Biomedical subjects

M Takata

Publications and source records attributed to M Takata.

At least 37 records · Page 2Linked to original sources

PubMed2002

Exertion of the suppressive effects of IFN-gamma on experimental immune mediated blepharoconjunctivitis in Brown Norway rats during the induction phase but not the effector phase.

BACKGROUND/AIMS: Interferon gamma (IFN-gamma) knockout mice exhibit severe allergic conjunctivitis (AC), indicating that IFN-gamma regulates the development of AC. The authors examined whether this inhibitory effect of IFN-gamma is exerted during the induction or effector phase of experimental AC. METHODS: Experimental immune mediated blepharoconjunctivitis (EC) was induced in Brown Norway (BN) rats, using ovalbumin (OVA) as the antigen. To investigate the role of IFN-gamma in the induction phase, EC was induced by active immunisation and IFN-gamma (10 micro g/time, total 70 micro g), or phosphate buffered saline (PBS) as a control, was injected intraperitoneally every other day from the day of immunisation. The rats were challenged with OVA eye drops 13 days after immunisation, and 24 hours later, the eyes were harvested for histology. To examine the effects of IFN-gamma in the effector phase, OVA specific T cells were transferred into syngeneic rats and IFN-gamma (10 micro g/time, total 50 micro g) or PBS was injected each day after the transfer until induction of EC 4 days later with an OVA challenge. To investigate the role of endogenous IFN-gamma during the effector phase, an anti-IFN-gamma monoclonal antibody (3 mg/time) was injected on days 3 and 4. RESULTS: Injection of IFN-gamma into actively immunised rats suppressed eosinophilic infiltration but not infiltration of mononuclear cells. In contrast, neither IFN-gamma nor anti-IFN-gamma affected EC in passively immunised rats. CONCLUSION: IFN-gamma is a suppressive cytokine for the development of EC and exerts this suppressive effect during the induction phase.

Adoptive Transfer↗

PubMed2001

Evidence for Pb-O covalency in tetragonal PbTiO3.

Accurate charge-density distributions of cubic and tetragonal PbTiO3 and BaTiO3 have been obtained by the MEM(maximum entropy method)/Rietveld analysis using synchrotron-radiation powder data. The Pb-O bonds in tetragonal PbTiO3 show rather strong covalency, while those in cubic PbTiO3 are ionic. This is the clear evidence of the Pb-O hybridization in tetragonal PbTiO3, which has been theoretically predicted as a key factor of much larger ferroelectricity of this substance than that of BaTiO3. Tetragonal PbTiO3 forms a layered structure of a two-dimensional covalent-bonding network consisting of the Ti-O5 pyramid.

Journal Article↗

PubMed2001

Loss of expression of the metastasis suppressor gene KiSS1 during melanoma progression and its association with LOH of chromosome 6q16.3-q23.

KiSS1 is a putative melanoma metastasis suppressor gene, the expression of which may be regulated by another gene(s) mapping to chromosome 6q16.3-q23. To additionally elucidate the role of KiSS1 in the progression of human melanoma in vivo, we examined KiSS1 mRNA expression in 51 melanocytic tumors with various stages of progression by in situ hybridization. We also examined a correlation between loss of KiSS1 mRNA expression and loss of heterozygosity (LOH) of 6q16.3-q23 in 27 melanoma metastases. All of the four nevocellular nevi and eight primary melanomas <4 mm in thickness showed KiSS1 mRNA expression, whereas only 50% (6 of 12) of primary melanomas >4 mm in thickness expressed KiSS1. Loss of KiSS1 mRNA was equally frequent in metastases; 44% (12 of 27) of tumors lost KiSS1 expression. LOH of 6q16.3-q23 was observed in 52% (14 of 27) of metastases. There was a strong association between LOH and loss of KiSS1 expression (P = 0.03); nine metastases with LOH of 6q16.3-q23 lost KiSS1 expression, whereas 10 tumors with no LOH showed positive KiSS1 mRNA expression. The findings in this study show, for the first time, KiSS1 down-regulation during the progression of melanoma in vivo and strongly suggest that inactivation of a tumor suppressor gene(s) mapping to 6q16.3-q23 by deletion or mutation coupled with LOH may lead to the down-regulation of KiSS1.

Adult↗

PubMed2001

Rad52 partially substitutes for the Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells.

