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The Japan Highway Public Corporation (JH) needs to work actively to further reduce the maintenance costs and is making efforts to computerize all maintenance work. JH is proposing early development of a system for supporting planned maintenance by determining bridge soundness and predicting deterioration, and selecting optimal timing and method for repair and/or reinforcement. JH is now conducting studies for building JH's version of BMS (JH-BMS). This paper discusses study themes for building JH-BMS, such as bridge soundness evaluation and deterioration prediction based on the inspection results, repair or reinforcement scenarios for life-cycle cost optimization and future directions or areas of research.

Men exhibit a worse survival rate than premenopausal women after intracerebral hemorrhage (ICH), however, no sex-specific management has been concerned. In a rat model involving infusion of ferrous citrate (FC) that simulates iron accumulation after hemorrhage, a higher degree of autophagy associated with higher injury severity was observed in striatum of males than in females. Since the imbalance between the levels of autophagy and energy demand may lead to cell death, we proposed that FC-induced autophagy is detrimental in a male specific manner and autophagy modulation affects injury severity in a sex-dependent manner. Rapamycin, an autophagy inducer, and conditional knockout gene of autophagy-related protein 7 (Atg7) in dopamine receptor D2 (DRD2) neurons were used to test our hypothesis using a mouse model with striatal FC infusion. The result showed that the levels of autophagic cell death and injury severity were higher in male than in female mice. Pre-treatment of FC-infused females with rapamycin increased the FC-induced behavioral deficit and DRD2 neuron death. However, DRD2 neuron-specific knockout of Atg7 decreased FC-induced injury severity and the number of TUNEL(+) DRD2 neurons in males. These results suggest that autophagy in FC-infusion males is overactive with maladaptive consequences and inhibition of autophagy decreases the severity of FC-induced striatal injury in males. These findings present prospects for male-specific therapeutic strategy that targets autophagy in patients suffering from iron overload.

Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit all-trans retinoic acid (RA) has been associated with axon outgrowth and nerve regeneration, the maintenance of differentiated neurons, the association with degenerative disease like Parkinson's disease, and its regulatory molecular mechanism from pluripotent stem cells to neural stem cells remain fragmented. We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson’s disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/β-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARβ/Gβ signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic evidence that human trophoblast stem cell-derived neural stem cells can potentially be used for neurobiological study, drug discovery, and as an alternative source of cell-based therapy in neurodegenerative diseases like Parkinson’s disease.

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. Disruption of normal imprinting leads to abnormal embryogenesis, certain inherited diseases, and is associated with various cancers. In the context of screening for the gene(s) responsible for the alteration of phenotype in cyclophilin A knockdown (CypA-KD) P19 cells, we observed a silent paternally expressed gene, Peg3. Treatment of CypA-KD P19 cells with the DNA demethylating agent 5-aza-dC reversed the silencing of Peg3 biallelically. Genomic bisulfite sequencing and methylation-specific PCR revealed DNA hypermethylation in CypA-KD P19 cells, as the normally unmethylated paternal allele acquired methylation that resulted in biallelic methylation of Peg3. Chromatin immunoprecipitation assays indicated a loss of acetylation and a gain of lysine 9 trimethylation in histone 3, as well as enhanced DNA methyltransferase 1 and MBD2 binding on the cytosine-guanine dinucleotide (CpG) islands of Peg3. Our results indicate that DNA hypermethylation on the paternal allele and allele-specific acquisition of histone methylation leads to silencing of Peg3 in CypA-KD P19 cells. This study is the first demonstration of the epigenetic function of CypA in protecting the paternal allele of Peg3 from DNA methylation and inactive histone modifications.

A case of radiation myelopathy after radioactive iodine therapy is reported. This is the first report to describe radiation myelopathy after I-131 therapy. A 62-year-old female with spinal metastasis of T10 received I-131 therapy. She presented with radiation myelopathy 34 months after the irradiation. We need to recognize the possibility of this serious complication even in the case of I-131 therapy. There is a risk of radiation myelopathy even after I-131 therapy, especially in cases with spinal cord compression such as this.

