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Biomedical subjects

I Okamoto

Publications and source records attributed to I Okamoto.

At least 19 recordsLinked to original sources
PubMed2006

The dynamics of imprinted X inactivation during preimplantation development in mice.

In the mouse, there are two forms of X chromosome inactivation (XCI), random XCI in the fetus and imprinted paternal XCI, which is limited to the extraembryonic tissues. While the mechanism of random XCI has been studied extensively using the in vitro XX ES cell differentiation system, imprinted XCI during early embryonic development has been less well characterized. Recent studies of early embryos have reported unexpected findings for the paternal X chromosome (Xp). Imprinted XCI may not be linked to meiotic silencing in the male germ line but rather to the imprinted status of the Xist gene. Furthermore, the Xp becomes inactivated in all cells of cleavage-stage embryos and then reactivated in the cells of the inner cell mass (ICM) that form the epiblast, where random XCI ensues.

Animals↗
PubMed2002

Protein kinase C isoforms in normal and transformed cells of the melanocytic lineage.

Enzymes belonging to the protein kinase C (PKC) family represent one of the major mediators of signal transduction in melanocytes. To identify PKC isoforms that may be associated with the process of malignant transformation and metastasis, we investigated the expression pattern of 11 different PKC isoforms (alpha, beta I, beta II, gamma, delta, epsilon, eta, theta, zeta, lambda, and iota) in melanoma lymph node metastases, in cell lines established from these metastases, in primary cell cultures from normal melanocytes, and in permanent cell lines established from spontaneously transformed melanocytes. PKC alpha, beta I, beta II, delta, epsilon, eta, zeta, lambda and iota were found to be expressed in total lysates from melanoma metastases. In permanent cell lines established from these metastases, the expression levels of PKC beta I, beta II, delta, epsilon, and eta were lower or undetectable when compared with initial expression in tumour lysates. In normal primary melanocyte cultures, the PKC isoforms beta II, delta, epsilon, eta and iota were undetectable. PKC gamma and theta isoforms were undetectable in all melanocytic cell types examined. PKC iota was the only isoform exclusively detected in tumour lysates, in spontaneously transformed melanoma cells and melanoma cell lines, but not in normal melanocytes, and may therefore be associated with the transformed phenotype in human melanoma in vitro and in vivo.

3T3 Cells↗
PubMed2002

Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells.

Inactivation of the X chromosome during early female development and the subsequent maintenance of this transcriptionally inert state through countless cell divisions remain a paradigm for epigenetic regulation in mammals. Nevertheless, the exact mechanisms underlying this chromosome-wide silencing process remain unclear. Using differentiating female embryonic stem (ES) cells as a model system, we recently found that histone H3 tail modifications are among the earliest known chromatin changes in the X inactivation process, appearing as soon as Xist RNA accumulates on the X chromosome, but prior to transcriptional silencing of X-linked genes (Heard et al., 2001). In this report we present an integrated analysis of the sequence of early events and chromatin modifications underlying X inactivation in differentiating female ES cells. We have extended our previous analysis concerning changes in histone tail modification states. We find that the hypomethylation of Arg-17 and that of Lys-36 on histone H3 also characterize the inactive X chromosome, and that these profiles show a similarly early onset during the initiation of X inactivation. In addition, we have investigated the kinetics of the shift in replication timing of the X chromosome undergoing inactivation. This event occurs slightly later than Xist RNA coating and the chromatin modifications. Finally, from an early stage in the X inactivation process, characteristic histone modification patterns can be found on the X chromosome at mitosis, suggesting that they represent true epigenetic marks of the inactive state.

Acetylation↗
PubMed2001

Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway.

CD44 is a widely distributed cell surface adhesion molecule and is implicated in diverse biological processes. However, the nature of intracellular signaling triggered by CD44 remains to be elucidated. Here, we show that CD44 undergoes sequential proteolytic cleavage in the ectodomain and intracellular domain, resulting in the release of a CD44 intracellular domain (ICD) fragment. Consequently, CD44ICD acts as a signal transduction molecule, where it translocates to the nucleus and activates transcription mediated through the 12-O-tetradecanoylphorbol 13-acetate-responsive element, which is found in numerous genes involved in diverse cellular processes. Expression of an uncleavable CD44 mutant as well as metalloprotease inhibitor treatment blocks CD44-mediated transcriptional activation. In search of the underlying mechanism, we have found that CD44ICD potentiates transactivation mediated by the transcriptional coactivator CBP/p300. Furthermore, we show that cells expressing CD44ICD produce high levels of CD44 messenger RNA, suggesting that the CD44 gene is one of the potential targets for transcriptional activation by CD44ICD. These observations establish a novel CD44 signaling pathway and shed new light on the functional link between proteolytic processing of an adhesion molecule at the cell surface and transcriptional activation in the nucleus.