Yeast Rad52 DNA-repair mutants exhibit pronounced radiation sensitivity and a defect in homologous re combination (HR), whereas vertebrate cells lacking Rad52 exhibit a nearly normal phenotype. Bio chemical studies show that both yeast Rad52 and Rad55-57 (Rad51 paralogs) stimulate DNA-strand exchange mediated by Rad51. These findings raise the possibility that Rad51 paralogs may compensate for lack of Rad52 in vertebrate cells, explaining the absence of prominent phenotypes for Rad52-deficient cells. To test this hypothesis, using chicken DT40 cells, we generated conditional mutants deficient in both RAD52 and XRCC3, which is one of the five vertebrate RAD51 paralogs. Surprisingly, the rad52 xrcc3 double-mutant cells were non-viable and exhibited extensive chromosomal breaks, whereas rad52 and xrcc3 single mutants grew well. Our data reveal an overlapping (but non-reciprocal) role for Rad52 and XRCC3 in repairing DNA double-strand breaks. The present study shows that Rad52 can play an important role in HR repair by partially substituting for a Rad51 paralog.

Animals↗

PubMed2001

Genetic analysis of the DNA-dependent protein kinase reveals an inhibitory role of Ku in late S-G2 phase DNA double-strand break repair.

Two major complementary double-strand break (DSB) repair pathways exist in vertebrates, homologous recombination (HR), which involves Rad54, and non-homologous end-joining, which requires the DNA-dependent protein kinase (DNA-PK). DNA-PK comprises a catalytic subunit (DNA-PKcs) and a DNA-binding Ku70 and Ku80 heterodimer. To define the activities of individual DNA-PK components in DSB repair, we targeted the DNA-PKcs gene in chicken DT40 cells. DNA-PKcs deficiency caused a DSB repair defect that was, unexpectedly, suppressed by KU70 disruption. We have shown previously that genetic ablation of Ku70 confers RAD54-dependent radioresistance on S-G(2) phase cells, when sister chromatids are available for HR repair. To test whether direct interference by Ku70 with HR might explain the Ku70(-/-)/DNA-PKcs(-/-/-) radioresistance, we monitored HR activities directly in Ku- and DNA-PKcs-deficient cells. The frequency of intrachromosomal HR induced by the I-SceI restriction enzyme was increased in the absence of Ku but not of DNA-PKcs. Significantly, abrogation of HR activity by targeting RAD54 in Ku70(-/-) or DNA-PKcs(-/-/-) cells caused extreme radiosensitivity, suggesting that the relative radioresistance seen with loss of Ku70 was because of HR-dependent repair pathways. Our findings suggest that Ku can interfere with HR-mediated DSB repair, perhaps competing with HR for DSB recognition.

Animals↗

PubMed2001

Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation.

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.

Animals↗

PubMed2001

Homologous DNA recombination in vertebrate cells.

The RAD52 epistasis group genes are involved in homologous DNA recombination, and their primary structures are conserved from yeast to humans. Although biochemical studies have suggested that the fundamental mechanism of homologous DNA recombination is conserved from yeast to mammals, recent studies of vertebrate cells deficient in genes of the RAD52 epistasis group reveal that the role of each protein is not necessarily the same as that of the corresponding yeast gene product. This review addresses the roles and mechanisms of homologous recombination-mediated repair with a special emphasis on differences between yeast and vertebrate cells.

Alleles↗

PubMed2001

Homologous-pairing activity of the human DNA-repair proteins Xrcc3.Rad51C.

The human Xrcc3 protein is involved in the repair of damaged DNA through homologous recombination, in which homologous pairing is a key step. The Rad51 protein is believed to be the only protein factor that promotes homologous pairing in recombinational DNA repair in mitotic cells. In the brain, however, Rad51 expression is extremely low, whereas XRCC3, a human homologue of Saccharomyces cerevisiae RAD57 that activates the Rad51-dependent homologous pairing with the yeast Rad55 protein, is expressed. In this study, a two-hybrid analysis conducted with the use of a human brain cDNA library revealed that the major Xrcc3-interacting protein is a Rad51 paralog, Rad51C/Rad51L2. The purified Xrcc3.Rad51C complex, which shows apparent 1:1 stoichiometry, was found to catalyze the homologous pairing. Although the activity is reduced, the Rad51C protein alone also catalyzed homologous pairing, suggesting that Rad51C is a catalytic subunit for homologous pairing. The DNA-binding activity of Xrcc3.Rad51C was drastically decreased in the absence of Xrcc3, indicating that Xrcc3 is important for the DNA binding of Xrcc3.Rad51C. Electron microscopic observations revealed that Xrcc3.Rad51C and Rad51C formed similar filamentous structures with circular single-stranded DNA.