CIDE-B [cell death-inducing DFF45 (DNA fragmentation factor 45)-like effector B] is a member of the CIDE family of apoptosis-inducing factors. The highly restricted pattern of expression of CIDE-B in the liver and spleen suggests that a mechanism exists for the tissue- and cell-specific regulation of transcription of this gene. We have analysed the promoters of the human CIDE-B gene, particularly the mechanism of cell-specific transcription. Expression of CIDE-B is driven by two promoters which are responsible for the synthesis of two types of transcript, and Sp1 and Sp3 are key regulators of basal transcription from both the upstream and the internal promoter, as indicated by EMSAs (electrophoretic mobility-shift assays) and site-directed mutagenesis. Bisulphite sequencing analysis demonstrated that the upstream promoter was hypermethylated in cells that did not express the long transcript of CIDE-B, but was hypomethylated in cells that expressed this transcript. Furthermore, methylation of this region in vitro reduced the promoter activity to approximately 5% of the control. Thus methylation at CpG sites in the upstream promoter region appeared to be important for cell-specific synthesis of the long transcript. By contrast, HNF4alpha (hepatocyte nuclear factor-4alpha) bound to the internal promoter and enhanced its activity. Moreover, the short transcript of CIDE-B gene was expressed in cells which do not normally express this transcript upon introduction of exogenous HNF4alpha, demonstrating the involvement of HNF4alpha in the cell-specific synthesis of the short transcript. Thus our analysis revealed a novel mechanism for the cell-specific transcription of the human CIDE-B gene, which involves epigenetic and genetic control at separate respective promoters.

Background Hepatolithiasis is a common disease in East Asia and its aggravating factor is bile duct stenosis because of refractory cholangitis. This study investigated the feasibility of gene therapy for bile duct stenosis by administration of p53 adenoviral vectors into the bile. Materials and methods Adenoviral vectors (AxCALacZ or AxCAhp53) were injected transpapillarily into the bile in the bile duct in a rat model of cholangitis. The extent and duration of the gene expression was evaluated with X-gal staining and p53 immunostaining. The bile duct tissue was examined to evaluate the inhibitory effect on the proliferative changes at 3 and 7 days after administration, and Ki-67 labeling index was determined. Results β-galactosidase was expressed in the bile duct epithelia, the bile duct wall and the surrounding connective tissue. The expression of β-galactosidase was detected at 4 weeks after the administration. Mean thickness of the bile duct wall at 7 days was 343.2 ± 14.0 μm for the AxCAhp53 group, 446.5 ± 25.3 μm for the AxCALacZ group and 447.1 ± 53.4 μm for the control group. The proliferation of the bile duct wall was significantly suppressed in the AxCAhp53 group ( P P P Conclusions P53 gene transfer by administration of the adenoviral vector into the bile suppressed the proliferative changes in the bile duct in a rat cholangitis model.

Background Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. Methods Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. Results The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. Conclusions The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world. Copyright © 2005 John Wiley & Sons, Ltd.

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 degrees C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35 x 10(10) pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5. (c) 2005 Elsevier Inc. All rights reserved.

To elucidate radiobiological effects of hypoxia on X-ray-induced apoptosis, MOLT-4 cells were treated under four set of conditions: (1) both X irradiation and incubation under normoxia, (2) X irradiation under hypoxia and subsequent incubation under normoxia, (3) X irradiation under normoxia and subsequent incubation under hypoxia, and (4) both X irradiation and incubation under hypoxia, and the induction of apoptosis was examined by fluorescence microscopy. About 28-33% apoptosis was observed in cells treated under conditions 1 and 2, but this value was significantly reduced to around 18-20% in cells treated under conditions 3 and 4, suggesting that post-irradiation hypoxic incubation rather than hypoxic irradiation mainly caused the reduction of apoptosis. The activation and expression of apoptosis signal-related molecules SAPK/JNK, Fas and caspase-3 were also suppressed by hypoxic incubation. Effects of hypoxic incubation were canceled when cells were treated under conditions 3 and 4 with an oxygen-mimicking hypoxic cell radiosensitizer, whereas the addition of N-acetyl-L-cysteine again reduced the induction of apoptosis. From these results it was concluded that hypoxia reduced the induction of apoptosis by changing the intracellular redox state, followed by the regulation of apoptotic signals in X-irradiated MOLT-4 cells.

In order to enhance the efficacy of conditionally replicating adenoviruses (CRAd) in the treatment of cancers of the biliary tract, we studied the efficacy in vitro and in vivo of AxE1CAUP, a CRAd vector that carries a gene for uracil phosphoribosyltransferase (UPRT), which converts 5-fluorouracil (5-FU) directly to 5-fluorouridine monophosphate and greatly enhances the cytotoxicity of 5-FU. AxE1CAUP replicated and induced an increased UPRT expression in biliary cancer cells more efficiently than AxCAUP, a nonreplicative adenovirus carrying the UPRT gene. Whereas AxCAUP and AxE1AdB, a CRAd without the UPRT gene, modestly increased the sensitivity of BC cells to 5-FU, AxE1CAUP markedly increased the sensitivity, especially when the timing of 5-FU administration was appropriately chosen. AxE1CAUP replicated much less efficiently in normal WI-38 fibroblasts without any change in the sensitivity to 5-FU. In nude mice with s.c. biliary cancer xenografts, i.t. AxE1CAUP/5-FU therapy inhibited tumor growth significantly more strongly than AxCAUP/5-FU or AxE1AdB/5-FU therapy. Furthermore, in mice with peritoneally disseminated biliary cancer, i.p. AxE1CAUP efficiently proliferated in the tumors, decreased the tumor burden, and prolonged the survival of the mice when 5-FU was started 10 or 15 days after the vector inoculation, whereas earlier initiation of 5-FU resulted in early eradication of the vector and no survival benefit. The present study shows that the CRAd expressing UPRT was a more potent sensitizer of biliary cancer to 5-FU, than was a nonreplicative UPRT-encoding vector or a CRAd without UPRT gene, even at a lower dose of the vector, and that timing of 5-FU administration was a key factor to maximize the efficacy. This gene therapy with appropriately timed administration of 5-FU should be useful in overcoming the resistance of biliary cancers to 5-FU.