Active Transport, Cell Nucleus↗
PubMed2001

Methotrexate poisoning with acute hepatorenal dysfunction.

A 17-year-old girl receiving high-dose methotrexate for the treatment of osteosarcoma developed complications of acute renal failure and liver dysfunction with a coagulation disorder. The methotrexate concentrations were quickly reduced from 600 micromol/L to 50 micromol/L by treatment with plasma exchange and hemodialysis at 72 hours after discontinuation of the drug. After this reduction, continuous hemodiafiltration was initiated to further lower the methotrexate concentrations because of the persistently high and then the actual rebound in the plasma concentrations after plasma exchange and hemodialysis treatment. Continuous hemodiafiltration was able to reduce the concentrations without any rebound, despite its low column clearance. The rebound in plasma methotrexate concentrations seems to be corrected by plasma methotrexate after plasma exchange and/or hemodialysis.

Acute Kidney Injury↗
PubMed2000

Ras oncoprotein induces CD44 cleavage through phosphoinositide 3-OH kinase and the rho family of small G proteins.

CD44 is a cell surface adhesion molecule for several extracellular matrix components. We previously showed that CD44 expressed in cancer cells is proteolytically cleaved at the ectodomain through membrane-anchored metalloproteases and that CD44 cleavage plays a critical role in cancer cell migration. Therefore, cellular signals that promote the migration and metastatic activity of cancer cells may regulate the CD44 ectodomain cleavage. Here, we demonstrate that the expression of the dominant active mutant of Ha-Ras (Ha-Ras(Val-12)) induces redistribution of CD44 to the newly generated membrane ruffling area and CD44 ectodomain cleavage. The migration assay revealed that the CD44 cleavage contributes to the Ha-Ras(Val-12)-induced migration of NIH3T3 cells on hyaluronate substrate. Treatment with LY294002, an inhibitor for phosphoinositide 3-OH kinase (PI3K), significantly inhibits Ha-Ras(Val-12)-induced CD44 cleavage, whereas that with PD98059, an inhibitor for MEK, does not. The active mutant p110 subunit of PI3K has also been shown to enhance the CD44 cleavage, suggesting that PI3K mediates the Ras-induced CD44 cleavage. Moreover, the expression of dominant negative mutants of Cdc42 and Rac1 inhibits the Ha-Ras(Val-12)-induced CD44 cleavage. These results suggest that Ras > PI3K > Cdc42/Rac1 pathway plays an important role in CD44 cleavage and may provide a novel molecular basis to explain how the activated Ras facilitates cancer cell migration.

3T3 Cells↗
PubMed2000

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine.

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.

Archaea↗
PubMed2000

IL-18 prevents the development of chronic graft-versus-host disease in mice.

The development of chronic graft-versus-host disease (GVHD), which is induced by the transfer of DBA/2 spleen cells into (C57BL/6 x DBA/2)F1 (BDF1) mice, is closely related to diminished donor anti-host CTL activity and host B cell hyperactivation. Therefore, an approach which activates donor CD8+ T cells or suppresses donor CD4+ T cell-host B cell interaction may have clinical utility in the treatment of chronic GVHD. We have previously demonstrated that IL-18 induces the development of naive CD8+ T cells into type I effector cells in DBA/2 anti-BDF1 MLC. In this paper we examined the effect of IL-18 administration on the development of chronic GVHD in mice. The treatment was started before or after the onset of clinical evidence of the disease. Regardless of the treatment schedule, IL-18 significantly decreased immunological parameters indicative of chronic GVHD, such as elevated serum IgG antinuclear Abs, IgG1, and IgE levels, and host B cell numbers and their activation. Importantly, IL-18-treated mice did not show the same acute GVHD-like symptoms reported for IL-12 treatment, because there was no weight loss, death, or severe immunodeficiency as indicated by a decrease in IL-2 and IFN-gamma production by Con A-stimulated spleen cells. In contrast, IL-18 treatment partially but significantly restored the production of these cytokines. Data further suggested that these IL-18-mediated therapeutic effects may be due to the induction of donor CD8+ CTL, the decrease in donor CD4+ T cell numbers, and a down-regulation of host B cell MHC class II expression. Thus, our results suggest that IL-18 has beneficial effects in the prevention and treatment of chronic GVHD.

