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Biomedical subjects

Masahiko Mori

Publications and source records attributed to Masahiko Mori.

15 recordsLinked to original sources
PubMed2006

Human RAD18 is involved in S phase-specific single-strand break repair without PCNA monoubiquitination.

Switching from a replicative to a translesion polymerase is an important step to further continue on replication at the site of DNA lesion. Recently, RAD18 (a ubiquitin ligase) was shown to monoubiquitinate proliferating cell nuclear antigen (PCNA) in cooperation with RAD6 (a ubiquitin-conjugating enzyme) at the replication-stalled sites, causing the polymerase switch. Analyzing RAD18-knockout (RAD18-/-) cells generated from human HCT116 cells, in addition to the polymerase switch, we found a new function of RAD18 for S phase-specific DNA single-strand break repair (SSBR). Unlike the case with polymerase switching, PCNA monoubiquitination was not necessary for the SSBR. When compared with wild-type HCT116 cells, RAD18-/- cells, defective in the repair of X-ray-induced chromosomal aberrations, were significantly hypersensitive to X-ray-irradiation and also to the topoisomerase I inhibitor camptothecin (CPT) capable of inducing single-strand breaks but were not so sensitive to the topoisomerase II inhibitor etoposide capable of inducing double-strand breaks. However, such hypersensitivity to CPT observed with RAD18-/- cells was limited to only the S phase due to the absence of the RAD18 S phase-specific function. Furthermore, the defective SSBR observed in S phase of RAD18-/- cells was also demonstrated by alkaline comet assay.

Camptothecin↗
PubMed2006

Replication-dependent and -independent responses of RAD18 to DNA damage in human cells.

Postreplication repair facilitates tolerance of DNA damage during replication, overcoming termination of replication at sites of DNA damage. A major post-replication repair pathway in mammalian cells is translesion synthesis, which is carried out by specialized polymerase(s), such as polymerase eta, and is identified by focus formation by the polymerase after irradiation with UVC light. The formation of these foci depends on RAD18, which ubiquitinates PCNA for the exchange of polymerases. To understand the initial processes in translesion synthesis, we have here analyzed the response to damage of RAD18 in human cells. We find that human RAD18 accumulates very rapidly and remains for a long period of time at sites of different types of DNA damage, including UVC light-induced lesions, and x-ray microbeam- and laser-induced single-strand breaks, in a cell cycle-independent manner. The accumulation of RAD18 at DNA damage is observed even when DNA replication is inhibited, and a small region containing a zinc finger motif located in the middle of RAD18 is essential and sufficient for the replication-independent damage accumulation. The zinc finger motif of RAD18 is not necessary for UV-induced polymerase eta focus formation, but another SAP (SAF-A/B, Acinus and PIAS) motif near the zinc finger is required. These data indicate that RAD18 responds to DNA damage in two distinct ways, one replication-dependent and one replication-independent, involving the SAP and zinc finger motifs, respectively.

Cell Cycle↗
PubMed2006

Regio- and stereoselective nickel-catalyzed homoallylation of aldehydes with 1,3-dienes.

Ni(acac)(2) catalyzes homoallylation of aldehydes with 1,3-dienes in the presence of triethylborane. Triethylborane serves as a reducing agent delivering a formal hydride to the C2 position of 1,3-dienes, thus generating a formal homoallyl anion species and enabling the novel homoallylation of aldehydes. The reaction proceeds smoothly at room temperature in the absence of any phosphane or nitrogen ligands and is highly regioselective and stereoselective for a wide variety combination of aldehydes and 1,3-dienes: e.g., isoprene and benzaldehyde combine to give a mixture of anti- and syn-1-phenyl-3-methyl-4-penten-1-ol (2.2) in a ratio of 15:1 in 90% yield. Under the conditions, sterically congested aliphatic aldehydes and ketones show low yields. In such cases, diethylzinc serves as a substitute for triethylborane and yields the expected products in good yields with similarly high regio- and stereoselectivity. 1,3-Cyclohexadiene is one exception among 24 kinds of dienes examined and undergoes allylation (not homoallylation) selectively.