Deuterium retentions in carbon dust and carbon–tungsten mixed dust (C–W dust) were investigated to estimate the tritium inventory in fusion experimental reactors. Several types of carbon dust or C–W dust were prepared by D 2 arc discharge using carbon or carbon–tungsten electrodes, respectively. These were prepared by changing substrate temperatures with discharge pressure of 1.6 Pa. Deuterium retained in carbon dust was desorbed in the form of D 2 , HD, CD 4 and C 2 D 4 . The peak temperature of the D 2 desorption rate was 1100 K. On the other hand, the deuterium retained in C–W dust was desorbed mainly in the form of D 2 and HD. The peak temperature of the D 2 desorption rate was 900 K. The deuterium concentrations in the atomic ratio, D/C, for carbon dust and C–W dust (C/W=70/30) were reduced from 0.33 to 0.12 and from 0.7 to 0.07, respectively, when the substrate temperature increased from 300 to 850 K.

Tritium retention of carbon dust co-deposited with fuel hydrogen is so large that it is required to evaluate the tritium inventory for estimating potential hazards of next step fusion reactors such as International Thermonuclear Experimental Reactor (ITER). In the present work, the authors prepared carbon dust samples by two methods. One was deuterium (D2) arc discharge with carbon electrodes to simulate co-deposition of carbon atoms with hydrogen atoms. The other was electron beam irradiation to carbon fiber composite (CFC) target in hydrogen gas to simulate disruptions. The dependence of retained hydrogen amount on hydrogen gas pressure and substrate temperature was investigated. The surface morphology and the crystal structure were also examined. In the case of D2 arc discharge, the concentration of retained deuterium was increased with the deuterium gas pressure, but did not depend on substrate temperature from 573 to 723 K. Under the anticipated ITER divertor condition, namely deuterium gas pressure and substrate temperature were less than 1 Pa and 573 K, the deuterium concentration was 0.2 in the atomic ratio, D/C. In the case of electron beam irradiation, the amount of retained hydrogen was the same as the background level.

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me(2)SO-induced mesodermal differentiation. This difference suggests that CypA is specifically required for the RA-induced neuronal pathway. In addition to the loss of RA-induced RA receptor beta expression and retinoic acid response element (RARE)-binding activity, a dramatic reduction in RA-induced RARE-mediated luciferase activity in the CypA knockdown cell line suggests that CypA affects RARE-mediated regulation of gene expression. Silent mutation of target sequences confirms the specificity of RNA interference in p19 embryonal carcinoma cells. Collectively, our data reveal that a novel function of CypA is required in the processing of RA-induced neuronal differentiation in p19 embryonal carcinoma cells.

Recombination activating gene-1 (RAG-1) and RAG-2 are expressed specifically in lymphocytes undergoing the antigen receptor gene rearrangement during the lymphocyte development. Our previous study showed that the -41 to -17 nucleotides (nt) 5' -upstream region of mouse RAG-2 were pre-requisite for the core promoter activity and that Pax-5/c-Myb/LEF-1 protein-protein complex was responsible for its activity in immature B cells. In this study, we show that the -65/-42 sequence, the non-conserved sequence between human and mouse RAG-2 promoter, is necessary for the full promoter activity for mouse RAG-2. Electrophoresis mobility shift assay revealed that Myc-associated zinc finger protein (MAZ) as well as SP1/3 binds a GA box in this region. Using chromatin immunoprecipitation, we show that MAZ binds the RAG-2 promoter region in pre-B cells. Furthermore, we show that MAZ synergistically activates the murine RAG-2 promoter with Pax-5/c-Myb/LEF-1 complex. These results first demonstrate that MAZ participates in activation of mouse RAG-2 promoter.

