DNA topoisomerase II (topo II) changes DNA topology by cleavage/re-ligation cycle(s) and thus contributes to various nuclear DNA transactions. It is largely unknown how the enzyme is controlled in a nuclear context. Several studies have suggested that its C-terminal domain (CTD), which is dispensable for basal relaxation activity, has some regulatory influence. In this work, we examined the impact of nuclear localization on regulation of activity in nuclei. Specifically, human cells were transfected with wild-type and mutant topo IIβ tagged with EGFP. Activity attenuation experiments and nuclear localization data reveal that the endogenous activity of topo IIβ is correlated with its subnuclear distribution. The enzyme shuttles between an active form in the nucleoplasm and a quiescent form in the nucleolus in a dynamic equilibrium. Mechanistically, the process involves a tethering event with RNA. Isolated RNA inhibits the catalytic activity of topo IIβ in vitro through the interaction with a specific 50-residue region of the CTD (termed the CRD). Taken together, these results suggest that both the subnuclear distribution and activity regulation of topo IIβ are mediated by the interplay between cellular RNA and the CRD.

CENP-U (CENP-50) is a component of the CENP-O complex, which includes CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized at kinetochores throughout the cell cycle in vertebrates. Although CENP-U deficiency results in some mitotic defects in chicken DT40 cells, CENP-U-deficient chicken DT40 cells are viable. To examine the functional roles of CENP-U in an organism-dependent context, we generated CENP-U-deficient mice. The CENP-U-deficient mice died during early embryogenesis (approximately E7.5). Thus, conditional CENP-U-deficient mouse ES cells were generated to analyze CENP-U-deficient phenotypes at the cell level. When CENP-U was disrupted in the mouse ES cells, all CENP-O complex proteins disappeared from kinetochores. In contrast, other kinetochore proteins were recruited in CENP-U-deficient mouse ES cells as CENP-U-deficient DT40 cells. However, the CENP-U-deficient ES cells died after exhibiting abnormal mitotic behavior. Although CENP-U was essential for cell viability during mouse early embryogenesis, CENP-U-deficient mouse embryonic fibroblast cells were viable, similar to the DT40 cells. Thus, although both DT40 and ES cells with CENP-U deficiency have similar mitotic defects, cellular responses to mitotic defects vary among different cell types.Electronic supplementary materialThe online version of this article (doi:10.1007/s10577-014-9404-1) contains supplementary material, which is available to authorized users.

In mammalian cells, two isoforms of DNA topoisomerase II (topo IIalpha and topo IIbeta) have been identified. Topo IIalpha is essential in mitotic cells, whereas the function of topo IIbeta remains unclear. In the present study, we investigated the developmental control of topo II isoforms in two different neuronal lineages, cerebellar Purkinje cells and granule cells, by immunohistochemical analysis with isoform-specific monoclonal antibodies. As expected, proliferating cells in the neuroepithelium and in the external germinal layer (EGL) were topo IIalpha immunopositive. The migrating as well as differentiating Purkinje cells and granule cells showed an enhanced topo IIbeta immunoreactivity. The postmitotic granule cells in the postnatal EGL showed an abrupt transition of expressed topo II isoforms from IIalpha to IIbeta. The transition was clearly coincident with the completion of final cell division and the initiation of terminal differentiation because no increase of the topo IIbeta immunoreactivity was observed in the spreading EGL cells that are still in the cell division cycle. The topo IIbeta signal was detected in both nucleoplasm and nucleolus of differentiating cells. However, the nucleoplasmic signal decreased significantly as the cells reached terminal differentiation. The residual topo IIbeta in nucleoli was shown to occupy an unique location with respect to other nucleolar proteins, nucleolin and DNA topoisomerase I. Our findings indicate that both Purkinje cells and granule cells express the topo II isoforms in a similar timing during the cerebellar development and also suggest that topo IIbeta localized in nucleoplasm is the functional entity involved in neuronal differentiation.

The diffusion coefficient (D) of peptide and protein drugs needs to be determined to examine the permeability through biological barriers and to optimize delivery systems. In this study, the D values of fluorescein isothiocyanate (FITC)-labelled dextrans (FDs) and peptides were determined and the permeability through a porous membrane was discussed. The observed D values of FDs and peptides, except in the case of insulin, were similar to those calculated based on a relationship previously reported between the molecular weight and D of lower-molecular-weight compounds, although the molecular weight range was completely different. The observed D value of insulin was between the calculated values for the insulin monomer and hexamer. The permeability of poly-lysine and insulin through the membrane was determined and the observed values were compared with predicted values by using the relationship between molecular weight and D and an equation based on the Renkin function. The observed permeability of insulin through the membrane was between that of the predicted permeability for the insulin monomer and hexamer. For the permeation of insulin, the determination of D was useful for estimating the permeability because of the irregular relationship between molecular weight and D. The methodology used in this study will be useful for a more quantitative evaluation of the absorption of peptide and protein drugs applied to mucous membranes.