Journal Article↗
PubMed2006

Calcification at the interface between titanium implants and bone: observation with confocal laser scanning microscopy.

It has not been previously possible to observe bone formation in undecalcified sections with titanium implants at high magnification because of the difficulty in sectioning bone together with implants. A method for examining the bone-implant interface in undecalcified sections is described in which implants are left in situ and confocal laser scanning microscopy (CLSM) is used to examine both the implant surface and adjacent bone. Pulsing of animals at different times with the fluorescent dyes calcein and alizarin red permitted assessment of temporal patterns of bone formation by CLSM. Reflectivity of the polished implant surface permitted accurate assessment of the position of the implant relative to labeled bone. The analysis showed that bone first formed as thin processes towards and across the implant surface, followed by further bone formation behind these processes. The interface between calcified bone tissue and the implant surface was characterized by a 10-microm space. The CLSM technique enabled detailed observations of new bone formation at the titanium implant interface.

Animals↗
PubMed2005

Severe growth retardation and short life span of double-mutant mice lacking Xpa and exon 15 of Xpg.

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause an early onset of Cockayne syndrome (CS) in some patients (XP-G/CS) with characteristics, such as growth retardation and a short life span. In the previous studies, we generated four Xpg mutant mice with two different C-terminal truncations, null, or a base substitution mutation to identify the protein region that causes the onset of CS, and found that the CS-causing mutations, null or a deletion of the last 360 amino acids, completely inhibited the NER activity of mouse XPG (Xpg), but the non-CS-causing mutations, XpgD811A (base substitution that eliminates the nuclease activity of Xpg) or XpgDeltaex15 (deletion of the exon 15 corresponding to the last 183 amino acids), resulted in the retention of residual NER activity. To understand why mutations that completely eliminate the NER activity of Xpg cause CS but those that abolish the nuclease activity without totally eliminating the NER activity of Xpg do not result in CS, we made a series of Xpg mutant mice with Xpa-null mutant allele and found that mice with the non-CS-causing deletion mutation (XpgDeltaex15) exhibited the CS phenotype when XPA was also absent but the base substitution mutation (XpgD811A) that eliminated the Xpg nuclease activity did not. These results indicate that Xpg has a second function, beside NER, and that the disruption of this second function (deletion of the last 183 amino acids) when combined with an NER defect causes CS. When we compared amino acid sequences corresponding to the exon 15 of Xpg, a significant homology was conserved among vertebrates, but not in Drosophila and Saccharomyces cerevisiae. These observations suggest that the second function of XPG may be conserved only in vertebrates and CS symptoms may occur in its absence.

Amino Acid Sequence↗
PubMed2005

Nickel-catalyzed addition of dimethylzinc to aldehydes across alkynes and 1,3-butadiene: an efficient four-component connection reaction.

In the presence of 10 mol % of Ni(acac)(2), four components comprising Me(2)Zn, alkynes, 1,3-butadiene, and carbonyl compounds combine in this order in 1:1:1:1 ratio to furnish (3E,6Z)-octadien-1-ols 1 in good yields. Similarly, the coupling reaction of Me(2)Zn, 1,omega-dienynes 5, and carbonyls furnishes 1-alkylidene-2-(4'-hydroxy-(1'E)-alkenyl)cyclopentanes and -cyclohexanes 6 and their oxygen and nitrogen heterocycle derivatives in good yield and an excellent level of 1,5-diastereoselectivity with respect to the cycloalkane methine carbon and the OH-bearing carbon of the C2 side chain. The reaction is completed in most cases within 1 h at room temperature under nitrogen, tolerates an ester, a hydroxy, an allyl and propargyl ethers, an allylamino, and a pyridyl functionalities, and accommodates a variety of aromatic and aliphatic alkynes and carbonyls (aldehydes and ketones).

Journal Article↗
PubMed2005

L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier.