Human LPTS/PinX1 is a telomerase-inhibitory protein, which binds to the telomere protein Pin2/TRF1 and the catalytic subunit hTERT of telomerase. To explore the proteins that might be involved in the telomerase pathway, we performed a yeast two-hybrid screening with LPTS/PinX1 as the bait. A novel gene, MCRS2, encoding for an isoform of MCRS1/p78 and MSP58 was isolated. The expression of MCRS2 protein is cell-cycle dependent, accumulating in the very early S phase. MCRS2 interacts with LPTS/PinX1 in vitro, in vivo and colocalizes with LPTS/PinX1 in cells. MCRS2 and its amino terminus inhibit telomerase activity in vitro and long-term overexpression of MCRS2 in SMMC-7721 cells results in a gradual and progressive shortening of telomeres. Our findings suggest that MCRS2 might be a linker between telomere maintenance and cell-cycle regulation.

Garnet-bearing ultramafic rocks including clinopyroxenite, wehrlite and websterite locally crop out in the Higashi-akaishi peridotite of the Besshi region in the Cretaceous Sanbagawa metamorphic belt. These rock types occur within dunite as lenses, boudins or layers with a thickness ranging from a few centimetres to 1 metre. The wide and systematic variation of bulk-rock composition and the overall layered structure imply that the ultramafic complex originated as a cumulate sequence. Garnet and other major silicates contain rare inclusions of edenitic amphibole, chlorite and magnetite, implying equilibrium at relatively low P-T conditions during prograde metamorphism. Orthopyroxene coexisting with garnet shows bell-shaped Al zoning with a continuous decrease of Al from the core towards the rim, consistent with rims recording peak metamorphic conditions. Estimated P-T conditions using core and rim compositions of orthopyroxene are 1.5-2.4 GPa ⁄ 700-800 � C and 2.9-3.8 GPa ⁄ 700-810 � C, respectively, implying a high P ⁄ T gradient (> 3.1 GPa ⁄ 100 � C) during prograde metamorphism. The presence of relatively low P-T conditions at an early stage of metamorphism and the steep P ⁄ T gradient together trace a concave upwards P-T path that shows increasing P ⁄ T with higher T, similar to P-T paths reported from other UHP metamorphic terranes. These results suggest either (1) down dragging of hydrated mantle cumulate parallel to the slab-wedge interface in the subduction zone by mechanical coupling with the subducting slab or (2) ocean floor metamorphism and ⁄ or serpentinization at early stage of subduction of oceanic lithosphere and ensuing HP-UHP prograde metamorphism.

1. The retinoblastoma susceptibility gene product, p105Rb (RB), is an important regulator in the control of cell proliferation, differentiation, and apoptosis. Several cellular factors that complex with RB and exert their cellular regulatory functions have been identified, such as the RB:cyclophilin A (CypA) complex. 2. CypA is a cytoplasmic immunophilin and known for its involvement in T-cell differentiation and proliferation. Although CypA has a pivotal role in the immune response, its function in other signaling pathways is largely unknown. 3. In this study, we used a model of neuronal differentiation to demonstrate that the nuclear translocation of CypA, the appearance of hypophosphorylated RB and the enhancement of RB: CypA complex formation correlates with retinoic acid induced neuronal differentiation. 4. Inhibition of CypA expression results in repression of both the hypophosphorylated RB and the neuron-specific differentiation marker, class III beta tubulin. 5. The evidence of enriched CypA and colocalization of RB with CypA in the nucleus of primary adult sensory neurons substantiated the important event of RB-mediated neuronal differentiation of p19 EC cells.

This study was designed to assess the efficacy of dynamic contrast-enhanced MRI (DCE-MRI), in comparison with power Doppler ultrasound (PDUS), for visualizing prostate cancer. 111 men suspected of having prostate cancer underwent imaging before undergoing octant biopsy. Subsequently, 31 cancer-positive patients were enrolled in this study. DCE-MRI was obtained using a three-dimensional fast-field echo sequence, which assured wide coverage of the prostate gland. The transrectal PDUS were scored according to the degree of power Doppler flow signals. The time intensity curve types for the DCE-MRI and the PDUS scores were compared with the histopathologic results for each region. The time intensity curves were correlated significantly with PDUS scores (p

The anisotropic thermal expansion Δ L / L was examined for melt-processed REBaCuO(Ag) bulk composites (RE=Sm, Y) as functions of not only the contents of the RE 2 BaCuO 5 second phase but also Ag metal added to the highly oriented REBa 2 Cu 3 O 7 matrix. The Δ L / L | c values in the c -direction of REBaCuO(Ag) bulks are about 1.5–1.8 times as large as the Δ L / L | ab values in the ab -plane. Δ L / L increases with increasing Ag content in both directions but Δ L / L | c is by far more sensitive to the Ag content. The resultant compressive strain due to Ag particles in the c -direction through the melt-processing was estimated and its possible contribution for the improvement of the superconducting characteristics of the matrix was discussed.