Base Sequence↗

PubMed2001

Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group.

Rejoining of ionizing radiation (IR) induced DNA DSBs usually follows biphasic kinetics with a fast (t(50): 5-30 min) component attributed to DNA-PK-dependent non-homologous endjoining (NHEJ) and a slow (t(50): 1-20 h), as of yet uncharacterized, component. To examine whether homologous recombination (HR) contributes to DNA DSB rejoining, a systematic genetic study was undertaken using the hyper-recombinogenic DT40 chicken cell line and a series of mutants defective in HR. We show that DT40 cells rejoin IR-induced DNA DSBs with half times of 13 min and 4.5 h and contributions by the fast (78%) and the slow (22%) components similar to those of other vertebrate cells with 1000-fold lower levels of HR. We also show that deletion of RAD51B, RAD52 and RAD54 leaves unchanged the rejoining half times and the contribution of the slow component, as does also a conditional knock out mutant of RAD51. A significant reduction (to 37%) in the contribution of the fast component is observed in Ku70(-/-) DT40 cells, but the slow component, operating with a half time of 18.4 h, is still able to rejoin the majority (63%) of DSBs. A double mutant Ku70(-/-)/RAD54(-/-) shows similar half times to Ku70(-/-) cells. Thus, variations in HR by several orders of magnitude leave unchanged the kinetics of rejoining of DNA DSBs, and fail to modify the contribution of the slow component in a way compatible with a dependence on HR. We propose that, in contrast to yeast, cells of vertebrates are 'hard-wired' in the utilization of NHEJ as the main pathway for rejoining of IR-induced DNA DSBs and speculate that the contribution of homologous recombination repair (HRR) is at a stage after the initial rejoining.

Animals↗

PubMed2001

Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40.

DT40 is an avian leucosis virus-transformed chicken B-lymphocyte line which exhibits high ratios of targeted to random integration of transfected DNA constructs. This efficient targeted integration may be related to the ongoing diversification of the variable segment of the immunoglobulin gene through homologous DNA recombination-controlled gene conversion. DT40s are a convenient model system for making gene-targeted mutants. Another advantage is the relative tractability of these cells, which makes it possible to disrupt multiple genes in a single cell and to generate conditionally gene-targeted mutants including temperature-sensitive mutants. There are strong phenotypic similarities between murine and DT40 mutants of various genes involved in DNA recombination. These similarities confirm that the DT40 cell line is a reasonable model for the analysis of vertebrate DNA recombination, despite obvious concerns associated with the use of a transformed cell line, which may have certain cell-line-specific characteristics. Here we describe our studies of homologous DNA recombination in vertebrate somatic cells using reverse genetics in DT40 cells.

Animals↗

PubMed2001

Pentagonal-dodecahedral la2 charge density in [80-ih ]fullerene: la2 @c80.

A double shell of Platonic solids: A trajectory that connects the six-membered rings of Ih -C80 is followed by the two La atoms of La2 @C80 according to maximum entropy/Rietveld analysis of the synchrotron powder-diffraction data. The result is a pentagonal-dodecahedral charge-density distribution for La2 (brown in the picture) inside the icosahedral C80 cage (green).

X-ray diffraction↗

PubMed2001

Arrest of cell division and nucleoid partition by genetic alterations in the sliding clamp of the replicase and in DnaA.

In Escherichia coli, an interaction between the replication initiator DnaA and the sliding clamp protein, the beta subunit (DnaN) of DNA polymerase III, is required to regulate the chromosomal replication cycle. We report here that colony formation by, and cell division of, the temperature (42 degrees C)-sensitive dnaN59 mutant are inhibited at 34-35 degrees C when DnaA is moderately (4-to 8-fold ) overexpressed, although chromosomal replication and the beta subunit-dependent regulation of DnaA activity are not significantly inhibited. Immunoblotting analysis revealed that the beta subunit is abundant (present at a level of about 5000 dimers per cell) at 34 degrees C, and its concentration per unit cell volume was practically unaffected in the dnaN59 mutant by the overexpression of DnaA. The dnaN mutant cells that overexpress DnaA become filamentous at 34 degrees C via an sfiA-independent pathway, different from that activated by the SOS response. This filamentation is accompanied by inhibition of nucleoid partition and FtsZ ring formation. In the dnaN59 mutant, oversupply of DnaA may disturb the coordinated action of cell cycle-regulating molecules, thus leading to the inhibition of these events.