PURPOSE: L-type amino acid transporters (LATs) prefer branched-chain and aromatic amino acids, including neurotransmitter precursors. The objective of this study was to clarify the expression and function of LAT at the inner blood-retinal barrier (BRB). METHODS: [3H]L-Leucine transport at the inner BRB was characterized by using in vivo integration plot analysis and a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2). The expression of the LAT1 was demonstrated by quantitative real-time RT-PCR, immunoblot, and immunohistochemical analyses. RESULTS: The apparent influx permeability clearance of [3H]L-leucine in the rat retina was found to be 203 microL/(min.g retina), supporting a carrier-mediated influx transport of L-leucine at the BRB. [3H]L-Leucine uptake by TR-iBRB2 cells was an Na+-independent and concentration-dependent process with a Km of 14.1 microM. This process was more potently cis inhibited by substrates of LAT1, D-leucine, D-phenylalanine, and D-methionine, than those of LAT2, L-alanine, and L-glutamine. [3H]L-Leucine efflux from TR-iBRB2 cells was trans-stimulated by substrates of LAT1. The expression of LAT1 mRNA was 100- and 15-fold greater than that of LAT2 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of LAT1 protein was observed in TR-iBRB2 and primary cultured human retinal endothelial cells and immunostaining of LAT1 was observed along the rat retinal capillaries. CONCLUSIONS: LAT1 is expressed at the inner BRB and mediates blood-to-retina L-leucine transport. This transport system plays a key role in maintaining large neutral amino acids as well as neurotransmitters in the neural retina.

Animals↗
PubMed2005

[Health behavioral factors related to the attitude toward quit-smoking among factory workers].

PURPOSE: It is important to focus on various characteristics of people to promote measures for quitting-smoking. Using the Health Belief Model, we here investigated social and psychological factors related to willingness to cease smoking among factory-workers in the northern part of Kyoto Prefecture. METHOD: From October to November 2001, the 810 subjects were surveyed about their smoking behavior and related factors using an unsigned self-administered questionnaire. Among the 534 respondents, we performed logistic regression analysis, considering 7 factors ((1) age, (2) susceptibility, (3) seriousness, (4) benefits, (5) barriers, (6) mass-media and (7) advice from others) as independent variables and interest in smoking cessation as a dependent variable. Four comparisons ((1) "no interest" vs "precontemplation or contemplation", (2) "no interest" vs "precontemplation", (3) "no interest" vs "contemplation", (4) "precontemplation" vs "contemplation") were conducted for 239 males who were current smokers at the time of the survey. RESULTS: Acceptance of the benefits of smoking cessation showed a significant positive association with willingness to quit smoking, with Ors for comparisons of "no interest" vs "preconemplation or contemplation", "no interest" vs "precontemplation", and "no interest" vs "contemplation" (odds ratios (95%CI) of 3.06 (1.76-5.31), 2.78 (1.58-4.90), 4.41 (1.75-11.15), respectively). Acceptance of the seriousness of health hazards from smoking had a significant positive association with the willingness to quit (odds ratio (95%CI) was 1.88 (1.02-3.46)). CONCLUSION: It is can be concluded that it is useful to enhance subjective recognition of the benefits of not-smoking and the seriousness of the harm caused by smoking, to increase the level of willingness of quitting from "no interest" to "precontemplation" or "contemplation".

Adult↗
PubMed2003

Identification of a new G-to-A transition mutation at nucleotide position 129 of the Xrcc4 gene in ionizing radiation-hypersensitive mutant LX830 cells.

The mouse lymphoma cell line LX830 is an X-ray-hypersensitive mutant. Complementation tests between LX830 cells and radiation-sensitive mutants of M10 (Xrcc4 deficient cells) or SX10 (DNA ligase IV deficient cells) cells showed that M10 cells did not complement LX830 cells, but SX10 cells did, suggesting that LX830 cells would belong to the X-ray-cross complementation group (XRCC4). A sequence analysis of Xrcc4 cDNA in LX830 cells disclosed a transition of G to A at nucleotide position 129, which resulted in a change of tryptophan (43) to a termination codon. Transfection of the mouse Xrcc4 cDNA rescued the X-ray sensitivity of the mutant cells. LX830 is an Xrcc4-deficient cell line bearing a termination codon in exon 2 of the Xrcc4 gene and no wild-type Xrcc4 gene.