Background: Statistical data of mortality and morbidity related to anesthesia have not been reported in Japan since World War II. The need to comprehensively examine the events of cardiac arrest as well as mortality prompted the first national study in Japan. Methods: Confidential questionnaires were sent to all Japan Society of Anesthesiologists Certified Training Hospitals every year from 1994 through 1998. Collected data were analyzed for incidence of cardiac arrest and other critical events during anesthesia and surgery, and their outcomes within 7 postoperative days. The principal causes of the critical incidents were also analyzed. Results: With an average response rate of 39.9%, a total of 2,363,038 cases were documented over 5 years. The average incidence per year of cardiac arrest during surgery due to all etiologies and that totally attributable to anesthesia was 7.12 [95%CI: 6.30,7.94] and 1.00 [0.88, 1.12]) per 10,000 cases, respectively. The average mortality per year in the operating room or within 7 postoperative days due to all etiologies and that totally attributable to anesthesia was 7.18 [6.22, 8.13] and 0.21 [0.15, 0.27] per 10,000 cases, respectively. The two principal causes of cardiac arrest during anesthesia and surgery due to all etiologies were massive hemorrhage (31.9%) and surgery (30.2%), and those totally attributable to anesthesia were drug overdose or selection error (15.3%) and serious arrhythmia (13.9%). Preventable human errors caused 53.2% of cardiac arrest and 22.2% of deaths in the operating room totally attributable to anesthesia. Conclusions: The rates in Japan of cardiac arrest and death during anesthesia and surgery due to all etiologies as well as those totally attributable to anesthesia are comparable to those of other developed countries.

In the divertor of next step fusion experimental devices such as ITER, simultaneous presence of carbon and tungsten material will lead to their mixing during the redeposition process. In the present paper, sputtering experiments on C–W mixed material by low energy hydrogen beam had been performed. The C–W mixed material was simulated by the deposits during the D2 arc discharge between carbon and tungsten electrode. Five types of C–W mixed materials having different W concentrations which ranged from 0 to 75 wt% were used as the specimens for the sputtering experiment. It was found that the sputtering yield strongly depends on the W concentration in the C–W mixed materials. From the results of electron probe micro-analysis, it was indicated that carbon material is preferentially sputtered by chemical sputtering. The formation of W–C bonding is considered to be the reason for the low sputtering yields of C–W mixed material layers.

Infectious recombinant adenovirus (rAd) is usually produced in human embryonic kidney 293 cells that harbor the E1 gene and rAd has been shown to be an efficient tool for gene transfer both in vivo and in vitro. It also has considerable potential in human gene therapy. However, rates of spontaneous mutations in genes introduced into host cells after multiple passages remain to be clarified. We have characterized the spontaneous mutation of genomes derived from human adenovirus type 5 (Ad5) and of human p53-rAd during multiple passages by two different methods, namely, a plaque assay and a molecular cloning assay, with subsequent direct nucleotide sequencing. Using the plaque assay, we found no mutations in the E1A and p53 genes derived from infectious Ad5 and p53-rAd, respectively. By contrast, we found spontaneous mutations in the E1A gene of Ad5, with a mutation rate of 9.28 x 10(-8) per base pair per plaque, in the molecular cloning assay. The rate of mutation of the p53 gene of p53-rAd, as determined by the molecular cloning assay, ranged from 1.50 x 10(-7) to 3.25 x 10(-7) per base pair per passage. The mutations in the p53 gene of p53-rAd were localized mainly in the transcriptional activation domain, the SH3 domain, and the regulation domain and they were rarely found in the DNA-binding domain, which is a major site for mutations in human cancers. Our results indicate that multiple passages can generate a heterogeneous population of p53-rAd and that the molecular cloning assay is an efficient technique with which to search for mutations in the genome of p53-rAd that cannot be detected by a plaque assay.

We have developed an X-ray microbeam with a small angular divergence by adopting X-ray optics with successive use of asymmetric Bragg reflection from silicon crystals for the both polarizations of the synchrotron X-rays. The microbeam actually obtained is several microns in size and possesses an angular divergence of less than 2 arcsec which enables us to measure the strain of 10−5–10−6. By scanning the sample against the microbeam, distribution of the minute local strain in various regions of semiconductor crystals for electronic devices, e.g., the strain around the SiO2/Si film edge in silicon devices, the strain in an InGaAsP/InP stripe laser were measured.

Crystallinity of bonded silicon-on-insulator (SOI) wafers has been investigated by means of diffractometry using a parallel X-ray microbeam. Peak shifts and extended tails were observed in the rocking curves of the SOI layer. Further, reciprocal lattice maps revealed that the SOI lattice planes were tilt dispersively within about 100 μrad. The typical sizes of the grains are ranging from a few μm to a several tens μm. The inferiority of the thin SOI crystal may arise from the extended force of buried oxide layer.