Bacterial Proteins↗

PubMed2001

Glucosamine enhances platelet-derived growth factor-induced DNA synthesis via phosphatidylinositol 3-kinase pathway in rat aortic smooth muscle cells.

Vascular smooth muscle cells play a key role in the development of atherosclerosis. Culture of vascular smooth muscle A10 cells with high glucose for 4 weeks enhanced platelet-derived growth factor (PDGF)-induced BrdU incorporation. Since a long period of high glucose incubation was required for the effect, and it was inhibited by co-incubation with azaserine, the role of hexosamine biosynthesis in the development of atherosclerosis in diabetes was studied in A10 cells. Addition of glucosamine to the culture media enhanced PDGF-stimulated BrdU incorporation, and PDGF-induced tyrosine phosphorylation of the PDGF beta-receptor was increased by glucosamine treatment. Of the subsequent intracellular signaling pathways, PDGF-induced PDGF beta-receptor association with PLC gamma was not affected, whereas tyrosine phosphorylation of Shc, subsequent association of Shc with Grb2, and MAP kinase activation were relatively decreased. In contrast, PDGF-induced PDGF beta-receptor association with the p85 regulatory subunit of PI3-kinase and PI3-kinase activation were increased by 20% (P<0.01) and 36% (P<0.01), respectively. The intracellular signaling molecules responsible for the glucosamine effect were further examined using pharmacological inhibitors. Pretreatment with PLC inhibitor (U73122) had negligible effects, and MEK1 inhibitor (PD98059) showed only a slight inhibitory effect on the PDGF-induced BrdU incorporation. In contrast, pretreatment with PI3-kinase inhibitor (LY294002) significantly inhibited glucosamine enhancement of PDGF-induced BrdU incorporation. These findings suggest that glucosamine is involved in the development of atherosclerosis by enhancing PDGF-induced mitogenesis specifically via the PI3-kinase pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Pravastatin suppresses the interleukin-8 production induced by thrombin in human aortic endothelial cells cultured with high glucose by inhibiting the p44/42 mitogen activated protein kinase.

1. 3-Hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) prevent the progression of atherosclerosis by lowering cholesterol. However, the effect of statins on the synthesis of pro-inflammatory cytokines from endothelial cells has not yet been fully investigated. Here, we examined the effect of pravastatin, one of the statins, on IL-8 synthesis induced by thrombin in human aortic endothelial cells (AoEC) cultured with high glucose concentrations. 2. Pravastatin significantly decreased the IL-8 synthesis induced by thrombin. 3. Pravastatin inhibited the p44/42 MAP kinase activity induced by thrombin, but did not inhibit the p38 MAP kinase activity. 4. Translocation of ras protein from the cytosol to plasma membrane was inhibited by pravastatin. 5. Pravastatin inhibit the activator protein-1 activity, but did not inhibit the activation of IkappaB-alpha. 6. Dominant negative ras inhibited the p44/42 MAP kinase activity induced by PMA. 7. Our results suggest that pravastatin inhibits IL-8 synthesis by blocking the ras-MAP (p44/42) kinase pathway rather than nuclear factor-kappaB. Pravastatin may prevent atherosclerosis not only by lowering cholesterol levels, but also by suppressing IL-8 synthesis in AoEC through the inhibition of p44/42 MAP kinase, and this may be more beneficial in diabetic patients than in non-diabetics.

Anticholesteremic Agents↗

PubMed2001

No evidence of deregulated patched-hedgehog signaling pathway in trichoblastomas and other tumors arising within nevus sebaceous.

