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Biomedical subjects

Y Kanakura

Publications and source records attributed to Y Kanakura.

At least 19 recordsLinked to original sources
PubMed2006

Role of DNA methylation for expression of novel stem cell marker CDCP1 in hematopoietic cells.

CDCP1, a novel stem cell marker, is expressed in hematopoietic cell line K562 but not in Jurkat. When CDCP1 promoter was transfected exogenously, Jurkat showed comparable promoter activity with K562, suggesting that the factor to enhance transcription was present but interfered to function in Jurkat. The reporter assay and si-RNA-mediated knockdown experiment revealed that zfp67, a zinc-finger protein, enhanced CDCP1 transcription. Amount of zfp67 in Jurkat was comparable with K562, but chromatin immunoprecipitation showed that zfp67 bound to CDCP1 promoter in K562 but not in Jurkat. There are CpG sequences around the promoter of CDCP1, which were heavily methylated in Jurkat but not in K562. Addition of demethylating reagent to Jurkat induced CDCP1 expression, and increased the zfp67 binding to CDCP1 promoter. Among normal hematopoietic cells such as CD34+CD38- cells, lymphocytes and granulocytes, inverse correlation between proportion of methylated CpG sequences and CDCP1 expression level was found. Demethylation of CpG sequences in lymphocytes, in which CpG sequences were heavily methylated, induced CDCP1 expression and its expression level further increased through zfp67 overexpression. The methylation of DNA appeared to regulate the cell-type-specific expression of CDCP1 through the control of interaction between chromatin DNA and transcription factors.

Antigens, CD↗
PubMed2006

Critical role of ADP interaction with P2Y12 receptor in the maintenance of alpha(IIb)beta3 activation: association with Rap1B activation.

OBJECTIVE: Platelet integrin alpha(IIb)beta3 plays a crucial role in platelet aggregation, and the affinity of alpha(IIb)beta3 for fibrinogen is dynamically regulated. Employing modified ligand-binding assays, we analyzed the mechanism by which alpha(IIb)beta3 maintains its high-affinity state. METHODS AND RESULTS: Washed platelets adjusted to 50 x 10(3) microL(-1) were stimulated with 0.2 U mL(-1) thrombin or 5 microm U46619 under static conditions. After the completion of alpha(IIb)beta3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated alpha(IIb)beta3 was then detected by fluorescein isothiocyanate (FITC)-labeled PAC1. The addition of 1 mum AR-C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained alpha(IIb)beta3 activation by approximately 92% and approximately 38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 microL(-1) also disrupted the sustained alpha(IIb)beta3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5'-diphosphate (ADP) in a dose-dependent fashion. The amounts of ADP released from activated platelets determined by high-performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL(-1) thrombin. Thrombin induced long-lasting PKC and Rap1B activation. AR-C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. CONCLUSIONS: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of alpha(IIb)beta3 activation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2005

Expression and subcellular localization of WAVE isoforms in the megakaryocyte/platelet lineage.

WAVE isoforms, which consist of WAVE-1, WAVE-2 and WAVE-3, are members of the Wiskott-Aldrich syndrome protein (WASP) family. They are implicated in the regulation of actin-cytoskeletal reorganization downsteam of the small GTPase, Rac. Since platelet attachment to extracellular matrices leads to filopodial and lamellipodial extension, we examined the expression and subcellular localization of WAVEs in platelets. Employing primary megakaryocytic cells derived from cord blood as well as megakaryocytic cell lines, we also examined their expression during megakaryocytic differentiation. Immunoblotting and immunohistochemical analysis revealed that platelets expressed WAVE-1 and WAVE-2, whereas WAVE-3 expression was hardly to be detected. WAVE-1 expression was associated with megakaryocytic differentiation, whereas WAVE-2 and WAVE-3 expression was not changed by the differentiation. In adhered platelets both WAVE-1 and WAVE-2 were localized at the edge of the lamellipodia and at the tips of filopodia. In WASP-deficient platelets we found normal lamellipodial formation and localization of WAVE-1 and WAVE-2 at the edges of lamellipodia. Furthermore, we demonstrated that WAVE-1 and WAVE-2 moved from a detergent-soluble cytosolic fraction to insoluble cytoskeleton fraction after platelet aggregation. These results suggest that WAVE-1 and WAVE-2 regulate actin reorganization during platelet spreading and aggregate formation.