The recombinant microbial transglutaminase from Streptoverticillium mobaraense var. (rMTGase) was expressed in Escherichia coli. Specific enzyme activity of rMTGase was comparable to native MTGase. However, the gelation of a sodium caseinate solution induced by rMTGase was slower than that induced by native MTGase. In addition, the mechanical property of kamaboko prepared with rMTGase was weaker than that with native MTGase. In SDS-PAGE analysis, α-casein monomers decreased more slowly during the incubation with rMTGase than MTGase. These results confirmed the difference of cross-linking activity between the 2 enzymes. Furthermore, thermal stability of rMTGase was lower compared to native MTGase. These results suggest that the difference of cross-linking activity and thermal stability between the 2 enzymes cause differences in gelation activity of protein.

The chemical vapor deposition (CVD) coating is considered as one of the possible methods for in situ repairing of the tungsten armor. In the present work, CVD coatings on eroded tungsten specimens were prepared to investigate the applicability of this method to repair the eroded tungsten surface. To simulate the damaged surface relevant to the disruption erosion, specimens were irradiated by an electron beam at a heat flux of 1250 MW/m2 before the CVD repairing procedure. From metallographic results, no pores or cracks were observed at the interface between the CVD layer and the eroded layer seemed to be successfully repaired by the CVD coating. However, the CVD layer was delaminated by thermal shock tests which simulate disruption heat loads. It was found that complete removal of the resolidified area before CVD coating is effective to repair the eroded surface.

Effects of Al atoms on the hydrogen exchange reactions of hydrogen molecules with surface hydroxyls in silicates are investigated by ab initio calculations at the HF/6-31G ∗∗ and MP2/6-31G ∗∗ levels with the model clusters H3SiOH and H3Si(OH)Al(H)2OSiH3. The direct interaction of Al atoms with surface hydroxyl is found to bring about the lowering in the potential energy barrier of exchange reactions between H2 and H3SiOH. The lowering is explained by the changes of the reaction mechanism and the weakening of the O–H bond in surface hydroxyl by the interaction of Al atoms.

Purpose: The transradial approach is not so popular in cerebral angiography. The purpose of this study was therefore to present our experience of success rate and safety of this method. Material and Methods: From December 1998 to June 2001, 526 carotid and vertebral angiographies with DSA were performed via the radial artery. A 1.4-mm catheter was used through a 1.4-mm introducer sheath. We evaluated the procedure as successful if sufficient images for diagnosis were obtained of the bilateral carotid arteries and unilateral vertebral artery. Each patient was reassessed for any complications, occurring until the next morning. The length of time needed for an examination was measured in the last 10 cases. Results: In all but 5 cases, the procedures were evaluated as successful (99.0%). Unsuccessful cases manifested severe pain at the radial puncture, angiospasm at the radial artery, loop formation at the radial artery, occlusion at the subclavian artery, and an aberrant right subclavian artery. No severe complications including neurological ones were encountered. Minor complications were noted in 17 cases (3.2%): 4 cases of thrombus at the ulnar artery, 1 angiospasm at the radial artery, and 12 cases of small hematoma at the puncture site. The radial approach took 14 min less in the common carotid study and 3 min 30 s less in the internal carotid study than by the femoral approach. Conclusion: The transradial approach enabled selective studies for carotid and vertebral angiography with a high success rate and safety with few complications.

Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells differentiated to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor and platelet-derived growth factor. We also developed a protocol that resulted in the differentiation of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell factor and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.

Recycling of Municipal Solid Waste is vigorously promoted in Japan and the necessity of energy recovery from organic waste is increasing. An anaerobic digestion demonstration plant for organic waste in Kyoto City, Japan has been operated for about two years. Three kinds of wastes (garbage and leftovers from hotels, yard waste and used paper) mixed at various ratios are used. The plant has maintained stable operations with each mixture, generating biogas by the decomposition of VS at the rate of about 820 m3N/ton-VS.

The Sp family is a family of transcription factors that bind to cis-elements in the promoter regions of various genes. Regulation of transcription by Sp proteins is based on interactions between a GC-rich binding site (GGGCGG) in DNA and C-terminal zinc finger motifs in the proteins. In this study, we characterized the GC-rich promoter of the gene for the DNA methyltransferase (Dnmt1) that is responsible for methylation of cytosine residues in mammals and plays a role in gene silencing. We found that a cis-element (nucleotides -161 to -147) was essential for the expression of the mouse gene for Dnmt1. DNA-binding assays indicated that transcription factors Sp1 and Sp3 bound to the same cis-element in this region in a dose-dependent manner. In Drosophila SL2 cells, which lack the Sp family of transcription factors, forced expression of Sp1 or Sp3 enhanced transcription from the Dnmt1 promoter. Stimulation by Sp1 and Sp3 were independent phenomena. Furthermore, cotransfection reporter assays with a p300-expression plasmid revealed the activation of the promoter of the Dnmt1 gene in the presence of Sp3. The transcriptional coactivator p300 interacted with Sp3 in vivo and in vitro. Our results indicate that expression of the Dnmt1 gene is controled by Sp1 and Sp3 and that p300 is involved in the activation by Sp3.