Amino Acid Sequence↗
PubMed2002

Elafin expression in human fetal and adult submandibular glands.

Elafin, a bifunctional protein, has the NH(2)-terminal domain functions as a transglutaminase substrate for crosslinking to lysine-containing proteins and the COOH-terminal whey acidic protein domain as a potent anti-elastase. Human fetal submandibular glands (n=100) and adult submandibular glands (n=10) were used to elucidate the expression pattern of elafin in the developmental processes of human submandibular gland by immunohistochemistry, in situ hybridization, and western blot analysis. Elafin mRNA was expressed both in the gland epithelium and intralobular mesenchymal tissue of fetal submandibular gland in an early developmental stage (10-18 weeks) and an early intermediate developmental stage (EIDS; 19-24 weeks). The elafin antigen was also found in the intralobular mesenchyme of submandibular gland in the same stages. Thereafter, elafin was transitionally expressed in the ducts and acini of submandibular glands. In the late intermediate developmental stage (LIDS; 25-32 weeks) and the late developmental stage (LDS; 33-40 weeks), elafin became markedly positive in the excretory and striated ducts but weakly positive in the intralobular mesenchymal tissue. The elafin was heavily present in the excretory and striated ducts of adult submandibular gland, while it was sparse in the intralobular mesenchymal tissues. Western blot analysis showed the protein extracts from submandibular glands of EIDS, LIDS, and LDS, adult submandibular gland, fetal tissues (8 weeks), and adult parotid saliva migrated into multiple bands, about 25, 50, 65, and 140 kDa, which were higher than the putative size of elafin protein, 12 kDa. These data suggest that elafin, anti-elastase, is an essential component highly utilized during the morphogenetic processes of fetal salivary gland development and continuously plays a role for the protection of the tubuloalveolar structures of adult salivary gland.

Blotting, Western↗
PubMed2002

Whole-body X-irradiation induces acute and transient expression of heme oxygenase-1 in rat liver.

Activation of the stress-inducible heme oxygenase-1 (HO-1) gene by X-irradiation was investigated in rat liver. When male Wistar MS strain rats (8 weeks) received whole-body irradiation of 17.0 Gy, 7 h later the activity of heme oxygenase in the liver was significantly enhanced (2.5 times). The level of HO-1 mRNA expression was increased by 2.3 and 4.0 times 2 and 4 h after radiation, and then declined at 7 and 10 h to the level of 2.0 and 1.6 times of the control. When the X-ray dose was varied from 4.0 to 21.7 Gy, the transcription of the gene was enhanced at all doses and the level of activation was dose-dependent. Finally, western blotting of irradiated liver demonstrated a significant increase in the level of HO-1 induced by X-rays, peaking at 4 h. Thus, X-rays were confirmed to be stressors that induce acute HO-1 expression transiently in the liver.

Animals↗
PubMed2002

Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene.

The catalytic subunit of DNA-dependent protein kinase plays critical roles in nonhomologous end joining in repair of DNA double-strand breaks and V(D)J recombination. In addition to the SCID phenotype, it has been suggested that the molecule contributes to the polymorphic variations in radiosensitivity and susceptibility to cancer in mouse strains. Here we show the nucleotide sequence of approximately 193-kbp and 84-kbp genomic regions encoding the entire Prkdc gene (also known as DNA-PKcs) in the mouse and chicken, respectively. A large retroposon was found in intron 51 in the mouse but not in the human or chicken. Comparative analyses of the genome strongly suggested that the region contains only two genes for Prkdc and Mcm4; however, several conserved sequences and cis elements were also predicted.

Animals↗