Maiko Furuya

XPC is involved in genome maintenance through multiple pathways in different tissues.

In an attempt to evaluate the role of the Xpc gene in maintaining genomic stability in vivo under normal conditions, the age-dependent accumulation of spontaneous mutations in different tissues was analyzed in Xpc-deficient lacZ-transgenic mice. Brain, testis, and small intestine revealed no effects from the Xpc-deficiency, whereas liver, spleen, heart, and lung showed an enhanced age-related accumulation of mutations in Xpc-deficient mice. In the spleen, the effect was not obvious at 2 and 12 months of age, but became apparent at 23 months. The magnitude of the observed effect at an advanced age was similar in the liver, spleen and heart, but was comparatively smaller in the lung. Haploinsufficiency was observed in liver and spleen but not in heart and lung. Analysis of DNA sequences in the mutants revealed that the frequency of G:C to T:A changes were elevated in the liver and heart of Xpc-deficient aged mice, supporting the possible involvement of XPC in base excision repair of oxidized guanine. The occurrence of two or more mutations within a single lacZ gene was termed a multiple mutation and was also elevated in old Xpc-deficient mice. Among the clones examined, two mutant clones showed as many as four mutations within a short stretch of DNA. This is the first demonstration to support suggestions for the existence of a role for XPC in the suppression of multiple mutations. These multiple mutations could conceivably be generated by error-prone trans-lesional DNA synthesis. Overall, these results indicate that there may be diverse roles or mechanisms through which XPC participates in genome maintenance in different tissues.

Role of the Msh2 gene in genome maintenance and development in mouse fetuses.

In an attempt to evaluate the roles of the mismatch repair gene Msh2 in genome maintenance and in development during the fetal stage, spontaneous mutations and several developmental indices were studied in Msh2-deficient lacZ-transgenic mouse fetuses. Mutation levels in fetuses were elevated at 9.5 dpc (days post coitum) when compared to wild-type mice, and the level of mutations continued to increase until the fetuses reached the newborn stage. The mutation levels in 4 different tissues of newborns showed similar magnitudes to those in the whole body. The levels remained similar after birth until 6 months of age. The molecular nature of the mutations examined in 12.5 dpc fetuses of Msh2(+/+) and Msh2(-/-) revealed unique spectra which reflect errors produced during the DNA replication process, and those corrected by a mismatch repair system. Most base substitutions and simple deletions were reduced by the presence of the Msh2 gene, whereas G:C to A:T changes at CpG sequences were not affected, suggesting that the latter change was not influenced by mismatch repair. On the other hand, analysis of developmental indices revealed that there was very little effect, including the presence of malformations, resulting from Msh2-deficiencies. These results indicate that elevated mutation levels have little effect on the development of the fetus, even if a mutator phenotype appears at the organogenesis stage.

Formation of bioactive N-doped TiO2 on Ti with visible light-induced antibacterial activity using NaOH, hot water, and subsequent ammonia atmospheric heat treatment.

Titanium (Ti) treated with NaOH and hot water, and heated in an ammmonia (NH3) gas atmosphere for 1 or 3h exhibited in vitro apatite formation within 7days when soaked in simulated body fluid (SBF). Moreover, the treated Ti decomposed methylene blue and showed excellent bactericidal activity against Escherichia coli under visible light irradiation. The surface treatment resulted in the formation of a fine network of N-doped anatase-type titania (TiO2-xNx) on the Ti surface, which was responsible for both the apatite formation in SBF and the visible light-induced antibacterial activity. These preliminary results highlight the efficacy of our simple method for producing novel bioactive Ti with visible light-induced antibacterial activity, which could be applied to orthopaedic and dental implants without the risk of infection.

Control of HAp Formation and Osteoconductivity on Nitrogen-Doped TiO 2 Scale Formed by Oxynitridation of Ti

The surface potential of the TiO2 scale formed on Ti was controlled by varying the Ti heat treatment conditions in a N2 atmosphere containing a trace amount of O2. The surface potential was attributed to the effective charge of nitrogen-related defects in the TiO2, where the positive and negative surface charges were associated with (N2)0+2 and (NO)0-1, respectively. The latter defects were formed only during the early stages of the heat treatment, and with increasing treatment time, this was followed by the formation of voids containing N2 in the scale rather than the disappearance of the defects in the TiO2 crystal lattice, resulting in zero surface charge. Hydroxyapatite (HAp) formation and osteoconductivity were enhanced on nitrogen-doped TiO2 scale with either a positive or negative surface potential. In contrast, for the unchanged TiO2 scale, no HAp formation was observed and the osteoconductivity was low.

TiO2 microspheres containing magnetic nanoparticles for intra‐arterial hyperthermia

Magnetic microspheres measuring 15-35 µm in diameter are believed to be useful for intra-arterial hyperthermia. In this study, we attempted to prepare titanium dioxide (TiO2 ) microspheres containing magnetic nanoparticles (MNPs). MNP-containing TiO2 microspheres with diameters of approximately 30 µm were successfully obtained by sol-gel reaction of titanium tetraisopropoxide in a water-in-oil emulsion with added cosurfactant of 1-butanol and subsequent heat treatment at 200°C. The microspheres showed ferrimagnetism owing to high content of MNPs in approximately 60 wt % and had a low-crystalline TiO2 matrix. Furthermore, the agar phantom was heated to above 43°C after approximately 1 min under an alternating magnetic field of 100 kHz and 300 Oe and showed in vitro biocompatibility similar to that of MNP-free TiO2 microspheres.

MC3T3-E1 Cellular Response and Protein Detection on Surface Potential-Controlled TiO2 Scale in Serum-Containing Medium

MC3T3-E1 cell differentiation and related surface potentials of rutile-type TiO2 scales formed on Ti are controlled by varying the Ti heat treatment conditions in a N2 atmosphere containing a trace amount of O2. The zeta potentials of the samples heated at 873 and 973 K for 1 h show large negative and positive values, respectively, while cell differentiation on the surface is enhanced in both cases (14 days incubation). In the case of untreated Ti, the cell differentiation diminishes and the zeta potential becomes more neutral. Protein detection by an immunogold-labeling technique and Ca and P detection by time-of-flight secondary ion mass spectrometry reveal that Ca and P, rather than an adhesive protein such as fibronectin, predominantly adsorbed on the scales formed in 1 h at 873 and 973 K, respectively. In the case of untreated Ti, both fibronectin and a non-adhesive protein such as albumin adsorbed, but no Ca and P were detected. The present findings illuminate the relationship between charged surfaces and MC3T3-E1 cellular response.

Effects of calorie restriction on the age-dependent accumulation of mutations in the small intestine of lacZ-transgenic mice

To understand the effect of calorie restriction on genome maintenance systems, the age-dependent accumulation of mutations in animals maintained on high and low calorie diets was examined using lacZ-transgenic mice. Mice were fed a diet of 95 kcal/w or 65 kcal/w from 2 to 17 months of age. The mutation frequencies in the lacZ gene in epithelial tissues from the small intestine were examined at 12 and 17 months. Mutation frequencies were found to be lower in mice fed with a low calorie diet than in mice fed with a high calorie diet at the two age points. The molecular nature of the mutations was examined with DNA sequencing. It showed a predominance of transversions from G:C to T:A, and this is a typical type of mutation induced by reactive oxygen species. The fraction of this type of mutation among the different types of mutations detected was not affected by calorie restriction. The percentage of the other types of mutation was not influenced either. These results suggest that calorie restriction reduces the age-dependent accumulation of mutations by stimulating or inducing various types of DNA protection and repair systems rather than protecting cells against any specific type of DNA alteration.