Lens epithelium-derived growth factor (LEDGF) or p75 is a co-activator of general transcription and also involved in insertion of human immunodeficiency virus type I (HIV-1) cDNA into host cell genome, which occurs preferentially to active transcription units. These phenomena may share an underlying molecular mechanism in common. We report here that LEDGF/p75 binds negatively supercoiled DNA selectively over unconstrained DNA. We identified a novel DNA-binding domain in the protein and termed it ‘supercoiled DNA-recognition domain’ (SRD). Recombinant protein fragments containing SRD showed a preferential binding to supercoiled DNA in vitro. SRD harbors a characteristic cluster of lysine and glutamic/aspartic acid residues. A polypeptide mimicking the cluster (K9E9K9) also showed this specificity, suggesting that the cluster is an essential element for the supercoil recognition. eGFP-tagged LEDGF/p75 expressed in the nucleus distributed partially in transcriptionally active regions that were identified by immunostaining of methylated histone H3 (H3K4me3) or incorporation of Br-UTP. This pattern of localization was observed with SRD alone but abolished if the protein lacked SRD. Thus, these results imply that LEDGF/p75 guides its binding partners, including HIV-1 integrase, to the active transcription site through recognition of negative supercoils generated around it.

We have developed a photoelectric dye-coupled polyethylene film as a prototype of retinal prosthesis, named "Okayama University-type retinal prosthesis. The purposes of this study are to conduct behavior tests to assess vision in Royal College of Surgeons (RCS) rats which underwent subretinal implantation of the dye-coupled film, and are also to reveal the retinal response to the dye-coupled film by immunohistochemistry.Polyethylene films were made of polyethylene powder at refined purity, and photoelectric dyes were coupled to the film surface at higher density, compared with the prototype. The dye-coupled film or the dye-uncoupled plain film, as a control, was implanted subretinally from a scleral incision in both eyes of a RCS rat at 6 weeks of the age. Behavior tests, 2, 4, 6, and 8 weeks after the implantation, were conducted by observing head-turning or body-turning in the direction, consistent with clockwise or anticlockwise rotation of a black-and-white-striped drum around a transparent cage, housed with a rat. After the behavior tests at 8 weeks, rats' eyes were enucleated to confirm the subretinal implantation of the films and were processed for immunohistochemistry. In the behavior tests, the number of head-turning, consistent with the direction of the drum rotation, was significantly larger in RCS rats with dye-coupled film implantation, compared with rats with plain film implantation (P<0.05, 3 repeated-measure ANOVA). The number of apoptotic neurons was significantly smaller in the eyes with the dye-coupled film implantation, compared to the eyes with the plain film (P<0.05, Mann-Whitney U-test). In conclusion, subretinal implantation of the photoelectric dye-coupled films restored the vision in RCS rats and prevented the remaining retinal neurons from apoptosis.

Photoelectric dye-coupled polyethylene film, designated Okayama University-type retinal prosthesis or OUReP™, generates light-evoked surface electric potentials and stimulates neurons. In this study, the vision was assessed by behavior tests in aged hereditary retinal dystrophic RCS rats with OUReP™, retinal apoptosis and electroretinographic responses were measured in dystrophic eyes with OUReP™. The dye-coupled films, or plain films as a control, were implanted in subretinal space of RCS rats. On behavior tests, RCS rats with dye-coupled films, implanted at the old age of 14 weeks, showed the larger number of head-turning, consistent with clockwise and anticlockwise rotation of a surrounding black-and-white-striped drum, compared with rats with plain films, under the dim (50 lux) and bright (150 lux) conditions in the observation period until the age of 22 weeks (n = 5, P < 0.05, repeated-measure ANOVA). The number of apoptotic cells in retinal sections at the site of dye-coupled film implantation was significantly smaller, compared with the other retinal sites, neighboring the film, or opposite to the film, 5 months after film implantation at the age of 6 weeks (P = 0.0021, Friedman test). The dystrophic eyes of RCS rats with dye-coupled films showed positive responses to maximal light stimulus at a significantly higher rate, compared with the eyes with no treatment (P < 0.05, Chi-square test). Electroretinograms in normal eyes of Wistar rats with dye-coupled or plain films showed significantly decreased amplitudes (n = 14, P < 0.05, repeated-measure ANOVA). In conclusions, vision was maintained in RCS rats with dye-coupled films implanted at the old age. The dystrophic eyes with dye-coupled films showed electroretinographic responses. Five-month film implantation caused no additional retinal changes.

Diffusion coefficients (D) of parabens and steroids in water and 1-octanol were determined by using the chromatographic broadening method at 37 degrees C, and the relationships between the D values and the physicochemical properties of the drugs were discussed. The D values in 1-octanol were lower than those in water because of the higher viscosity of 1-octanol. The D values depend on not only the molecular weight (MW), but also the lipophilicity of the drugs in water and on the ability for hydrogen-bonding in 1-octanol. When the lipophilic index (LI), calculated from the retention time using in a reverse-phase column, was used as a parameter of drug lipophilicity, the following equation was obtained for D in water (D(w)); log D(w)=-0.215.log MW-0.077.log LI-4.367. When the hydrogen bond index (HI), the logarithm of the ratio of the partition coefficient of the drugs in 1-octanol and cyclohexane, was used as an index of hydrogen-bonding, the following equation was obtained for D in 1-octanol (D(o)); log D(o)=-0.690.log MW-0.074.log HI-4.085.