Acute Disease↗
PubMed2000

Karyotypic findings in two cases of male breast cancer.

Male breast cancer is uncommon; so far, only 10 cases with chromosome banding analysis have been published. We report the cytogenetic findings of two invasive breast cancers in two Caucasian men lacking a history of familial breast cancer and more than 70 years of age. Both had ductal carcinomas with lymphangiosis carcinomatosa and positive lymph nodes at diagnosis. Strong expression of estrogen receptor, weak expression of progesterone receptor, and lack of expression of androgen receptor by both tumors were demonstrated by immunohistochemistry, as well as lack of expression of p53 and C-ERB-B-2. The karyotypes were 45 approximately 46,XY,-Y[4],-7[2],+8[2],t(8;12)(q21;q24)[3], del(9)(q22)[3],del(11)(p11p14)[5],del(18)(q21)[7], t(19;20)(p10;q10)[8] [cp13] and 61 approximately 69,XXXY,-Y[3], del(2)(p21)[4],del(3)(p22q26)[3],-4,-4[5],+5,+5[5], dic(5;11)(p14;q23)[3],del(6)(q23)[4],del(8)(p21)[3],-9[4],-11[4],+ i(12)(p10)[4],-16[3],del(17)([13)[5],del(18)(q21)[4],+19[5], +20[4][cp7], respectively. Although the available data on male breast cancer are still very limited, our findings confirm that gain of an X chromosome, loss of the Y chromosome, gain of chromosome 5, and loss of material from chromosomes 17 and 18 are nonrandom aberrations in male breast cancer. Trisomy 8, characteristic of ductal carcinomas, was found in one case.

Aged↗
PubMed2000

Prevalence and distribution of hepatitis C subtypes in patients with opioid dependence.

UNLABELLED: AIM AND SETTING: The drug addiction out-patient clinic at the University Hospital for Psychiatry in Vienna performed a study to identify the prevalence of hepatitis C virus (HCV) infections in a group of opiate-dependent patients, to detect the distribution of HCV subtypes and to calculate the comorbidity of human immunodeficiency virus (HIV) and hepatitis B virus (HBV). DESIGN AND PARTICIPANTS: We consecutively investigated unselected patients (n = 173) during an observation period of 2 months with the diagnosis of opioid dependence (DSM-IV: 304.0) and polysubstance dependence (DSM-IV: 304.9). MEASUREMENTS: Blood was investigated focusing on liver enzymes and on viral status including HIV, hepatitis B and hepatitis C, followed by subtyping of the virus. FINDINGS: In 80.3% hepatitis C antibodies were found, 66.5% were HCV RNA (PCR) positive. 3a was the most frequent subtype (35.6%), followed by 1a (28.8%) and 1b (22.0%). Four patients had both subtypes 1a and 1b (6.8%), 3 were 2b positive (5.1%) and 1 patient had subtypes 2a/2c (1.7%). No significant difference in aspartate (AST) and alanine aminotransferases (ALT) concerning the different subtypes (AST: p = 0.290; ALT: p = 0.260) could be calculated; 11.6% showed co-infection with HIV, 2 patients had a chronic infection with hepatitis B. CONCLUSIONS: The rate of HCV infection in substance-dependent patients at our drug addiction out-patient clinic is extremely high. The distribution of subtypes showed a relatively homogeneous distribution of the types 1a, 1b and 3a. The recommended therapy with alpha-interferon should be initiated in drug-dependent patients under considerations of an enrollment in oral maintenance with synthetic opioids.

Adolescent↗
PubMed2000

Evaluating the role of inducible nitric oxide synthase using a novel and selective inducible nitric oxide synthase inhibitor in septic lung injury produced by cecal ligation and puncture.