Tritium retention of carbon dust co-deposited with fuel hydrogen is large, and then it is required to evaluate the tritium inventory as a safety issue of ITER. Several species of co-deposited carbon dust were prepared by D 2 arc discharge with carbon electrodes. The dependence of D 2 gas pressure on the retained deuterium amount of the co-deposited dust was investigated. The structure and the surface morphology were also examined. The retained deuterium amount increased with the discharge gas pressure. The deuterium concentrations of the co-deposited carbon dust samples prepared at 1.3 Pa and 6.8 Pa were 0.12 and 0.3 in the atomic ratio, D/C, respectively. No clear dependence of the substrate temperature on retained deuterium amount was observed, perhaps due to the coarse temperature control. In the environment of gas pressure in ITER, approximately 1 Pa, the tritium concentration is estimated approximately T/C 0.06, which is several times smaller than the value estimated so far, T/C 0.2.

The cAMP-response element-binding protein-binding protein (CBP) and p300 are common coactivators for several transcriptional factors. It has been reported that both CBP and p300 are significant for the activation of peroxisome proliferator-activated receptor gamma (PPARgamma), which is a crucial nuclear receptor in adipogenesis. However, it remains unclear whether CBP and/or p300 is physiologically essential to the activation of PPARgamma in adipocytes and adipocyte differentiation. In this study, we investigated the physiological significance of CBP/p300 in NIH3T3 cells transiently expressing PPARgamma and CBP and in 3T3-L1 preadipocytes stably expressing CBP- or p300-specific ribozymes. In PPARgamma-transfected NIH3T3 cells, induction of expression of PPARgamma target genes such as adipocyte fatty acid-binding protein (aP2) and lipoprotein lipase (LPL) by adding thiazolidinedione was enhanced, depending on the amount of a CBP expression plasmid transfected. Expression of aP2 and LPL genes, as well as glycerol-3-phosphate dehydrogenase activity and triacylglyceride accumulation after adipogenic induction, was largely suppressed in 3T3-L1 adipocytes expressing either the CBP- or p300-specific active ribozyme, but not in inactive ribozyme-expressing cells. These data suggest that both CBP and p300 are indispensable for the full activation of PPARgamma and adipocyte differentiation and that CBP and p300 do not mutually complement in the process.

We have developed an X-ray microbeam with a narrow energy bandwidth and a small angular divergence in both horizontal and vertical directions normal to the X-rays. Utilizing synchrotron radiation from the so-called third-generation ring like SPring-8 (Super Photon ring, 8 GeV), the X-ray microbeam possesses sufficient photons even after beam size compression. Using the X-ray microbeam, we have demonstrated to measure very local and minute lattice strain in silicon materials after some wafer preparation processes or device fabrication processes. Local strain in an Si crystal around the SiO 2 /Si film edge and also strain at silicon-on-insulator (SOI) interface have been measured as sample materials.

The mechanism of the reaction of a water molecule with surface isolated hydroxyl of silica and silicates is investigated by ab initio molecular orbital calculations within a model reaction, H2O+H3SiOH, to figure out the role of water molecules in tritium release process. The hydrogen release from the surface isolated hydroxyl is found to proceed as a result of the Si–O bond breaking. Namely, tritium would be released by the hydroxyl-exchange reaction between water and surface hydroxyl of silicate, not by the hydrogen-exchange reaction. Prior to the exchange reaction, water molecules are found to prefer adsorbing as proton acceptors in the water – silanol complex, whereas the hydroxyl-exchange reaction occurs from a complex with a different form, in which the water molecule adsorbs as a proton donor. The overall potential energy barrier is calculated as 24.4 kcal mol−1 for the hydrogen release from the silicate in the presence of water molecules.