Nevus sebaceous is a congenital malformation of the skin within which a number of neoplasms showing adnexal differentiation may arise. Recently, deletions in the patched gene region were reported in nevus sebaceous and constitutive activation of the patched-hedgehog signaling pathway was implicated in the development of tumors arising within nevus sebaceous. To substantiate further a role of the patched-hedgehog signaling pathway in secondary tumors arising within nevus sebaceous, we examined 11 nevus sebaceous associated with secondary tumors for loss of heterozygosity of the patched gene region by microsatellite polymerase chain reaction and patched mRNA expression by in situ hybridization. Unexpectedly, however, none of the tumors (including eight trichoblastomas) and nevus sebaceous lesions showed loss of heterozygosity at any polymorphic loci close to the patched gene. Further more, none of the nevus sebaceous lesions and secondary tumors gave detectable signals for patched mRNA. In contrast, four of 11 sporadic basal cell carcinomas, that were examined for comparison, showed loss of heterozygosity at the patched gene locus (p <0.05), and moderate to strong signals for patched mRNA was observed in all seven basal cell carcinoma tumors examined (p <0.0001). Additional investigation by reverse transcription-polymerase chain reaction in four basal cell carcinomas and two nevus sebaceous tumors also showed the expression of Gli-1, another target gene in the patched-hedgehog signaling pathway, in all the basal cell carcinomas samples but not in any of the nevus sebaceous tumors examined. The findings in this study do not support the view that the deregulation of the patched-hedgehog signaling pathway is involved in the pathogenesis of nevus sebaceous and associated tumors, and show that, although morphologically similar, trichoblastomas and basal cell carcinomas have a different molecular pathogenesis.

Adult↗

PubMed2001

Antisense oligodeoxynucleotides to latent membrane protein 1 induce growth inhibition, apoptosis and Bcl-2 suppression in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells, but not in EBV-positive natural killer cell lymphoma cells.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP-1) is essential for immortalization of B cells by EBV, protects the infected cells from apoptotic cell death and induces Bcl-2 expression. Suppression of LMP-1 expression by antisense oligodeoxynucleotides (AS-oligo) to LMP-1 inhibits proliferation, promotes apoptosis and suppresses Bcl-2 expression in EBV-transformed B cells. However, the function of LMP-1 expression in EBV-positive natural killer (NK) cell lymphoma cells has not been reported previously. We examined the function of LMP-1 in two EBV-positive NK cell lymphoma cell lines (NK-YS and YT) through suppressing LMP-1 expression by AS-oligo to LMP-1. The AS-oligo to LMP-1 suppressed LMP-1 mRNA and protein expression in two EBV-positive NK cell lymphoma cell lines, as well as in an EBV-transformed B-cell line (CMG-1). Proliferation was inhibited, apoptosis was induced and Bcl-2 expression was suppressed in CMG-1 cells, but none of these events were observed in NK-YS or YT cells. These results suggest that proliferation, inhibition of apoptosis and Bcl-2 expression in EBV-positive NK cell lymphoma cells are not directly regulated by LMP-1 as in EBV-transformed B-cell lines, but are probably mediated through other signal transducing systems.

Adaptor Proteins, Signal Transducing↗

PubMed2001

DNA replication-coupled inactivation of DnaA protein in vitro: a role for DnaA arginine-334 of the AAA+ Box VIII motif in ATP hydrolysis.

The DnaA protein, which initiates chromosomal replication in Escherichia coli, is negatively regulated by both the sliding clamp of DNA polymerase III holoenzyme and the IdaB protein. We have found that, when the amount of minichromosome is limited in an in vitro replication system, minichromosomal replication-stimulated hydrolysis of DnaA-bound ATP yields the ADP-bound inactive form. The number of sliding clamps formed during replication was at least five per minichromosome, which is 2.7-fold higher than the number formed during incubation without replication. These results support the notion that coupling of DnaA-ATP hydrolysis to DNA replication is the outcome of enhanced clamp formation. We have also found that the amino acid substitution R334H in DnaA severely inhibits the hydrolysis of bound ATP in vitro. Whereas ATP bound to wild-type DnaA is hydrolysed in a DNA-dependent intrinsic manner or in a sliding clamp-dependent manner, ATP bound to DnaA R334H protein was resistant to hydrolysis under the same conditions. This arginine residue may be located in the vicinity where ATP binds, and therefore may play an essential role in ATP hydrolysis. This residue is highly conserved among DnaA homologues and also in the Box VIII motif of the AAA+ protein family.

Adenosine Triphosphate↗

PubMed2001

Pemphigus foliaceus developing after metastasis of cutaneous squamous cell carcinoma to regional lymph nodes.

We describe a patient in whom pemphigus foliaceus developed after cutaneous squamous cell carcinoma (SCC) metastasized to regional lymph nodes. Immunologic analysis revealed that production of anti-desmoglein 1 autoantibodies started when SCC metastasized, and the SCC expressed desmoglein 1, suggesting a pathogenic role of metastasized SCC in developing pemphigus foliaceus.

Aged↗