We studied the role of inducible nitric oxide synthase (iNOS) in septic lung injury using a novel and selective iNOS inhibitor (a fused piperidine derivative; ONO-1714) following a cecal ligation and puncture (CLP) procedure. All rats that received CLP died within 48 h after the intervention. The subcutaneous injection of ONO-1714 at 0.03 mg/kg every 12 h resulted in a significantly longer survival time than the saline control only when administration was started 12 h after the CLP procedure. The other administration schedules, which started immediately or 6 h after the intervention, did not show any improvement in the survival rates in comparison with the saline control. The administration of ONO-1714 at higher (0.1 mg/ kg) or lower (0.01 mg/kg) doses when given anytime after the intervention did not improve the survival rates. The NO(x) (NO(2)(-) + NO(3)(-)) levels in the plasma significantly increased 12 h after intervention in comparison with NO(x) at 0 h and thereafter further increased in parallel with the time elapsed. The CLP rats that were initially treated with ONO-1714 (0.03 mg/kg subcutaneously every 12 h) 12 h after intervention showed significantly reduced NO(x) levels in the plasma in comparison with the saline control. The NO synthase activity in lung homogenates increased from 6 to 24 h after the CLP and thereafter decreased to 42 h. The administration of ONO-1714 inhibited iNOS activity (under calcium-free conditions) in preference to total iNOS activity (under calcium-dependent conditions) in lung homogenates, which thus suggested that this compound selectively inhibited iNOS in lung tissue. The iNOS protein expression in the lung and liver homogenates showed a similar time course with alterations of NOS activity, namely a maximum level at 24 h after the intervention followed by decreasing levels to 42 h. On the other hand, other isozymes of NOS, eNOS, and nNOS in lung homogenates, were constantly expressed over the time course after the CLP. Since the iNOS mRNA expression in lung homogenates continued to elevate until 42 h, the decrease in iNOS activity and protein expression later than 24 h after the CLP was thus considered to be due to some posttranscriptional mechanism during the late phase of sepsis. In conclusion, intervention with a potent and selective iNOS inhibitor seemed to improve survival in CLP rats when used at the appropriate doses and time points. However, the self-limited mechanism of iNOS regulation in the lungs may also indicate that iNOS plays only a limited role in sepsis and septic shock.

Amidines↗
PubMed2000

X-chromosome inactivation in XX androgenetic mouse embryos surviving implantation.

Using genetic and cytogenetic markers, we assessed early development and X-chromosome inactivation (X-inactivation) in XX mouse androgenones produced by pronuclear transfer. Contrary to the current view, XX androgenones are capable of surviving to embryonic day 7.5, achieving basically random X-inactivation in all tissues including those derived from the trophectoderm and primitive endoderm that are characterized by paternal X-activation in fertilized embryos. This finding supports the hypothesis that in fertilized female embryos, the maternal X chromosome remains active until the blastocyst stage because of a rigid imprint that prevents inactivation, whereas the paternal X chromosome is preferentially inactivated in extra-embryonic tissues owing to lack of such imprint. In spite of random X-inactivation in XX androgenones, FISH analyses revealed expression of stable Xist RNA from every X chromosome in XX and XY androgenonetic embryos from the four-cell to morula stage. Although the occurrence of inappropriate X-inactivation was further suggested by the finding that Xist continues ectopic expression in a proportion of cells from XX and XY androgenones at the blastocyst and the early egg cylinder stage, a replication banding study failed to provide positive evidence for inappropriate X-inactivation at E6. 5.

Animals↗
PubMed2000

Iron(III)picolinate-induced oxygenation and subsequent rearrangement of triterpenoid derivatives with hydrogen peroxide.

The reaction of oleanane triterpenoid 1b with a FeIII(PA; picolinate)3/H2O2/MeCN system (reagent system A), a simple model system for mono-oxygenase, gave the 11 alpha-hydroxyl derivative 3 as major product, along with 11-oxo derivative 4 and 12-oxo derivative 6. The reaction of lupane triterpenoid 2b with reagent system A gave only oxidative rearrangement compounds, (20R)-aldehyde 8 and (20S)-aldehyde 9 were epimeric isomers. Then, we have found that iron(III) picolinate complex, FeIII(PA)3 is efficient in effecting the rearrangement of triterpenoid epoxides 5 and 7 into the corresponding carbonyl compounds, 6, 8 and 9 with 1,2-shift of the hydride.

Acetylation↗
PubMed1999

Human ECT2 is an exchange factor for Rho GTPases, phosphorylated in G2/M phases, and involved in cytokinesis.