Blood Platelets↗
PubMed2005

Impaired platelet function in a patient with P2Y12 deficiency caused by a mutation in the translation initiation codon.

In this study, we have identified a patient (OSP-1) with a congenital P2Y12 deficiency showing a mild bleeding tendency from her childhood and examined the role of P2Y12 in platelet function. At low concentrations of agonists OSP-1 platelets showed an impaired aggregation to several kinds of stimuli, whereas at high concentrations they showed a specifically impaired platelet aggregation to adenosine diphosphate (ADP). ADP normally induced platelet shape change and failed to inhibit PGE1-stimulated cAMP accumulation in OSP-1 platelets. Molecular genetic analysis revealed that OSP-1 was a homozygous for a mutation in the translation initiation codon (ATG to AGG) in the P2Y12 gene. Heterologous cell expression of wild-type or mutant P2Y12 confirmed that the mutation was responsible for the deficiency in P2Y12. OSP-1 platelets showed a markedly impaired platelet spreading onto immobilized fibrinogen. Real-time observations of thrombogenesis under a high shear rate (2000 s(-1)) revealed that thrombi over collagen were small and loosely packed and most of the aggregates were unable to resist against high shear stress in OSP-1. Our data suggest that secretion of endogenous ADP and subsequent P2Y12-mediated signaling are critical for platelet aggregation, platelet spreading, and as a consequence, for stabilization of thrombus.

Aged↗
PubMed2003

Oncogenic receptor tyrosine kinase in leukemia.

Growth, survival and differentiation of hematopoietic cells are regulated by the interaction between hematopoietic growth factors and their receptors. While the defect in this interaction results in an insufficient hematopoiesis, the aberrantly elevated activation leads to the transformation of hematopoietic cells. The constitutive active mutations of receptor tyrosine kinase, such as c-Kit platelet-derived growth factor receptor (PDGFR) or fins-like tyrosine kinase 3 (Flt3), play a major role in the development of hematopoietic neoplasia. The constitutive activation is provoked by several mechanisms, such as making fusion genes by chromosomal translocations, or various mutations involving regulatory regions of the receptor. The chromosomal translocation brings the receptor intracytoplasmic domain juxtaposed to an unrelated molecule which has dimerization or multimerization motif, resulting in the constitutive dimerization of the receptor. The missense, insertion or deletion mutations in the regulatory regions, such as juxtamembrane domain, activation loop and extracellular domain, cause constitutive activation by releasing the respective auto-inhibitory functions of each regulatory region. Constitutive active receptors generate different signals quantitatively and qualitatively from wild type receptor, which mediate the oncogenic phenotype. Given the frequent involvement of constitutive active receptor tyrosine kinase in hematopoietic malignancies, targeted inhibitions of active tyrosine kinase and downstream aberrant signaling are rapidly developing novel therapeutic modality with much promise.

Animals↗
PubMed2001

Efficient retrovirus-mediated PIG-A gene transfer and stable restoration of GPI-anchored protein expression in cells with the PNH phenotype.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder characterized by complement-mediated hemolysis due to deficiencies of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in subpopulations of blood cells. Acquired mutations in the X-linked phosphatidylinositol glycan-class A (PIG-A) gene appear to be the characteristic and pathogenetic cause of PNH. To develop a gene therapy approach for PNH, a retroviral vector construct, termed MPIN, was made containing the PIG-A complementary DNA along with an internal ribosome entry site and the nerve growth factor receptor (NGFR) as a selectable marker. MPIN transduction led to efficient and stable PIG-A and NGFR gene expression in a PIG-A-deficient B-cell line (JY5), a PIG-A-deficient K562 cell line, an Epstein-Barr virus-transformed B-cell line (TK-14(-)) established from a patient with PNH, as well as peripheral blood (PB) mononuclear cells from a patient with PNH. PIG-A expression in these cell lines stably restored GPI-AP expression. MPIN was transduced into bone marrow mononuclear cells from a patient with PNH, and myeloid/erythroid colonies and erythroid cells were derived. These transduced erythroid cells restored surface expression of GPI-APs and resistance to hemolysis. These results indicate that MPIN is capable of efficient and stable functional restoration of GPI-APs in a variety of PIG-A-deficient hematopoietic cell types. Furthermore, MPIN also transduced into PB CD34(+) cells from a normal donor, indicating that MPIN can transduce primitive human progenitors. These findings set the stage for determining whether MPIN can restore PIG-A function in multipotential stem cells, thereby providing a potential new therapeutic option in PNH.

