Photocycle features of heterologously expressed and assembled eukaryotic flavin-binding BLUF domains of photoactivated adenylyl cyclase (PAC), a blue-light receptor in Euglena gracilis

Photoactivated adenylyl cyclase (PAC) is a recently discovered blue-light photoreceptor that mediates photomovement in Euglena gracilis (Iseki et al., Nature, 2002, 415, 1047-1051). PAC appears to be a heterotetramer composed of two FAD-binding subunits (PACα and PACβ). Both subunits have a pair of homologous regions (F1 and F2) which show homology with prokaryotic "sensors of blue-light using FAD" (BLUF) domains. The F1 and F2 domains of PAC are the only eukaryotic BLUF domains found thus far. We obtained soluble recombinant F1 and F2 proteins in PACα by heterologous expression with fused glutathione-S-transferase (GST) in E. coli. The expressed F1 samples did not bind flavins, but the F2 samples contained both FAD and FMN with trace amounts of riboflavin. We also assembled the histidine-tagged recombinant F2 (6His-F2) from inclusion bodies in E. coli with exogenous FAD or FMN. Blue-light-induced changes in absorption spectra of these assembled samples were highly similar to those reported for prokaryotic BLUF domains. The FAD- or FMN-assembled 6His-F2 photocycled with nearly the same rate constants of light-reaction and dark-relaxation, which were slightly lower than those of GST-cleaved F2. The estimated quantum efficiency for the phototransformation was 0.28-0.32, and the half-life was 34-44 s at 25 °C for the recombinant PACα F2, whereas that reported for prokaryotic BLUF domains varied from ca. 3.5 s (Tl10078) to ca. 900 s (AppA). The mutated recombinant Y472F and Q514G of PACα F2 and the F2 domain of the PACα homologue from Eutreptiella gymnastica, which lacks the Gln residue conserved in other BLUF domains, showed no photoinduced transformation

UVA1 genotoxicity is mediated not by oxidative damage but by cyclobutane pyrimidine dimers in normal mouse skin

UVA1 induces the formation of 8-hydroxy-2′-deoxyguanosines (8-OH-dGs) and cyclobutane pyrimidine dimers (CPDs) in the cellular genome. However, the relative contribution of each type of damage to the in vivo genotoxicity of UVA1 has not been clarified. We irradiated living mouse skin with 364-nm UVA1 laser light and analyzed the DNA damage formation and mutation induction in the epidermis and dermis. Although dose-dependent increases were observed for both 8-OH-dG and CPD, the mutation induction in the skin was found to result specifically from the CPD formation, based on the induced mutation spectra in the skin genome: the dominance of C → T transition at a dipyrimidine site. Moreover, these UV-specific mutations occurred preferentially at the 5′-TCG-3′ sequence, suggesting that CpG methylation and photosensitization-mediated triplet energy transfer to thymine contribute to the CPD-mediated UVA1 genotoxicity. Thus, it is the CPD formation, not the oxidative stress, that effectively brings about the genotoxicity in normal skin after UVA1 exposure. We also found differences in the responses to the UVA1 genotoxicity between the epidermis and the dermis: the mutation induction after UVA1 irradiation was suppressed in the dermis at all levels of irradiance examined, whereas it leveled off from a certain high irradiance in the epidermis

Bcl-2 protein expression is associated with p27 and p53 protein expressions and MIB-1 counts in breast cancer