The retinoblastoma susceptibility gene product, p105Rb (RB), is generally believed to be an important regulator in the control of cell growth, differentiation, and apoptosis. Several cellular factors that form complexes with RB and exert their cellular regulatory functions have been identified, such as the newly identified RB:cyclophilin A (CypA) complex. The physical interactions between RB and CypA were demonstrated by glutathione S-transferase affinity matrix binding assays and immunoprecipitation, followed by Western blot analyses. The N-terminal region of CypA mediated the interaction with RB, whereas the region upstream of the A-pocket of RB was required for binding to CypA. Ectopic expression of RB into Jurkat cells partially blocks the function of cyclosporin (CsA) to inhibit nuclear factor for activation of T cell (NFAT) activation by phorbol ester (PMA) plus ionomycin A (IA), suggesting that RB may prevent CsA inhibition of T lymphocyte activation. These results are further evidenced by the effect of RB on both calcineurin (CN) and NFAT binding activity in vitro, suggesting that the interaction of RB with CypA interferes with the CsA:CypA complex and blocks CsA-inhibited CN activity. These data reveal the functional link between RB and CypA and their involvement in T cell activation signaling.

Activating transcription factor 2 (ATF2) and its homolog ATF7 are phosphorylated at Thr-69/Thr-71 and at Thr-51/Thr-53, respectively, by stress-activated MAPKs regulating their transcriptional functions in G1 and S phases. However, little is known about the role of ATF2 and ATF7 in G2/M phase. Here, we show that Cdk1-cyclin B1 phosphorylates ATF2 at Thr-69/Thr-71 and ATF7 at Thr-51/Thr-53 from early prophase to anaphase in the absence of any stress stimulation. Knockdown of ATF2 or ATF7 decreases the rate of cell proliferation and the number of cells in M-phase. In particular, the knockdown of ATF7 severely inhibits cell proliferation and G2/M progression. The inducible expression of a mitotically nonphosphorylatable version of ATF7 inhibits G2/M progression despite the presence of endogenous ATF7. We also show that mitotic phosphorylation of ATF7 promotes the activation of Aurora kinases, which are key enzymes for early mitotic events. These results suggest that the Cdk1-mediated phosphorylation of ATF7 facilitates G2/M progression, at least in part, by enabling Aurora signaling.

Because of their pluripotent characteristics, human induced pluripotent stem cells (iPSCs) possess great potential for therapeutic application and for the study of degenerative disorders. These cells are generated from normal somatic cells, multipotent stem cells, or cancer cells. They express embryonic stem cell markers, such as OCT4, SOX2, NANOG, SSEA-3, SSEA-4, and REX1, and can differentiate into all adult tissue types, both in vitro and in vivo. However, some of the pluripotency-promoting factors have been implicated in tumorigenesis. Here, we describe the merits of tumor suppresser genes as reprogramming factors for the generation of iPSCs without tumorigenic activity. The initial step of reprogramming is induction of the exogenous pluripotent factors to generate the oxidative stress that leads to senescence by DNA damage and metabolic stresses, thus inducing the expression of tumor suppressor genes such as p21 CIP1 and p16 INK4a through the activation of p53 to be the pre-induced pluripotent stem cells (pre-iPSCs). The later stage includes overcoming the barrier of reprogramming-induced senescence or cell-cycle arrest by shutting off the function of these tumor suppressor genes, followed by the induction of endogenous stemness genes for the full commitment of iPSCs (full-iPSCs). Thus, the reactive oxygen species (ROS) produced by oxidative stress might be critical for the induction of endogenous reprogramming-factor genes via epigenetic changes or antioxidant reactions. We also discuss the critical role of tumor suppressor genes in the evaluation of the tumorigenicity of human cancer cell-derived pluripotent stem cells, and describe how to overcome their tumorigenic properties for application in stem cell therapy in the field of regenerative medicine.

Chromatin is subject to proofreading and repair mechanisms during the process of DNA replication, as well as repair to maintain genetic and epigenetic information and genome stability. The dynamic structure of chromatin modulates various nuclear processes, including transcription and replication, by altering the accessibility of the DNA to regulatory factors. Structural changes in chromatin are affected by the chemical modification of histone proteins and DNA, remodeling of nucleosomes, incorporation of variant histones, noncoding RNAs, and nonhistone DNA-binding proteins. Phenotypic diversity and fidelity can be balanced by controlling stochastic switching of chromatin structure and dynamics in response to the environmental disruptors and endogenous stresses. The dynamic chromatin remodeling can, therefore, serve as a sensor, through which environmental and/or metabolic agents can alter gene expression, leading to global cellular changes involving multiple interactive networks. Furthermore its recent evidence also suggests that the epigenetic changes are heritable during the development. This review will discuss the environmental sensing system for chromatin regulation and genetic and epigenetic controls from developmental perspectives.

The differential cross sections of the 9 Be + α inelastic scattering at 30 MeV were measured at the tandem of Tsukuba University. All the known states of 9 Be up to energies ~ 12 MeV were observed and decomposed into three rotational bands, each of them having a cluster structure consisting of a 8 Be core plus a valence neutron in one of the sub-shells: p3/2-, s1/2+ and p1/2-. Existence of a neutron halo in the positive parity states was confirmed.