3T3 Cells↗
PubMed2001

Downregulation of an AIM-1 kinase couples with megakaryocytic polyploidization of human hematopoietic cells.

During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3-dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-ras(G12V)), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes.

Animals↗
PubMed2001

Homogenous enzyme immunoassay for cyclosporine in whole blood using the EMIT 2000 cyclosporine specific assay with the COBAS MIRA-plus analyzer.

We describe the evaluation of the EMIT 2000 cyclosporine specific assay kit, an enzyme-multiplied immunoassay for cyclosporine in whole blood, with a COBAS MIRA-plus analyzer. The enzyme used for the assay was glucose-6-phosphate dehydrogenase (EC 1.1.1.49 G6PDH) from Leuconostoc mesenteroides; the monoclonal antibody is fairly specific for cyclosporine, and is not reactive with most metabolites. The assay principle is based on competitive immunoassay with G6PDH-labeled cyclosporine and cyclosporine in sample to the anticyclosporine mouse monoclonal antibody binding site. The within-assay coefficient of variation (CV) of this method was 2.7-4.2% (n = 10) at the levels of 56.2-339.7 microg/L. Day-to-day CVs ranged from 4.2-8.1% at the levels of 47.2-350.2 microg/L. The within-day CVs ranged from 2.0-6.4% at the levels of 43.3-330.5 microg/L. The functional detection limit was 24.9 microg/L. Samples treated with pretreatment reagent were stable at least 5 hr. Calibration was stable at least 10 days. The analytical recovery was 81-109%. The correlation between values obtained with the EMIT 2000 cyclosporine specific assay kit (y) and fluorescence polarization immunoassay (FPIA) (TDxFLx) (x) was: y = 0.880x - 13.053 microg/L (r = 0.984, Sy/x = 15.968, n = 71) with a mean difference of 31.42 +/- 19.89 microg/L ((TDxFLx - EMIT 2000) +/- SD); for the FPIA (AxSYM) (x): y = 0.989 - 4.144 microg/L (r = 0.981, Sy/x = 17.478, n = 71) with a mean difference of 5.56 +/- 17.38 microg/L ((AxSYM - EMIT 2000) +/- SD); and for the radioimmunoassay (RIA, CYCLO-Trac SP) (x): y = 0.893 - 6.764 microg/L (r = 0.993, Sy/x = 10.582, n = 71) with a mean difference of 22.18 +/- 14.98 microg/L ((RIA - EMIT 2000) +/- SD) using the Bland-Altman technique.

Chromatography, High Pressure Liquid↗
PubMed2001

Anti-prothrombin antibodies combined with lupus anti-coagulant activity is an essential risk factor for venous thromboembolism in patients with systemic lupus erythematosus.

Anti-prothrombin antibodies (anti-prothrombin) and anti-beta2-glycoprotein I antibodies (anti-beta2-GP I) are the most common and characterized anti-phospholipid antibodies (aPL) detected using specific enzyme-linked immunosorbent assay (ELISA) systems. Recently, lupus anti-coagulant (LA) activity detected by a phospholipid-dependent coagulation assay was reported to be associated with anti-prothrombin and/or anti-beta2-GP I. Here we show that the co-existence of IgG anti-prothrombin and LA activity might be an essential risk factor for venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). We examined not only the levels of antibodies to prothrombin and anti-beta2-GP I (both IgG and IgM isotypes) using an ELISA system, but also LA activity detected using both diluted Russell's viper venom time (dRVVT) and STACLOT LA test in 124 patients with SLE. The SLE patients were divided into four groups according to the results of ELISA and LA assay results for each aPL: group A, ELISA+ and LA+ group B, ELISA+ and LA-; group C, ELISA- and LA+ group D, ELISA- and LA-. Regarding IgG anti-prothrombin, the prevalence of VTE was significantly higher in group A (16/35 cases, 45.7%, P < 0.001, Fisher's exact probability test) than in the other groups (B, 2/30, 6.7%; C, 1/22, 4.5%; D, 1/37, 2.7%). With respect to IgM anti-prothrombin and IgG or IgM anti-beta2-GP I, the prevalence of VTE was higher in both groups A and C than in group D, but no statistical difference in prevalence was found between groups A and C. Multivariate logistic regression analysis of risk factors for VTE confirmed that the co-existence of IgG anti-prothrombin and LA activity was the only significant risk factor for VTE (odds ratio, 19.13; 95% confidence intervals, 4.74-77.18).