Animal cells divide into two daughter cells by the formation of an actomyosin-based contractile ring through a process called cytokinesis. Although many of the structural elements of cytokinesis have been identified, little is known about the signaling pathways and molecular mechanisms underlying this process. Here we show that the human ECT2 is involved in the regulation of cytokinesis. ECT2 catalyzes guanine nucleotide exchange on the small GTPases, RhoA, Rac1, and Cdc42. ECT2 is phosphorylated during G2 and M phases, and phosphorylation is required for its exchange activity. Unlike other known guanine nucleotide exchange factors for Rho GTPases, ECT2 exhibits nuclear localization in interphase, spreads throughout the cytoplasm in prometaphase, and is condensed in the midbody during cytokinesis. Expression of an ECT2 derivative, containing the NH(2)-terminal domain required for the midbody localization but lacking the COOH-terminal catalytic domain, strongly inhibits cytokinesis. Moreover, microinjection of affinity-purified anti-ECT2 antibody into interphase cells also inhibits cytokinesis. These results suggest that ECT2 is an important link between the cell cycle machinery and Rho signaling pathways involved in the regulation of cell division.

Antibodies↗
PubMed1999

Calcium influx triggers the sequential proteolysis of extracellular and cytoplasmic domains of E-cadherin, leading to loss of beta-catenin from cell-cell contacts.

Cadherins are major cell-cell adhesion molecules in both tumor and normal tissues. Although serum levels of soluble E-cadherin have been shown to be higher in the cancer patients than in healthy volunteers, the detail mechanism regulating release of soluble E-cadherin remains to be elucidated. Here we show that the ectodomain of E-cadherin is proteolytically cleaved from some cancer cells by a membrane-bound metalloprotease to yield soluble form, and the residual membrane-tethered cleavage product is subsequently degraded by intracellular proteolytic pathway. Futhermore, we show that extracellular calcium influx, that is induced by mechanical scraping of cells or ionomycin treatment, enhances the metalloprotease-mediated E-cadherin cleavage and the subsequent degradation of the cytoplasmic domain. Immunocytochemical analysis demonstrates that the sequential proteolysis of E-cadherin triggered by the calcium influx results in translocation of beta-catenin from the cell-cell contacts to cytoplasm. Our data suggest that calcium influx-induced proteolysis of E-cadherin not only disrupts the cell-cell adhesion but also activates beta-catenin-mediated intracellular signaling pathway, potentially leading to alterations in motility and proliferation activity of cells.

Base Sequence↗
PubMed1999

Regulated CD44 cleavage under the control of protein kinase C, calcium influx, and the Rho family of small G proteins.

CD44 is a cell surface receptor for several extracellular matrix components and is implicated in tumor cell invasion and metastasis. Our previous studies have shown that CD44 expressed in cancer cells is proteolytically cleaved at the extracellular domain through membrane-associated metalloproteases and that CD44 cleavage plays a critical role in CD44-mediated tumor cell migration (Okamoto, I., Kawano, Y., Tsuiki, H., Sasaki, J., Nakao, M., Matsumoto, M., Suga, M., Ando, M., Nakajima, M., and Saya, H. (1999) Oncogene 18, 1435-1446). In the present study, we first demonstrate rapid degradation of the membrane-tethered CD44 cleavage product through intracellular proteolytic pathways, and it occurs only after CD44 extracellular cleavage. To address the mechanisms regulating CD44 cleavage at the extracellular domain, we show that 12-O-tetradecanoylphorbol 13-acetate (TPA) and the calcium ionophore ionomycin rapidly enhance metalloprotease-mediated CD44 cleavage in U251MG cells via protein kinase C-dependent and -independent pathways, respectively, suggesting the existence of multiple distinct pathways for regulation of CD44 cleavage. Concomitant with TPA-induced CD44 cleavage, TPA treatment induces redistribution of CD44 and ERM proteins (ezrin, radixin, and moesin) to newly generated membrane ruffling areas. Treatment with lysophosphatidic acid, which is known to activate the Rho-dependent pathway, inhibits TPA-induced CD44 redistribution and CD44 cleavage. Furthermore, overexpression of Rac dominant active mutants results in the redistribution of CD44 to the Rac-induced ruffling areas and the enhancement of CD44 cleavage. These results suggest that the Rho family proteins play a role in regulation of CD44 distribution and cleavage.

Calcium↗