BACKGROUND: Recent experimental studies have shown that Bcl-2, which has been established as a key player in the control of apoptosis, plays a role in regulating the cell cycle and proliferation. The aim of this study was to investigate the relationship between Bcl-2 and p27 protein expression, p53 protein expression and the proliferation activity as defined by the MIB-1 counts. The prognostic implication of Bcl-2 protein expression in relation to p27 and p53 protein expressions and MIB-1 counts for breast cancer was also evaluated. METHODS: The immunohistochemical expression of Bcl-2 protein was evaluated in a series of 249 invasive ductal carcinomas of the breast, in which p27 and p53 protein expressions and MIB-1 counts had been determined previously. RESULTS: The Bcl-2 protein expression was found to be decreased in 105 (42%) cases. A decreased Bcl-2 protein expression was significantly correlated with a nuclear grade of III, a negative estrogen receptor, a decreased p27 protein expression, a positive p53 protein expression, positive MIB-1 counts and a positive HER2 protein expression. The incidence of a nuclear grade of III and positive MIB-1 counts increased as the number of abnormal findings of Bcl-2, p27 and p53 protein expressions increased. A univariate analysis indicated a decreased Bcl-2 protein expression to be significantly (p = 0.0089) associated with a worse disease free survival (DFS), while a multivariate analysis indicated the lymph node status and MIB-1 counts to be independently significant prognostic factors for the DFS. CONCLUSION: The Bcl-2 protein expression has a close correlation with p27 and p53 protein expressions and the proliferation activity determined by MIB-1 counts in invasive ductal carcinoma of the breast. The prognostic value of Bcl-2 as well as p27 and p53 protein expressions was dependent on the proliferation activity in breast cancer

Electronic Effects of Nitrogen Atoms of Supports on Pt-Ni Rhombic Dodecahedral Nanoframes for Oxygen Reduction

Pt-based nanostructures immobilized on carbon supports have been widely used as electrocatalysts. Their catalytic activity can be improved by support modification including nitrogen doping and coating with nitrogen-containing polymers, where nitrogen atoms possibly interact with surface Pt atoms at a catalyst/support interface. To understand electronic effects of nitrogen-doped and polymer-coated carbon supports on the catalytic activity of Pt-based nanostructured catalysts, we prepared Pt3Ni nanoframes (NFs) supported on polybenzimidazole (PBI)-coated and uncoated carbon nanotubes for the oxygen reduction reaction (ORR), and then compared their catalytic activities and electronic properties with those of NFs immobilized on nitrogen-doped and undoped carbon supports. Although both PBI-coating and nitrogen-doping approaches improved the catalytic activity of NFs, ex situ X-ray photoelectron spectroscopy and in situ X-ray absorption spectroscopy revealed that nitrogen doping showed electronic effects on NFs, whereas PBI-coating showed almost no impact on the electronic state of NFs but stabilized Pt(OH)ad species under electrochemical conditions. Our studies demonstrate that difference in microscopic environments of nitrogen atoms at the catalyst/support interface is highly sensitive to the electronic effects of supports on Pt-based electrocatalysts

Micro CT Analysis on the Correlation of a Cervical Defect and the Reparative Dentin in Human Permanent Teeth

The cervical defect (CD), the so-called wedge-shaped defect in Japan, or the non-carious cervical tooth lesion of human permanent teeth may be eroded by cross tooth-brushing with and without occlusal stress, or by attachment of dental clasp for a long time. Such a CD occasionally causes dentin hypersensitivity although the reparative dentin (RD) is deposited towards the dental pulp cavity. However, the correlation of a CD and the RD in volume has not been elucidated yet except for the difference of their areas on the ground sections reported previously. In this study, we analyzed the area and volume correlations of a CD and the RD in each teeth (n=10) by micro CT analysis. The area ratio (RD/CD) was 72.4±28.6 % (r=0.856, p<0.01) on the longitudinal cut plane showing the maximum areas of a CD and the RD, which were similar to those of the ground section assumed longitudinal central line of a tooth. On the other hand, the volume ratio (RD/CD) was 18.0±8.2 % (r=0.792, p<0.01). Therefore, it is clearly illustrated by micro CT analysis that the volume ratio (RD/CD) should be extremely lower than the area ratio in the longitudinal X-ray slices and also the ground sections previously reported. As the clinical consideration, the RD formation will be alleviated dentin hypersensitivity. On the basic observations, it was accuracy illustrated that micro CT analysis was useful for the volume measurement of the RD as well as the CD

Differentiation of photocycle characteristics of flavin-binding BLUF domains of α-And β-subunits of photoactivated adenylyl cyclase of Euglena gracilis

Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PACα and PACβ), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PACαF2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PACβF2 domain, which like PACαF2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PACβF2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PACαF2 were 0.28-0.32 and 34-44 s. The remarkable differences between PACαF2 and PACβF2 may be related to the sensitivity of the photoactivation. In PACαF2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PACβF2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PACβF2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PACβF2 plays a main role in suppressing the quantum efficiency