Adolescent↗
PubMed2001

Frequency of natural coagulation inhibitor (antithrombin III, protein C and protein S) deficiencies in Japanese patients with spontaneous deep vein thrombosis.

One hundred and thirteen consecutive Japanese patients with deep venous thrombosis (DVT) were studied for the incidences of antithrombin III (AT-III), protein C (PC) and protein S (PS) deficiencies, and the results were compared with those of normal subjects. Ten of the 392 normal Japanese subjects were found with PS deficiency (n = 8, 2.02%) or PC deficiency (n = 2, 0.5%). PS deficiencies comprised type I (1/8, 12.5%), type 11 (4/8, 50%), and type III (3/8, 37.5%). All PC deficiencies were type I. Among patients with DVT, 32 (28.3%) were deficient in AT-III, PC and PS. These patients consisted of two AT-III deficiency (1.77%), nine PC deficiency (7.96%), 20 PS deficiency (17.7%), and one combined deficiency of PC and PS (0.88%). Both of the patients with AT-III deficiency were classified as type II, all those with PC deficiency as type I, and those with PS deficiency as type I in 25% (5/20), type II in 55% (11/20) and type III in 20% (4/20). The frequency of PC and PS deficiencies in patients with DVT were 15.6 and 7.38 times the control population frequency, respectively, and this difference was statistically significant (P < 0.05). These data suggest that the Japanese population has a high frequency of PC and PS deficiencies. We recommend that PS activity should be measured for screening of thrombosis since type II deficiency accounted for approximately 50% of PS deficiency cases in both patients and the normal group in the Japanese.

Adolescent↗
PubMed2001

Moxibustion treatment of breech presentation.

Breech presentation was successfully corrected by stimulating acupuncture points with moxibustion or low-frequency electrical current. Only patients with breech pregnancies at the 28th week or later were entered into the study. With moxibustion treatment, the control group had a spontaneous correction rate of 165/224 (73.66%), and the treatment group had a correction rate of 123/133 (92.48%) (P<0.0001, x2 test). With low-frequency percutaneous electrical stimulation, the correction rate was 20/941 (83.87%) in the control group and 171/191 (89.52%) in the treatment group (P=0.094, x2 test). The controls in the moxibustion study did no exercises and received no external manipulation to correct breech presentation whereas those in the electrical stimulation study experienced both. Acupuncture stimulation, especially with moxibustion, is expected to serve as a safe and effective modality in the management of breech presentation in a clinical setting.

Acupuncture Points↗
PubMed2001

[Informed consent for the use of the remaining portion of laboratory specimens for education, research, and quality control of laboratory tests].

The remaining portion of a laboratory specimen is usually used for education, research, and quality control of laboratory tests in hospitals, but informed consent has not been obtained because of the high volume of patients who undergo laboratory tests. However, patients must be informed in some manner. Therefore, we decided to inform patients that any remaining specimen would be used for various purposes by placing such a notice on walls in the central clinical laboratory and hospital lobby. We then obtained a signature on a dissent document, instead of a consent document, from any patient who dissented from such use. This indirect process for obtaining informed consent was approved by the ethics committee of Osaka University Medical School. The number of dissent documents sent in to the director was 54 of about 400,000 patients who underwent laboratory tests over the last 3 years, and there was no complaint against this "informed consent process".

Clinical Laboratory Techniques↗
PubMed2001

Whole-body hybrid PET with 18F-FDG in the staging of non-Hodgkin's lymphoma.

