED of 5000 μmol/kg.The dose-response studies in the host-mediated assay and the results obtained in the in vitro spot test demonstrate similarities in the mutagenic action of the cyclic sulfates PSF and BSF and the respective sulfones, while the stronger alkylating compound ESF was a weak mutagen both in vitro and in vivo.     

Transcription of the human microsomal epoxide hydrolase gene (EPHX1) is regulated by an HNF-4α/CAR/RXR/PSF complex

Microsomal epoxide hydrolase (mEH) is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes where they are involved in cholesterol excretion and metabolism, lipid digestion and regulating numerous signaling pathways. Previous studies have demonstrated the critical role of GATA-4 and a C/EBPα–NF/Y complex in the regulation of the mEH gene (EPHX1). In this study we show that HNF-4α and CAR/RXR also bind to the proximal promoter region and regulate EPHX1 expression. Bile acids, which inhibit the expression of HNF-4α also decrease the expression of EPHX1. Studies also established that the binding of HNF-4α was essential for the activation of EPHX1 activity by CAR suggesting the formation of a complex between these adjacent factors. The nature of this regulatory complex was further explored using a biotinylated oligonucleotide of this region in conjunction with BioMag beads and mass spectrometric analysis which demonstrated the presence of an additional inhibitory factor (PSF), confirmed by co-immunoprecipitation and ChIP analyses, which interacted with DNA-bound CAR/RXR/HNF-4α forming a 4-component regulatory complex.     

Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam

The lateral resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy is enhanced about 12% by applying annular‐shaped amplitude modulation to the radially polarized excitation beam. A focused annularly filtered radially polarized excitation beam provides a more condensed point spread function (PSF), which contributes to enhance effective STED resolution of CW STED microscopy. Theoretical analysis shows that the FWHM of the effective PSF on the detection plane is smaller than for conventional CW STED. Simulation shows the donut‐shaped PSF of the depletion beam and confocal optics suppress undesired PSF sidelobes. Imaging experiments agree with the simulated resolution improvement.      

Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research

A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39 μm along the lateral direction and 1.3 μm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94 K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90 K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790 nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800 nm spectral region.     

Determination of protein mobility in mitochondrial membranes of living cells

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F₁F₀ ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D = 0.7 μm²/s and 0.6 μm²/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F₁F₀ ATPase: 0.4 μm²/s and 0.0005 μm²/s; COX: 0.3 μm²/s and 0.007 μm²/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.     

RRM proteins interacting with the cap region of topoisomerase I

RNA recognition motif (RRM) domains bind both nucleic acids and proteins. Several proteins that contain two closely spaced RRM domains were previously found in protein complexes formed by the cap region of human topoisomerase I, a nuclear enzyme responsible for DNA relaxation or phosphorylation of SR splicing proteins. To obtain molecular insight into specific interactions between the RRM proteins and the cap region of topo I we examined their binary interactions using the yeast two-hybrid system. The interactions were established for hnRNP A1, p54(nrb) and SF2/ASF, but not for hnRNP L or HuR. To identify the amino acid pattern responsible for binding, experimental mutagenesis was employed and computational modelling of these processes was carried out. These studies revealed that two RRM domains and six residues of the consensus sequence are required for the binding to the cap region. On the basis of the above data, a structural model for the hnRNP A1-topoisomerase I complex was proposed. The main component of the hnRNP A1 binding site is a hydrophobic pocket on the beta-surface of the first RRM domain, similar to that described for Y14 protein interacting with Mago. We demonstrated that the interaction between RRM domains and the cap region was important for the kinase reaction catalyzed by topoisomerase I. Together with the previously described inhibitory effect of RRM domains of SF2/ASF on DNA cleavage, the above suggests that the binding of RRM proteins could regulate the activity of topoisomerase I.     

Full field spatially-variant image-based resolution modelling reconstruction for the HRRT

Accurate characterisation of the scanner's point spread function across the entire field of view (FOV) is crucial in order to account for spatially dependent factors that degrade the resolution of the reconstructed images. The HRRT users' community resolution modelling reconstruction software includes a shift-invariant resolution kernel, which leads to transaxially non-uniform resolution in the reconstructed images. Unlike previous work to date in this field, this work is the first to model the spatially variant resolution across the entire FOV of the HRRT, which is the highest resolution human brain PET scanner in the world. In this paper we developed a spatially variant image-based resolution modelling reconstruction dedicated to the HRRT, using an experimentally measured shift-variant resolution kernel. Previously, the system response was measured and characterised in detail across the entire FOV of the HRRT, using a printed point source array. The newly developed resolution modelling reconstruction was applied on measured phantom, as well as clinical data and was compared against the HRRT users' community resolution modelling reconstruction, which is currently in use. Results demonstrated improvements both in contrast and resolution recovery, particularly for regions close to the edges of the FOV, with almost uniform resolution recovery across the entire transverse FOV. In addition, because the newly measured resolution kernel is slightly broader with wider tails, compared to the deliberately conservative kernel employed in the HRRT users' community software, the reconstructed images appear to have not only improved contrast recovery (up to 20% for small regions), but also better noise characteristics.     

人PSF蛋白的原核表达及分析鉴定

PSF(polypyrimidine tract binding protein-associated splicing factor)蛋白是一种多功能的DNA/RNA结合蛋白,能够抑制原癌基因的表达,在人和小鼠中具有肿瘤抑制的作用.以人成纤维细胞总RNA为材料,利用PSF特异性引物通过RT-PCR的方法扩增得到PSF...     

Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.