UNLABELLED: PET with a double-head gamma camera (hybrid PET) is a new approach to tumor imaging with 18F-FDG. This study was conducted to clarify the feasibility of whole-body FDG hybrid PET in the staging of non-Hodgkin's lymphoma (NHL) in comparison with PET with a dedicated camera (dedicated PET) and to compare the results of both FDG studies with those of CT and 67Ga scanning as conventional imaging studies (CIS). METHODS: Thirty patients with NHL were prospectively evaluated. The results of the imaging studies regarding detection of the sites involved and staging were compared with each other and with those of the reference standard based on the final overall clinical evaluation. RESULTS: Of the total of 206 sites, whole-body FDG hybrid PET and dedicated PET detected 159 sites (77.2%) and 179 sites (86.9%), respectively. Eighteen of the 20 sites missed by hybrid PET alone consisted of lesions < 1.5 cm. Both FDG studies provided concordant staging results in all but 2 patients. CIS, on the other hand, detected 164 (79.6%) of the 206 sites, 137 of which were also detected by hybrid PET. Hybrid PET detected an additional 22 sites not found by CIS, whereas CIS detected 27 additional sites. Hybrid PET and CIS provided concordant staging results in 19 patients. Hybrid PET correctly staged NHL in 5 additional patients, whereas CIS correctly staged NHL in only 1 additional patient. CONCLUSION: Whole-body FDG hybrid PET appeared to be an accurate method of staging NHL. Despite its poorer image quality compared with dedicated PET, hybrid PET provided NHL staging results comparable with those of dedicated PET. Hybrid PET also yielded results comparable with those of CIS. However, whole-body FDG hybrid PET is currently inadequate as a single modality for staging NHL and is complementary to CT.

Adolescent↗
PubMed2001

Association between the prevalence of antibodies to beta(2)-glycoprotein I, prothrombin, protein C, protein S, and annexin V in patients with systemic lupus erythematosus and thrombotic and thrombocytopenic complications.

BACKGROUND: Anti-phospholipid (aPL) antibodies (Abs) frequently found in the plasma of patients with systemic lupus erythematosus (SLE) have been associated with thrombotic complications. Our aim was to clarify the roles in thrombosis of aPL Abs that react with complexes of phospholipids and plasma proteins such as beta(2)-glycoprotein I (beta(2)-GPI), prothrombin, protein C, protein S, and annexin V. METHODS: We determined the prevalence of aPL Abs to various phospholipid-binding plasma proteins in SLE patients with arterial thrombosis (30 cases), venous thrombosis (19 cases), thrombocytopenia (14 cases), fetal loss (14 cases), and patients without complications (91 cases). The aPL Abs were measured by an ELISA system in which human plasma proteins (beta(2)-GPI, prothrombin, protein C, protein S, and annexin V) were immobilized on gamma-irradiated or plain polystyrene plates. RESULTS: All types of aPL Abs were frequently observed in the patients with SLE when gamma-irradiated polystyrene plates were used (51 of 168 cases positive for anti-beta(2)-GPI, 94 of 168 cases positive for anti-prothrombin, 36 of 168 cases positive for anti-protein C, 47 of 168 cases positive for anti-protein S, and 50 of 168 cases positive for anti-annexin V), whereas no Abs to these plasma proteins were detected when plain polystyrene plates were used. Multivariate analysis confirmed that both anti-beta(2)-GPI and anti-prothrombin Abs were significant risk factors for arterial thrombosis [odds ratios (ORs), 8.8 and 14.5, respectively; 95% confidence intervals (CIs), 3.2-25 and 1.8-116, respectively] but not for venous thrombosis. The presence of anti-protein S Abs was a significant risk factor for venous thrombosis (OR, 30.4; CI, 3.3-281) but not for arterial thrombosis. The only significant risk factor for fetal loss was the presence of anti-annexin V Abs (OR, 5.9; CI, 1.4-14.8). CONCLUSIONS: Patients with SLE frequently have some aPL Abs to beta(2)-GPI, prothrombin, protein C, protein S, and annexin V. Thrombotic complications in SLE may depend on the antigenic specificities of these Abs, alone or in combination.

Adolescent↗
PubMed2000

Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways.

Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD-mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways. (Blood. 2000;96:3907-3914)

Acute Disease↗